Aims

Bone marrow-derived mesenchymal stem cells obtained from bone marrow aspirate concentrate (BMAC) with platelet-rich plasma (PRP), has been used as an adjuvant to hip decompression. Early results have shown promise for hip preservation in patients with osteonecrosis (ON) of the femoral head. The purpose of the current study is to examine the mid-term outcome of this treatment in patients with precollapse corticosteroid-induced ON of the femoral head.

Autografts containing bone marrow (BM) are current gold standard in the treatment of critical size bone defects, delayed union and bone nonunion defects. Although reaching unprecedented healing rates in bone reconstruction, the mode of action and cell-cell interactions of bone marrow mononuclear cell (BM-MNC) populations have not yet been described. BM-MNCs consist of a heterogeneous mixture of hematopoetic and non-hematopoetic lineage fractions. Cell culture in a 3D environment is necessary to reflect on the complex mix of these adherend and non-adherend cells in a physiologically relevant context. Therefore, the main aim of this approach was to establish conditions for a stable 3D BM-MNC culture to assess cellular responses on fracture healing strategies. BM samples were obtained from residual material after surgery with positive ethical vote and informed consent of the patients. BM-MNCs were isolated by density gradient centrifugation, and cellular composition was determined by flow cytometry to obtain unbiased data sets on contained cell populations. Collagen from rat tail and human fibrin was used to facilitate a 3D culture environment for the BM-MNCs over a period of three days. Effects on cellular composition that could improve the regenerative potential of BM-MNCs within the BM autograft were assessed using flow cytometry. Cell-cell-interactions were visualized using confocal microscopy over a period of 24 hours. Cell localization and interaction partners were characterized using immunofluorescence labeled paraffin sectioning. Main BM-MNC populations like Monocytes, Macrophages, T cells and endothelial progenitor cells were determined and could be conserved in 3D culture over a period of three days. The 3D cultures will be further treated with already clinically available reagents that lead to effects even within a short-term exposure to stimulate angiogenic, osteogenic or immunomodulatory properties. These measures will help to ease the translation from “bench to bedside” into an intraoperative protocol in the end

Aims. Minimally manipulated cells, such as autologous bone marrow concentrates (BMC), have been investigated in orthopaedics as both a primary therapeutic and augmentation to existing restoration procedures. However, the efficacy of BMC in combination with tissue engineering is still unclear. In this study, we aimed to determine whether the addition of BMC to an osteochondral scaffold is safe and can improve the repair of large osteochondral defects when compared to the scaffold alone. Methods. The ovine femoral condyle model was used. Bone marrow was aspirated, concentrated, and used intraoperatively with a collagen/hydroxyapatite scaffold to fill the osteochondral defects (n = 6). Tissue regeneration was then assessed versus the scaffold-only group (n = 6). Histological staining of cartilage with alcian blue and safranin-O, changes in chondrogenic gene expression, microCT, peripheral quantitative CT (pQCT), and force-plate gait analyses were performed. Lymph nodes and blood were analyzed for safety. Results. The results six months postoperatively showed that there were no significant differences in bone regrowth and mineral density between BMC-treated animals and controls. A significant upregulation of messenger RNA (mRNA) for types I and II collagens in the BMC group was observed, but there were no differences in the formation of hyaline-like cartilage between the groups. A trend towards reduced sulphated glycosaminoglycans (sGAG) breakdown was detected in the BMC group but this was not statistically significant. Functional weightbearing was not affected by the inclusion of BMC. Conclusion. Our results indicated that the addition of BMC to scaffold is safe and has some potentially beneficial effects on osteochondral-tissue regeneration, but not on the functional endpoint of orthopaedic interest. Cite this article: Bone Joint Res 2021;10(10):677–689

Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice. Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1. +. ) and stem cell factor receptor, CD117 (c-kit. +. ) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1. +. mononuclear cell (MNC. sca-1+. ) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC. sca-1+,c-kit+. counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours. Sca-1. +. mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1′-dioctadecyl- 3,3,3,’3′-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium. Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures

Introduction and Objective. The early pro-inflammatory hematoma phase of bone healing is characterized by platelet activation followed by growth factor release. Bone marrow mesenchymal stromal cells (MSC) play a critical role in bone regeneration. However, the impact of the pro-inflammatory hematoma environment on the function of MSC is not fully understood. We here applied platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of MSC in comparison to a non-inflammatory control environment simulated by fibrin (FBR) hydrogels. Materials and Methods. MSC were isolated from acetabular bone marrow of patients undergoing hip arthroplasty. PRP was collected from pooled apheresis thrombocyte concentrates. The phenotype of MSC was analyzed after encapsulation in hydrogels or exposure with platelet-derived factors with regards to gene expression changes, cell viability, extracellular vesicle (EV) release and immunomodulatory effects utilizing cellular and molecular, flow cytometry, RT-PCR, western blot and immunofluorescence stainings. Results. Our results showed that encapsulation of MSC in PRP induced changes in cell metabolism increasing lactate production and reducing mitochondria membrane potential. This was followed by significantly decreased mTOR phosphorylation and differential gene regulation. While PRP-released factors could support EV-biogenesis and immunoregulation-related gene expression, FBR hydrogel reduced CD63+ and CD81+ EV release by MSC. In co-cultures with mitogen stimulated PBMC, pre-exposure of MSC with PRP reduced the proliferation rate and frequency of peripheral blood CD4. +. and favored the persistence of FOXP3. +. regulatory T lymphocytes (32±4.7% compared to 9±2.3% in control co-cultures where MSC were exposed to FBR). Conclusions. Our data indicate that exposure of MSC with a hematoma environment causes metabolic adaptation of MSC followed by increased immune regulatory functions, which in turn might contribute to resolution of inflammation required for successful bone healing

Aim. Autologous-labeled leukocytes combined with sulfur colloid bone marrow scan is the current imaging modality of choice for diagnosing prosthetic joint infection (PJI). Although this technique is reliable, in-vitro leukocyte labeling raises technical difficulties that limit its widespread use and sulfur colloid is increasingly difficult to obtain. Therefore, valid alternatives are needed. The purpose of our study was to determine the clinical value of 99mTc-sulesomab combined with 99mTc-colloidal rhenium sulphide (nanocolloid) bone marrow imaging in the diagnosis of infection in painful total joint arthroplasties. Materials and methods. A retrospective study was conducted on a cohort of 53 patients with painful hip or knee prostheses that underwent 99mTc-sulesomab and 99mTc-nanocolloids sequentially, between January 2008 and December 2016. The combined images were interpreted as positive for infection when there was activity on the sulesomab scan without corresponding activity on the bone marrow scan. The final diagnosis was made with microbiological findings or by clinical follow up of at least 12 months. Results. There were 49 total knee and 4 total hip replacements. Forty of them were women, with an average age of 65 years. Infections were diagnosed in 5 of the 53 patients. An isolated 99mTc-sulesomab scan shows 100% sensitivity but only 29.4% specificity. Combining it with a 99mTc-nanocolloid bone marrow scan, the overall sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 100%, 95.8%, 81.4%, 100% and 96.2% respectively. Conclusion. 99mTc-sulesomab combined with 99mTc-nanocolloid showed to be a useful method for diagnosing prosthetic joint infections. These technically simpler and ready-to-use products may be an alternative to autologous-labeled leukocytes/sulfur colloid marrow scan, although it needs validation at a larger scale

Objectives. Long bone defects often require surgical intervention for functional restoration. The ‘gold standard’ treatment is autologous bone graft (ABG), usually from the patient’s iliac crest. However, autograft is plagued by complications including limited supply, donor site morbidity, and the need for an additional surgery. Thus, alternative therapies are being actively investigated. Autologous bone marrow (BM) is considered as a candidate due to the presence of both endogenous reparative cells and growth factors. We aimed to compare the therapeutic potentials of autologous bone marrow aspirate (BMA) and ABG, which has not previously been done. Methods. We compared the efficacy of coagulated autologous BMA and ABG for the repair of ulnar defects in New Zealand White rabbits. Segmental defects (14 mm) were filled with autologous clotted BM or morcellized autograft, and healing was assessed four and 12 weeks postoperatively. Harvested ulnas were subjected to radiological, micro-CT, histological, and mechanical analyses. Results. Comparable results were obtained with autologous BMA clot and ABG, except for the quantification of new bone by micro-CT. Significantly more bone was found in the ABG-treated ulnar defects than in those treated with autologous BMA clot. This is possibly due to the remnants of necrotic autograft fragments that persisted within the healing defects at week 12 post-surgery. Conclusion. As similar treatment outcomes were achieved by the two strategies, the preferred treatment would be one that is associated with a lower risk of complications. Hence, these results demonstrate that coagulated BMA can be considered as an alternative autogenous therapy for long bone healing. Cite this article: Z. X. H. Lim, B. Rai, T. C. Tan, A. K. Ramruttun, J. H. Hui, V. Nurcombe, S. H. Teoh, S. M. Cool. Autologous bone marrow clot as an alternative to autograft for bone defect healing. Bone Joint Res 2019;8:107–117. DOI: 10.1302/2046-3758.83.BJR-2018-0096.R1

Bone marrow is an environment rich in its diversity of cell types and niches. Both hematopoietic and osteogenic stromal cells are present and have been studied extensively. Less is known about the function of one of the most abundant cell types in the bone marrow: adipocytes. There are several hypotheses that have been proposed including: passive role as a space filler; active role in the body's general lipid metabolism; role in providing a localized energy reservoir for emergency situations affecting the bone or hematopoiesis; support of differentiation or function of other cell types (such as bone, endothelial, and other stromal cells). There are several human pathologies associated with increases in adipocyte hypertrophy or proliferation including changes associated with aging, osteoporosis, and osteonecrosis. The reasons for these changes are poorly understood. One etiology associated with both osteoporosis and osteonecrosis, corticosteroid therapy, has been shown to increase the lipid content of osteoblasts and adipocytes. With osteonecrosis, several pathogenetic mechanisms involving adipocytes have been proposed:. Mechanical - increased size and number cause increased intraosseous pressure and decreased venous outflow. Direct precursor cells away from osteoblastogenesis towards adipogenesis. Liquid fat causing a hypercoagulable state. Osteocyte dysfunction or apoptosis. Adipocyte and bone marrow necrosis. Release adipokines and other factors that have an effect on the cells within the bone marrow (inhibiting angiogenesis, e.g.). The possibility that adipocytes may actually play an active role in propagating specific pathologic features has only recently been discussed. This is in part due to our increasing understanding that adipocytes have an endocrine role in metabolism. Only recently have scientists tried to identify specific cellular mechanisms that may be involved in the pathogenesis of osteonecrosis. Results from these studies will not only contribute to our understanding of the disease of osteonecrosis (and other diseases such as osteoporosis) but will also help us to appreciate the multiple functionalities of the heretofore unappreciated adipocyte

This study was done to determine the effectiveness of percutaneous autologous bone marrow injection in fracture healing and to determine if centrifuged bone marrow is more effective in bone healing as compared to uncentrifuged marrow. This is a randomized interventional trial of 106 patients who had bone marrow injection. The study was done in 2 parts. In the first part, 51 patients were divided into three groups – a) Fresh fractures,(within 6 weeks of injury) b) Delayed union – (8 to 12 weeks after injury) c) Non union – more than 16 weeks after injury. All patients in the first part of the study underwent percutaneous autologous bone marrow injection and were followed up at 6,8,10 and 12 weeks and every 4th week thereafter. Forty seven out of 51 patients united. The second part of the study was done to compare centrifuged and uncentrifuged bone marrow injections. Fifty five patients having either tibial or femoral fractures were divided into two groups, centrifuged and uncentri-fuged and appropriate marrow injection was done. All patients were followed up every 6 weeks till 36 weeks. 48 patients out of 55 united. Equal number of patients united in the centrifuged and uncentrifuged group. We conclude that percutaneous autologous bone marrow injection is a simple and effective tool which can be used for fracture healing and centrifugation of bone marrow yields no added advantage in bone healing

Summary Statement. In articular cartilage defects, chemokines are upregulated and potentially induce the migration of bone marrow cells to accelerate the healing processes. Introduction. The treatment of damaged articular cartilages is one of the most challenging issues in sports medicine and in aging societies. In the microfracture technique for the treatment of articular cartilage defects, bone marrow cells are assumed to migrate from the bone marrow. Bone marrow cells are well-known for playing crucial roles in the healing processes, but how they can migrate from underlying bone marrow remains to be investigated. We have previously shown that SDF-1, one of chemokines, play crucial roles in the recruitment of mesenchymal stem cells in bone healing processes, and the induction of SDF-1 can induce a successful bone repair. If the migration can be stimulated by any means in the cartilage defects, a better result can be expected. The aim of this study was to elucidate the mechanisms of the migration of bone marrow cells and which factors contribute to the processes. Materials & Methods. Articular cartilage defects of 2 mm of diameter were created by drilling the cartilage with a wire to just the subchondral bone in 5-week-old SD rats. The width and depth of the created defects were confirmed by HE staining in histology. The healing tissues were harvested at days 2, 6, and 14 after the operation, and total RNAs were entracted. PCR array was conducted according to the manufacturer's instruction. Quantitative PCR (qPCR) was performed using cDNA of the healing tissues. Bone marrow cells were harvested from 5-week-old SD rat, and a standard migration assay was performed using chemokines. Results. CCL2, CCL3, CCL7 and CCL12 were upregulated in the healing tissues of cartilage defects shown by PCR array. The expression pattterns were confirmed by an expression analysis by qPCR. Both CCL2 and CCL3 induced the migration of bone marrow cells in the in vitro migration assay. Discussion/Conclusion. This study showed for the first time that CCL chemokines are upregulated in the articular cartilage defects and induce the migration of bone marrow cells. These results lead to an innovative measures along with an appropriate delivery method in induction the migration of bone marrow cells from the underlying bone marrow to stimulate articular cartilage healing processes

Cutting rodent's bone ends and irrigation of the medullary canal is the common method used for cells collection in allogenic transplantation, however it does not yield sufficient cells for autologous transplantation. The aim of this experiment was to establish and validate a method for bone marrow collection for autologous MSCs transplantation. Two collection methods were examined: 1) Transection of the bone ends and irrigation of the medullary canal, 2) Trephining of the bone with a hypodermic needle without aspiration. Then cell harvesting was compared in the idealised laboratory situation and under simulated surgery. First, two lower limbs were harvested from the same rat cadaver for comparison, bone marrow in one limb was collected by cutting the femoral head and the distal tibia and irrigation of the canal through drilled holes at the distal end of the femur and proximal end of the tibia. Other limb, hypodermic needle was used as a trephining tool into the medullary canal multiple times without applying negative pressure and rinsed from inside and outside. Second, bone marrow was harvested from another rat's cadaver in the surgery room to simulate the conditions needed for autologous transplantation. The number of cells from irrigation method was 1.28*106 cells, whereas that from trephining method reached 17*106. The number cells from the bone marrow harvested in the surgery room was found 29.6*106. We report a novel technique for harvesting cells for autologous cell therapy from only one limb. A significantly larger number of cells from bone marrow could be collected using the needle trephining method. There is no negative effect on the viability of cells after bone marrow harvesting in the surgery room

Fracture nonunion is a severe clinical problem for the patient, as well as for the clinician. About 5-20% of fractures does not heal properly after more than six months, with a 19% nonunion rate for tibia, 12% for femur and 13% for humerus, leading to patient morbidity, prolonged hospitalization, and high costs. The standard treatment with iliac crest-derived autologous bone filling the nonunion site may cause pain or hematoma to the patient, as well as major complications such as infection. The application of mesenchymal autologous cells (MSC) to improve bone formation calls for randomized, open, two-arm clinical studies to verify safety and efficacy. The ORTHOUNION * project (ORTHOpedic randomized clinical trial with expanded bone marrow MSC and bioceramics versus autograft in long bone nonUNIONs) is a multicentric, open, randomized, comparative phase II clinical trial, approved in the framework of the H2020 funding programme, under the coordination of Enrique Gòmez Barrena of the Hospital La Paz (Madrid, Spain). Starting from January 2017, patients with nonunion of femur, tibia or humerus have been actively enrolled in Spain, France, Germany, and Italy. The study protocol encompasses two experimental arms, i.e., autologous bone marrow-derived mesenchymal cells after expansion (‘high dose’ or ‘low dose’ MSC) combined to ceramic granules (MBCP™, Biomatlante), and iliac crest-derived autologous trabecular bone (ICAG) as active comparator arm, with a 2-year follow-up after surgery. Despite the COVID 19 pandemic with several lockdown periods in the four countries, the trial was continued, leading to 42 patients treated out of 51 included, with 11 receiving the bone graft (G1 arm), 15 the ‘high dose’ MSC (200x10. 6. , G2a arm) and 16 the ‘low dose’ MSC (100x10. 6. , G2b arm). The Rizzoli Orthopaedic Institute has functioned as coordinator of the Italian clinical centres (Bologna, Milano, Brescia) and the Biomedical Science and Technologies and Nanobiotechnology Lab of the RIT Dept. has enrolled six patients with the collaboration of the Rizzoli’ 3rd Orthopaedic and Traumatological Clinic prevalently Oncologic. Moreover, the IOR Lab has collected and analysed the blood samples from all the patients treated to monitor the changes of the bone turnover markers following the surgical treatment with G1, G2a or G2b protocols. The clinical and biochemical results of the study, still under evaluation, are presented. * ORTHOUNION Horizon 2020 GA 733288

Introduction: Until recently adult stem cells were presumed to be committed to differentiation of specific tissues. Adult hematopoietic stem cells (HSCs, CD34+) for example, originally believed to be limited to hematopoiesis are capable of transdifferentiation to generate cells of different lineages. This capability is referred to as stem cell plasticity. Studies in cardiac and peripheral vascular disease and nonunions and osteonecrosis in orthopedics have demonstrated that concentrated bone marrow is an effective and safe method of treatment. The present study evaluated a methodology to concentrate a small sample of bone marrow at point of care to compare with described techniques. Methods: Sixty or 120 mL of bone marrow was withdrawn from the posterior iliac crest. The concentration process utilized the standard SmartPReP-2/DePuy Symphony Centrifuge (Harvest Technologies, Plymouth, MA). The shape and density of the floating shelf was modified to enhance collection of nuclear cells. The bone marrow was analyzed for cell counts, morphology, and flow cytometry. Hematopoietic stem cells (CD34+) were used as a marker for stem cell concentration. Bone marrow stem cells were cultured using specialized media supplements. The systems were also compared using the hind limb ischemia (HLI) model. Results: Using the Harvest BMC System™ system, the results for the Colony Forming Unit (CFU’s) Analysis were as follows (Mean ± S.D.): the aspirate volume: 120 mL, CFU’s/cm. 3. : 3040±1251, BMC volume delivered: 20mL, and Progenitor cells delivered: 60,800±29,200. The cultures demonstrated viable hMSCs that were identical to a commercially available cell line. The cultures were transferred into osteogenic media; after 10 days the bone marrow derived cells and the commercial cell lines were stained with Von Kossa silver stain and for alkaline phosphatase demonstrating osteoblastic differentiation. Hind limb ischemia studies have demonstrated that laser doppler blood flow was significantly better following BMAC infusion as compared to cells concentrated with Ficoll. These results were confirmed by a Boyden chamber migratory assay. Discussion: A bone marrow concentrate can be prepared at point of care within 15 minutes of collection. The Harvest BMAC system is capable of producing a concentration of stem cells equivalent to or greater than those used in successful clinical studies. Successful clinical results can be obtained using one-third (1/3) of the aspirate volume required by other methods. Ongoing clinical and animal studies are confirming its clinical application

Purpose: Local factors such as poor vascular supply, open fracture, or infection can affect the potential for bone formation after fracture, arthrodesis or distraction. The fundamental principal for the treatment of late healing or nonunion is to supplement the local supply of the elements necessary for bone maturation. Centrifuged bone marrow is known to have a osteogenic effect in the treatment of femoral head necrosis or as a complement to conventional grafts. We examined the effect of bone marrow grafts used with conventional grafts. Material and methods: This retrospective analysis included 14 cases where centrifuged bone marrow graft was used as complementary treatment for post-traumatic nonunion (10 cases), distraction callus (three cases) or late healing after arthrodesis (one case). Bone marrow (300 ml) was harvested from the posterior iliac crest then centrifuged to isolate the maximum number of nucleated cells and stem cells. The centrifugate (60–80 ml) was injected into the fracture site with a trocar during the same operative time. Cell concentrations (total nucleated cells, stem cells (CFU-GM), fibroblastic colonies) were noted. Patients were followed at regular visits. Bone healing was considered to be acquired when weight-bearing was possible without fixation or immobilisation. Results: Definitive bone healing was achieved rapidly in two cases. Two patients required a conventional graft of a nonunion to achieve consolidation. For six patients, consolidation could not be achieved (three nonunions and three distraction calluses). Final outcome was good or very good in 57% of the cases. Mean delay to bone healing was 6.5 months. The infectious context had no effect on the method. The mean number of nucleated cells injected was 3.9•109 cells in successful cases and 2.8•109 cells in unsuccessful cases. These concentrations affected outcome. Discussion: This technique for stimulating bone maturation by supplying bone generating cells is indicated for late healing or recent nonunion. It is less effective for distraction calluses or for very old nonunions. Morbidity and iatrogenic effects are minimal. A rigorous harvesting method is required since the result is highly dependent on the cell concentrations and the number of injected cells. Bone marrow injections after centrifugation should be greater than 85 ml and have a cell concentration around 45•106 cells/ml. The method is less successful for old injuries and in patients with arteritis. Conclusion: Bone marrow grafts are indicated for the treatment of late healing or recent nonunion. Morbidity is low but a rigorous harvesting method is required. The method should be implemented shortly after the fracture without waiting for potential signs of nonunion

Stabilization and bone grafting are the basic principles in the treatment of fracture non-union, however, infection is always a concern. Percutaneous bone marrow grafting has been suggested as an alternative, which provides a source of osteogenic cells with osteoinductive effect. This prospective study evaluates the efficacy of percutanous bone marrow grafting in patients with tibial non-union while on the waiting list for open surgical procedures. 21 adult patients with established tibial non-union were recruited. The average age of fracture non-union was 12 months (range 6–36). Infected cases, deformed non-unions and gap non-unions were excluded. Eleven were hypertrophic and ten atrophic type of non-union. Under local anaesthesia, bone marrow was aspirated from the iliac crests using a 16 G sternal puncture needle. 3–5ml marrow was aspirated and injected immediately into and about the non-union site. Subsequent aspirations were performed 1 cm posterior to the previous site until a maximum of 15 ml marrow was injected. Patients were immobilised in a plaster cast. Radiographs were repeated at 6 weeks interval. A second injection was repeated at 6 weeks if there was no evidence of callus formation. The procedure was considered a failure, if there was no union at six weeks following the third injection. Bone marrow could not be aspirated in one patient. 19 patients were followed up clinically and radiologically until there was definite bone union or failure. Bone union was achieved in 15 patients out of 20 (75%), with an average time to union following the first injection 14 weeks (range 6–22 ). Two of the patients needed only one injection, nine needed two injections, and four patients needed three injections to unite. 4 patients (20%) showed no evidence of union. There were no complications at the donor or recipient site. We conclude that percutanous bone marrow grafting is a safe, simple, and reliable method of treating tibial non-union with minimal deformity

Aims. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been reported to be a promising cellular therapeutic approach for various human diseases. The current study aimed to investigate the mechanism of BMSC-derived exosomes carrying microRNA (miR)-136-5p in fracture healing. Methods. A mouse fracture model was initially established by surgical means. Exosomes were isolated from BMSCs from mice. The endocytosis of the mouse osteoblast MC3T3-E1 cell line was analyzed. CCK-8 and disodium phenyl phosphate microplate methods were employed to detect cell proliferation and alkaline phosphatase (ALP) activity, respectively. The binding of miR-136-5p to low-density lipoprotein receptor related protein 4 (LRP4) was analyzed by dual luciferase reporter gene assay. HE staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry were performed to evaluate the healing of the bone tissue ends, the positive number of osteoclasts, and the positive expression of β-catenin protein, respectively. Results. miR-136-5p promoted fracture healing and osteoblast proliferation and differentiation. BMSC-derived exosomes exhibited an enriched miR-136-5p level, and were internalized by MC3T3-E1 cells. LRP4 was identified as a downstream target gene of miR-136-5p. Moreover, miR-136-5p or exosomes isolated from BMSCs (BMSC-Exos) containing miR-136-5p activated the Wnt/β-catenin pathway through the inhibition of LRP4 expression. Furthermore, BMSC-derived exosomes carrying miR-136-5p promoted osteoblast proliferation and differentiation, thereby promoting fracture healing. Conclusion. BMSC-derived exosomes carrying miR-136-5p inhibited LRP4 and activated the Wnt/β-catenin pathway, thus facilitating fracture healing. Cite this article: Bone Joint Res 2021;10(12):744–758

Invertebral disc degeneration (IDD) is a degenerative disease involving a variety of musculoskeletal and spinal disorders such as lower back pain (LBP). Secretome derived from mesenchymal stem cells (MSCs) have exerted beneficial effect on tissue regeneration. In this study, the goal was to investigate the paracrine and the anti-inflammatory effects of secretome from interleukin IL1β preconditioned Bone Marrow MSCs (BMSCs) on human nucleus pulposus cells (hNPCs) in a 3D in vitro model. Secretome was collected from BMSCs (BMSCs-sec) after preconditioning with 10 ng/mL IL1β. hNPCs were isolated from surgical specimens, culture expanded in vitro, encapsulated in alginate beads and treated with: growth medium; IL1β 10 ng/mL; IL1β 10 ng/mL for 24 hours and then BMSCs-sec. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess assay) and ROS quantification (Immunofluorescence) iii) glycosaminoglycan (GAG) amount (DMBB) and iv) gene expression levels of extracellular matrix (ECM) components and inflammatory mediators (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D. In vitro tests showed an enhancement of hNPCs proliferation after treatment with BMSCs-sec (p ≤ 0.05) compared to IL1β group. After 24 hours, the percentage of dead cells was higher in IL1β treated hNPCs compared to control group and decreased significantly in combined IL1β and BMSCs-sec sample group (p ≤ 0.01). Nitrite and ROS production were significantly mitigated and GAGs content was improved by preconditioned BMSCs-sec (p ≤ 0.05). Furthermore, gene expression levels were modulated by BMSCs-sec treatment compared to controls. Our results supported the potential use of BMSCs' secretome as a cell-free strategy for IDD, overcoming the side effects of cell-therapy. Moreover, secretome derived from IL1β preconditioned BMSCs was able to reduce hNPCs death, attenuate ECM degradation and oxidative stress counteracting IDD progression. Acknowledgements: Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects

Introduction: Articular cartilage injuries are very common, and if untreated can become symptomatic and progressively lead to premature osteoarthritis. It is well known that damaged cartilage has very limited potential to heal itself, and repair and regeneration of hyaline cartilage remain a clinical and scientific challenge. There are no pharmacological methods that can regenerate cartilage, and currently clinical treatments of debridement, chondrocyte transplantation and marrow stimulation have not been shown to restore consistently a durable articular surface. Tissue engineering and gene therapy concepts may improve cartilage repair by introducing cells, scaffolds, growth factors and other potential modulators of cartilage healing process. When analyzing cartilage treatment outcomes, traditionally we use macro- and microscopic assessment, immunohistochemistry, biochemical characterization etc. Recently, it has been postulated that biomechanical properties of newly formed cartilage are just as important, and novel methods of measurements have been proposed. Materials and methods: 38 defects were created on weight-bearing part of the medial femoral condyle in sheep. The sheep were randomly assigned to one of four groups. In the bone marrow clot (BMC) group, the sheep were implanted with untreated autologous bone marrow clot that was aspirated from iliac crest of respected animal. In the bone marrow transduced with Ad. GFP (GFP) group, the sheep were implanted with autologous bone marrow clots genetically modified to over express green fluorescent protein (GFP). In the bone marrow transduced with Ad. TGF-β1 (TGF) group, the sheep were implanted with autologous bone marrow clots genetically modified to over express transforming growth factor-β1. Untreated sheep served as a control (defect without implant), and native cartilage served as positive control. Specimens were collected after 6 months and analyzed by single-impact micro-indentation (SIMI), atomic force microscope (AFM) and scanning electron microscope (SEM). Results: SIMI and AFM measurements showed that repair tissue has greater Young’s elastic modulus then native cartilage. There was a statistically significant difference between TGF-β1, GFP and BMC groups. SEM analysis showed presence of structurally organized collagen molecules in TGF-β1, GFP and BMC groups. Conclusion: The results of this study showed that it is possible to enhance cartilage repair process by means of genetically modified bone marrow. Furthermore, biomechanical data obtained with SIMI, AFM and SEM provided more detailed insight into articular cartilage function and structure, and in future may be of practical importance for a better understanding of both cartilage mechanics and cartilage disease progression

Aims. Despite recent advances in arthroscopic rotator cuff repair, re-tear rates remain high. New methods to improve healing rates following rotator cuff repair must be sought. Our primary objective was to determine if adjunctive bone marrow stimulation with channelling five to seven days prior to arthroscopic cuff repair would lead to higher Western Ontario Rotator Cuff (WORC) scores at 24 months postoperatively compared with no channelling. Methods. A prospective, randomized controlled trial was conducted in patients undergoing arthroscopic rotator cuff repair. Patients were randomized to receive either a percutaneous bone channelling of the rotator cuff footprint or a sham procedure under ultrasound guidance five to seven days prior to index surgery. Outcome measures included the WORC, American Shoulder and Elbow Surgeons (ASES), and Constant scores, strength, ultrasound-determined healing rates, and adverse events. Results. Overall, 94 patients were randomized to either bone channelling or a sham procedure. Statistically significant improvements in all clinical outcome scores occurred in both groups from preoperative to all timepoints (p < 0.001). Intention-to-treat analysis revealed no statistical differences in WORC scores between the two interventions at 24 months postoperatively (p = 0.690). No differences were observed in secondary outcomes at any timepoint and healing rates did not differ between groups (p = 0.186). Conclusion. Preoperative bone channelling one week prior to arthroscopic rotator cuff repair was not associated with significant improvements in WORC, ASES, Constant scores, strength, or ultrasound-determined healing rates. Cite this article: Bone Joint J 2021;103-B(1):123–130

In 79 consecutive patients with unicameral bone cysts we compared the results of aspiration and injection of bone marrow with those of aspiration and injection of steroid. All were treated by the same protocol. The only difference was the substance injected into the cysts. The mean radiological follow-up to detect activity in the cyst was 44 months (12 to 108). Of the 79 patients, 14 received a total of 27 injections of bone marrow and 65 a total of 99 injections of steroid. Repeated injections were required in 57% of patients after bone marrow had been used and in 49% after steroid. No complications were noted in either group. In this series no advantage could be shown for the use of autogenous injection of bone marrow compared with injection of steroid in the management of unicameral bone cysts

Aims: to evaluate the efþcacy of this technique in 46 tibias and 22 femurs with a delayed bone healing (> 6 months) with a minimum follow-up of one year after injection. Methods: Forty-þve injections were performed in a one-day clinic. At least 300 ml autologeous bone marrow is aspirated from the iliac crest using multiple aspiration sites. Through isopyknic centrifugation the mixture of bone marrow and phosphateÐbuffered saline was layered over undiluted Ficoll-Paque. Centrifugation was done for 35 min. at 400 times gravity. An average of 52 cc of mainly myeloid cells were obtained with a nucleated recovery rate of average 62% (27–90%). In 21 cases additional surgery was performed at the moment of bone marrow grafting. Eleven times an implant exchange, seven dynamisations and 3 additional bonegrafting. The bone marrow grafting was performed through cannulated screws seated in the medullary cavity below and above the fracture site. Results: we encounter one postoperative irritation of the pes anserinus tendons due to inþltration. Despite the fact that we aspirated an average of 340 cc of bone marrow no adverse reaction was seen from this nor from the sometimes forceful injection of 50 cc concentrated bone marrow. In eight cases no bone healing occurred. In þve cases, probably due to a lack of stability and implant failure. Conclusions: the use of concentrated bone marrow injected in the medullary cavity near the fracture site is cost effective and seems to give favorable results

Aims. Treatment of chronic osteomyelitis (COM) for young patients remains a challenge. Large bone deficiencies secondary to COM can be treated using induced membrane technique (IMT). However, it is unclear which type of bone graft is optimal. The goal of the study was to determine the clinical effectiveness of bone marrow concentrator modified allograft (BMCA) versus bone marrow aspirate mixed allograft (BMAA) for children with COM of long bones. Methods. Between January 2013 and December 2017, 26 young patients with COM were enrolled. Different bone grafts were applied to repair bone defects secondary to IMT procedure for infection eradication. Group BMCA was administered BMCA while Group BMAA was given BMAA. The results of this case-control study were retrospectively analyzed. Results. Patient infection in both groups was eradicated after IMT surgery. As for reconstruction surgery, no substantial changes in the operative period (p = 0.852), intraoperative blood loss (p = 0.573), or length of hospital stay (p = 0.362) were found between the two groups. All patients were monitored for 12 to 60 months. The median time to bone healing was 4.0 months (interquartile range (IQR) 3.0 to 5.0; range 3 to 7) and 5.0 months (IQR 4.0 to 7.0; range 3 to 10) in Groups BMCA and BMAA, respectively. The time to heal in Group BMCA versus Group BMAA was substantially lower (p = 0.024). Conclusion. IMT with BMCA or BMAA may attain healing in large bone defects secondary to COM in children. The bone healing time was significantly shorter for BMCA, indicating that this could be considered as a new strategy for bone defect after COM treatment. Cite this article: Bone Joint Res 2021;10(1):31–40

Introduction: Stabilization and bone grafting are the basic principles in the treatment of fracture non-union. Percutaneous bone marrow grafting has been suggested as an alternative source of osteogenic cells with an osteoindutive effect. Our aim is to assess prospectively, the efþcacy of percutanous bone marrow grafting in atrophic tibial non-union. Methods: 20 patients with established atrophic tibial non-union on the waiting list for surgical treatment were recruited. Under local anaesthesia bone marrow was aspirated from the iliac crest and injected into the fracture site. All patients were immobilized in above knee casts. A second injection was repeated at 6 weeks if there was no evidence of callous formation. The procedure was considered a failure if there was no union at six weeks following a third injection. Results: 19 patient were followed up clinically and radiologically until deþnite bone union or failure. Union occurred in 15 patients (75%), with an average time to union following the þrst injection of 14 weeks (range 6–22). Four patients showed no evidence of union. There were no cases of infection or complication at the donor or recipient site. Discussion: Percutanous bone marrow grafting is effective in inducing bone union. It is a minimally invasive technique and could be performed under local anaesthesia, with minimal cost and the potential to avoid a larger surgical procedure. All our patients were on the waiting list for open bone grafting but only 20% of them needed this. We recommend this technique for atrophic tibial non-unions with minimal deformity

Introduction. Bone marrow stimulation has been a successful treatment option in cartilage repair and microfracture was the procedure of choice since the late 1980s. Despite its success in young and active patients, microfracture has inherent shortcomings such as shallow channels, wall compression, and non-standardized depth and diameter. This in vitro study assessed bone marrow access comparing microfracture, 1 and 2mm K-Wires, 1mm drill, and a recently introduced standardized subchondral bone needling procedure (Nanofracture) that creates 9mm deep and 1mm wide channels. Methods. An adult ovine model was used to assess access to bone the marrow spaces as well as effects on bone following microfracture, nanofracture, K-wire, and drilling following ethical clearance. All bone marrow stimulation techniques were conducted on a full thickness articular cartilage defect on the medial femoral condyles by the same surgeon. The same groups were repeated in vitro in 4 paired ovine distal femurs. MicroCT (Inveon Scanner, Siemens, Germany) was performed using 3D reconstruction and 25 micron slice analysis (MIMICS, Materialise, Belgium). Results. Microfracture elicited shallow depth with bone compression surrounding the channels. Trabecular channel access was limited; the channel depth and diameter were non-standardized and highly user and instrument dependent. Nanofracture demonstrated deep cancellous bone perforation with a high number of open trabecular channels. K-Wire drilling with both diameters resulted in well-defined channel walls, outlined by fine osseous deposits. Trabecular channel access was limited. The diameter of bone perforation is standardized, but depth is defined by visual controls. 1mm drill bit reaming demonstrated better osseous evacuation, but still limited trabecular marrow access. Discussion and Conclusion. Nanofracture resulted in thin, fragmented cancellous bone channels without rotational heat generation. Compared to microfracture, drilling and K-Wire stimulation, nanofracture showed superior bone marrow access with multiple trabecular access channels extending 9mm into subchondral bone

Aims: This study investigates the use of novel autologous bone marrow plugs as a biological ÒmatrixÒ to support transgene expression following genetic modiþcation in vitro, and to deliver gene vectors to cartilage defects in vivo. Methods: Adenoviral vectors encoding marker genes (luciferase, green ßuorescent protein (GFP)) and bioactive genes (TGF-?) as well as genetically modiþed mesenchymalstem cells were used to characterize an autologous delivery system using clots of bone marrow aspirates in vitro, and within rabbit osteochondral defects in vivo. Results: Bone marrow clots were able to support expression of luciferase and TGF-? transgenes for up to 21d. In addition incubation of bone marrow clots with rTGF-? demonstrated, that the clots have chondrogenic potential, as evidenced by type II collagen and proteogly-can staining. Bone marrow clots seeded with cells genetically modiþed to express luciferase were able to support transgene expression following implantation into rabbit osteochondral defects for up to 14 days. Implanted clots were able to remain within the defects without þxation, and considerable integration with surrounding tissue was observed after 3 days. The bone marrow clots were also able to effectively localize transgene expression within the defects without leakage to surrounding tissue. Conclusion: These results demonstrate that genetically modiþed bone marrow plugs can support persistent transgene expression in vitro and within osteochondral defects in vivo. They provide an effective delivery system with chondrogenic potential

Abstract. Purpose. It is becoming apparent that mesenchymal stem cells (MSCs) do not directly contribute to mesenchymal tissue regeneration. Pre-clinical attempts to repair large bone defects in big animal models have been hampered by poor MSCs survival after implantation which impedes their direct or indirect effects. Based on previous work, we hypothesized that a venous axial vascularization of the scaffold supporting MSCs or their combination with fresh bone marrow (BM) aspirate would improve their in vivo survival. Methods. Cross-shape profile tubular microporous monetite implants (12mm long, 5mm large) as two longitudinal halves were produced by 3D powder printing. They were implanted around the femoral veins of Wistar rats and loaded with 1mL of BM aspirate either alone or supplemented by 10. 7. MSCs. This was compared with BM-free scaffolds loaded only with 10. 7. MSCs. After 8 weeks bone formation were investigated by micro-CT, scanning electron microscopy, histology and immunohistochemistry. Results. Little bone formation was observed within the scaffold when it was only loaded with MSCs surprisingly. Coupling MSCs, autologous BM and venous perfusion of the scaffold led to a higher volume of new bone than BM alone suggesting that MSCs augmented the bone formation capacity of BM aspirate or enhanced its survival post implantation. Conclusion. Subcutaneous bone formation within 3D-printed implant that mixed of BM with or without MSCs was successfully achieved for the first time by venous perfusion. The inability of MSCs to form differentiated tissues by their own was confirmed in this study; however, contact between MSCs and BM cells and MSCs paracrine secretome (e.g., cytokines, chemokines, extracellular vesicles) may have induced immunomodulatory effects (e.g., macrophages polarization, Treg cells) that triggered bone formation. This approach, if translatable to large animal models, offers immediate clinical value as well as an insight into the role of immune system in tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported: I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Purpose of the study: The current approach for improving the performance of compact bone substitutes is to seed them with selected mesenchymatous stem cells amplified and differentiated to the osteoblastic line in vitro. We hypothesized that the preservation of all these elements in the bone marrow would be most effective for bone tissue formation. Material and methods: Subcutaneous and intramuscular implantation in C57BL/6 mice. We developed a new approach for bone tissue engineering based on an extemporaneous incorporation of total bone marrow into an injectable bone substitute (IBS2). IBS2 is a new polymerizable hydrogel associated with beads of calcium phosphate (BCP) which can be used to implant total bone marrow. A subcutaneous and intramuscular implantation model in mice was tested to analyze the feasibility of this type of graft. Total bone marrow cells from C57BL/6 male mice were seeded in IBS (10 million cells per 100 microliters). This implant was injected subcutaneously (dorsal position) and intramuscularly (left hind foot) in C57BL/6 female mice. TRAP activity was measured under optical microscopy on paraffin embedded HES stained slices at 4 and 8 weeks. Results and discussion: Incorporation of total bone marrow cells in injectable IBS2 produced implants which were rich in mesenchymatous cells, vessels, osteo-clasts, collagen fibers, and osteoid tissue. This demonstrated the great potential of this new approach. In addition, this method is simple and can be performed in the operative room without ex vivo culture. Comparison of this model of extemporaneous cell therapy with a graft of meschymatous cells amplified ex vivo is currently under way

Purpose: Adding bone marrow cells to ceramic materials provides an osteoprogenic capacity favouring bony regrowth. Likewise, addition of platelets, which contain growth factors, might increase the rate of bone formation. The purpose of this work was to quantify in vitro the osteogenic potential obtained by adding platelet-rich plasma (PRP) to the bone marrow culture on ceramic materials. Material and methods: PRP was obtained by centrifugation of blood and added to bone marrow cells harvested from the iliac crest and cultured on biphasic macroporous ceramic materials. Addition of PRP was repeated with platelet counts every two days. Differentiation of bone marrow cell into cells with osteogenic potential was evaluated by quantifying alkaline phosphatase activity after 15 days culture. Results: Proliferation of mesenchymatous cells was clearly enhanced in cultures with PRP (+31%). Mean prevalence of phasphatase-alkaline-positive colonies was also improved after addition of PRP (+38%). Similarly, alkaline phosphatase activity was higher after addition of PRP (+31%). Discussion: Adjunction of PRP to bone marrow cells cultured on ceramic materials stimulates proliferation of osteoblast-like cells. Increased cell proliferation and differentiation observed in vitro provides quantitative elements favouring the combination of platelets with bone grafts using bone substitutes

Mesenchymal stem cells (MSC) have a well recognised potential for tissue repair. This potential is two pronged: they can differentiate into the functional cell types of the damaged tissues and they can support tissue recovery by secreting trophic factors, depositing an extracellular matrix (ECM) and dampening inflammation. Three-dimensional microscopy recently shown that MSCs in the bone marrow create an intricate proteo-cellular scaffold with the ECM forming an interconnected cellular continuum whose structure is guided by the deposited ECM. This proteo-cellular scaffold controls bone marrow functions from hematopoiesis to osteogenesis. In the current study we aimed to optimise ECM production under in vitro conditions by immortalised MSCs with the view that the generated ECM can be utilised for tissue repair. With immunocytochemistry we determined the deposition of bone marrow-characteristic ECM proteins: collagen I, III, IV, V, VI, laminin and fibronectin. While primary MSCs produced slightly higher amount ECM proteins than immortalised MSCs, the relative abundancy of the ECM proteins was very similar. In order to isolate the ECM, we optimised a decellularisation method based on gentle lysis with sodium-deoxycholate and DNase digestion. Immunostaining for collagen I, III, VI and fibronectin and labelling the nuclei with Hoechst-33342 confirmed removal of all cells while retaining the ECM in its original architecture. Ideally, the decellularised ECM retains associated cytokines and chemokines, such as CXCL12, important for attracting MSCs. To test this, we seeded Molm-13 leukemia cells on decellularised ECM as MSC-produced CXCL12- and other cytokines protect leukemia cells against chemotherapeutics. We found that the decellularisation process however removed these factors and thus for therapeutic purposes, the decellularised ECM would need to be re-loaded with the essential chemo/cytokines. Overall, we developed a system for decellularised ECM production by immortalised MSCs and the results warrant further exploration of this avenue

Osteoporosis is characterised by an uncoupling of bone formation and resorption resulting in a net reduction in bone density. Stem cells derived from bone marrow in osteoporotic patients typically contain more adipocytes,. Intermittent Parathyroid hormone (iPTH), has been shown to cause the preferential differentiation of mesenchymal stem cells (MSCs) to osteoblasts. We isolated rat bone marrow derived MSCs, investigating the effect of iPTH on adipocyte differentiation. MSCs were harvested from the femora of 6–10week oldWT rats and cultured to induce adipogenesis for 21 days. Subsequently, cells were continually cultured in adipogenic media, osteogenic media or in osteogenic media supplemented with PTH 1–34 either continuously or intermittently for 6hours in every 72hour cycle. ALP and Alizarin Red assessed osteogenic differentiation, and Oil Red O used to assess intracellular microdroplet formation. A student t-test was used to analyse results, and a p value<0.05 considered significant. Quantitatively measurements of Alizarin Red staining significantly increased in all adipocytes grown in osteogenic media compared to the cells continually cultured in adipogenic media. Calcium phosphate deposition continued to increase significantly in these groups up to day 14. At day 14, Alizarin Red staining from cells cultured in iPTH were significantly higher than osteogenic media alone. ALP expression was significantly higher for cells cultured in osteogenic media and iPTH compared to adipogenic media at days 3–14. Expression peaked at day 7, at this timepoint cells cultured in iPTH expressed significantly more ALP than other groups. Oil Red O measurements were significantly reduced from days 7–14 for all osteogenic groups, this significance was greatest for the iPTH group at day 7. iPTH increased the transdifferentiation of adipocytes derived from MSCs into osteoblasts, this effect was most significant after 7 days. Ultimately, the role of iPTH on adipocytes may lead to improved bone formation with many orthopaedic applications

Introduction: Cigarette smoking is associated with musculoskeletal degenerative disorders and increased risk of fracture delayed- and non-union. A lower-than-average concentration of mesenchymal stem cells may be the reason for the reduced regenerative potential. The aim of this study was to compare the concentration of bone marrow MSC of smokers and non-smokers. Methods: As part of a larger IRB approved clinical trial, 20ml bone marrow samples were processed and MSC were isolated. FACS analysis was used both to assess the purity of the separation process and to evaluate the number of MSC recovered from each sample. Differences in continuous outcomes between smoking and non-smoking groups were assessed by two tailed t test and difference between categorical outcomes was measured by chi square test. Results: Twenty six subjects participated in the study. Thirteen were smokers and thirteen were non-smokers. Groups were not significantly different with regard to age and gender. The average concentration of MSC was 352.04x10. 3. /ml for non smokers and 131.23x10. 3. /ml for smokers (SD’s were 245.72 x10. 3. /ml and 161.54 x10. 3. / ml respectively. The difference between the smokers and nonsmokers was significant (t=3.2 p=0.004). Discussion: The present study indicates that cigarette smokers have lower-than-average concentration of MSC in their bone marrow. Since MSC are a key element in every regenerative process of the musculoskeletal system, our findings may contribute to understanding and prevention of delayed and non-union. Further investigation is undertaken to address the issue of bone marrow recovery after smoking cessation

Osteoporosis is characterised by an uncoupling of bone formation and resorption resulting in net resorption. Stem cells derived from bone marrow in osteoporotic patients typically contain more adipocytes. Intermittent Parathyroid hormone (iPTH), has been shown to cause the preferential differentiation of mesenchymal stem cells (MSCs) to osteoblasts. We isolated rat bone marrow derived MSCs, investigating the effect of iPTH on adipocyte differentiation. MSCs were harvested from the femora of 6–10week oldWT rats and cultured to induce adipogenesis for 21 days. Subsequently, cells were continually cultured in adipogenic media, osteogenic media or in osteogenic media supplemented with PTH 1–34 either continuously or intermittently for 6hours in every 72hour cycle. ALP and Alizarin Red assessed osteogenic differentiation, and Oil Red O used to assess intracellular microdroplet formation. A student t-test was used to analyse results, and a p value<0.05 considered significant. Quantitatively measurements of Alizarin Red staining significantly increased in all adipocytes grown in osteogenic media compared to the cells continually cultured in adipogenic media. Calcium phosphate deposition continued to increase significantly in these groups up to day 14. At day 14, Alizarin Red staining from cells cultured in iPTH were significantly higher than osteogenic media alone. ALP expression was significantly higher for cells cultured in osteogenic media and iPTH compared to adipogenic media at days 3–14. Expression peaked at day 7, at this timepoint cells cultured in iPTH expressed significantly more ALP than other groups (Figure 2). Oil Red O measurements were significantly reduced from days 7–14 for all osteogenic groups, this significance was greatest for the iPTH group at day 7. iPTH increased the transdifferentiation of adipocytes derived from MSCs into osteoblasts, this effect was most significant after 7 days. Ultimately, the role of iPTH on adipocytes may lead to improved bone formation with many orthopaedic applications

Autologous bone grafting is a standard procedure for the clinical repair of skeletal defects, and good results have been obtained. Autologous vascularized bone grafting is currently the procedure of choice because of high osteogenic potential and resistance against reabsorption. Disadvantages of this procedure include limited availability of donor sites, clinical difficulty in handling, and a failure rate exceeding 10%. Allografts are often used for massive bone loss, but since only the marginal portion is newly vascularized after the implantation non healing fractures are often reported, along with a graft reabsorption. To overcome these problems, some studies in literature tried to conjugate bone graft and vascular supply, with encouraging results. On the other side, several studies in literature reported the ability of bone marrow derived cells to promote neo-vascularization. In fact, bone marrow contains not only hematopoietic stem cells (HSCs) and MSCs as a source for regenerating tissues but also accessory cells that support angiogenesis and vasculogenesis by producing several growth factors. In this scenario a new procedure was developed, consisting in an allogenic bone graft transplantation in a critical size defect in rabbit radius, plus a deviation at its inside of the median artery and vein with a supplement of autologous bone marrow concentrate on a collagen scaffold. Twenty-four New Zealand male white rabbits (2500–3000 g) were divided into 2 groups, each consisting of 12 animals. Surgeries were performed as follow:. −. Group 1 (#12): allogenic bone graft (left radius) / allogenic bone graft + vascular pedicle + autologous bone marrow concentrate (right radius). −. Group 2 (#12): sham operated (left radius)/ allogenic bone graft + vascular pedicle (right radius). For each group, 3 experimental time: 8, 4 and 2 weeks (4 animals for each time). The bone used as graft was previously collected from an uncorrelated study. An in vitro evaluation of bone marrow concentrate was performed in all cases, and at the time of sacrifice histological and histomorphometrical assessment were performed with immunohistochemical assays for VEGF, CD31 e CD146 to highlight the presence of vessels and endothelial cells. Micro-CT Analysis with quantitative bone evaluation was performed in all cases. The bone marrow concentrate showed a marked capability to differentiate into osteogenic, chondrogenic and agipogenic lineages. No complications such as infection or intolerance to the procedure were reported. The bone grafts showed only a partial integration, mainly at the extremities in the group with vascular and bone marrow concentrate supplement, with a good and healthy residual bone. immunohistochemistry showed an interesting higher VEGF expression in the same group. Micro CT analysis showed a higher remodeling activities in the groups treated with vascular supplement, with an area of integration at the extremities increasing with the extension of the sacrifice time. The present study suggests that the vascular and marrow cells supplement may positively influence the neoangiogenesis and the neovascularization of the homologous bone graft. A longer time of follow up and improvement of the surgical technique are required to validate the procedure

Introduction: Alcohol can induce osteoporosis and osteonecrosis. Studies have demonstrated that alcohol contributed to abnormal lipid metabolism in cells in bone marrow but the mechanisms have not been defined. The purpose of this study was to evaluate the effect of alcohol on the differentiation of pluripotential cells cloned from bone marrow. Materials and Methods: The cells were maintained in culture and treated with either increasing concentrations of ethanol (0.09, 0.15, and 0.21 mol/L) or without alcohol to serve as controls. Morphologic features of the cells were monitored using a phase-contrast microscope. Alkaline phosphatase activity was determined using a colorimetric assay. Gene expression of adipogenesis [422 (aP2), PPAR y] and osteogenesis (osteocalcin) was evaluated using the Northern blot technique and reverse transcription-polymerase chain reaction (RT-PCR). ANOVA was used for statistical analysis. Results: The cells treated with ethanol started to accumulate triglyceride vesicles at Day 7; the number of adipocytes and the percentage of the area that contained the cells with fat vesicles increased significantly; and the level of alkaline phosphatase activity diminished with longer durations of exposure and with higher concentrations of ethanol. Analysis of gene expression showed diminished expression of osteocalcin without a significant increase in the expression of the fat cell specific gene, 422 (aP2), and PPAR y, in cells treated with ethanol. This suggested that adipogenesis may occur at a point downstream in the fatty acid metabolism pathway. Discussion: Alcohol induces bone marrow fatty changes in patients and in animal models contributing to osteoporosis and osteonecrosis. This study demonstrated that alcohol treatment decreased osteogenesis while enhancing adipogenesis by bone marrow stromal cells, which may be one of the mechanisms leading to osteoporosis and osteonecrosis. Inhibition of adipogenesis may lead to the prevention of the disease. Clinical relevance: This is a novel finding that alcohol induces adipogenesis in a cloned bone marrow stromal cell. The results explain the clinical observation that there is increased adipogenesis in alcohol-induced osteoporosis and osteonecrosis

Background. Periprostetic joint infections (PJI) are often difficult to diagnose, to treat and often leave the patient with severe impaired function. The presence of low virulent bacteria is frequently discovered in apparent aseptic revisions of shoulder arthroplasties and pose a challenge to diagnose preoperatively. Dual Isotope In111 Leucocyte/ Tc99 Bone Marrow SPECT CT scan (L/BMS) is considered the radionuclide gold standard in preoperative diagnosing PJI with reported high specificity and sensitivity in hip and knee arthroplasties. Unfortunately, it is labour-intensive and expensive to perform and documentation using L/BMS on shoulder arthroplasties lack. Aim. To investigate if L/BMS succeeds in detecting shoulder PJI compared to tissue cultures obtained perioperatively. Method. All patients referred to a highly-specialised shoulder department with a painful or stiff shoulder-arthroplasty were included in the cohort. To diagnose infection as a possible cause of arthroplasty failure a L/BMS was planned for all patients. If the arthroplasty was revised, 5 tissue biopsies were obtained from the most infection-suspicious site during revision. Biopsies were cultured in broth and on plates for 14 days due to the high frequency of low virulent infection in shoulder revisions. Infection was defined as growth of the same bacteria in 3 or more of 5 the biopsies. Results. During the observation period 71 patients were referred. Revision surgery was performed in 62% of the patients (44/71) of which 29 also had been examined by L/BMS. A microbiological diagnose was available for all. The most predominant organism isolated was P. Acnes. Two patients both had a positive L/BMS and positive cultures. Negative L/BMS and negative cultures were found in 20 patients. The remaining 7 patients had negative L/BMS, but positive cultures. The two patients with a positive L/BMS both showed overt clinical signs of infection. L/BMS show a sensitivity 0.22 95%CI(0–0.49) and specificity 1.00 95%CI(1.00–1.00) in detecting shoulder PJI. The Positive Predictive Value is 1.00 95%CI(1.00–1.00) and Negative Predictive Value 0.74 95%CI(0.57–0.90). No patients infected with P. Acnes resulted in a positive scintigraphy nor had they preoperative or perioperative signs of infection. Conclusion. Only patients with severe infectious symptoms of shoulder PJI resulted in positive L/BMS. Hence, the scan added nothing to the preoperative clinical diagnose. In111 Leucocyte/ Tc99 Bone Marrow SPECT CT scan cannot be recommended as a standard screening procedure when evaluating failed shoulder arthroplasties for possible infection

Background: High doses of local antibiotics are used to treat infected acute fractures or chronic osteomyelitis. In the U.S.A., tobramycin is one of the most commonly used antibiotics in trauma surgery. It is an aminoglycoside antibiotic with a broad spectrum of action. However, its effect on the osteogenic potential of bone marrow derived mesenchymal stem cells (MSC’s) is unknown. We hypothesised that high concentrations of tobramycin would be detrimental to the osteogenic potential of multipotent stem cells derived from the bone marrow. Methods: MSC’s were derived in vitro from reamings obtained in patients undergoing hip hemiarthroplasties. Following subculture, these cells were exposed to various concentrations of tobramycin for 15 days, with a change of media every other day. The amount of bone formed under each condition was assessed by solubilising the mineral content in hydrochloric acid overnight and then measuring the change in colour induced by Calcium exposed to a commercial reagent. The amount of calcium detected was then determined using a standard curve. This experiment was repeated in cells from 3 patients. Results: The amount of calcium formed was as follows Tobramycin concentration of 0 microg/ml. There was a statistically significant impairment in osteogenesis at a concentration of tobramycin of 400 microg/ml and above. Conclusion: A high local dose of tobramycin affects negatively the osteogenic potential of stem cells derived from the bone marrow

Purpose of the study: Nonunion, which is a biological failure, requires revision, usually an aggressive operation. Haematopoietic bone marrow contains colony forming unit fibroblasts (CFU-F) which could favour bone healing. The purpose of this work was to determine whether a minimally invasive procedure, injection of CFU-F into the nonunion space, could favour bone healing without further procedure. Material and methods: Our series included 43 patients: 36 male and 7 female, mean age 39.9 years. Forty-seven atrophic and aseptic nonunions of long bones were treated with percutaneous injection of concentrated autologous bone marrow: 27 tibias, 17 femurs, 3 humeri. Bone marrow was harvested from the posterior iliac crests (346 ml) then centrifuged to keep the leuko-platelet fraction (78 ml). This concentrate was injection into the nonunion space under radioscopic guidance. Efficacy was assessed on the basis of clinical criteria (complete pain-free weight-bearing, absence of contention, absence of mobility) and on radiographic criteria (healing of 3/4 corticals). Results: Thirty nonunions healed: 19 tibias (70%, 11 femurs (65%) and 0 humerus. Mean time to healing was 5.9 months (2.4–15.6). Factors of poor prognosis were: smoking, alcohol, diabetes, corticosteroids, radiotherapy, history of sepsis (p=0.01). Early grafting increased the chances of success (p=0.04). Age, initial skin opening, type of fixation did not have a significant impact on healing. The number of CFU-F had an effect on the rate of healing. Discussion: This technique is effective for the treatment of nonunion of the lower limb, allowing bone healing in two thirds of the cases with a minimally aggressive procedure. The method is easy to perform but requires a rigorous technique for the different phases of puncture, concentration and reinjection. Nonunions unresponsive to conventional methods, and thus corresponding to multifactorial problems, probably constitute the limitation of this method. Cell expansion or differentiation techniques could be helpful in improving the success rate but at the present time the osteogenic potential of these cells remains to be elucidated as a function of their stage of maturation. Conclusion: Percutaneous grafts of concentrated autologous bone marrow can be a useful contribution to the therapeutic armamentarium for nonunion. Morbidity is low and the method does not compromise future options. It can be proposed as a first-intention solution for the treatment of long bone nonunion

Aims. Osteoporosis is characterized by decreased trabecular bone volume, and microarchitectural deterioration in the medullary cavity. Interleukin-19 (IL-19), a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. The aim of our study was to investigate the effect of IL-19 on osteoporosis. Methods. Blood and femoral bone marrow suspension IL-19 levels were first measured in the lipopolysaccharide (LPS)-induced bone loss model. Small interfering RNA (siRNA) was applied to knock down IL-19 for further validation. Thereafter, osteoclast production was stimulated with IL-19 in combination with mouse macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The effect of IL-19 was subsequently evaluated using tartrate-resistant acid phosphatase (TRAP) staining and quantitative real-time polymerase chain reaction (RT-qPCR). The effect of IL-19 on osteoprotegerin (OPG) was then assessed using in vitro recombinant IL-19 treatment of primary osteoblasts and MLO-Y4 osteoblast cell line. Finally, transient transfection experiments and chromatin immunoprecipitation (ChIP) experiments were used to examine the exact mechanism of action. Results. In the LPS-induced bone loss mouse model, the levels of IL-19 in peripheral blood serum and femoral bone marrow suspension were significantly increased. The in vivo results indicated that global IL-19 deletion had no significant effect on RANKL content in the serum and bone marrow, but could increase the content of OPG in serum and femoral bone marrow, suggesting that IL-19 inhibits OPG expression in bone marrow mesenchymal stem cells (BMSCs) and thus increases bone resorption. Conclusion. IL-19 promotes bone resorption by suppressing OPG expression in BMSCs in a LPS-induced bone loss mouse model, which highlights the potential benefits and side effects of IL-19 for future clinical applications. Cite this article: Bone Joint Res 2023;12(11):691–701

Introduction: Our previous study found that glucocorticoids shifted the properties of osteogenesis to adipogenesis in murine marrow stem cells. These effects may be one of the important mechanisms in the pathogenesis of osteonecrosis. Statins prevented these steroid effects. In this study, we investigated the effects of dexamethasone and lovastatin on the expressions of bone morphogenetic protein-2 (BMP2) in the bone marrow stroma cells cultured from osteonecrotic patients. Materials and Methods: Bone marrow fluid aspiration from iliac crest was performed in osteonecrosis (ON) and non-ON patients after surgical treatment for their hip disorder. The mean age of the patients was 59 years in the ON group and 63 years in the non-ON group. Nucleated stroma cells were isolated from bone marrow fluid by percol separation. The third passage cultures were used for experiments. Drug treatments for cultures included dexamethasone (10. −7. M), lovastatin (10. −6. M), and dexamethasone plus lovastatin for 4 days. BMP-2 mRNA expression was evaluated by RT-PCR. Different responses to drugs between the ON group and the non-ON group were compared. Results: Bone marrow stroma cells of ON patients were found to be more susceptible to the suppressive effect of dexamethasone on BMP2 expression. Discussion: Lovastatin stimulated the osteogenesis and reversed the steroid suppressive effect in bone marrow stroma cells in non-ON cases. However, this reverse effect was found to be mild in ON cases

INTRODUCTION. There is no effective therapy available today that alters the pathobiologic course of osteoarthritis. Recent advances have shown Mesenchymal stem cells to be a potential disease modifying treatment. Considering the tissue differentiation property and vast paracrine effects of MSCs we proposed the present study to find out the safety and efficacy of Mesenchymal stem cells in osteoarthritis of knee joint. METHODS. 12 patients with grade 1and2 bilateral osteoarthritis knee (Ahlbacks radiological grading) were selected. 8–10 ml of bone marrow was aspirated under strict aseptic precautions from the iliac spine. After the stem cell culture and expansion for 4–6 weeks the MSC suspension in 10xPBS was injected directly into the 24 knees by lateral approach. The outcome was evaluated by modified VAS score, WOMAC score, KOOS and MRI measurement of knee articular cartilage integrity by the modified WORMS score. RESULTS. Statistically significant improvement in VAS score, total WOMAC score and total KOOS score was observed from pre injection to 1st follow up at 6 weeks, 2nd follow up at 6 months and final follow up of mean 26.7 months. There was also a significant improvement from 1st follow up to 2nd and final follow up. The modified WORMS score showed a statistically significant decrease of 1.49 %. CONCLUSION. Intra-articular injection of autologous bone marrow derived culture-expanded MSCs can be considered a potential treatment of early osteoarthritis knee which relieves pain, stiffness, improves physical functions, and improves the articular cartilage integrity

Bone grafts are frequently used to augment bone healing. Autologous bone graft is the gold standard for osteogenesis but is limited by availability and donor site morbidity. The processing required to lower the immunogenicity of allograft also reduces the osteogeneic properties. Bone marrow contains mesenchymal stem cells (MSCs) which differentiate into osteoblasts, forming bone. Our study examined the use of bone marrow to enhance the osteogenic properties of allograft. Bioactive proteins within allogenic bone graft stimulate marrow-derived MSCs to differentiate into osteoblasts, thereby increasing the osteogenic nature of the graft. After informed consent, bone marrow aspirates were taken from five patients during orthopaedic operations. Freeze-dried ethylene oxide treated allograft, from a number of donors, was obtained from the bone bank. MSCs isolated from each marrow aspirate were grown on eight samples of test allograft. Further allograft was heated to 70°C to denature the osteogenic proteins and MSCs from each aspirate were grown on 8 samples, as a negative control. Osteoblastic differentiation of MSCs cultured on the types of allograft was compared. Scanning electron microscopy confirmed that MSCs covered the allograft after 14 days. Transmission electron microscopy showed that cells on the test allograft were characteristic of osteoblasts and produced collagen extracellular matrix. The levels of osteoblastic proteins, ALP, osteopontin and Type I pro-collagen, produced by cells on test allograft were significantly greater compared with heat-treated control (P< 0.005), after days 7 and 14. Our study showed that marrow-isolated MSCs could be successfully cultured on allograft. As the levels of osteoblastic proteins increased significantly when MSCs were grown on allograft, osteogenic proteins within allograft caused MSCs to change into osteoblasts. This confirms that autologous marrow MSCs could be grown on allograft to increase its osteogenic prior to grafting, resulting in increased rate of bony healing

Objectives. To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Methods. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. Results. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. Conclusions. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis. Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1

Introduction: Ethanol is one of risk factors associated with osteonecrosis, it has been demonstrated that ethanol induces adipogenesis, decreases osteogenesis in bone marrow stroma cells and produces intracellular lipid deposits, resulting in the death of osteocytes. Materials and Methods: In this approach, we isolated human bone marrow stroma cells and triggered for different differentiations. Results: These cells could be induced for osteogenesis, adipogenesis, and chondrogenesis. We also evaluated cell surface markers of isolated human bone marrow stromal cells that were found to express CD29, CD49d, CD62 CD90, CD105/SH2, SH3, CD133, and CD166, but not CD31, CD34, CD45, or CD56. Discussion: We demonstrated that ethanol decreases the expression of osteogenic genes, but increases adipogenic genes expressions. Moreover, we found that ethanol decreases the beta-catenin-dependent canonical Wnt signaling pathway related gene expressions, including Wnt 3a and LRP5 genes. Interestingly, ethanol also diminishes the intra-nuclear translocation of β-catenin in human bone marrow stromal cells. Therefore, these results indicate that ethanol might decrease osteogenic gene expressions through Wnt signaling pathway

A combination of stem cell therapy and tissue engineering is emerging as one of the most promising approaches for skeletal tissue repair and regeneration. Osteoinduction of human bone marrow mesenchymal stem cells (MSCs) is initiated through local signals or growth factors, of which the bone morphogenetic proteins (BMPs) are the best characterised. Cytomodulin-1 (CM-1), a synthetic heptapeptide with functional similarity to members of the TGF-B super family, has been classified as a novel growth factor associated with osteoinduction of MSCs. However, the effects of CM-1 on human bone MSCs are still unclear. The aim of this study was to determine any effects for CM-1 and its scrambled control (CM-1 SCRAM) on the proliferation and differentiation of human bone marrow MSCs along the osteogenic lineage. Primary human bone marrow MSCs were cultured in the presence of CM-1 and CM-1 SCRAM at a range of concentrations (10-8M – 10-6M) in vitro for up to three weeks. 100 ng/mL of recombinant human BMP-2 (rhBMP-2) was used as a positive control. At the end of the culture period, histological and biochemical assays were carried out on the cultures. Biochemical assays revealed that 10-7M of CM-1 significantly stimulated alkaline phosphatase specific activity compared with the negative control group (P< 0.05) in a similar way to the rhBMP-2 positive control group. These data were supported by an observed increase in positive alkaline phosphatase staining in the 10-7M of CM-1 and rhBMP-2 treated cells. However, total DNA content was not significantly different between any of the groups. This study indicated the potential of using CM-1 as an osteogenic growth factor for skeletal tissue regeneration which may provide an alternative approach to meet the major clinical need in orthopaedics and craniofacial surgery. * Cytomodulin-1 and the scrambled control were genuine gifts from Professor (emeritus) Rajendra S. Bhatnagar at the Department of Bioengineering, University California Berkley, USA

Introduction: Human bone marrow stromal stem cells(BMSSC’s) have the ability to differentiate into a variety of mesenchymal cell types including osteoblasts, fibroblasts, adipiocytes and myocytes. These stromal cells are involved in the process of bone formation during the healing of fractures. Collagen lyophilisate is a sterile extract of bovine demineralised bone matrix. This material contains proteins removed from bone that may control the differentiation of osteoblasts from BMSSC’s. These proteins are localised within collagen type 1. The aim of this study was to determine the effects of collagen lyophilisate on the osteogenic differentiation of bone marrow stromal stem cells cultured in vitro. Methods: Bone marrow was aspirated from the iliac crest of a human donor who was undergoing an unrelated elective orthopadic surgical procedure. Stromal stem cells were isolated from marrow, cultured and then characterised using immunofluorescent antibodies to Stro −1, a stromal stem cell marker. 3x10. 4. BMSSC’s were seeded into each of 3 culture wells and incubated with standard growth medium or standard medium with collagen lyophilisate diluted 1:50 or 1:100. Cells were cultured for a maximum duration of 30 days. At selected time intervals until day 30, osteogenic differentiation was assessed by determination of alkaline phosphatase, osteopontin, pro collagen carboxyterminal (type 1 collagen synthesis) and calcium in cultures using specific assays. Results: Cells cultured in collagen lyophilisate displayed a polygonal morphology early in the culture period and later formed complex aggregates. Cells in control cultures maintained a fibroblstic morphology until confluence. On day 21 alkaline phosphatase activity was significantly higher in collagen lyophilisate containing cultures than control cultures. Osteopontin levels were not enhanced in the collagen lyophilisate containing cultures. Type 1 collagen synthesis was higher in the collagen lyophilisate 1:50 group than all other groups at day 14. No differences in type 1 collagen synthesis were detected between cultures at other time periods. Calcium was not detected in any of the control cultures for the duration of the culture period. In contrast, calcium was detected in collagen lyophilisate containing cultures on day 15. Conclusion: Collagen lyophilisate resulted in changes in cellular morphology and arrangement. The ability of collagen lyophilisate to enhance alkaline phosphatase activity, increase collagen type 1 expression and stimulate the deposition of calcium in stromal stem cell cultures provides evidence that it has osteogenic properties

Introduction. Problematic bone defects are encountered regularly in orthopaedic practice particularly in fracture non-union, revision hip and knee arthroplasty, following bone tumour excision and in spinal fusion surgery. At present the optimal source of graft to ‘fill’ these defects is autologous bone but this has significant drawbacks including harvest site morbidity and limited quantities. Bone marrow has been proposed as the main source of osteogenic stem cells for the tissue-engineered cell therapy approach to bone defect management. Such cells constitute a minute proportion of the total marrow cell population and their isolation and expansion is a time consuming and expensive strategy. In this study we investigated human bone marrow stem cells as a potential treatment of bone defect by looking at variability in patient osteogenic cell populations as a function of patient differences. We produced a model to predict which patients would be more suited to cell based therapies and propose possible methods for improving the quality of grafts. Methods. Bone marrow was harvested from 30 patients undergoing elective total hip replacement surgery in Musgrave Park Hospital, Belfast (12 males, 18 females, age range 52-82 years). The osteogenic stem cell fraction was cultured and subsequently analysed using colony forming efficiency assays, flow cytometry, fluorescence activated cell sorting and proteomics. Results. The number and proliferative capacity of osteogenic stem cells varied markedly between patients. Statistical analysis revealed significantly better osteogenic capacity in:. male patients. samples in which the growth hormone Fibroblastic Growth Factor-2 was added to culture medium. patients who used the cholesterol lowering agent simvastatin. Patient use of inhaled steroids and NSAIDs were found to have detrimental effects. A statistical model to predict marrow profiles based on these variables was produced. Conclusions. Stem cell based tissue engineering represents the future of the treatment of bone defect. This study provides evidence that inter-patient variability in marrow cell colony forming and proliferation ability can in some way be explained by patient associated factors. Using this knowledge, we can identify which patients would be best suited to this method of treatment and propose techniques for enhancement of their graft profiles

Limited success in regenerating large bone defects has been achieved by bridging them with osteoconductive materials. These substitutes lack the osteogenic and osteoinductive properties of bone autograft. A direct approach would be to stimulate osteogenesis in these biomaterials by the addition of fresh bone-marrow cells (BMC). We therefore created osteoperiosteal gaps 2 cm wide in the ulna of adult rabbits and either bridged them with coral alone (CC), coral supplemented with BMC, or left them empty. Coral was chosen as a scaffold because of its good biocompatibility and resorbability. In osteoperiosteal gaps bridged with coral only, the coral was invaded chiefly by fibrous tissue. It was insufficient to produce union after two months. In defects filled with coral and BMC an increase in osteogenesis was observed and the bone surface area was significantly higher compared with defects filled with coral alone. Bony union occurred in six out of six defects filled with coral and BMC after two months. An increase in the resorption of coral was also observed, suggesting that resorbing cells or their progenitors were present in bone marrow and survived the grafting procedure. Our findings have shown that supplementation of coral with BMC increased both the resorption of material and osteogenesis in defects of a clinical significance

Abstract

Aims: It is well known that the success of an orthopedic implant is determined by a close apposition between bone and implant surface. The excellent physical properties and the controlled degradation of poly-ε-caprolactone (PCL) has been shown, however the suitability for bone engineering applications of a material is critically influenced by the interactions between cells and scaffold. The aim of this study was to evaluate the interaction between bone marrow cells and PCL surface. Bone marrow cells were obtained from femurs of New Zealand rabbits and seeded on PCL directly (WBMC) or after gradient centrifugation (MSC), mimicking the in vivo colonization of PCL after implantation and the pre-seeding strategy. Methods: PCL was dissolved in chloroform (3% w/v solution) and spin coated as a thin (100nm) film onto p-doped silicon wafers. The surface wettability and roughness were analyzed by SFE measurements and AFM. Cells were seeded on PCL and adhesion/proliferation evaluated at 1, 7, 14, 21 and 28 days. Fluorescence microscopy and SEM imaging were performed at defined time endpoints. Results: At 2 wks adherence-selected MSC had already formed confluent multilayers, whereas WBMC were still semi-confluent. At 4 wks a consistent layer of ECM was observed underneath the cell layers of both cultures. Conclusions: PCL is a proper substrate for bone cell attachment and growth, as cell confluence was reached at 2 wks for MSC and at 3–4 wks for WBMC. To avoid any risk of bacterial contamination, the seeding of WBMC on PCL scaffold, which implies reduced handling of cells outside the body, was shown to be effective and may be recommended in the clinical practice

INTRODUCTION. Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. MATERIALS AND METHODS. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. RESULTS. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. DISCUSSION. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

The reconstruction of large bone segments is a major goal in orthopaedic surgery. Autologous cancellous bone is recognized as the most biologically active graft material, but autologous bone harvest is associated with significant morbidity and founds its limit in the available quantity. Biomaterials or allografts do not encounter these limitations, but have no osteogenic and limited osteinductive potential. In order to enhance tissue regeneration and healing we have tried to obtain a graft with osteconductive, inductive and osteogenic properties. The day before operation 350 cc of autologous blood is donated from the patient and centrifuged to obtain a platelet-rich plasma. Bone marrow is aspirated from the posterior iliac crests with the patient under spinal anaesthesia and is processed to increase its stem cell content. The structural scaffold used is morcellized cancellous bone provided from our Bone Bank. At operation bone is mixed with bone marrow buffy coat and Platelet Rich Plasma in a sterile glass becker with addition of CaCl2 till clot formation to produce a gel-like component that is handled easily. We have utilized this technique from November 2000 till January 2004 for 68 patients: 41 of these patients required healing of large bone defects: 22 males and 19 females. Fresh bone marrow alone was used for a percutaneous injection in 11 cases; open surgery with autologous growth factors, bone marrow buffy coat and allograft was used in 30 patients. The radiological and clinical results showed early healing of the defects treated with this technique and no complications related to the procedure at an average follow up of 23 months (3–40)

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

We present our clinical experience in treating atrophic non-union of long bones by injecting, percutaneously, autologous bone marrow aspirate concentrated as a source of progenitors stem cells. Bone marrow aspirated from the iliac crest contains progenitor cells that can be used to obtain bone-healing of non-union. However, its efficacy appears to be related to the number and concentration of progenitors in the graft. The last three-year period, 11 patients (8 men-3 women) with established atrophic non-union were treated in our department. In all cases, the gap between the fragments was smaller than 5 mm. A constant volume of 60+60 ml of marrow were aspirated from both iliac crests and centrifuged for 15 minutes aiming at the increase of concentration of progenitor-mononucleotide cells. An average volume of 20 ml (+/− 2) concentrated bone marrow was injected percutaneously, under C-arm, at the site of non-union. The graft contained an average of 272.64 x 10(6)/ ml mononucleotide cells. The evaluation of treatment was based on the clinical and radiological findings after 3, 6, 9 and 12 months. However, prior to administration of bone marrow stem cells, there was no case with evidence of ongoing deep sepsis. Bone union was obtained in 10 out of 11 patients (full weight bearing, callus formation in 3 out of 4 cortices). In one case a second operation was needed due to impaired indications of treatment. However, in all cases, there were no signs of local or systematic complications. Percutaneous concentrated bone marrow grafting is an efficient and safe method, for treating atrophic non-unions, with a minimal invasion technique. Contraindications for the above technique are a gap larger than 5 mm and a preexisting angular and axial deformity

Background. Aseptic loosening remains the primary reason for failure of orthopaedic implants. Therefore a prime focus of Orthopaedic research is to improve osteointegration and outcomes of joint replacements. The topography of a material surface has been shown to alter cell adhesion, proliferation and growth. The use of nanotopography to promote cell adhesion and bone formation is hoped to improve osteointegration and outcomes of implants. We have previously shown that 15nm high features are bioactive. The arrangement of nanofeatures has been shown to be of importance and block-copolymer separation allows nanopillars to be anodised into the titania layer, providing a compromise of control of order and height of nanopillars. Osteoblast/osteoclast stem cell co-cultures are believed to give the most accurate representation of the in vivo environment, allowing assessment of bone remodelling related to biomaterials. Aims. To assess the use of nanotopography on titania substrates when cultured in a human bone marrow derived co-culture method. Hypothesis. Under co-culture conditions 15nm high nanopillars on titania substrate will induce significantly increased levels of osteogenic differentiation, producing a method of enhancing secondary implant fixation. Methods/Design. Bone marrow was aspirated from patients undergoing elective arthroplasty. Co-culture of adherent osteoprogenitors and osteoclast progenitors on polished titania and titania patterned with 15nm nanopillars fabricated by the block copolymer technique was performed. Histochemical staining has been performed to identify and quantify osteoclasts and bone nodule formation. Scanning electron microscopy (SEM) has been conducted to morphologically examine the effect on differentiation of untreated and nanopatterned titania substrates on osteoprogenitors and osteoclasts. Real-Time, quantitative reverse-transcription polymerase chain reaction is currently being utilised to quantify expression of osteoblast, osteoclast and inflammatory response related genes. Results. SEM has shown an increase in bone deposition on titania substrates with 15nm nanopillars. The remaining results of this project will be presented with a discussion of their clinical significance

Hematopoietic stem cells (HSCs) reside within a specialised niche area in the bone marrow (BM). They have tremendous clinical relevance, although HSC expansion and culture ex vivo is not currently possible, reducing BM transplant success. This project expands a novel 3D MSC niche model developed in our lab to include HSCs. MSCs were loaded with green fluorescent magnetic iron oxide (FeO. 3. ) nanoparticles (200 nm diameter) at a concentration of 0.1 mg ml. −1. , and incubated for 30 min over a magnet to enhance cellular uptake. The cells were washed, detached and resuspended, then transferred to a plate with magnets above. Spheroids formed within hours and were implanted into 2 mg ml. −1. collagen gel. HSCs were loaded with nanoparticles via incubation with suspension, and then introduced to the gel containing the spheroid. Immunostaining, BrdU and Calcein/ ethidium homodimer viability assays were performed to characterise the cells. Cells in both monolayers and spheroids remain viable up to 7 days in culture. MSCs in monolayers and spheroids were stained with antibodies for: STRO-1, an MSC marker; SDF-1 (CXCL-12), a secreted HSC homing factor; and nestin, a marker for HSC-supportive MSCs in vitro. MSCs in spheroids retain a higher level of expression of all three for 7 days compared to MSCs in monolayers. BrdU assay results show that the MSCs are more quiescent in spheroids compared to monolayers. Proof of principle studies are promising for the success of the proposed niche model. MSCs express a higher level of MSC markers and retain quiescence when they are in spheroids as compared to monolayers. They also express a higher level of HSC niche factor SDF-1α, which facilitates HSC migration and retention

Introduction: Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. Material and Methods: Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. Results: Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. Discussion: Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

Tissue engineering can be deþned as any effort to create or induce the formation of a speciþc tissue in a speciþc location through the selection and manipulation of cells, matrices, and biologic stimuli. The biologic concepts and the biochemical and biophysical principles on which these efforts are based have become a rapidly evolving þeld of biomedical research. More importantly, tissue engineering is becoming a clinical reality in the practice of orthopaedic surgery, providing patients and physicians with an expanding set of practical tools for effective therapy. The efþcacy of all current clinical tools depends entirely on the cells in the grafted site, particularly the small subset of stem cells and progenitor cells that are capable of generating new tissue. The current author reviews a series of key biologic concepts related to the rational design and selection of cells in contemporary bone grafting and tissue engineering efforts. The functional paradigms of stem cell biology are reviewed and sources for autogenous stem cells for connective tissues are discussed. Finally a technique to obtain stem cells for the treatment of non unions is described. We included 48 patients: 38 cases of post-traumatic non union (12 of them with infection); 4 non unions following arthrodesis (3 knees, 1 tibiotarsal); 4 cases with Illizarov technique; 2 patients with congenital abnormalities. The source of bone marrow was the iliac crest. The marrow was reduced in volume (50 ml) in order to increase the concentration in stem cells by elimination of erythrocytes and polynuclear cells. The number of nucleated cells was counted in the marrow transplanted and the þbroblast colony forming cells (CFU-F) and the osteoblast colony forming cells (CFU-Ost) were cloned to appreciate the number and the activity of progenitor in the marrow transplanted

Tissue engineering can be defined as any effort to create or induce the formation of a specific tissue in a specific location through the selection and manipulation of cells, matrices, and biologic stimuli. The biologic concepts and the biochemical and biophysical principles on which these efforts are based have become a rapidly evolving field of biomedical research. More importantly, tissue engineering is becoming a clinical reality in the practice of orthopaedic surgery, providing patients and physicians with an expanding set of practical tools for effective therapy. The efficacy of all current clinical tools depends entirely on the cells in the grafted site, particularly the small subset of stem cells and progenitor cells that are capable of generating new tissue. The current author reviews a series of key biologic concepts related to the rational design and selection of cells in contemporary bone grafting and tissue engineering efforts. The functional paradigms of stem cell biology are reviewed and sources for autogenous stem cells for connective tissues are discussed. Finally a technique to obtain stem cells for the treatment of non unions is described. We included 48 patients: 38 cases of posttraumatic non union (12 of them with infection); 4 non unions following arthrodesis (3 knees, 1 tibiotarsal); 4 cases with Illizarov technique; 2 patients with congenital abnormalities. The source of bone marrow was the iliac crest. The marrow was reduced in volume (50 ml) in order to increase the concentration in stem cells by elimination of erythrocytes and polynuclear cells. The number of nucleated cells was counted in the marrow transplanted and the fibroblast colony forming cells (CFU-F) and the osteoblast colony forming cells (CFU-Ost) were cloned to appreciate the number and the activity of progenitor in the marrow transplanted

Summary. Randomised controlled study evaluating new bone formation in vivo in fracture non-unions by bone marrow derived stromal cells (BMSC). These cells do not show statistically significant new bone formation. Age of the patient during fracture, diabetes and doubling time had been observed to be correlated with fracture healing. Introduction. Regenerating new bone by cell therapy could provide therapeutic options in many conditions such as fracture non-unions and osteo-chondral defect regeneration in advance OA. In this randomised controlled study we evaluated the efficacy of new bone formation by bone marrow derived stromal cells (BMSC) in patients with non-union. Methods. An ethically approved and adequately powered single centre randomised control trial recruited 35 patients for treatment of non-unions with BMSC. Bone marrow was harvested and autologous BMSC were culture expanded in autologous serum at our local MHRA-licensed facility (Oscell, Oswestry, UK). Following selection by adherence and in vitro culture expansion using autologous serum, cells in serum and serum alone was randomised for insertion at one of the two fracture sides by StratOs® computer software. Patients and the operating surgeon were blinded to the side of cell insertion. Such method of randomisation created internal controls at the fracture sites- one side receiving the cell (‘test side’) and other, not (‘control’). Serial radiographs extending up to an average of twelve months were evaluated by four independent assessors blinded to side of cell insertion. Callus formation and bridging of fracture was compared for ‘test’ and ‘control’ side. Radiological and clinical outcome at final follow-up was also noted. Results. Thirty five patients were recruited (21 males, 14 females; mean age 51.2±13.2SD). The mean duration of non-union was 3±2SD years, with a mean 3.5 (range 1–12) surgical interventions prior to BMSC insertion. Five patients had diabetes. New callus formation and fracture bridging was slow, with no significant difference between the cell-insertion and control side although a substantial improvement in fracture bridging/formation of new callus was noted at 9–12 months. Fracture union was achieved in 21 patients at final follow-up with failure to progress to union in 14 patients. Age at accident, having diabetes and cell doubling time during culture predicted union (r2=0.63, p=0.017). There was no reported adverse effects from the trial. Conclusion. The study concluded that patient biology predicts the final outcome in cases with non-union of fracture. Slower doubling time during in vitro expansion can be significantly correlated with failure to unite in addition to diabetes and age of the patient. BMSC's are safe option for cell therapy in a setting of non-union although it failed to show statistically significant difference of new bone formation or fracture bridging for up to one year

Background and aim: Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into osteogenic and chondrogenic pathways. MSCs are among the key repair cells in fracture healing and implant osseointegration. They are also an attractive tool of cell therapy in reconstruction procedures of bone. Minipigs are a large-animal model recommended for preclinical studies of orthopaedic bone implants. Minipigs are claimed to have bone physiology close to humans, but their MSC characteristics are poorly defined. The aim of this study was to isolate and characterize minipig bone marrow and peripheral blood derived MSCs in comparison of human MSCs. Methods: Five male minipigs (weight 36.2 ± 2.2 kg) were subjected for experimental femoral osteotomy, which was fixed with either compression plate or intra-medullary nailing. Before surgery, bone marrow (BM) sample (2–4 ml) was aspirated from the posterior iliac crest and a peripheral blood (PB) sample (20 ml) was also collected. Mononuclear cells (MNC) were isolated by Ficoll gradient centrifugation. MSCs were cultured and selected by plastic adherence. Cell morphology was evaluated during the whole culture period and proliferation capacity was examined by determining the number of population doublings (PDs) at the end of each passage. Osteoblastic differentiation capacity was investigated by culturing MSCs in the presence of beta-glycerophosphate, dexamethasone and ascorbic acid. The lineage phenotype was studied by alkaline phosphatase and von Kossa staining. Results: MNC were successfully isolated from all BM and PB samples. Plastic adherent cells obtained fibroblast-like morphology and proliferated over time in culture. The maximum PDs were 3.4 ± 0.7 and 4.3 ± 0.5 for BM- and PB-derived cells, respectively. The maximum PD capacity of PB-derived cells was significantly higher than that of BM-derived cells (p=0.027). However, when cultured in osteoblastic induction medium, only BM-derived cells were capable of differentiating into alkaline phosphatase positive osteoblasts with an occasional presence of von Kossa-stained mineralized bone nodules. The maximum PDs of minipig BM-derived MSCs were similar to those of human BM-MSCs isolated from young adult fracture patients. Conclusion: We successfully isolated plastic adherent MSCs from minipig bone marrow samples, which proliferated and differentiated into cells of osteoblastic lineage. BM-derived porcine MSCs had similar morphology to human MSCs. There were marked inter-individual variations in the proliferation and differentiation capacity of minipig MSCs, resembling the observations in humans. No circulating MSCs could be detected in minipigs before surgery and this confirmed our previous observation in humans

During remodelling, osteoclasts produce discrete bone cavities filled with bone and this is associated with the dimensions of the cavity. The aim of this study is to investigate the effect of pores of similar size to those produced by osteoclasts on the morphology, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. The hypothesis is that a porous surface similar in morphology to a bone surface prepared by osteoclasts will increase cell proliferation and osteogenic differentiation of MSCs. Sheep BMSCs were seeded onto plain titanium surfaces and 100µm, 250µm and 500µm discrete pores surfaces. Cell metabolic activity was investigated using Presto Blue on days 3, 7 and 10. Bone mineralisation was quantified by Alizarin red staining at days 3, 7 and 14. Cell morphology was observed by scanning electron microscopy (SEM). Data was statistically analysed using one-way analysis of variance and a Bonferroni correction method. Cells on porous discs had a three dimensional phenotype and aligned on the circumference of each pore. Metabolic activity was significantly higher by day 10 on plain discs compared to all porous discs. Bone mineralization was significantly higher on 100µm pores by day 3 (0.545mM±0.66; p=0.047) than plain discs and significantly higher on both 100µm and 250µm pores by day 7(p=0.000 and p=0.005) than plain discs. Substantial mineralised bone matrix was found on 100µm discs without being treated with osteogenic supplements, compared to other control disc types (p=0.043, p=0.003, p=0.000). The different topographies altered cell behaviour and migration.100µm pores demonstrated earlier and enhanced bone mineralisation even in the absence of osteogenic supplements. This pore size is aligned to the size of individual resorption bays that osteoclasts produce on bone surfaces and is considerably lower than the pore sizes used to enhance osteo-integration of implant surfaces

Introduction: The treatment of bone defects that occurs following fractures, the excision of bone tumours and at revision arthroplasty surgery, often involves the use of either autologous or allogenous bone grafts. However, both grafts have limitations. The aim of tissue engineering is to produce cells within an extracellular matrix that resembles tissue, which can be implanted into a patient to heal a tissue defect. The potential to engineer bone tissue grafts from patients’ autologous cells would improve the treatment of bone defects. Bone marrow contains cells, known as mesenchymal stem cells (MSCs), which have the ability to differentiate into osteoblasts. To create a 3-dimensional structure necessary for the reconstruction of tissue, cells need to be grown on a scaffold, for which hydroxyapatite (HA) was used, as it is osteoconductive. In living bone, increased extravascular perfusion increases new bone formation. Thus, these physiological conditions were reproduced in our novel bioreactor by perfusing MSCs seeded on porous HA scaffolds at a rate of 6ml/hr. Hypotheses: 1. Culture in this bioreactor improves cell penetration through a HA scaffold. 2. MSCs cultured on HA in this bioreactor differentiated into osteoblasts. Method: MSCs were isolated from 8 bone marrow aspirates, which were taken from patients during orthopaedic procedures following informed consent. For each experiment, MSCs from each patient were seeded onto 2 x 1cm. 3. scaffolds. To test cell penetration, the HA scaffolds were cultured for 7 days, then sectioned longitudinally and the number of cells were counted at increasing depths. Observations of MSCs on HA were compared under scanning (SEM) and transmission (TEM) electron microscopy. The HA scaffolds were cultured with MSCs in the bioreactor for 5, 10 & 15 days, after which time alkaline phosphatase (ALP) and type I pro-collagen protein levels were measured. Results: Penetration of cells through the porous HA scaffold was significantly greater when the cells had been cultured in the bioreactor (P< 0.05). Observing MSCs after 7 days in bioreactor culture under SEM, adherent fibroblastic cells formed a network over the HA. However, by 14 days the HA was covered with cuboidal cells, consistent with osteoblasts. TEM results showed that MSCs cultured on HA in the bioreactor produced organised collagen matrix after 28 days. Osteoblastic protein levels were significantly greater at each time point when MSCs were cultured in bioreactor conditions: ALP (P< 0.005) and type I pro-collagen (P< 0.05). Discussion and Conclusions: These results show that when cultured in our novel bioreactor, MSCs penetrated uniformly through the porous HA scaffold, whereas few cells penetrated in static culture conditions. Thus, our bio-reactor significantly improves the 3-dimensional growth of cells, resembling tissue. Moreover, in this study MSCs grown on HA in the bioreactor produced significantly larger amounts of ALP and type I pro-collagen, indicating that the MSCs differentiated into osteoblasts. Observations under TEM showed extracellular collagen matrix production which, when mineralized, produces bone. Therefore, this culture method could potentially be used to convert MSCs, isolated from patients’ bone marrow, into tissue-engineered bone

Multiple myeloma (MM) is an incurable hematological tumor stemming from malignant plasma cells. MM cells accumulate in the bone marrow (BM) and shape the BM niche by establishing complex interactions with normal BM cells, boosting osteoclasts (OCLs) differentiation and causing bone disease. This unbalance in bone resorption promotes tumor survival and the development of drug resistance. The communication between tumor cells and stromal cells may be mediated by: 1) direct cell-cell contact; 2) secretion of soluble factors, i.e. chemokines and growth factors; 3) release of extracellular vesicles/exosomes (EVs) which are able to deliver mRNAs, miRNAs, proteins and metabolites in different body district. Primary CD138+ MM cells were isolated from patients BM aspirates. MM cell lines were cultured alone in complete RPMI-1640 medium or co-cultured with murine (NIH3T3) or human (HS5) BMSC cell lines or murine Raw264.7 monocytes in DMEM medium supplemented with 10% V/V FBS. Silencing of Jagged1 and Jagged2 was obtained by transient expression of specific siRNAs or by lentiviral transduction using a Dox-inducible system (pTRIPZ). EVs were isolated using differential ultracentrifugation. EVs concentration and size were analyzed using Nano Track Analysis (NTA) system. The uptake of PKH26-labelled MM-derived EVs by HS5 or Raw264.7 was measured after 48 hours by confocal microscopy and flow cytometry. Osteoclast (OCL) differentiation of Raw264.7 cells was induced by 50ng/ml mRANKL, co-culturing with MM cells, CM or EVs. OCLs were stained by TRAP Kit and counted. Bone resorption was assessed by Osteo Assay Surface plates. Flow cytometric detection of apoptotic cells was performed after staining with Annexin V. Gene expression was analyzed by qRT-PCR, while protein levels were determined using flow cytometry ELISA or WB. Notch oncogenic signaling is dysregulated in several hematological and solid malignancies. Notch receptors and ligands are key players in the crosstalk between tumor cells and BM cells. We have demonstrated that: 1) the dysregulated Jagged ligands on MM cells trigger the activation of Notch receptors in the nearby stromal cells by cell-cell contact. This results in the release of anti-apoptotic and growth stimulating factors, i.e. IL6 and SDF1; 2) MM cells promote the development of bone lesions boosting osteoclast differentiation by secreting soluble factors (i.e. RANKL) and by the activation of Notch signaling mediated by direct contact with osteoclast precursors; 3) Finally, we present evidences that EVs play a crucial role in the dysregulated interactions of MM cells with the microenvironment and that Notch signaling regulates their release and participate in this cross-talk. These evidences supports the hypothesis that Jagged targeting on MM cells may interrupt the communication between tumor cells and the surrounding milieu, blocking the activation of the oncogenic Notch pathway and finally resulting in the a reduction of MM-associated bone disease and drug resistance

Aims. The aim of this study was to investigate the global and local impact of fat on bone in obesity by using the diet-induced obese (DIO) mouse model. Methods. In this study, we generated a diet-induced mouse model of obesity to conduct lipidomic and 3D imaging assessments of bone marrow fat, and evaluated the correlated bone adaptation indices and bone mechanical properties. Results. Our results indicated that bone mass was reduced and bone mechanical properties were impaired in DIO mice. Lipidomic sequencing and bioinformatic analysis identified 373 differential lipids, 176 of which were upregulated and 197 downregulated. Functional enrichment analysis revealed a significant downregulation of the pathways: fat digestion and absorption (ko04975) and lipolysis regulation in adipocytes (ko04923) in DIO mice, leading to local fat accumulation. The use of 3D imaging confirmed the increase in fat accumulation within the bone marrow cavity of obese mice. Conclusion. Our study sheds light on the intricate interplay between fat and bone, and provides a non-toxic and non-invasive method for measuring marrow adipose tissue. Cite this article: Bone Joint Res 2023;12(9):580–589

Purpose of the study: Drilling along yields disappointing results for osteonecrosis of the femoral head due to the high failure rate despite prolonged rest and also because of the risk of fracture. To prevent these problems, we have developed a new drilling technique which was evaluated prospectively. Material and methods: The procedure performed percutaneously uses a lateral cortical orifice measuring 5mm, non-concentrated autologous bone marrow was injected after drilling. Osteoinductive protein (BMP7) was associated in random fashion (groups BMP+ and BMP−). Ficat stage 1 and 2 necrosis was included. Outcome was the rate or revision for prosthesis. Results: Forty hips (36 patients) were included and assessed at mean four years (range 2–6). The necrosis was related to: alcoholism (n=5), cortisone (n=25), barotraumas (n=2), metabolic disease (n=4), idiopathic condition (n=4). Group BMP- (drilling+bone marrow) included 24 hips and group BMP+ (drilling+bone marrow+BMP7) 16 hips. The groups were comparable regarding necrosis stage (15% stage 3, 65% stage 2, 20% stage 1) and mean Koo index (27 BMP+ vs 34 BMP-; NS). There were no infections and no fractures despite immediate and complete weight-bearing. The revision rate for prosthesis was higher in the BMP- group (67%) than in the BMP+ group (43%) but the difference did not reach significance (p=0.10). The failure rate was not affected by the severity of the necrosis in the BMP+ group: all stage 3 hips were revised in the BMP- group versus none in the BMP+ group. The only variable predictive of revision for prosthesis was the Koo index (p=0.02). Discussion: Adjunction of BMP did not improve significantly the success rate of drilling with bone marrow adjunction but adding BMP appeared to limit the unfavourable impact of server necrosis observed in the BMP- group. To reach a statistical power of 80%, 40 cases would be needed in each arm. This threshold has not yet been reached. It can be noted however that the proposed method does ensures early weight bearing without the risk of complications. Similarly, since it is a percutaneous procedure, later arthroplasty is not compromised. The principle confounding factor, the richness of the bone marrow, was not assessed, motivating a new randomized trial with measurement of CFU-F

Aims: The aim of the study is to evaluate the clinical application in veterinary orthopedics of the bone stromal cells loaded on three-dimensional resorbable osteogenic scaffolds. Methods: On the basis of the results obtained after an experimental study on 54 adult sheep (data in process), the Authors have carried out a clinical study on 9 dogs of different breed, age,sized with the different orthopaedic lesion associated to large bone defects (from 2 to 4,8 cm) (bone cyst of glenoid rime, non-union of the tibia and of the femur, osteosarcoma of the radio and the proximal humerus, lenghtening of the radius, bone large defect of the distal radius).With the local anesthesia performed with 2% lidocaine the marrow samples were collected from the iliac crest two hours before the surgery. The bone marrow nucleated cells were then isolated from the bone marrow by gradient centrifugation and loaded on the scaffold on biomaterial, which size and shape was defined before performing the surgery. The cells separated were added with some drops of thrombin. The material used for the study was Osteostim Skelite resorbable bone graft substitute (manufactured by Millenium Biologic Inc-.Canada) which chemical composition and size facilitates the ingrowth of bone. X-ray exams were performed immediately after the surgery. Clinical, ultrasound and x-ray exams were performed after 20 days and then every month. Results: 7 of 9 treated dogs have shown very good clinical and x-ray results. Conclusions: One of the objective of the study was to use the fresh bone stromal cells (BMSCs) in clinical applications in large bone defects in the dog. The advantages of using the cells are: they don’t need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with bone and lastly, the technique is easier and the costs are lower. We use a fully resorbable biomaterial with BMSCs to obtain a complete substitution of large bone defects since the final goal is the complete substitution of the biomaterial scaffold with new formed bone. Persistency of biomaterial, in fact, limits the complete integration of the two (old and new) interfaces and may represent a weak spot in functionality when tensions and loads are fully applied to the bone, in spite of a satisfactory surgical recovery

Aim: To assess the efþcacy of percutaneous reaming of simple bone cysts followed by administration of a mixture of autologous bone marrow combined with demineralized bone matrix. Material-methods: 10 patients (7 boys aged 10 years on average and 3 girls aged 12 years on average) with a bone cyst (3 patients at the femur, and 7 at the humerus), were treated with the same surgical technique. The diagnosis was based exclusively on imaging methods (X-ray, CT, MRI) and no biopsy was performed. Under general analgesia and image intensi-þer control the cystic cavity was aspirated. Should ßuid was encountered then a mixture of bone marrow and demineralized bone matrix (DBM) was injected after thorough reaming of the canal proximally and distally. None of the patients required overnight hospital stay. The limb was protected till the patient became asymptomatic. Results: The average follow-up time was 24 months (9–29 months). No perioperative complications were noted. All patients were asymptomatic at the last examination, with normal limb movement. All bone cysts were classiþed from an independent observer, according to the Neer classiþcation, as type I and II. Conclusion: Percutaneous administration of DBM combined with autologous bone marrow after restoring the continuity of the medullary canal with reaming is a simple surgical technique requiring no hospitalization and a short period of immobilization while the clinical and radiological outcome is excellent

Although success has been achieved with implantation of bone marrow mesenchymal stem cells (bMSCs) in degenerative discs, its full potential may not be achieved if the harsh environment of the degenerative disc remains. Axial distraction has been shown to increase hydration and nutrition. Combining both therapies may have a synergistic effect in reversing degenerative disc disease. In order to evaluate the effect of bMSC implantation, axial distraction and combination therapy in stimulating regeneration and retarding degeneration in degenerative discs, we first induced disc degeneration by axial loading in a rabbit model. The rabbits in the intervention groups performed better with respect to disc height, morphological grading, histological scoring and average dead cell count. The groups with distraction performed better than those without on all criteria except the average dead cell count. Our findings suggest that bMSC implantation and distraction stimulate regenerative changes in degenerative discs in a rabbit model

Purpose: The gravity of osteonecrosis in patients with sickle cell anaemia is well known, but the spontaneous course of grade I and II necrosis is not. The first MRI studies performed in these patients were made in 1985. This study compared the spontaneous course in 45 cases of grade I and II necrosis diagnosed between 1985 and 1990 with that in 43 cases of hip necrosis with the same grades I and II diagnosed between 1990 and 1995 in adult patients with sickle cell anaemia treated by medullary drilling with autologous bone marrow grafts. Material and methods: The 45 cases of necrosis followed were diagnosed between 1985 and 1990. These patients did not undergo conservative treatment until the sphericity of the femoral head was lost. They were followed clinically and radiographically up through 2000. The second group of 43 cases of hip necrosis were diagnosed between 1990 and 1995. These patients were treated by meduallary drilling with an autologous bone marrow graft. The bone marrow as harvested from the iliac crests, concentrated and reinjected in the osteonecrotic area. The patients were followed clinically and radiographically until 2000. All patients had an x-ray of the hip (AP and lateral views) at last follow-up. As the follow-up was different for the two groups, comparisons were made using the survival curves; all patients were followed for at least five years. Results: In group I where the clinical course was spontaneous, the spherical shape of the head was lost in 100% of the patients at five years (30% at one year, 60% at two years and 100% at five years), leading to surgery for 80%, usually with prosthesis implantation. In group II where the patients were treated by drilling and autologous bone marrow transplantation, two patients (5%) lost femoral head sphericity at five years. Ten percent of the patients (4 patients) had lost the femoral head sphericity at the current mean follow-up of eight years (minimum five, maximum ten) and required reoperation for prosthesis implantation. MRI and CT imaging in the non-reoperated patients demonstrated a spherical head and remodelling or disappearance of the osteonecrosis at five years. Discussion and conclusion: The spontaneous course of necrosis in adults with sickle cell anaemia is unfavourable after five years. This spontaneous course can be modified (at least the rate of progression) by drilling associated with autologous bone marrow transplantation, if it is performed early enough

Study Design: Retrospective review of patients treated with mineralized collagen matrix hydrated with bone marrow in Anterior Cervical Decompression and Fusion (ACDF). Background: Autologous iliac crest bone is regarded as the gold standard graft material for ACDF. Postoperative donor site pain and morbidity made us seek for an alternative while still providing a high rate of fusion as attained with autologous bone. Methods: All patients who underwent ACDF between August 2005 and May 2007 by the senior author for cervical myelopathy or radiculopathy secondary to degenerative changes or trauma were included. Patients with an additional posterior fusion were excluded. A total of 80 patients were observed for 16 months (range, 6–27 months). In all patient we used a mineralized collagen matrix hydrated with bone marrow aspirate from the left iliac crest as our cage graft material. All patients had PEEK interbody cages and anterior cervical plating. Clinical and radiographic follow-up data were obtained. X-rays were reviewed by both the senior author and an independent radiologist. Clinical outcomes were measured as described by Robinson and with the Nurick scale. Results: 31 single-level fusions, 35 two-level, 13 three-level and 1 four-level fusion were performed. No revision surgery was needed. One patient had a subsequent laminectomy for residual cord compression. All patients achieved a solid fusion. No symptomatic pseudoarthrosis was noted. No donor site morbidities were noted. With a successful clinical result defined as an excellent or good outcome accompanied by significant pain relief, 73 patients had a successful clinical result. Conclusion: The use of a mineralized collagen matrix in combination with bone marrow aspirate in ACDF eliminates iliac crest donor site morbidities while still providing high rates of fusion

Aim. To evaluate the efficacy of bone marrow derived stromal cells (BMSC) for the treatment of non-unions in fractures. Methods. An ethically approved single centre randomised control trial recruited 35 patients for treatment of non-unions with BMSC during 2006–2010. Autologous BMSC were culture expanded at the Good Manufacturing Practice (GMP) standard Oscell® laboratory in the hospital. Following in vitro expansion- cells in autologous serum and serum alone were randomised for insertion at one of the two fracture sides by StratOs® computer software. Patients and the operating surgeon were blinded to the side of cell insertion. Such method of randomisation created internal controls at the fracture sites- one side receiving the cell (‘test side’) and other, not (‘control’). Serial radiographs extending up to an average of twelve months were evaluated by six independent assessors blinded to side of cell insertion. Callus formation and bridging of fracture was compared for ‘test’ and ‘control’ side. Radiological and clinical outcome at final follow-up was also noted. Results. The study had 21 males and 14 females with a mean age of 51.2±13.2 years (range 18–76). The average duration of non-union was 3±2 years (range 1–10 years) with mean 3.5 (range 1–12) surgical interventions prior to BMSC insertion. Independent assessment of ‘test’ and ‘control’ side revealed that the callus formation and fracture bridging was slow although a trend to improvement on the side of the BMSC insertion was observed at 9–12 months. At final follow-up 22 patients progressed to bony union; 13 patients had persisting non union. Conclusion. BMSC can achieve progression to union in substantial number of cases of resistant non-unions where the alternative is extensive reconstructive procedures or amputations. Larger trials are required to study the pattern of early healing following cell therapy in such cases

Introduction: Bone marrow derived stromal stem cells (BMSSC’s) have the ability to differentiate into a variety of mesenchymal tissues including bone. The objective of this study was to evaluate the use a hydroxyapatite – BMSSC (HA-BMSSC) composite graft for posterior spinal fusion in a rabbit model. Method: The HA- BMSSC composite graft was prepared by seeding rabbit marrow derived BMSSC’s onto 5 grams of HA granules which were cultured for a further 7 days prior to implantation. Bilateral posterior L4–L5 interlamina spinal fusion was performed using the HA- BMSSC composite graft (4 Rabbits), hydroxyapatite(HA) granules (6 rabbits) or autologous bone graft obtained from the iliac crest (6 rabbits). Rabbits were sacrificed at 5 weeks. Fusion was assessed by manual palpation. Quantitative histological analysis of cartilage, fibrous tissue and bone in the mid portion of the graft was performed using image analysis software. Results: Three of four of the HA- BMSSC grafts fused successfully compared to 5 of 6 of the autologous bone grafts and 0 of 6 of the HA control grafts. The fusion rate was significantly higher in the iliac crest and HA- BMSSC groups than the HA control group (p< 0.05). In both the HA control and HA stem cell composite grafts there was ingrowth of new bone and encasement of HA granules by new trabecular bone at the graft – host interface. Within the mid region of the grafts there was bone formation in 2 of four fusion masses in the HA- BMSSC group comprising 26% and 45% of tissue in the area examined. In contrast bone formation was seen in the centre of only one of the six 6 HA fusion masses and amounted to only 2% of tissue. There was no significant difference in average percentage area of new bone, cartilage or fibrous tissue within the central region of the HA and HA-BMSSC grafts. There was a higher mean percentage area of new bone formation within the autologous bone graft (27%) than the HA control group (0.3%). p< 0.02. Discussion: The BMSSC –HA composite was as effective as autologous graft and superior to HA in promoting fusion, but HA when used alone was ineffective. A positive finding to support the osteogenic potential of the stem cell loaded HA granules was the presence of moderate amounts of enchondral new bone isolated within the central regions of the graft away from the graft host interface in 2 of 4 fusion masses. In contrast the HA control grafts only supported significant amounts of bone formation in the periphery, adjacent to the host bed

Background. Posterolateral fusion (PLF) is a commonly accepted surgical procedure and overall the most common technique performed to obtain fusion in the lumbar spine. Harvesting autologous bone from the iliac crest is associated with increased operation time, blood loss, and chronic donor site pain. Allograft material has an insufficient osteoinductive potential. Bone marrow concentrate (BMC) could be an option how to promote allograft PLF healing. The purpose of the presented study was to investigate the validity of BMC addition to allografts in instrumented lumbar PLF surgery. Methods. The study was prospective, randomised, controlled and blinded. Eighty patients with degenerative disease of the lumbar spine underwent instrumented (S. 4. , Aesculap, Tuttlingen, Germany) lumbar or lumbosacral PLF. In forty cases, the PLF was done with spongious allograft chips alone (Group I). In another forty cases, spongious allograft chips were mixed with BMC (Group II), where the mesenchymal stem cell (MSCs) concentration was 1.74 × 10. 4. /L at average (range, 1.06–1.98 × 10. 4. /L). Patients were scheduled for anteroposterior and lateral radiographs at 12 and 24 months after the surgery and for CT scanning at 24 months after the surgery. Fusion status and the degree of mineralization of the fusion mass were evaluated separately by two radiologists blinded to patient group affiliation. Results. In Group I at 12 months, the bone graft mass was assessed in X-rays as fused in no case (0 %) and at 24 months in 4 cases (10 %). In Group II, 6 cases (15 %) achieved fusion at 12 months and 14 cases (35 %) at 24 months. The statistically significant difference between both groups was proven for complete fusion at 12 months (p = 0.041) and at 24 months (p = 0.011), too. CT scans showed that 16 cases (40 %) in Group I and 32 cases (80 %) in Group II had evidence of at least unilateral continuous bridging bone between neighboring vertebrae at 24 months (p < 0.05). We have confirmed the hypothesis that the autologous BMC together with the allograft is a better alternative for the PLF than the allograft alone. Conclusions. The use of autologous MSCs in form of the BMC in combination with allograft is an effective option how to enhance the PLF healing. Allograft by itself is not an effective material as a posterior onlay graft for the PLF in adult surgery

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs).

ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates.

Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components.

Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients.

Background. A cell-based tissue-engineered construct can be employed for treating meniscal lesions occurring in the non-vascularized inner two-thirds. The objective of this study was to test the hypothesis that both pre-differentiation of human bone marrow derived stromal cells (hBMSCs) into chondrogenic lineage before cell seeding and platelet-rich plasma (PRP) pretreatment on a PLGA mesh scaffold enhances the healing capacity of the meniscus with hBMSCs-seeded scaffolds in vivo. Methods. PRP of 5 donors was mixed and used for the experiments. The woven PLGA mesh scaffold (VicrylTM, Ethicon) measuring 20×8 mm (thickness, 0.2 mm) was prepared. The scaffolds were immersed into 1,000 μl of PRP and were centrifuged at 150g for 10 min. Then, the scaffold was flipped 180° and the same procedure was done for the other side. After washing, the scaffolds were soaked into 1,000 μl of DMEM media. hBMSCs from an iliac crest of 10 patients after informed consent and approval of our IRB were induced into chondrogenic differentiation with chondrogenic media containing 10 ng/ml rhTGF-ß3 in 1.2% alginate bead culture system for 7 days. Then, 2×10. 5. hBMSCs were recovered, seeded onto the scaffold, and cultured under dynamic condition. Based on the presence of pre-differentiation into chondrogenic lineage and the PRP pretreatment, 4 study groups were prepared. (no differentiation without PRP, no differentiation with PRP, chondrogenic differentiation without PRP, chondrogenic differentiation with PRP) Cell number for each cell-seeded scaffold was determined at 24 hours after seeding. Then, scaffolds were placed between human meniscal discs and were implanted subcutaneously in nude mice for 6 weeks (n=10 per group). Results. Cell attachment analysis revealed no significant difference among groups (p>0.05). The average cell number attached on the scaffold was ranged 1.1×10. 5. to 1.2×10. 5. among groups after 24 hours, so the initial cell seeding efficiency was ranged 55 to 60%. Histologic results from the 10 constructs containing hBMSCs undifferentiated and seeded onto non-PRP treated scaffolds revealed none had healed at all. Of the constructs containing hBMSCs undifferentiated and seeded onto PRP-pretreated scaffolds, three menisci healed and seven did not heal. Of the constructs containing hBMSCs pre-differentiated into chondrogenic lineage and seeded onto non-PRP treated scaffolds, six menisci healed and four did not heal. Of the constructs containing hBMSCs pre-differentiated into chondrogenic lineage and seeded onto PRP-pretreated scaffolds, seven menisci healed and three did not heal. Histological evaluation demonstrated a continuous hypercellular new fibrous tissue integrating into the native devitalized meniscus disc tissue in healed samples. The histological outcome between the groups was significant (p<0.05) (Table 1) (Figure 1). Conclusion. hBMSCs, which were differentiated into chondrogenic lineage before cell seeding and attached PRP-pretreated PLGA mesh scaffolds, demonstrated enhanced healing capacity of human meniscus in a meniscal repair mouse model. These findings demonstrate that both pre-differentiation of hBMSCs into chondogenesis and the PLGA scaffold modified by PRP pretreatment provides more biomimetic and biocompatible strategy for cell-mediated meniscal repair. Acknowledgements. This study was supported by Basic Science Research Program through the National Research Foundation of Korea (#2015-01004099)

Introduction. Failures in fracture healing are mainly caused by a lack of neovascularization. We have previously demonstrated that G-CSF-mobilized peripheral blood (GM-PB) CD34+ cells, an endothelial progenitor enriched cell population, contributed to fracture healing via vasculogenesis and osteogenesis. We postulated the hypothesis that local transplantation of culture expanded bone marrow (cEx-BM) CD34+ cells could exhibit therapeutic potential for fracture healing. Materials. BM CD34+ cells were cultured in specific medium with 5 growth factors for 1week. A reproducible model of femoral fracture was created in nude rats with periosteum cauterization, which leads to nonunion at 8 weeks post-fracture. Rats received local administration of the following cells or PBS alone(1)cEx-BM, (2)BM, (3)GM-PB CD34+ cells or (4)PBS. Results. Our 7-day culture expansion technique allowed us to obtain 23 times of BM CD34+ cells maintaining 60% purity of CD34 positivity. cEx-BM CD34+ cells exhibited striking therapeutic efficacy for unhealing fracture promoting neovascularization and osteogenesis in sites of fracture. Moreover, cEx-BM CD34+ cells showed high capacity of colony formation and osteogenic differentiation. Conclusion. BM CD34+ cells can be obtained from the fracture site at the time of primary operation and stored for further use, autologous culture expanded BM CD34+ cell transplantation therapy would be not only a simple but also powerful therapeutic strategy for unhealing fracture

Mesenchymal Stromal Cells (MSC) are promising therapies for fracture healing. However, undifferentiated MSC may act only through an inductive paracrine effect without direct bone formation. Here, we developed an injectable product constituted of human bone-forming cells derived from bone marrow (BM)-MSC (ALLO-P2) that display more potent bone repair properties not only by stimulating host osteoinduction but also by direct bone formation. In vitro, ALLO-P2 overexpressed markers such as ALP compared to BM-MSC isolated from the same donors, suggesting their engagement into the osteogenic lineage. In vivo, a single dose of ALLO-P2 significantly enhanced bone neoformation 14 days post-administration over the calvaria of NMRI-Nude mice compared to the control excipient. Histological analyses and mouse/human type I collagen double-immunolabelling revealed the presence of mineralized bone nodules of mixed host and donor origins in mice administered with ALLO-P2. Together, these results show that ALLO-P2 is a potential promising clinical candidate to promote bone repair, since it can be produced at high yields, is injectable and boosts ossification mechanisms involved in the physiological repair process

Summary. Methotrexate chemotherapy (commonly used in treating cancers and rheumatoid arthritis) creates an inflammatory condition in bone, decreasing osteogenesis, enhancing adipogenesis, increasing osteoclastogenesis, leading to bone loss and marrow adiposity; treatment with fish oil or folinic acid counteracts these negative effects and prevents bone loss. Introduction. Chemotherapy with anti-metabolite methotrexate (MTX) is commonly used in treating cancers and rheumatoid arthritis; however it is known to cause bone loss for which currently there are no adjunct preventative treatments. Methods and Materials. Using a rat model, this study investigated the damaging effects in bones caused by daily MTX injections (0.75mg/kg) for 5 consecutive days (mimicking induction phase treatment for childhood leukaemia) and also the potential protective benefits of omega-3 fatty acid-rich fish oil at different doses (0.25, 0.5 or 0.75 mL/100g BW) in comparison to antidote folinic acid (given i.p at 0.75mg/kg 6 hours post MTX, which is clinically used to reduce MTX toxicities in soft tissues). Results. Histological analysis showed that MTX significantly reduced primary spongiosa bone height and metaphyseal trabecular bone volume. MTX also significantly reduced density of osteoblasts at the secondary spongiosa. Ex vivo differentiation assays with bone marrow stromal cell populations of treated rats revealed a significant reduction in osteogenic differentiation but an increase in adipogenesis. Consistently, RT-PCR gene expression study within the stromal cell population revealed a lower expression of osteogenic transcription factors Runx2 and Osx and bone matrix protein osteocalcin but a significantly upregulated adipogenesis-related genes FABP4 and PPARγ, indicating that MTX chemotherapy induces a switch in the differentiation potential towards adipogenesis at the expense of osteogenesis. MTX increased the density of osteoclasts within the metaphyseal bone as revealed by histological analysis and osteoclast precursor cell pool as shown by ex vivo osteoclastogenesis assay with bone marrow samples. Consistently, mRNA expression of proinflammatory and osteoclastogenic cytokines IL-1, IL-6, TNF-α, and the RANKL/OPG ratio were significantly upregulated by MTX. Supplementary treatment with fish oil (0.5mL/100g BW) or folinic acid significantly preserved metaphyseal trabecular bone volume, osteoblast density, and bone marrow stromal cell osteogenic differentiation and suppressed MTX-induced adipogenesis. These supplements also prevented MTX-induced increased osteoclast density, osteoclastogenesis, and expression of proinflammatory and osteoclastogenic cytokines. Conclusion. These results suggest that MTX chemotherapy creates an inflammatory condition in bone resulting in increased osteoclast formation and decreased osteoblast formation thus leading to bone loss, and that supplementary treatment with fish oil at 0.5mL/100g BW or folinic acid counteract these negative effects, helping to conserve bone formation, suppress bone resorption and bone marrow adiposity, and thus prevent bone loss during MTX chemotherapy

In 16 mature New Zealand white rabbits mesenchymal stem cells were aspirated from the bone marrow, cultured in monolayer and implanted on to a full-thickness osteochondral defect artificially made on the patellar groove of the same rabbit. A further 13 rabbits served as a control group. The rabbits were killed after 14 weeks. Healing of the defect was investigated histologically using haematoxylin and eosin and Safranin-O staining and with immunohistochemical staining for type-II collagen. We also used a reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA of type-I and type-II collagen. The semiquantitative histological scores were significantly higher in the experimental group than in the control group (p < 0.05). In the experimental group immunohistochemical staining on newly formed cartilage was more intense for type-II collagen in the matrix and RT-PCR from regenerated cartilage detected mRNA for type-II collagen in mature chondrocytes. These findings suggest that repair of cartilage defects can be enhanced by the implantation of cultured mesenchymal stem cells

Introduction. Nonunions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing, although the effects of different clinical ES waveforms available in clinical practice on BMMSCs cellular activities is unknown. Materials and Methods. We compared Direct Current (DC), Capacitive Coupling (CC), Pulsed Electromagnetic wave (PEMF) and Degenerate Wave (DW) by stimulating human-BMMSCs for 5 days for 3 hours a day. Cytotoxicity, cell proliferation, cell-kinetics and cell apoptosis were evaluated after ES. Migration and invasion were assessed using fluorescence microscopy and affected gene and protein expression were quantified. Results. DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms and unstimulated cells after 5 days of ES (p < 0.001). DC, DW and CC resulted in significantly more cells in S phase and G2/M phase (p < 0.01) compared to the unstimulated BMMSCs. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression (p < 0.001) compared to the other waveforms and unstimulated BMMSCs. DC increased cellular migration in a scratch-wound assay and all ES waveforms increased migration gene expression with DC having the greatest effect (p < 0.01). Conclusion. The ES waveform is vital in influencing BMMSCs cellular activities. Migration and invasion were increased by ES which suggests that the recruitment of BMMSCs to the healing site during a fracture could be increased by ES

Aims. Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells. Methods. Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of cell viability and ALP activity were examined by in vitro studies. Results. The live cells rate (the ratio of total number of cells in the well plate to the absorbance-measured number of live cells) was significantly decreased at ≥ 500 μg/ml of gentamicin on day 14; apoptosis rate was significantly increased at ≥ 750 μg/ml, and ALP activity was significantly decreased at ≥ 750 μg/ml. Real-time reverse transcription-polymerase chain reaction results showed no significant decrease in the ALP and activating transcription factor 4 transcript levels at ≥ 1,000 μg/ml on day 7. Mineralization potential was significantly decreased at all concentrations. Restoration of cell viability was significantly decreased at 750 and 1,000 μg/ml on day 21 and at 500 μg/ml on day 28, and ALP activity was significantly decreased at 500 μg/ml on day 28. Conclusion. Our findings suggest that the exposure concentration and duration of antibiotic administration during CLAP could affect cell functions. However, further in vivo studies are needed to determine the optimal dose in a clinical setting. Cite this article: Bone Joint Res 2024;13(3):91–100

Introduction: Aseptic loosening at the bone-implant interface of THA acetabular components is a significant cause of implant failure. This loosening has been attributed either to wear particle-induced osteolysis or to the effects of joint fluid-pressure. It may be possible to prevent the loosening of implants by improving fixation between the bone and implant, or promoting the growth of a biological bony seal, in order to prevent the influx of wear particles or pressurized joint fluid. Additionally in revision implants it is important to promote osseointegration in situations where bone stock may be limited. The hypothesis of this study was spraying autologous BMSCs in fibrin glue onto the surface of HA-coated acetabular components would increase bone formation around the implant and improve bone-implant contact. Materials and Methods: Bone marrow was aspirated from the iliac crest of six goats, and BMSCs isolated and expanded in vitro. 10 x 10e6 BMSCs were suspended in reconstituted thrombin pre-operatively. A standard posterior approach was used. The acetabular shell was then coated with 2 ml of fibrin glue, with (n=6) or without 10 x 10e6 autologous BMSCs (n=6), and the acetabular component impacted into position. Antibiotic and analgesic prophylaxes were carried out. All animals were weight bearing within 48 hours post-operatively. Walking and ground reaction forces were assessed pre-operatively, as well as 6 and 12 weeks post-operatively. Results were expressed as a percentage of force transmitted through the right leg versus the left leg. After 12 weeks, the acetabulae were retrieved, and processed for histology. The percentage of new bone around the cups was measured within 5 radial zones, using image analysis. Bone-implant contact was also analysed between the new bone and implant surface. Mann Whitney U test was used to show statistical significance. Results: New bone formation in Zone 5 showed a significant increase in the BMSC group (71.97±10.91%), when compared to the controls (23.85±15.13%, p=0.028). The other zones did not show a significant difference. Overall new bone growth in the BMSC group was 30% greater than the control group (71.42±8.97% and 54.22±16.56%, respectively, p=0.58). Bone-implant contact was significantly improved in the BMSC group (20.03±4.64%), in contrast to the control group (13.71±8.32%, p=0.027). With regards to the force plate analysis, there was no significant difference in loading between groups at both 6 weeks (Controls-79.74±3.63%, BMSCs-59.39±9.33%, p=0.086) and 12 weeks (Controls-86.0%±2.85%, BMSCs-62.33±5.12%, p=0.055). Discussion and Conclusions: In this study, overall bone growth was greater when cups were treated with BMSCs. Bone-implant contact was significantly improved as well. This study has clinical applications, as using MSCs in fibrin glue promotes a bony seal in contact with the implant which may prevent the migration of particles, or joint fluid, decreasing the likelihood of aseptic loosening of THAs, and improving their longevity. Also, this technique may improve fixation in situations where bone stock is reduced

Abstract

Background. Despite promising results have shown by osteogenic cell-based demineralized bone matrix composites, they need to be optimized for grafts that act as structural frameworks in load-bearing defects. The purpose of this experiment is to determine the effect of bone marrow mesenchymal stem cells seeding on partially demineralized laser-perforated structural allografts that have been implanted in critical femoral defects. Materials and Methods. Thirty-two wistar rats were divided into four groups according to the type of structural bone allograft; the first: partially demineralized only (Donly), the second: partially demineralized stem cell seeded (DST), the third: partially demineralized laser-perforated (DLP), and the fourth: partially demineralized laser-perforated and stem cell seeded (DLPST). Trans-cortical holes were achieved in four rows of three holes approximated cylindrical holes 0.5 mm in diameter, with centres 2.5 mm apart. P3 MSCs were used for graft seeding. Histologic and histomorphometric analysis were performed at 12 weeks. Results. DLP grafts had the highest woven bone formation, where most parts of laser pores were completely healed by woven bone. DST and DLPST grafts surfaces had extra vessel-ingrowth-like porosities. Furthermore, in the DLPST grafts, a distinct bone formation at the interfaces was noted. Conclusion. This study indicated that surface changes induced by laser perforation, accelerated angiogenesis induction by MSCs, which resulted in endochondral bone formation at the interface. Despite non-optimal results, stem cells showed a tendency to improve osteochondrogenesis, and the process might have improved, if they could have been supplemented with the proper stipulations

Summary. The findings demonstrate that culture expanded human mesenchymal stem cells (MSCs) incorporated and proliferated in clinically relevant cell scaffolds better than freshly isolated bone marrow mononucleated cells (MNCs); in fact, only in MSC cultures were cells present for longer term chondrogenic inductions. Introduction. The treatment of chondral defects poses a significant clinical problem and a variety of cell sources and techniques have been studied and practiced to regenerate cartilage. Preclinical and clinical evidence suggests that MSCs can help regenerate cartilage when transplanted into cartilage lesions. However, the uptake of MSCs for cell therapies is limited due to the need for their culture expansion to generate subsequent numbers for transplantation. An alternative is to use minimally manipulated MNCs, which avoids the costs and regulatory implications of culture expansion and would enable the treatment of cartilage defects in a one-step procedure. Therefore, this study has focused on comparing these two cell types within three different scaffolds that can currently be used as cell delivery systems. Methods. Culture expanded human MSCs and MNCs freshly isolated from bone marrow were seeded at a density of 50,000 cells in 3mm. 2. scaffolds of Chondro-Gide® (type I/III collagen), Alpha Chondro Shield® (polyglycolic acid) and Hyalofast™ (hyaluronic acid). The cell-seeded scaffolds were incubated for 2 hours to permit initial cell adhesion and then treated with or without chondrogenic inducers (100nM dexamethasone, 10ng/ml TGF-β1, 37.5µg/ml ascorbic acid and ITS-X in DMEM/10% serum) for 28 days at 37°C. The Cell incorporation, growth and viability was assessed using Live/Dead staining and confocal microscopy, along with histological stains of the sectioned scaffolds. Proteoglycan synthesis was measured using DMMB assay of glycosaminoglycan (GAG) into the harvested culture medium. Results. MSCs adhered to the scaffolds to a much greater extent than the MNCs. In fact, the low number of MNCs initially incorporated into the scaffolds diminished over time such that no viable MNCs were seen during long term cultures and in all cases. MSCs incorporated into the Chondro-Gide® scaffold better than into the Alpha Chondro Shield® or Hyalofast™, and during long term cultures the MSCs in Chondro-Gide® proliferated to become significantly greater in number than those in the other two scaffolds. There was no clear matrix deposition. However, the MSCs in Hyalofast™ were rounded in shape, which is consistent with the morphology of chondrocytes, in the presence of chondrogenic inducers only. Furthermore, a significantly greater level of GAG was detected in the medium harvested from Chondro-Gide® and Hyalofast™ cultures under chondrogenic conditions compared with non chondrogenic conditions. Discussion/Conclusion. This study has shown that human MSCs incorporated, adhered and proliferated better in clinically utilised cell scaffolds compared to MNCs, enabling the induction of chondrogenesis in the longer term. Freshly isolated MNCs from bone marrow contain only 0.01–0.001% of MSCs in addition to non-adherent cell types, e.g. hematopoietic cells, which may account for their low cellular incorporation and decreased cell proliferation in the scaffolds. This outcome for MNCs may be improved using prospective MSC isolation techniques, where in vivo studies are also required to properly examine the chondrogenic potential. Nonetheless, our initial work suggests that culture expanded MSCs are a better option than minimally manipulated cells for cartilage repair

Once damaged, articular cartilage has limited capacity for self-repair due to their avascular and acellular nature. Tissue engineering approaches using cultured chondrocytes and biomaterials as scaffoldings hold promises for repairing cartilage defects. However, the source of articular chondrocytes is limited and the chon-drocytes may de-differentiate when cultured for a prolonged period. Bone marrow derived mesenchymal stem cells (BMSCs) have multi-differentiation potentials and autologous BMSCs are easy to obtain and culture with no/little immunological reaction when re-implanted. 24 NZW rabbits were used. Rabbit autologous BMSCs were obtained through marrow aspirations and expanded in culture under the chondrogenic induction media (DMEM, 10% FCS, plus 10ng/ml TGF-β1) for 3 weeks. A full-thickness articular cartilage defect (3 mm in diameter and 3 mm in depth) was created on both medial condyles in the rabbit. For experimental group (16 joints), the defects were filled immediately with alginate capsules containing autologous chondrogenic cells (8.5 x 104); for the control groups, the defects were filled with either alginate capsules alone (16 joints) or left untreated (16 joints). All the animals were terminated at 6 and 12 weeks after surgery and the cartilage samples were harvested for histology, immunochemistry and in situ hybridization examinations. For histology, in the experimental group the defects were filled with immature hyaline-like cartilaginous tissues at 6 weeks; by 12 weeks the newly formed cartilage showing signs of remodeling and integrating into the surrounding articular cartilage. The expression of type II collagen in the newly formed cartilaginous tissues was confirmed by immunohistochemistry and by in situ hybridization methods. In the control groups, the defects were mainly filled with fibrous tissues in all the animals at the two time points examined. We have used Wakitani cartilage grading system for semi-quantitative histological evaluation. Significant lower scores (with superior histology) were found in the experimental group comparing to the two control groups. Our results confirmed that full-thickness articular cartilage defects can be repaired by chondrogenically differentiated autologous BMSCs seeded into alginate capsules. Further studies are ongoing to explore the long term outcome of this treatment approach as well as using new scaffolds for cartilage tissue engineering

Purpose. Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2. Method. Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses. Results. The mean number of cell colonies was 1.25-fold higher after hypoxia culture relative to normoxia. There were no differences in colony diameters. A panel of common protein signatures (CD29, CD90, CD105 and CD151) for stem cells declined in expression after expansion in hypoxia. However, other signatures (CD13, CD34 and CD44) expression level increased under hypoxia, whilst CD73 expression was unchanged. Pellets from hypoxia-expanded MSCs showed on average a 1.4-fold increase in chondrogenic capacity as judged by glycosaminoglycan (GAG) matrix per DNA content relative to normoxia pellets. The gene expression of collagen II, SOX9, aggrecan and matrillin-3 increased by 1.2-, 2-, 1.3- and 1.5-fold, respectively, in pellets formed from hypoxia-expanded stem cells relative to their normoxia counterparts. Conclusion. Expansion of stem cells under hypoxia potentiates their capacity to form cartilage with improved cartilage properties. However, there is a need for signatures to identify stem cells with propensity to form cartilage

Aims. Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. Methods. Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) analysis; the legs were removed for micro-CT imaging and subsequent histology. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and purified bone marrow collected during surgeries by using density gradient centrifugation. After a series of interventions such as knockdown or overexpressing BRD4, Alizarin red staining, RT-qPCR, and Western Blot (Runx2, alkaline phosphatase (ALP), Osx) were performed on hBMSCs. Results. Overexpression of BRD4 enhanced while inhibition of Brd4 suppressed the osteogenic differentiation of hBMSCs in vitro. Overexpression of Brd4 increased the expression of mitotically associated long non-coding RNA (Mancr). Downregulation of Mancr suppressed the osteoinductive effect of BRD4. In vivo, inhibition of BRD4 by JQ1 significantly attenuated pathological bone formation in the ATP model (p = 0.001). Conclusion. BRD4 was found to be upregulated in HO and Brd4-Mancr-Runx2 signalling was involved in the modulation of new bone formation in HO. Cite this article: Bone Joint Res 2021;10(10):668–676

Objective: Bone marrow stromal cells (BMSC) represent an interesting target for novel strategies in the gene and cell therapy of skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. Materials and Methods: Stromal cells were obtained from healthy donors. For the first 2 weeks, culture medium was supplemented only with human recombinant fibroblast growth factor 2 (FGF-2) to promote cell proliferation and maintain cells in a more immature state. Confluent cultures were detached with trypsin-EDTA. Cells were replated for the in vitro differentiation experiments and for determination of BMSC growth kinetics. Cultures were stimulated with appropriate inductive media and the chondro-/osteo-/adipo-diferentiations were tested by staining with alizarin red, alcian blue, Sudan black and by immunostaining for osteocalcina or collagen II. Results: After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Conclusion: Culture expansion causes BMSC gradually to lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion. There are numerous potential applications of this novel strategy, for example: reconstruction of extensive long-bone defects, osteochondral defect repair, treatment of bone cyst, bioactivable scaffolds, etc

Background: Traumatic brachial plexus (BP) injuries may cause loss of elbow flexion. After nerve surgery active elbow flexion often remains insufficient. Muscle strength improvement via cell therapy would be a potential option and could avoid muscle transfer surgery. The primary objective of this pilot study was to assess the safety and feasibility of autologous bone marrow (BM)-derived mononuclear cell (MNC) injection in partly denervated m. biceps brachii of BP patients. Secondary, this study has focused on the myogenic potential of BM-derived MNC by assessing the morphological and functional improvement of the biceps. Methods: Nine adult BP patients with insufficient force recovery of elbow flexion were included. Three escalating doses (0.9, 4 and 8 * 108) of MNCs were injected in the m. biceps brachii (group A, B and C). In group A, BM was aspirated under local anesthesia (60 ml). In group B and C, BM was aspirated in combination with a muscle tendon transfer (Steindler flexorplasty) under general anesthesia (350 and 650 ml respectively). A muscle biopsy was performed before and 3 months after transplantation. Furthermore, quantitative needle EMG, CT-scan and clinical function was obtained at pre-transplantation and at 3 and 6 months follow-up. The EMG and CT-scan data were blinded during analysis. Results: No negative side effects were observed. Biopsies showed an increase of 80% in myofiber diameter (P = 0.007), 51% in satellite cells (P = 0.045), 83% in capillary to myofiber ratio (P < 0.001) and a decrease of 51% in fibrosis (P = 0.012). Histological changes were most apparent in group B with an increase of 126% in myofiber diameter (P = 0.019), 100% in capillary to myofiber ratio (P = 0.027), and a decrease of 70% in fibrosis (P = 0.023). EMG demonstrated an increase of 36% in amplitude (P = 0.045), 29% in duration (P = 0.005) and 29% in number of phases of the motor unit potentials (P = 0.002). CT-scan analysis showed a decrease of 48% in mean muscle density (P = 0.009). Discussion: This study shows that BM-derived MNC transplantation in a partly denervated muscle of traumatic PB patients is safe and feasible. Muscle improvement was observed in muscle biopsies. Furthermore, changes in EMGs and CT-scans were also suggestive for muscle regeneration. The BM dose applied in group B could represent the optimal dose to enhance partly denervated muscles. The results of the present study require confirmation in a placebo-controlled study

Articular cartilage repair remains a challenge in orthopedic surgery, as none of the current clinical therapies can regenerate the functional hyaline cartilage tissue. In this study, we proposed a one-step surgery strategy that uses autologous bone marrow mesenchymal stem cells (MSCs) embedded in type II collagen (Col-II) gels to repair the full thickness chondral defects in minipig models. Briefly, 8 mm full thickness chondral defects were created in both knees separately, one knee received Col-II + MSCs transplantation, while the untreated knee served as control. At 1, 3 and 6 months postoperatively, the animals were sacrificed, regenerated tissue was evaluated by magnetic resonance imaging, macro- and microscopic observation, and histological analysis. Results showed that regenerated tissue in Col-II + MSCs transplantation group exhibited significantly better structure compared with that in control group, in terms of cell distribution, smoothness of surface, adjacent tissue integration, Col-II content, structure of calcified layer and subchondral bone. With the regeneration of hyaline-like cartilage tissue, this one step strategy has the potential to be translated into clinical application

Introduction. According to proposal of Noble, the femoral bone marrow cavity form of patients who underwent Total Hip Arthroplasty (THA) can be classified under 3 categories; those are Stovepipe, Normal and Champagne-fluted. We developed typical sodium chloride femoral model was created by 3D prototyping technique. The purpose was to identify the relationship of pressure zone of the surrounding areas between femoral bone marrow cavity form and hip stem. Materials and Method. As opponent clarified stem design concept Zweymüller type model was used. According to CT data with the patients who underwent THA, the sodium chloride femoral model was custom-made and selected as the representative model based on Noble's 3 categories. Eight models of each category were used to performed mechanical test. Result. In mechanics test, the result of comparison between the contact pressure zones of zone 1–7, significant differences of contact pressure zones were identified between the Stovepipe group and Normal group in zone 3, 4 and 5. In zone 3 and 5, such significant differences were also identified between Champagne-fluted group and Normal group. In Stovepipe group, a significant difference of the contact pressure zone was observed at the proximal and distal. In Champagne-fluted group and the Normal group, a significant difference was observed in the contact pressure in distal femur (3, 4, 5 Zone) and (Zone1, 2, 6, 7) proximal femur. Discussion. Although in most studies Sawbone® is used for femoral models, the focus of this research is of those who possess a characteristic femur with marrow cavity form. Therefore, sodium chloride bone model was used instead. In comparison in terms of applicability between sodium chloride bone model and regular model, the failure of all 24 joints of sodium chloride bone model were unconfirmed in mechanics test. Moreover, the possibility that its performance in mechanics test is equivalent to Sawbone®is considered. The design concept for Zweymüller type achieves the ability to load distribute within a wide range of cortical bone across the middle position to distal femur. It's determined by the concept that a wide range of contact pressure was admitted at middle position and distal femur in the Champagne-fluted group and the Normal group. On the other hand, the contact pressure zone of Stovepipe was not able to meet the expected level at distal femur. The method of this research is control its stress condition within the stem design. By this point, it is considered possible that the stability of various stem design was able to be forecasted and the assessment of stableness was positive. Conclusion. On the basis of Noble's categories, 3 types of bone models were created by 3D prototyping technique, and pressure distribution measurement were performed. The result from the pressure distribution indicated that even in Zweymüller stem had anxiety of securing force in Champagne-fluted type and Stovepipe type canal. We believe the method of in vivo study can develop to assess the stability of implant preoperatively

The repair of cartilage defects remains a significant clinical challenge. The use of mesenchymal stem cells for cell-based tissue-engineering strategies represents a promising alternative for the repair of the defects. In this study, we investigated the TGF-bate1 dose and cellular density-dependent effect on chondrogenic differentation of human bone marrow-derived mesenchymal stem cells (MSCs) cultured in alginate beads in vitro. Methods A volume of 6 ml bone marrow was collected from six volunteer donors respectively. MSCs were cultured in different cellular density (1×104, 1×105, 1×106 and 5×106/ml) and treated with different doses of TGF-beta1 (0, 1, 10, 50 and 100 ng/ml). Immunohistochem-istry and in situ hybridization were applied to detect the expression of collagen type II and assay proteoglycan in different time internal. Results 95% cellss were alive after density gradient centrifugation. BMSCs had a similar spindle-like morphology. Type II collagen and proteoglycan were showed positive staining in the 10 ng/ml TGF-beta1 group, weakly positive in the 50 ng/ml and 100 ng/ml group, negative in the 0 ng/ml and 1 ng/ml group. With time, the proteoglycan quantity increased. All cell density groups except 1×104/ml showed positive expression of collagen type II and proteoglycan synthesis, and better staining with increase of cellular density. Proteoglycan synthesis did not increased until the fifth weeks. Conclusions The chondrogenesis differentiation of human MSCs is dose-dependent. 10ng/ml TGF-beta1 is a suitable concentration for such inducing. The cellular density is also important for the differentiation of MSCs. Too small density is ineffective. The more cells, the better differentiation. And the time of in vitro culture should not be longer than 4 weeks

Spontaneous muscle regenerative potential is limited, as severe injuries incompletely recover and result in chronic inflammation. Current therapies are restricted to conservative management, not providing a complete restitutio ad integrum; therefore, alternative therapeutic strategies are welcome, such as cell-based therapies with stem cells or Peripheral Blood Mononuclear Cells (PBMCs). Here, we described two different in vitro myogenic models: a 2D perfused system and a 3D bioengineered scaffold within a perfusion bioreactor. Both models were assembled with human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human primary skeletal myoblasts (hSkMs) to study induction and maintenance of myogenic phenotype in presence of PBMCs. When hBM-MSCs were cultured with human primary skeletal myoblasts (hSkMs) in medium supplemented with 10 ng/mL of bFGF; cells showed increased expression of myogenic-related gene, such as Desmin and Myosin Heavy Chain II (MYH2) after 21 days, and a prevalent expression of anti-inflammatory cytokines (IL10, 15-fold). Next, PBMCs were added in an upper transwell chamber and hBM-MSCs significantly upregulated myogenic genes throughout the culture period, while pro-inflammatory cytokines (e.g., IL12A) were downregulated. In 3D, hBM-MSCs plus hSkMs embedded in fibrin-based scaffolds, cultured in dynamic conditions, showed that all myogenic-related genes tended to be upregulated in the presence of PBMCs, and Desmin and MYH2 were also detected at protein level, while pro-inflammatory cytokine genes were significantly downregulated in the presence of PBMCs. In conclusion, our works suggest that hBM-MSCs have a versatile myogenic potential, enhanced and modulated by PMBCs. Moreover, our 3D biomimetic approach seemed to better resemble the tissue architecture allowing an efficient in vitro cellular cross-talk.

Introduction. The concept of “bone graft expanders” has been popularised to increase the volume and biological activity of the implanted Material. HYPOTHESIS. Orthoss® granules support exogenously seeded MSCs and attract neighbouring host MSCs. Methods. In 3-D cultures’ Orthoss® granules were seeded with 2×10. 5. bone marrow MSCs/granule and maintained in MSC expansion or differentiation media for 21 days. In homing experiments’ bone autografts were placed in close proximity to Orthoss®. Scaffold colonisation and MSC differentiation were assessed by confocal microscopy’ standard electron microscopy’ and energy-dispersive X-ray spectroscopy. Results. Long-term incubation of MSC/scaffold resulted in formation of multiple cell-matrix layers lining the scaffold pores as well as outer surfaces. MSC differentiation to osteoblasts was evident as strong deposition of Calcium and Phosphorus was detected in both MSC expansion and osteogenic conditions. Cell egress experiments demonstrated the migration of cells from neighbouring autografts and their attachment and re-settlement on Orthoss®. Discussion & Conclusions. Orthoss® scaffolds support MSC attachment’ growth and osteogenic differentiation whereas resident bone subpopulations can rapidly migrate towards’ attach’ and expand on them. These results indicate that Orthoss® can serve as a graft expander for repairing large bone defects in trauma patients

Introduction. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors and are known to regulate proliferation and expression of the differentiated phenotype of chondrocytes, osteoblasts, and osteoclasts. To investigate the osteoblastic differentiation gene expressions that contribute to BMP-7 dependent ostogenesis, we performed gene expression profiling of BMP-7-treated mouse bone marrow stromal cells. Methods. D1 cells (mouse bone marrow stromal cells) were cultured in osteogenic differentiation medium (ODM) for 3 days, and then treated with BMP-7 for 24 hr. Total RNA was extracted using Trizol, purified using RNeasy columns. Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit to yield biotinylated cRNA. The data analysis up- and down-regulation developmental processes (anterior/posterior patterning, ectoderm development, embryogenesis, gametogenesis, mesoderm development, other development process, and segment specification) genes expression fold. Results. We detected 18 mRNAs (Id2, Igf2, Pparg, S100a10, Foxn3, Tulp3, Mycbp2, Notch3, Ptk7, Lrp4, Tnfrsf11b, Ogn, Cyr61, Mglap, Akp2, Ltbp4, Ibsp, and Thbs1) that were differentially up-regulated after BMP-7 stimulation. 3 mRNAs (Wars, Adss and Trim35) were differentially down-regulated after BMP-7 stimulation. Conclusion. The data indicate that BMP-7 regulate various developmental processes genes expression during osteoblastic differentiation. Though further studies are needed in relation to each expression gene profiles and osteoblastic differentiation, this information may serve as a point of comparison for osteoblastic differentiation of BMP-7. Furthermore, the data should facilitate the informed use of BMP-7 as a therapeutic agent and tissue engineering tool. Acknowledgement. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-10089-0)

Introduction: Oxidative stress occurs when reactive oxygen species (ROS) are produced faster than they can be removed by cellular defence mechanisms contributing to ageing, many chronic diseases, such as atherosclerosis, RA, Parkinson and Alzheimer’s disease and skeletal pathologies. Here we address the impact of ROS on the viability of early osteogenic precursors in the bone marrow and study the influence of estrogen on this interaction. Cells have a number of mechanisms to protect themselves from ROS, which are constantly being formed in the cell through normal metabolic pathways, such as Vitamin E, C and estrogen. Estrogen has been shown to prevent intracellular accumulation of peroxide and to attenuate oxidant-induced death of neuronal and endothelial cells. In addition, it contributes significantly to bone turnover and relieves postmenopausal symptoms. This study has focused on the potential anti-oxidant properties of estrogen against oxidative on bone marrow stromal cells. stress induced by H. 2. O. 2. Methods: Primary bone marrow stromal cells were pre-treated with several different doses between 10. −6. M – 10. −8. M of estrogen prior to H. 2. O. 2. administration at 0.08–0.4 mM 30% (v/v) for 2–24h. The cellular production of ROS was determined by using the free radical indicator DCFH-DA. Apoptosis was determined by morphological criteria. Results: H. 2. O. 2. induced an increase in apoptosis of osteoprogenitor cells (p< 0.05). Determination of apoptosis and cell number by nuclear staining, indicated that pre-treatment of bone marrow stromal cells with 17-beta estradiol reduced the apoptotic response induced by H. 2. O. 2. (p< 0.05) and restored cell number to control levels. In order to test the anti-oxidant activity of estrogen, the dye DCFH-DA was introduced in a cell free system in the presence or absence of 17-beta estradiol and H. 2. O. 2. The same experiment was repeated in the presence of bone marrow stromal cells. H. 2. O. 2. increased both intracellularly and extracellularly oxidant activity and estradiol has the capacity of modifying this activity both inside and outside the cell. Discussion: These data demonstrate the ability of estrogen, used at physiological doses, to block oxidant-induced apoptosis of osteoprogenitor cells. Estrogen appears to reduce the generation of ROS in these cells. These data could have important implications on the maintenance of osteogenic stem cells during fractures, ageing and disease

Introduction: Recent studies have shown that MSCs can be isolated from the peripheral blood of many different species. Hematopoietic stem cell (HSC) mobilization from the bone marrow to the circulating bloodstream can be induced using granulocyte colony stimulating factors (G-CSF). As it has been shown that HSCs and MSCs have positive interactions with each other, it may be possible that G-CSF also promotes the release of circulating peripheral blood MSCs (PBMSCs). The hypothesis of this study was that G-CSF would increase the mobilization of peripheral blood-derived stromal-like cells. Materials and Methods: Six sheep with normal hematological profiles were given 5& #956;g/kg Neupogen& #63721; (filgrastim, G-CSF) subcutaneously for five days. Pre- and post-G-CSF treatment, blood was taken 4, 12, 24, and 2 weeks post-treatment. PBMSCs were isolated from the blood and cells plated at a cell density of 4.0 x 10e4 nucleated cells/cm2. Fibroblastic colony forming units (CFU-F) were counted 7 and 14 days after initial culture. The cells were tested for their multipotency by treating them with osteogenic, adipogenic, and chondrogenic supplements, and staining with the Von Kossa, Oil Red ‘O,’ and Alcian Blue stains, respectively, to show differentiation down the different lineages. Results: No CFU-F formation was observed in all blood samples taken before G-CSF therapy (0 CFU-F) after 7 and 14 days in culture. After G-CSF treatment, CFU-Fs were observed in blood samples taken 4, 12, and 336 hours (2 weeks) post-G-CSF. The CFU-F count was highest after 14 days in culture in the blood samples obtained 2 weeks post-G-CSF administration (1.027 ± 30.1353 CFU-F/cm2), compared to the lowest count, which was at 12 hours post-G-CSF treatment (0.064 ± 0.064 CFU-F/cm2). Hematology showed an increase in white blood cell (WBC), neutrophil, and eosinophil counts 24 hours after G-CSF administration. Two weeks post-G-CSF treatment, WBC, neutrophil, lymphocyte, and monocyte counts dropped back to normal range values. The highest number of CFU-F/cm2 were observed at this time. When WBC numbers were correlated with CFU-F counts using Pearson’s correlation co-efficient, the result was 0.523, a significant value (p=0.023) indicating that 27.4% of the WBC counts were related to CFU-F counts and vice versa. When time was accounted for as a third variable using the test for partial correlation coefficients, the co-efficient was found to be −0.0063, and was not significant (p=0.492). Expanded cells were fibroblastic in morphology, and upon differentiation were positive for the Von Kossa, Oil Red ‘O,’ and Alican Blue stains, indicating differentiation down the osteogenic, adipogenic, and chondrogenic lineages, respectively. Discussion and Conclusions: We have shown that PBMSCs can be isolated after G-CSF administration in sheep, and that the numbers of CFU-F increase after WBC levels have returned to normal. A previous in vitro study proposed that the increased BMSC growth observed when co-cultured with CD45+ HSCs was due to positive interactions between HSCs and MSCs, indicating a possible steady-state balance. PBMSCs may have important future applications in bone tissue regeneration

Aims

Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation.