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. 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

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

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