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

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

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

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

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: 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

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