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.

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

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

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

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

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. 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

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. 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

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

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

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

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

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

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

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

Aims. This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. Methods. A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process. Results. Mean callus volume was larger in the elastic fixation group (1,755 mm. 3. (standard error of the mean (SEM) 297)) than in the stiff fixation group (258 mm. 3. (SEM 65)). Pathological observation found that the expression levels of osterix (OSX), collagen, type I, alpha 1 (COL1α1), and alkaline phosphatase (ALP) in the callus of the elastic fixation group were higher than those of the stiff fixation group. The protein sequence of the callus revealed 199 DEPs, 124 of which were highly expressed in the elastic fixation group. In the in vitro study, it was observed that a stress of 200 g led to upregulation of thrombospondin 1 (THBS1) and osteoglycin (OGN) expression in bone marrow mesenchymal stem cells (BMSCs). Additionally, these genes were found to be upregulated during the osteogenic differentiation process of the BMSCs. Conclusion. Elastic fixation can promote fracture healing and osteoblast differentiation in callus, and the ability of elastic fixation to promote osteogenic differentiation of BMSCs may be achieved by upregulating genes such as THBS1 and OGN. Cite this article: Bone Joint Res 2024;13(10):559–572

Aims

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

Aims. Successful cell therapy in hip osteonecrosis (ON) may help to avoid ON progression or total hip arthroplasty (THA), but the achieved bone regeneration is unclear. The aim of this study was to evaluate amount and location of bone regeneration obtained after surgical injection of expanded autologous mesenchymal stromal cells from the bone marrow (BM-hMSCs). Methods. A total of 20 patients with small and medium-size symptomatic stage II femoral head ON treated with 140 million BM-hMSCs through percutaneous forage in the EudraCT 2012-002010-39 clinical trial were retrospectively evaluated through preoperative and postoperative (three and 12 months) MRI. Then, 3D reconstruction of the original lesion and the observed postoperative residual damage after bone regeneration were analyzed and compared per group based on treatment efficacy. Results. The mean preoperative lesion volume was 18.7% (SD 10.2%) of the femoral head. This reduced to 11.6% (SD 7.5%) after three months (p = 0.015) and 3.7% (SD 3%) after one year (p < 0.001). Bone regeneration in healed cases represented a mean 81.2% (SD 13.8%) of the initial lesion volume at one year. Non-healed cases (n = 1 stage progression; n = 3 THAs) still showed bone regeneration but this did not effectively decrease the ON volume. A lesion size under mean 10% (SD 6%) of the femoral head at three months predicted no ON stage progression at one year. Regeneration in the lateral femoral head (C2 under Japanese Investigation Committee (JCI) classification) and in the central and posterior regions of the head was predominant in cases without ON progression. Conclusion. Bone regeneration was observed in osteonecrotic femoral heads three months after expanded autologous BM-hMSC injection, and the volume and location of regeneration indicated the success of the therapy. Cite this article: Bone Joint Res 2022;11(12):881–889

Aims. The role of N,N-dimethylformamide (DMF) in diabetes-induced osteoporosis (DM-OS) progression remains unclear. Here, we aimed to explore the effect of DMF on DM-OS development. Methods. Diabetic models of mice, RAW 264.7 cells, and bone marrow macrophages (BMMs) were established by streptozotocin stimulation, high glucose treatment, and receptor activator of nuclear factor-κB ligand (RANKL) treatment, respectively. The effects of DMF on DM-OS development in these models were examined by micro-CT analysis, haematoxylin and eosin (H&E) staining, osteoclast differentiation of RAW 264.7 cells and BMMs, H&E and tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA) of TRAP5b and c-terminal telopeptides of type 1 (CTX1) analyses, reactive oxygen species (ROS) analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Cell Counting Kit-8 (CCK-8) assay, and Western blot. Results. The established diabetic mice were more sensitive to ovariectomy (OVX)-induced osteoporosis, and DMF treatment inhibited the sensitivity. OVX-treated diabetic mice exhibited higher TRAP5b and c-terminal telopeptides of type 1 (CTX1) levels, and DMF treatment inhibited the enhancement. DMF reduced RAW 264.7 cell viability. Glucose treatment enhanced the levels of TRAP5b, cathepsin K, Atp6v0d2, and H. +. -ATPase, ROS, while DMF reversed this phenotype. The glucose-increased protein levels were inhibited by DMF in cells treated with RANKL. The expression levels of antioxidant enzymes Gclc, Gclm, Ho-1, and Nqo1 were upregulated by DMF. DMF attenuated high glucose-caused osteoclast differentiation by targeting mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signalling in BMMs. Conclusion. DMF inhibits high glucose-induced osteoporosis by targeting MAPK and NF-κB signalling. Cite this article: Bone Joint Res 2022;11(4):200–209

Aims. One of the main causes of tibial revision surgery for total knee arthroplasty is aseptic loosening. Therefore, stable fixation between the tibial component and the cement, and between the tibial component and the bone, is essential. A factor that could influence the implant stability is the implant design, with its different variations. In an existing implant system, the tibial component was modified by adding cement pockets. The aim of this experimental in vitro study was to investigate whether additional cement pockets on the underside of the tibial component could improve implant stability. The relative motion between implant and bone, the maximum pull-out force, the tibial cement mantle, and a possible path from the bone marrow to the metal-cement interface were determined. Methods. A tibial component with (group S: Attune S+) and without (group A: Attune) additional cement pockets was implanted in 15 fresh-frozen human leg pairs. The relative motion was determined under dynamic loading (extension-flexion 20° to 50°, load-level 1,200 to 2,100 N) with subsequent determination of the maximum pull-out force. In addition, the cement mantle was analyzed radiologically for possible defects, the tibia base cement adhesion, and preoperative bone mineral density (BMD). Results. The BMD showed no statistically significant difference between both groups. Group A showed for all load levels significantly higher maximum relative motion compared to group S for 20° and 50° flexion. Group S improved the maximum failure load significantly compared to group A without additional cement pockets. Group S showed a significantly increased cement adhesion compared to group A. The cement penetration and cement mantle defect analysis showed no significant differences between both groups. Conclusion. From a biomechanical point of view, the additional cement pockets of the component have improved the fixation performance of the implant. Cite this article: Bone Joint Res 2022;11(4):229–238

Objectives

Traumatic brachial plexus injury causes severe functional impairment of the arm. Elbow flexion is often affected. Nerve surgery or tendon transfers provide the only means to obtain improved elbow flexion. Unfortunately, the functionality of the arm often remains insufficient. Stem cell therapy could potentially improve muscle strength and avoid muscle-tendon transfer. This pilot study assesses the safety and regenerative potential of autologous bone marrow-derived mononuclear cell injection in partially denervated biceps.

For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.

This paper examines the fate of decalcified allografts (homografts) of iliac cancellous bone impregnated with autologous red marrow and implanted intermuscularly into the anterior abdominal wall of rabbits. In contrast to the findings of Urist and other workers that cortical bone decalcified with hydrochloric acid (HCl) and then freeze-dried is inductive to new bone formation in various heterotopic sites, evidence is presented that iliac bone decalcified by HCl and grafted alone to a muscular site is itself very weakly inductive to bone formation. However, when combined with autologous bone marrow the HCl-decalcified bone provides a better substrate for bone formation by marrow cells than does either undecalcified iliac bone, or iliac bone decalcified with ethylene-diamine-tetra-acetic acid. The freezing or freeze-drying of decalcified bone does not affect new bone formation when implanted alone or with autologous marrow. The differences between the cortical and cancellous bone as inductive substrates for osteogenesis are discussed and the interrelationship of bone and marrow in combined bone grafts are re-evaluated

1. This paper reports a histological study of the fate of sheep and calf cancellous bone grafts impregnated with autologous red marrow of Wistar rats and implanted intramuscularly as composite xenograft-autografts for two to twelve weeks. It also includes some biochemical estimations of certain types of sheep and calf bone used to prepare these composite grafts.

2. Only one of 223 devitalised bone xenografts implanted without autologous marrow formed new bone; in contrast 216 of 223 transplanted with marrow formed new bone.

3. The new bone formed by the composite grafts is derived from the autologous marrow. There was no evidence for an inductive effect upon the marrow of the various types of xenograft bone studied as described previously for allograft bone (Burwell 1966).

4. The highest score of new bone formation was found in composite grafts based on fully deproteinised sheep iliac bone prepared at Oswestry. Statistically this score was significantly higher than those registered by composite grafts prepared from intact (frozen and freeze-dried), decalcified (frozen and freeze-dried) and Kiel sheep bone, and by Kiel and Oswestry calf bone (Table II).

5. The histological evidence reported suggests that the high score with the sheep Oswestry composite grafts is because Oswestry bone is feebly immunogenic, if at all; and that such feeble or absent immunogenicity permits more marrow cells to differentiate into osteoblasts and lay down new bone without impediment.

6. The lower scores of new bone formation in most of the undeproteinised composite grafts of sheep origin–intact frozen, intact freeze-dried and Kiel–are attributed to residual immunogenicity within the organic material of the donor bone, because each type evoked the formation of mature plasma cells.

7. The Kiel bone grafts appeared to evoke less of a plasma cell reaction and may be less immunogenic than the intact and decalcified bone xenografts.

8. The sheep Oswestry CXA's formed significantly more new bone than did the calf Oswestry CXA's. This difference may be due to the different physical properties of the bone obtained from old sheep compared with the bone obtained from a young calf.

Aims. The purpose of this study was to determine whether intracellular Staphylococcus aureus is associated with recurrent infection in a rat model of open fracture. Methods. After stabilizing with Kirschner wire, we created a midshaft femur fracture in Sprague-Dawley rats and infected the wound with green fluorescent protein (GFP)-tagged S. aureus. After repeated debridement and negative swab culture was achieved, the isolation of GFP-containing cells from skin, bone marrow, and muscle was then performed. The composition and viability of intracellular S. aureus in isolated GFP-positive cells was assessed. We suppressed the host immune system and observed whether recurrent infection would occur. Finally, rats were assigned to one of six treatment groups (a combination of antibiotic treatment and implant removal/retention). The proportion of successful eradication was determined. Results. Green fluorescent protein-containing cells were successfully isolated after the swab culture was negative from skin (n = 0, 0%), muscle (n = 10, 100%), and bone marrow (n = 10, 100%) of a total of ten rats. The phagocytes were predominant in GFP-positive cells from muscle (73%) and bone marrow (81%) with a significantly higher viability of intracellular S. aureus (all p-values < 0.001). The recurrent infection occurred in up to 75% of rats after the immunosuppression. The proportion of successful eradication was not associated with implant retention or removal, and the efficacy of linezolid in eradicating intracellular S. aureus is significantly higher than that of vancomycin. Conclusion. Intracellular S. aureus is associated with recurrent infection in the rat model of open fracture. Usage of linezolid, a membrane-permeable antibiotic, is an effective strategy against intracellular S. aureus. Cite this article:Bone Joint Res. 2020;9(2):71–76

Aims. The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. Methods. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Results. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. Conclusion. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction. Cite this article: Bone Joint Res 2023;12(1):9–21

Aims. Implantation of ultra-purified alginate (UPAL) gel is safe and effective in animal osteochondral defect models. This study aimed to examine the applicability of UPAL gel implantation to acellular therapy in humans with cartilage injury. Methods. A total of 12 patients (12 knees) with symptomatic, post-traumatic, full-thickness cartilage lesions (1.0 to 4.0 cm. 2. ) were included in this study. UPAL gel was implanted into chondral defects after performing bone marrow stimulation technique, and assessed for up to three years postoperatively. The primary outcomes were the feasibility and safety of the procedure. The secondary outcomes were self-assessed clinical scores, arthroscopic scores, tissue biopsies, and MRI-based estimations. Results. No obvious adverse events related to UPAL gel implantation were observed. Self-assessed clinical scores, including pain, symptoms, activities of daily living, sports activity, and quality of life, were improved significantly at three years after surgery. Defect filling was confirmed using second-look arthroscopy at 72 weeks. Significantly improved MRI scores were observed from 12 to 144 weeks postoperatively. Histological examination of biopsy specimens obtained at 72 weeks after implantation revealed an extracellular matrix rich in glycosaminoglycan and type II collagen in the reparative tissue. Histological assessment yielded a mean overall International Cartilage Regeneration & Joint Preservation Society II score of 69.1 points (SD 10.4; 50 to 80). Conclusion. This study provides evidence supporting the safety of acellular UPAL gel implantation in facilitating cartilage repair. Despite being a single-arm study, it demonstrated the efficacy of UPAL gel implantation, suggesting it is an easy-to-use, one-step method of cartilage tissue repair circumventing the need to harvest donor cells. Cite this article: Bone Joint J 2023;105-B(8):880–887

Aims. Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA). Methods. Mesenchymal stem/stromal cells (MSCs) were isolated from rat bone marrow (BM) and used to evaluate the impact of 29-mer on chondrogenic differentiation of BM-MSCs in culture. Knee OA was induced in rats by a single intra-articular injection of monosodium iodoacetate (MIA) in the right knees (set to day 0). The 29-mer dissolved in 5% hyaluronic acid (HA) was intra-articularly injected into right knees at day 8 and 12 after MIA injection. Subsequently, the therapeutic effect of the 29-mer/HA on OA was evaluated by the Osteoarthritis Research Society International (OARSI) histopathological scoring system and changes in hind paw weight distribution, respectively. The regeneration of chondrocytes in damaged AC was detected by dual-immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and chondrogenic markers. Results. The 29-mer promoted expansion and chondrogenic differentiation of BM-MSCs cultured in different defined media. MIA injection caused chondrocyte death throughout the AC, with cartilage degeneration thereafter. The 29-mer/HA treatment induced extensive chondrocyte regeneration in the damaged AC and suppressed MIA-induced synovitis, accompanied by the recovery of cartilage matrix. Pharmacological inhibitors of PEDF receptor (PEDFR) and signal transducer and activator of transcription 3 (STAT3) signalling substantially blocked the chondrogenic promoting activity of 29-mer on the cultured BM-MSCs and injured AC. Conclusion. The 29-mer/HA formulation effectively induces chondrocyte regeneration and formation of cartilage matrix in the damaged AC. Cite this article: Bone Joint Res 2024;13(4):137–148

Aims. Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear. Methods. Six-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis. Results. Abnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice. Conclusion. Abnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ. Cite this article: Bone Joint Res 2022;11(7):453–464

Aims. Cartilage injuries rarely heal spontaneously and often require surgical intervention, leading to the formation of biomechanically inferior fibrous tissue. This study aimed to evaluate the possible effect of amelogenin on the healing process of a large osteochondral injury (OCI) in a rat model. Methods. A reproducible large OCI was created in the right leg femoral trochlea of 93 rats. The OCIs were treated with 0.1, 0.5, 1.0, 2.5, or 5.0 μg/μl recombinant human amelogenin protein (rHAM. +. ) dissolved in propylene glycol alginate (PGA) carrier, or with PGA carrier alone. The degree of healing was evaluated 12 weeks after treatment by morphometric analysis and histological evaluation. Cell recruitment to the site of injury as well as the origin of the migrating cells were assessed four days after treatment with 0.5 μg/μl rHAM. +. using immunohistochemistry and immunofluorescence. Results. A total of 12 weeks after treatment, 0.5 μg/μl rHAM. +. brought about significant repair of the subchondral bone and cartilage. Increased expression of proteoglycan and type II collagen and decreased expression of type I collagen were revealed at the surface of the defect, and an elevated level of type X collagen at the newly developed tide mark region. Conversely, the control group showed osteoarthritic alterations. Recruitment of cells expressing the mesenchymal stem cell (MSC) markers CD105 and STRO-1, from adjacent bone marrow toward the OCI, was noted four days after treatment. Conclusion. We found that 0.5 μg/μl rHAM. +. induced in vivo healing of injured articular cartilage and subchondral bone in a rat model, preventing the destructive post-traumatic osteoarthritic changes seen in control OCIs, through paracrine recruitment of cells a few days after treatment. Cite this article: Bone Joint Res 2023;12(10):615–623

Aims. The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Methods. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. Results. LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. Conclusion. LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism. Cite this article: Bone Joint Res 2024;13(12):779–789

Aims. The use of 3D-printed titanium implant (DT) can effectively guide bone regeneration. DT triggers a continuous host immune reaction, including macrophage type 1 polarization, that resists osseointegration. Interleukin 4 (IL4) is a specific cytokine modulating osteogenic capability that switches macrophage polarization type 1 to type 2, and this switch favours bone regeneration. Methods. IL4 at concentrations of 0, 30, and 100 ng/ml was used at day 3 to create a biomimetic environment for bone marrow mesenchymal stromal cell (BMMSC) osteogenesis and macrophage polarization on the DT. The osteogenic and immune responses of BMMSCs and macrophages were evaluated respectively. Results. DT plus 30 ng/ml of IL4 (DT + 30 IL4) from day 3 to day 7 significantly (p < 0.01) enhanced macrophage type 2 polarization and BMMSC osteogenesis compared with the other groups. Local injection of IL4 enhanced new bone formation surrounding the DT. Conclusion. DT + 30 IL4 may switch macrophage polarization at the appropriate timepoints to promote bone regeneration. Cite this article: Bone Joint Res 2021;10(7):411–424

Stem cells are defined by their potential for self-renewal and the ability to differentiate into numerous cell types, including cartilage and bone cells. Although basic laboratory studies demonstrate that cell therapies have strong potential for improvement in tissue healing and regeneration, there is little evidence in the scientific literature for many of the available cell formulations that are currently offered to patients. Numerous commercial entities and ‘regenerative medicine centres’ have aggressively marketed unproven cell therapies for a wide range of medical conditions, leading to sometimes indiscriminate use of these treatments, which has added to the confusion and unpredictable outcomes. The significant variability and heterogeneity in cell formulations between different individuals makes it difficult to draw conclusions about efficacy. The ‘minimally manipulated’ preparations derived from bone marrow and adipose tissue that are currently used differ substantially from cells that are processed and prepared under defined laboratory protocols. The term ‘stem cells’ should be reserved for laboratory-purified, culture-expanded cells. The number of cells in uncultured preparations that meet these defined criteria is estimated to be approximately one in 10 000 to 20 000 (0.005% to 0.01%) in native bone marrow and 1 in 2000 in adipose tissue. It is clear that more refined definitions of stem cells are required, as the lumping together of widely diverse progenitor cell types under the umbrella term ‘mesenchymal stem cells’ has created confusion among scientists, clinicians, regulators, and our patients. Validated methods need to be developed to measure and characterize the ‘critical quality attributes’ and biological activity of a specific cell formulation. It is certain that ‘one size does not fit all’ – different cell formulations, dosing schedules, and culturing parameters will likely be required based on the tissue being treated and the desired biological target. As an alternative to the use of exogenous cells, in the future we may be able to stimulate the intrinsic vascular stem cell niche that is known to exist in many tissues. The tremendous potential of cell therapy will only be realized with further basic, translational, and clinical research. Cite this article: Bone Joint J 2019;101-B:361–364

The management of symptomatic osteochondral lesions of the talus (OLTs) can be challenging. The number of ways of treating these lesions has increased considerably during the last decade, with published studies often providing conflicting, low-level evidence. This paper aims to present an up-to-date concise overview of the best evidence for the surgical treatment of OLTs. Management options are reviewed based on the size of the lesion and include bone marrow stimulation, bone grafting options, drilling techniques, biological preparations, and resurfacing. Although many of these techniques have shown promising results, there remains little high level evidence, and further large scale prospective studies and systematic reviews will be required to identify the optimal form of treatment for these lesions. Cite this article: Bone Joint J 2021;103-B(2):207–212

Bone marrow mesenchymal stromal cells were aspirated from immature male green fluorescent protein transgenic rats and cultured in a monolayer. Four weeks after the creation of the osteochondral defect, the rats were divided into three groups of 18: the control group, treated with an intra-articular injection of phosphate-buffered saline only; the drilling group, treated with an intra-articular injection of phosphate-buffered saline with a bone marrow-stimulating procedure; and the bone marrow mesenchymal stromal cells group, treated with an intra-articular injection of bone marrow mesenchymal stromal cells plus a bone marrow-stimulating procedure. The rats were then killed at 4, 8 and 12 weeks after treatment and examined. The histological scores were significantly better in the bone marrow mesenchymal stromal cells group than in the control and drilling groups at all time points (p < 0.05). The fluorescence of the green fluorescent protein-positive cells could be observed in specimens four weeks after treatment

Open surgery is rarely justified for the initial treatment of a unicameral bone cyst, but there is some debate concerning the relative effectiveness of closed methods. This study compared the results of steroid injection with those of autologous bone marrow grafting for the treatment of unicameral bone cysts. Between 1990 and 2001, 30 patients were treated by steroid injection and 28 by grafting with autologous bone marrow. The overall success rates were 86.7% and 92.0%, respectively (p > 0.05). The success rate after the initial procedure was 23.3% in the steroid group and 52.0% in those receiving autologous bone marrow (p < 0.05), and the respective cumulative success rates after second injections were 63.3% and 80.0% (p > 0.05). The mean number of procedures required was 2.19 (1 to 5) and 1.57 (1 to 3) (p < 0.05), the mean interval to healing was 12.5 months (4 to 32) and 14.3 months (7 to 36) (p > 0.05), and the rate of recurrence after the initial procedure was 41.7% and 13.3% in the steroid and in the autologous bone marrow groups, respectively (p < 0.05). Although the overall rates of success of both methods were similar, the steroid group had higher recurrence after a single procedure and required more injections to achieve healing

Aims. A growing number of fractures progress to delayed or nonunion, causing significant morbidity and socioeconomic impact. Localized delivery of stem cells and subcutaneous parathyroid hormone (PTH) has been shown individually to accelerate bony regeneration. This study aimed to combine the therapies with the aim of upregulating fracture healing. Methods. A 1.5 mm femoral osteotomy (delayed union model) was created in 48 female juvenile Wistar rats, aged six to nine months, and stabilized using an external fixator. At day 0, animals were treated with intrafracture injections of 1 × 10. 6. cells/kg bone marrow mesenchymal stem cells (MSCs) suspended in fibrin, daily subcutaneous injections of high (100 μg/kg) or low (25 μg/kg) dose PTH 1-34, or a combination of PTH and MSCs. A group with an empty gap served as a control. Five weeks post-surgery, the femur was excised for radiological, histomorphometric, micro-CT, and mechanical analysis. Results. Combination therapy treatment led to increased callus formation compared to controls. In the high-dose combination group there was significantly greater mineralized tissue volume and trabecular parameters compared to controls (p = 0.039). This translated to significantly improved stiffness (and ultimate load to failure (p = 0.049). The high-dose combination therapy group had the most significant improvement in mean modified Radiographic Union Score for Tibia fractures (RUST) compared to controls (13.8 (SD 1.3) vs 5.8 (SD 0.5)). All groups demonstrated significant increases in the radiological scores – RUST and Allen score – histologically compared to controls. Conclusion. We demonstrate the beneficial effect of localized MSC injections on fracture healing combined with low- or high-dose teriparatide, with efficacy dependent on PTH dose. Cite this article: Bone Joint Res 2021;10(10):659–667

Aims. Bone metastasis ultimately occurs due to a complex multistep process, during which the interactions between cancer cells and bone microenvironment play important roles. Prior to colonization of the bone, cancer cells must succeed through a series of steps that will allow them to gain migratory and invasive properties; epithelial-to-mesenchymal transition (EMT) is known to be integral here. The aim of this study was to determine the effects of G protein subunit alpha Q (GNAQ) on the mechanisms underlying bone metastasis through EMT pathway. Methods. A total of 80 tissue samples from patients who were surgically treated during January 2012 to December 2014 were used in the present study. Comparative gene analysis revealed that the GNAQ was more frequently altered in metastatic bone lesions than in primary tumour sites in lung cancer patients. We investigated the effects of GNAQ on cell proliferation, migration, EMT, and stem cell transformation using lung cancer cells with GNAQ-knockdown. A xenograft mouse model tested the effect of GNAQ using micro-CT analyses and histological analyses. Results. GNAQ-knockdown showed down-regulation of tumour growth through mitogen-activated protein kinase (MAPK) signalling in lung cancer cells, but not increased apoptosis. We found that GNAQ-knockdown induced EMT and promoted invasiveness. GNAQ-knockdown cells injected into the bone marrow of murine tibia induced tumour growth and bone-to-lung metastasis, whereas it did not in control mice. Moreover, the knockdown of GNAQ enhanced cancer stem cell-like properties in lung cancer cells, which resulted in the development of resistance to chemotherapy. Conclusion. The present study reveals that the GNAQ-knockdown induced cancer stem cell-like properties. Cite this article: Bone Joint Res 2021;10(5):310–320

Aims. For cementless implants, stability is initially attained by an interference fit into the bone and osteo-integration may be encouraged by coating the implant with bioactive substances. Blood based autologous glue provides an easy, cost-effective way of obtaining high concentrations of growth factors for tissue healing and regeneration with the intention of spraying it onto the implant surface during surgery. The aim of this study was to incorporate nucleated cells from autologous bone marrow (BM) aspirate into gels made from the patient’s own blood, and to investigate the effects of incorporating three different concentrations of platelet rich plasma (PRP) on the proliferation and viability of the cells in the gel. Methods. The autologous blood glue (ABG) that constituted 1.25, 2.5, and 5 times concentration PRP were made with and without equal volumes of BM nucleated cells. Proliferation, morphology, and viability of the cells in the glue was measured at days 7 and 14 and compared to cells seeded in fibrin glue. Results. Overall, 2.5 times concentration of PRP in ABG was capable of supporting the maximum growth of cells isolated from the BM aspirate and maintain their characteristics. Irrespective of PRP concentration, cells in ABG had statistically significantly higher viability compared to cells in fibrin glue. Conclusion. In vitro this novel autologous gel is more capable of supporting the growth of cells in its structure for up to 14 days, compared to commercially available fibrin-based sealants, and this difference was statistically significant. Cite this article: Bone Joint Res 2020;9(7):402–411

Aims. The purpose of our study was to determine whether mesenchymal stem cells (MSCs) are an effective and safe therapeutic agent for the treatment of knee osteoarthritis (OA), owing to their cartilage regeneration potential. Methods. We searched PubMed, Embase, and the Cochrane Library, with keywords including “knee osteoarthritis” and “mesenchymal stem cells”, up to June 2019. We selected randomized controlled trials (RCTs) that explored the use of MSCs to treat knee OA. The visual analogue scale (VAS), Western Ontario and McMaster University Osteoarthritis Index (WOMAC), adverse events, and the whole-organ MRI score (WORMS) were used as the primary evaluation tools in the studies. Our meta-analysis included a subgroup analysis of cell dose and cell source. Results. Seven trials evaluating 256 patients were included in the meta-analysis. MSC treatment significantly improved the VAS (mean difference (MD), –13.24; 95% confidence intervals (CIs) –23.28 to –3.20, p = 0.010) and WOMAC (MD, –7.22; 95% CI –12.97 to –1.47, p = 0.010). The low-dose group with less than 30 million cells showed lower p-values for both the VAS and WOMAC. Adipose and umbilical cord–derived stem cells also had lower p-values for pain scores than those derived from bone marrow. Conclusion. Overall, MSC-based cell therapy is a relatively safe treatment that holds great potential for OA, evidenced by a positive effect on pain and knee function. Using low-dose (25 million) and adipose-derived stem cells is likely to achieve better results, but further research is needed. Cite this article: Bone Joint Res 2020;9(10):719–728

Aims. Extracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold. Methods. Rabbit bone marrow mesenchymal stem cells (MSCs) were differentiated into the chondrogenic lineage on scaffolds. Quality of in vitro regenerated cartilage was assessed by cell viability, growth, matrix synthesis, and differentiation. Bilateral osteochondral defects were created in 15 four-month-old male New Zealand white rabbits and segregated into three treatment groups with five in each. The groups were: 1) untreated and allogeneic chondrocytes; 2) multi-layered scaffold with and without cells; and 3) randomly aligned scaffold with and without cells. After four months of follow-up, the outcome was assessed using histology and immunostaining. Results. In vitro testing showed that the secreted ECM oriented itself along the fibre in multi-layered scaffolds. Both types of CG scaffolds supported cell viability, growth, and matrix synthesis. In vitro chondrogenesis on scaffold showed an around 400-fold increase in collagen type 2 (COL2A1) expression in both CG scaffolds, but the total glycosaminoglycan (GAG)/DNA deposition was 1.39-fold higher in the multi-layered scaffold than the randomly aligned scaffold. In vivo cartilage formation occurred in both multi-layered and randomly aligned scaffolds treated with and without cells, and was shown to be of hyaline phenotype on immunostaining. The defects treated with multi-layered + cells, however, showed significantly thicker cartilage formation than the randomly aligned scaffold. Conclusion. We demonstrated that MSCs loaded CG scaffold with multi-layered zonal architecture promoted superior hyaline AC regeneration. Cite this article: Bone Joint Res 2020;9(9):601–612

Objectives. Mesenchymal stem cells have the ability to differentiate into various cell types, and thus have emerged as promising alternatives to chondrocytes in cell-based cartilage repair methods. The aim of this experimental study was to investigate the effect of bone marrow derived mesenchymal stem cells combined with platelet rich fibrin on osteochondral defect repair and articular cartilage regeneration in a canine model. Methods. Osteochondral defects were created on the medial femoral condyles of 12 adult male mixed breed dogs. They were either treated with stem cells seeded on platelet rich fibrin or left empty. Macroscopic and histological evaluation of the repair tissue was conducted after four, 16 and 24 weeks using the International Cartilage Repair Society macroscopic and the O’Driscoll histological grading systems. Results were reported as mean and standard deviation (. sd. ) and compared at different time points between the two groups using the Mann-Whitney U test, with a value < 0.05 considered statistically significant. Results. Higher cumulative macroscopic and histological scores were observed in stem cell treated defects throughout the study period with significant differences noted at four and 24 weeks (9.25, . sd. 0.5 vs 7.25, . sd. 0.95, and 10, . sd. 0.81 vs 7.5, . sd. 0.57; p < 0.05) and 16 weeks (16.5, . sd. 4.04 vs 11, . sd. 1.15; p < 0.05), respectively. Superior gross and histological characteristics were also observed in stem cell treated defects. Conclusion. The use of autologous culture expanded bone marrow derived mesenchymal stem cells on platelet rich fibrin is a novel method for articular cartilage regeneration. It is postulated that platelet rich fibrin creates a suitable environment for proliferation and differentiation of stem cells by releasing endogenous growth factors resulting in creation of a hyaline-like reparative tissue. Cite this article: D. Kazemi, K. Shams Asenjan, N. Dehdilani, H. Parsa. Canine articular cartilage regeneration using mesenchymal stem cells seeded on platelet rich fibrin: Macroscopic and histological assessments. Bone Joint Res 2017;6:98–107. DOI: 10.1302/2046-3758.62.BJR-2016-0188.R1

Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. Results. TGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. Conclusion. A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades. Cite this article: Bone Joint Res 2020;9(7):412–420

Objectives. Mesenchymal stem cells (MSCs) are of growing interest in terms of bone regeneration. Most preclinical trials utilize bone-marrow-derived mesenchymal stem cells (bMSCs), although this is not without isolation and expansion difficulties. The aim of this study was: to compare the characteristics of bMSCs and adipose-derived mesenchymal stem cells (AdMSCs) from juvenile, adult, and ovarectomized (OVX) rats; and to assess the effect of human parathyroid hormone (hPTH) 1-34 on their osteogenic potential and migration to stromal cell-derived factor-1 (SDF-1). Methods. Cells were isolated from the adipose and bone marrow of juvenile, adult, and previously OVX Wistar rats, and were characterized with flow cytometry, proliferation assays, osteogenic and adipogenic differentiation, and migration to SDF-1. Experiments were repeated with and without intermittent hPTH 1-34. Results. Juvenile and adult MSCs demonstrated significantly increased osteogenic and adipogenic differentiation and superior migration towards SDF-1 compared with OVX groups; this was the case for AdMSCs and bMSCs equally. Parathyroid hormone (PTH) increased parameters of osteogenic differentiation and migration to SDF-1. This was significant for all cell types, although it had the most significant effect on cells derived from OVX animals. bMSCs from all groups showed increased mineralization and migration to SDF-1 compared with AdMSCs. Conclusion. Juvenile MSCs showed significantly greater migration to SDF-1 and significantly greater osteogenic and adipogenic differentiation compared with cells from osteopenic rats; this was true for bMSCs and AdMSCs. The addition of PTH increased these characteristics, with the most significant effect on cells derived from OVX animals, further illustrating possible clinical application of both PTH and MSCs in bone regenerative therapies. Cite this article:L. Osagie-Clouard, A. Sanghani-Kerai, M. Coathup, R. Meeson, T. Briggs, G. Blunn. The influence of parathyroid hormone 1-34 on the osteogenic characteristics of adipose- and bone-marrow-derived mesenchymal stem cells from juvenile and ovarectomized rats. Bone Joint Res 2019;8:397–404. DOI: 10.1302/2046-3758.88.BJR-2019-0018.R1

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with limited coding potential, which have emerged as novel regulators in many biological and pathological processes, including growth, development, and oncogenesis. Accumulating evidence suggests that lncRNAs have a special role in the osteogenic differentiation of various types of cell, including stem cells from different sources such as embryo, bone marrow, adipose tissue and periodontal ligaments, and induced pluripotent stem cells. Involved in complex mechanisms, lncRNAs regulate osteogenic markers and key regulators and pathways in osteogenic differentiation. In this review, we provide insights into the functions and molecular mechanisms of lncRNAs in osteogenesis and highlight their emerging roles and clinical value in regenerative medicine and osteogenesis-related diseases. Cite this article: J. Zhang, X. Hao, M. Yin, T. Xu, F. Guo. Long non-coding RNA in osteogenesis: A new world to be explored. Bone Joint Res 2019;8:73–80. DOI: 10.1302/2046-3758.82.BJR-2018-0074.R1

Objectives. Cellular movement and relocalisation are important for many physiologic properties. Local mesenchymal stem cells (MSCs) from injured tissues and circulating MSCs aid in fracture healing. Cytokines and chemokines such as Stromal cell-derived factor 1(SDF-1) and its receptor chemokine receptor type 4 (CXCR4) play important roles in maintaining mobilisation, trafficking and homing of stem cells from bone marrow to the site of injury. We investigated the differences in migration of MSCs from the femurs of young, adult and ovariectomised (OVX) rats and the effect of CXCR4 over-expression on their migration. Methods. MSCs from young, adult and OVX rats were put in a Boyden chamber to establish their migration towards SDF-1. This was compared with MSCs transfected with CXCR4, as well as MSCs differentiated to osteoblasts. Results. MSCs from OVX rats migrate significantly (p < 0.05) less towards SDF-1 (9%, . sd. 5%) compared with MSCs from adult (15%, . sd. 3%) and young rats (25%, . sd. 4%). Cells transfected with CXCR4 migrated significantly more towards SDF-1 compared with non-transfected cells, irrespective of whether these cells were from OVX (26.5%, . sd. 4%), young (47%, . sd. 17%) or adult (21%, . sd. 4%) rats. Transfected MSCs differentiated to osteoblasts express CXCR4 but do not migrate towards SDF-1. Conclusions. MSC migration is impaired by age and osteoporosis in rats, and this may be associated with a significant reduction in bone formation in osteoporotic patients. The migration of stem cells can be ameliorated by upregulating CXCR4 levels which could possibly enhance fracture healing in osteoporotic patients. Cite this article: A. Sanghani-Kerai, M. Coathup, S. Samazideh, P. Kalia, L. Di Silvio, B. Idowu, G. Blunn. Osteoporosis and ageing affects the migration of stem cells and this is ameliorated by transfection with CXCR4. Bone Joint Res 2017;6:–365. DOI: 10.1302/2046-3758.66.BJR-2016-0259.R1