Objective

The aim of this study was to investigate PDGF release in the peripheral circulation following trauma and to correlate it with the numbers of MSCs in iliac crest bone marrow (BM) aspirate.

Osteogenesis Imperfecta (OI) is a heritable bone disorder characterized by bone fragility and often caused by mutations in the Type I collagen-encoding genes COL1A1 and COL1A2. The pathophysiology of OI, particularly at the cellular level, is still not well understood. This contributes to the lack of a cure for this disorder as well as an effective preventive or management options of its complications. In the bone environment, mesenchymal stem cells (MSCs) and osteoblasts (Ob) exert their function, at least partially, through the secretion of extracellular vesicles (EV). EV is a heterogeneous group of nanosized membrane-enclosed vesicles that carry/transfer a cargo of proteins, lipid and nucleic acids from the secreting cell to its target cells. Our objective is to characterize EVs secreted by human control (HC)- and OI-MSCs and their derived Obs, with focus on their protein content. We hypothesize that there will be differences in the protein content of EVs secreted by OI-Obs compared to HC-Ob, which may indicate a deviation from healthy Ob behavior and, thus, a role in OI pathophysiology. MSCs were harvested from the adipose tissue of four COL1A1-OI and two HC patients. They were proliferated in an EV-depleted media, then induced to differentiate to extracellular matrix (ECM)-producing osteoblasts, which then gets mineralized. EVs secreted by MSCs (MSC-EV) and Obs (Ob-EV) were then purified and concentrated. Using liquid chromatography- tandem mass spectrometry, proteomic analysis of the EV groups was done. A total of 384 unique proteins were identified in all EVs, 373 were found in Vesiclepedia indicating a good enrichment of our samples with EV proteins. 67 proteins of the total 384 were exclusively or significantly upregulated (p-value < 0 .05) in OI-Ob-EV and 28 proteins in the HC-Ob-EVs, relative to each other. These two groups of differentially expressed proteins were compared by Gene Ontology (GO) analysis of their cellular compartment, molecular functions and biological processes. We observed that there were differences in the cellular origin of EV-proteins, which may indicate heterogeneity of the isolated EVs. Molecular function and biological process analyses of the HC-Ob-EV proteins showed, as expected, predominantly calcium-related activities such as extracellular matrix (ECM) mineralization. OI-Ob-EV proteins were still predominantly exhibiting ECM organization and formation functions. Annexins A1,2,4,5 and 6 were differentially and significantly upregulated by the HC-Ob-EVs. Fibronectin (FN), Fibulin-1 and −2, and Laminins (α4 & γ1), which are amongst the early non-collagenous proteins to form the ECM, were differentially and significantly upregulated in the OI-Ob-EVs. We concluded that the persistent expression of Fibronectin (FN), Fibulin-1 and −2, and Laminins in OI-Ob-EVs might indicate the presence of an immature ECM that the OI-Obs are trying to organize. ECM mineralization is largely dependent on the presence of an organized mature ECM, and this being compromised in OI bone environment, may be a contributor to the bone fragility seen in these patients. Annexins, which are calcium-binders that are vital for ECM mineralization, were significantly downregulated in the OI-Ob-EVs and this may be a further contributor to ECM mineralization impairment and bone fragility

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

Osteoarthritis (OA) is the fastest growing global health problem, with a total joint replacement being the only effective treatment for patients with end stage OA. Many groups are examining the use of bone marrow or adipose derived mesenchymal stem cells (MSCs) to repair cartilage, or modulate inflammation to promote healing, however, little efficacy in promoting cartilage repair, or reducing patient symptoms over temporary treatments such as micro-fracture has been observed. There is a growing body of literature demonstrating that MSCs derived from the synovial lining of the joint are superior in terms of chondrogenic differentiation and while improvements in clinical outcome measures have been observed with synovial MSCs, results from clinical studies are still highly variable. Based on our results, we believe this variability in clinical studies with MSCs results in part from the isolation, expansion and re-injection of distinct MSCs subtypes in normal vs. OA tissues, each with differing regenerating potential. However, it remains unknown if this heterogeneity is natural (e.g. multiple MSC subtypes present) or if MSCs are influenced by factors in vivo (disease state/stage). Therefore, in this study, we undertook an ‘omics’ screening approach on MSCs from normal and OA knee synovial tissue. Specifically, we characterized their global proteome and genomic expression patterns to determine if multiple MSC from normal and OA joints are distinct at the protein/gene expression level and/if so, what proteins/genes are differentially expressed between MSCs derived from normal and OA synovial tissue. Synovium tissue was collected from OA patients undergoing joint replacement and normal cadaveric knees. The in vitro adipogenic, chondrogenic and osteogenic differentiation potential of the MSCs was analyzed via qPCR and histology. Fully characterized MSC populations where then analyzed through an unbiased shotgun proteomics, and microarray analysis. Synovial MSCs isolated from both OA and normal knees demonstrated similar multipotent differentiation capacity. Likewise, both OA and normal MSCs display the typical MSCs cell surface marker profile in vitro (CD90+, CD44+, CD73+, CD105+). Using shotgun proteomics, 7720 unique peptides corresponding to 2183 proteins were identified and quantified between normal and OA MSCs. Of these 2183 proteins, 994 were equally expressed in normal and OA, MSCs, 324 were upregulated in OA MSCs (with 50 proteins exclusively expressed in OA MSCs), 630 proteins were upregulated in normal MSCs (with 16 proteins exclusively expressed in normal MSCs). Microarray analysis of normal and OA MSCs demonstrated a similar result in where, 967 genes were differentially expressed between normal and OA MSCs, with 423 genes upregulated in OA, and 544 genes upregulated in normal MSCs. In this project, we have demonstrated that although normal and OA synovial derived MSCs demonstrate similar multipotent differentiation potential and cell surface markers expression, these cells demonstrated significant differences at the molecular level (protein and gene expression). Further research is required to determine if these differences influence functional differences in vitro and/or in vivo and what drives this dramatic change in the regulatory pathways within normal vs. OA synovial MSCs

Mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and other mesenchymal tissues but are also important modulators of innate and adaptive immune responses. We have capitalized on these important functions to mitigate adverse responses when bone is exposed to pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or prolonged pro-inflammatory cytokines. Our goal was to optimize osteogenesis and mitigate persistent undesired inflammation by: 1. preconditioning MSCs by short term exposure to lipopolysaccharide (LPS) and Tumor Necrosis Factor alpha (TNF-α), 2. genetic modification of MSCs to overexpress Interleukin 4 (IL-4) either constitutively, or as NFκB-responsive IL-4 over-expression cells, and 3. training the MSCs (innate immune memory) by repeated stimulation with LPS. In the first experiment, bone marrow MSCs and macrophages were isolated from femurs and tibias of C57BL/6 mice. MSCs (1×104 cells) were seeded in 24-well transwell plates in the bottom chamber with MSC growth medium. MSCs were treated with 20 ng/ml TNF-α and 1–20 μg/ml LPS for three days. Primary macrophages (2 × 103 cells) were seeded to the insert of a separate transwell plate and polarized into the M1 phenotype. At day four, MSCs and macrophages were washed and the inserts with M1 macrophages were moved to the plates containing preconditioned MSCs at the bottom of the well. Co-culture was carried out in MSC growth medium for 24h. In the second experiment, bone marrow derived macrophages and MSCs were isolated from femora and tibiae of Balb/c male mice. 5×104 macrophages and 1×104 MSCs were seeded in the bottom well of the 24-well transwell plate. The upper chambers were seeded with unmodified MSCs, MSCs preconditioned with 20 ng/ml TNF-α and 20 mg/ml LPS for 3 days, NFκB-IL4 secreting MSCs (all 5×104 cells), or controls without MSCs. Co-culture was carried out in mixed osteogenic-macrophage media with clinically relevant polyethylene or titanium alloy particles. In the third experiment, bone marrow MSCs and macrophages were collected from femurs and tibias of C57BL/6 male mice. The MSCs were stimulated by LPS, washed out for five days, and re-stimulated by LPS in co-culture with macrophages. First, preconditioned MSCs enhanced anti-inflammatory M2 macrophage (Arginase 1 and CD206) expression, decreased pro-inflammatory M1 macrophage (TNF-α/IL-1Ra ratio) expression, and increased osteogenic markers (alkaline phosphatase expression and matrix mineralization) in co-culture. Second, NFκB-IL4 secreting MSCs decreased pro-inflammatory M1 (TNF-α), increased anti-inflammatory M2 (Arg1, IL-1ra) expression, and enhanced the expression of osteogenic factors Runx2 and alkaline phosphatase, in the presence of particles, compared to other groups. Third, LPS-trained MSCs increased anti-inflammatory (Arginase1 and CD206), and decreased the proinflammatory (TNF-α, IL1b, iNOS, and IL6) marker expression in MSC/macrophage co-culture. Transforming MSCs via the techniques of preconditioning, genetic modification, or training (innate immune memory) can modulate/convert a potentially injurious microenvironment to an anti-inflammatory pro-reconstructive milieu. These effects are highly relevant for bone healing in the presence of adverse stimuli. These concepts using transformed MSCs could also be extended to other organ systems subjected to potentially damaging agents

Bone is capable of regeneration, and defects often heal spontaneously. However, cartilage, tendon, and ligament injuries usually result in replacement if the site by organized scar tissue, which is inferior to the native tissue. The osteogenic potential of mesenchymal stem cells (MSCs) has already been verified. MSCs hold great potential for the development of new treatment strategies for a host of orthopedic conditions. The multi-lineage potential and plasticity of MSCs allow them to be building blocks for a host of nonhematopoietic tissues, including bone. More recently, several groups have reported on the successful clinical application of tissue engineering strategies in the repair of bony defects in patients secondary to trauma and tumor resection. Advances in fabrication of biodegradable scaffolds that serve as beds for MSC implantation will hopefully lead to better biocompatibility and host tissue integration. Current strategies for bone tissue engineering include the use of osteoconductive matrix devices that promote bony ingrowth, and the delivery of osteoinductive growth factors, including bone morphogenetic protein (BMP) family, BMP-2 and BMP-7, to bony defect sites. Minimal toxicity has been observed in animal models involving genetically-manipulated stem cells transduced with retroviral and adenoviral vectors. Gene therapy using stem cells as delivery vehicles is a powerful weapon that can be used in a plethora of clinical situations that would benefit from the osteoinductive, proliferative, and angiogenic effects of growth factors. With better understanding of the biology of stem cells in the future and with enhancement of technologies that are capable to influence, modify, and culture these cells, a new field of regenerative skeletal medicine may emerge

Epidemiological studies have shown that accumulated mechanical stress is a risk factor for the development of osteoarthritis (OA). This debilitating progressive clinical condition affects a broad spectrum of patients and will ultimately lead to definitive arthroplasty surgery as the endpoint treatment option in many cases. The aim of this study is to establish a graded murine model of OA by medial meniscotibial destabilisation of the knee joint and in phase two, to investigate the migration and engraftment of radioisotope labeled mesenchymal stem cells (MSCs) at varying points of disease progression. The first phase of the study was to establish the murine model, an Irish first. All procedures were performed aseptically under general anaesthesia via a midline medial parapatellar approach on a murine fracture table. Microsurgical dissection was performed through necropsy analysed layers to the joint space and the meniscotibial ligament identified and transected. Validated histopathological analysis was performed at two, four, eight and twelve weeks postoperatively. The results showed a gradation of OA changes from mild unicondylar changes at two weeks, moderate unicompartmental change at four, severe unicompartmental change at eight and severe bicompartmental change at twelve weeks post-operatively. In vivo Bazooka-Single Photon Emission Computed Tomography (SPECT) (Phase 2) imaging studies are currently ongoing following the model establishment

Cell-scaffold based cartilage tissue engineering strategies provide the potential to restore long-term function to damaged articular cartilage. A major hurdle in such strategies is the adequate (uniform and sufficient) population of porous 3D scaffolds with cells, but more importantly, the generation of engineered tissue of sufficient quality of clinically relevant size. We describe a novel approach to engineer cartilage grafts using pre-differentiated micro-mass cartilage pellets, integrated into specifically designed 3D plotted scaffolds. Expanded (P2) human nasal chondrocytes (HNCs) or bone marrow-derived mesenchymal stem cells (MSCs) from 3 donors (age 47–62 years) were micro-mass cell pellet cultivated at 5 × 105 cells/pellet for 4 days. Subsequently, pellets were integrated into degradable 3D Printed polymer (PEGT/PBT) scaffolds with 1mm fibre spacing. Constructs were cultured dynamically in spinner flasks for a total of 21 days. As a pellet-free control, expanded HNCs were spinner flask seeded into PEGT/PBT fibre plotted scaffolds. Constructs were assessed via histology (Safranin-O staining), biochemistry (glycosaminoglycan (GAG) and DNA content) and collagen type I and II mRNA expression. After 4 days, micro-mass cultured pellets could be successfully integrated into the fibre plotted scaffolds. DNA content of pellet integrated constructs was 4.0-fold±1.3 higher compared to single seeded constructs. At day 21, cartilage tissue was uniformly distributed throughout pellet integrated scaffolds, with minimal cell necrosis observed within the core. GAG/DNA and collagen type II mRNA expression were significantly higher (2.5-fold±0.5 and 3.1-fold±0.4 respectively) in pellet versus single cell seeded constructs. Furthermore, compared to single cell, the pellet seeded constructs contained significantly more total GAG and DNA (1.6-fold±0.1 and 3.1-fold±1.0 respectively). We developed a novel 3D tissue assembly approach for cartilage tissue engineering which significantly improved the seeding efficiency (∼100%), as well as tissue uniformity and integrity compared to more traditional seeding approaches using single cell suspensions. Furthermore, the integration of micro-mass cell pellets into 3D plotted PEGT/PBT scaffolds significantly improved the amount and quality of tissue engineered cartilage

Purpose. The data regarding the effects of noggin on bone morphogenetic protein (BMP)-induced osteogenesis of mesenchymal stem cells (MSCs) are controversial. Most studies performed in rodent cells/models indicated that noggin was a negative regulator of BMP-2-induced osteogenesis; however, one study conducted with human MSCs in culture showed that the addition of noggin induced osteogenesis in vitro. To clear the controversy, we designed this study to evaluate the effects of knocking down noggin gene expression on BMP-2-induced osteogenesis of human bone marrow-derived primary MSCs in vitro. Method. MSCs were isolated from human tibial bone marrow by density gradient centrifugation. Two noggin small interfering RNAs (siRNAs) were used in this study to knockdown noggin gene expression. There were four study groups: MSCs with no transfection of siRNA (named as NT group), MSCs transfected with non-targeting negative control siRNA (named as control group), MSCs transfected with noggin siRNA1 (named as NOGsi1 group), and MSCs transfected with noggin siRNA2 (named as NOGsi2 group). After transfection, MSCs were induced to undergo osteogenic differentiation by incubating in basal medium containing 0.1 μg/ml BMP-2 for 35 days. The expression levels of osteoblastic marker genes were measured by real-time quantitative PCR on day 14. Also assessed was alkaline phosphatase (ALP) activity by a colorimetric kinetic assay and Fast Blue B staining on day 14. Calcium deposition was determined by the calcium assay on day 35. Results. The expression levels of integrin binding sialoprotein (IBSP) and osteocalcin (OC) were significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT and control groups (all p<0.038). Although the expression level of runt-related transcription factor 2 (RUNX2) was also reduced in NOGsi1 and NOGsi2 groups compared with NT and control groups, it did not reach statistical significance. ALP activity was significantly lower in NOGsi1 and NOGsi2 groups than that of NT group (both p<0.024). The same pattern was also observed in ALP Fast Blue B staining. Calcium deposition was also significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT group (both p<=0.048). Conclusion. Noggin suppression by siRNA inhibits BMP-2-induced osteogenesis of human bone marrow-derived MSCs. Our results, contrary to the extensive studies conducted in rodent cells/models, corroborated with the previous study that the addition of noggin in the cell culture increased osteogenesis of human MSCs. This suggests that the effects of noggin on BMP-2-induced osteogenesis of MSCs might be species-specific

Purpose. A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results. Results. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N (55 vs. 78 % of control OA cells). Results also showed that the decreased type X collagen mRNA level was maintained not only when cells were removed from the PPE:N surfaces and transferred to new polystyrene culture dishes in the presence of chondrogenic media, but also when transferred to pellet cultures. Culturing MSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. Conclusion. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results may lead us one step closer to the production of large amounts of reprogrammed MSCs for tissue engineering applications

Purpose. Internal fixation of fractures in the presence of osteopenia has been associated with a failure rate as high as 25%. Enhancing bone formation and osseointegration of orthopaedic hardware is a priority when treating patients with impaired bone regenerative capacity. Fibroblast Growth Factor (FGF) 18 regulates skeletal development and could therefore have applications in implant integration. This study was designed to determine if FGF 18 promotes bone formation and osseointegration in the osteopenic FGFR3−/− mouse and to examine its effect on bone marrow derived mesenchymal stem cells (MSCs). Method. In Vivo: Intramedullary implants were fabricated from 0.4 × 10mm nylon rods coated with 300nm of titanium by physical vapour deposition. Skeletally mature, age matched female FGFR3−/− and wild type mice received bilateral intramedullary femoral implants. Left femurs received an intramedullary injection of 0.1μg of FGF 18 (Merck Serono), and right femurs received saline only. Six weeks later, femurs were harvested, radiographed, scanned by micro CT, and processed for undecalcified for histology. In Vitro: MSCs were harvested from femurs and tibiae of skeletally mature age matched FGFR3−/− and wild type mice. Cells were cultured in Alpha Modified Eagles Medium (αMEM) to monitor proliferation or αMEM supplemented with ascorbic acid and sodium beta-glycerophosphate to monitor differentiation. Proliferation was assessed through cell counts and metabolic activity at days 3, 6 and 9. Differentiation was assessed through staining for osteoblasts and mineral deposition at days 6, 9 and 12. Results. Wild type mice exhibited more peri-implant bone formation compared to FGFR3−/− mice. Peri-implant bone formation at the proximal metaphyseal-diaphyseal junction was increased in FGF18 treated femurs compared with contralateral control femurs in wild type (p = NS) and FGFR3−/− (p = 0.04) mice. Histological analysis corroborated micro CT findings, with FGF 18 treated FGFR3−/− femurs forming peri-implant bone instead of the fibrous response seen in controls. In vitro studies showed that FGF18 significantly increased MSC proliferation and metabolism in a dose dependent manner in wild type and FGFR3−/− mice. Osteoblast differentiation was inhibited by FGF18 in wild type MSCs, but was increased at physiological concentrations in cells harvested from FGFR3−/− mice. Conclusion. FGF 18 increases bone formation and osseointegration of intramedullary implants in osteopenic mice and increases MSC proliferation in both the presence and absence of FGFR3. FGF18 also promoted osteoblast differentiation in the absence of FGFR3 signalling, most likely via FGFR1 or 2. Additional work is needed to confirm the identity of the alternate FGFR and to evaluate its capacity to improve osseous healing in unfavourable in-vivo environments

Purpose. Whilst it is known that oxidative stress can cause early degenerative changes observed in experimental osteoarthritis and that a major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients express type X collagen, a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification), little is known whether the expression of type X collagen in MSCs from OA patients can be related to oxidative stress or inflammatory reactions that occur during this disease. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Bone marrow aspirates were processed essentially as previously described. Briefly, non-adherent cells were discarded after 72h of culture and the adherent ones were expanded for 2–3 passages. MSCs from normal donor (control) were obtained from Lonza. Cells were then lysed and protein expression was detected by Western blot using specific antibodies directed against type X collagen, as well as the antioxidant enzymes Mn-superoxide dismutase (MnSOD), catalase (CAT) and glutathione peroxidase-1 (GPx-1) and inflammation related proteins cyclooxygenase-1 (COX-1) and intercellular adhesion molecule-1 (ICAM-1). GAPDH was used as a housekeeping gene and served to normalize the results. Correlations between the expressions of the different proteins were realized using the correlation Z test with StatView (SAS Institute). Results. Results confirmed that type X collagen was over-expressed in MSCs from OA patients when compared to expression in cells of normal donors. MnSOD, CAT, and COX-1 were also over-expressed. Results showed that the expression of MnSOD strongly correlated to the expression of type X collagen (r=0.79; p=0.03). The expression of CAT weakly correlated to the expression of type X collagen (r=0.67; p=0.10) whereas GPx was not expressed in MSCs from OA patients. Regarding inflammatory reaction, results showed that COX-1 expression strongly correlated to type X collagen expression (r=0.77; p=0.004). ICAM-1 was weakly expressed and no correlation with the expression of type X collagen was observed. Interestingly, COX-1 expression was highly correlated to the expression MnSOD (r=0.92; p=0.0001) and the expression of CAT (r=−0.82; p=0.02). Conclusion. We showed that the level of anti-oxidant enzymes correlates with type X collagen expression in MSCs from OA patients. This suggests that oxidative stress may lead to the up-regulation of stem cell hypertrophy. Results also suggest that prostaglandin production though COX-1 activity is associated with anti-oxidant enzyme expression (MnSOD) and hypertrophy (type X collagen expression). Further studies are however necessary to better understand whether the increased expression of these proteins is the cause or the effect of type X collagen over-expression in MSCs from OA patients

Osteoarthritis is a global problem and the treatment of early disease is a clear area of unmet clinical need. Treatment strategies include cell therapies utilising chondrocytes e.g. autologous chondrocyte implantation and mesenchymal stem/stromal cells (MSCs) e.g. microfracture. The result of repair is often considered suboptimal as the goal of treatment is a more accurate regeneration of the tissue, hyaline cartilage, which requires a more detailed understanding of relevant biological signalling pathways. In this study, we describe a modulator of regulatory pathways common to both chondrocytes and MSCs. The chondrocytes thought to be cartilage progenitors are reported to reside in the superficial zone of articular cartilage and are considered to have the same developmental origin as MSCs present in the synovium. They are relevant to cartilage homeostasis and, like MSCs, are increasingly identified as candidates for joint repair and regenerative cell therapy. Both chondrocytes and MSCs can be regulated by the Wnt and TGFβ pathways. Dishevelled Binding Antagonist of Beta-Catenin (Dact) family of proteins is an important modulator of Wnt and TGFβ pathways. These pathways are key to MSC and chondrocyte function but, to our knowledge, the role of DACT protein has not been studied in these cells. DACT1 and DACT2 were localised by immunohistochemistry in the developing joints of mouse embryos and in adult human cartilage obtained from knee replacement. RNAi of DACT1 and DACT2 was performed on isolated chondrocytes and MSCs from human bone marrow. Knockdown efficiency and cell morphology was confirmed by qPCR and immunofluorescence. To understand which pathways are affected by DACT1, we performed next-generation sequencing gene expression analysis (RNAseq) on cells where DACT1 had been reduced by RNAi. Top statistically significant (p < 0 .05) 200 up and downregulated genes were analysed with Ingenuity® Pathway Analysis software. We observed DACT1 and DACT2 in chondrocytes throughout the osteoarthritic tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 was localised to the articular surface. Furthermore, in mouse embryos (E.15.5), we observed DACT2 at the interzones, sites of developing synovial joints, suggesting that DACT2 has a role in cartilage progenitor cells. We subsequently analysed the expression of DACT1 and DACT2 in MSCs and found that both are expressed in synovial and bone marrow-derived MSCs. We then performed an RNAi knockdown experiment. DACT1 knockdown in both chondrocyte and MSCs caused the cells to undergo apoptosis within 24 hours. The RNA-seq study of DACT1 silenced bone marrow-derived MSCs, from 4 different human subjects, showed that loss of DACT1 has an effect on the expression of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways. In summary, we describe for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. Loss of DACT1 induced cell death in both chondrocytes and MSCs, with RNA-seq analysis revealing a direct impact on transcript levels of genes involved in the Wnt and TFGβ signalling, key regulatory pathways in skeletal development and repair

The ability of mesenchymal stem cells (MSCs) to differentiate in vitro into chondrocytes, osteocytes and myocytes holds great promise for tissue engineering. Skeletal defects are emerging as key targets for treatment using MSCs due to the high responsiveness of bone to interventions in animal models. Interest in MSCs has further expanded in recognition of their ability to release growth factors and to adjust immune responses.

Despite their increasing application in clinical trials, the origin and role of MSCs in the development, repair and regeneration of organs have remained unclear. Until recently, MSCs could only be isolated in a process that requires culture in a laboratory; these cells were being used for tissue engineering without understanding their native location and function. MSCs isolated in this indirect way have been used in clinical trials and remain the reference standard cellular substrate for musculoskeletal engineering. The therapeutic use of autologous MSCs is currently limited by the need for ex vivo expansion and by heterogeneity within MSC preparations. The recent discovery that the walls of blood vessels harbour native precursors of MSCs has led to their prospective identification and isolation. MSCs may therefore now be purified from dispensable tissues such as lipo-aspirate and returned for clinical use in sufficient quantity, negating the requirement for ex vivo expansion and a second surgical procedure.

In this annotation we provide an update on the recent developments in the understanding of the identity of MSCs within tissues and outline how this may affect their use in orthopaedic surgery in the future.

Cite this article: Bone Joint J 2014;96-B:291–8.

Construction of a functional skeleton is accomplished through co-ordination of the developmental processes of chondrogenesis, osteogenesis, and synovial joint formation. Infants whose movement in utero is reduced or restricted and who subsequently suffer from joint dysplasia (including joint contractures) and thin hypo-mineralised bones, demonstrate that embryonic movement is crucial for appropriate skeletogenesis. This has been confirmed in mouse, chick, and zebrafish animal models, where reduced or eliminated movement consistently yields similar malformations and which provide the possibility of experimentation to uncover the precise disturbances and the mechanisms by which movement impacts molecular regulation. Molecular genetic studies have shown the important roles played by cell communication signalling pathways, namely Wnt, Hedgehog, and transforming growth factor-beta/bone morphogenetic protein. These pathways regulate cell behaviours such as proliferation and differentiation to control maturation of the skeletal elements, and are affected when movement is altered. Cell contacts to the extra-cellular matrix as well as the cytoskeleton offer a means of mechanotransduction which could integrate mechanical cues with genetic regulation. Indeed, expression of cytoskeletal genes has been shown to be affected by immobilisation. In addition to furthering our understanding of a fundamental aspect of cell control and differentiation during development, research in this area is applicable to the engineering of stable skeletal tissues from stem cells, which relies on an understanding of developmental mechanisms including genetic and physical criteria. A deeper understanding of how movement affects skeletogenesis therefore has broader implications for regenerative therapeutics for injury or disease, as well as for optimisation of physical therapy regimes for individuals affected by skeletal abnormalities.

Cite this article: Bone Joint Res 2015;4:105–116