Macromolecular crowding (MMC) is a biophysical phenomenon that accelerates thermodynamic activities and biological processes by several orders of magnitude. Herein, we ventured to identify the optimal crowder and to assess the influence of MMC in umbilical cord mesenchymal stem cell. 7 types of carrageenan (κ&λ, κ-LV1, κ-LV2, λ-MV, λ-HV, ι-MV, ι-HV) acted as crowder and biophysical properties were assessed respectively. Human umbilical cord mesenchymal stem cells were seeded at 15,000 cells/cm. 2. in 24 well plates and allowed to attach for 24 h. Subsequently, the medium was changed to medium with 7 types of carrageenan (10, 50, 100, 500 μg/ml) and 100 μM L-ascorbic acid phosphate (Sigma Aldrich). Medium without carrageenan was used as control. Cell morphology and SDS-PAGE analysis were conducted after 3, 5 and 7 days. Biophysical assessment showed 7 types of carrageenan have increased particle size with concentration, good polydispersity and negative charges. SDS-PAGE and densitometric analyses revealed significant increase (p < 0.001) in collagen deposition in the presence of 10 μg/ml carrageenan λ and ι at all the time points. SDS-PAGE and densitometric analysis also showed that the highest collagen deposition was observed in culture at 50 μg/ml carrageenan λ. No significant difference was observed in cell morphology between the groups. Collectively, these data primarily illustrate the beneficial effect of carrageenan λ in human umbilical cord mesenchymal stem cell culture

Introduction. Mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. We have previously demonstrated that the infrapatellar synovial fat pad is a rich source of mesenchymal stem cells and these cells are able to undergo chondrogenic differentiation. Although synovial fat pad derived mesenchymal stem cells may represent a heterogenous population, clonal populations derived from the synovial fat pad have not previously been studied. Materials and Methods. Mesenchymal stem cells were isolated from the infrapatellar synovial fat pad of a patient undergoing total knee arthroplasty and expanded in culture. Six clonal populations were also isolated before initial plating using limiting dilution and expanded. The cells from the mixed parent population and the derived clonal populations were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium for 14 days. Gene expression analyses; glycosoaminoglycan and DNA assays; and immunohistochemical staining were determined to assess chondrogenic responses. Results. Cells from the mixed parent population and the derived clonal populations stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic marker CD34 and for the neural and myogenic marker CD56. Interestingly, a variable number of cells were also positive for the pericyte marker 3G5 both in the mixed parent and clonal populations. The clonal populations exhibited a variable chondrogenic response; one clonal cell population exhibited a significantly greater chondrogenic response when compared with the mixed parent population. Discussion. Pericytes are a candidate stem cell in many tissue and our results show that all six clonal populations derived from the heterogenous synovial fat pad population express the pericyte marker 3G5. The variable chondrogenic responses suggest inherent differences between these populations. The chondrogenic potential of the synovial fat pad could be optimised by the identification of clonal populations with a propensity to differentiate down particular differentiation pathways, and this has implications on the future tissue engineering applications of these cells for cartilage repair

In this presentation, the response of mesenchymal stem cells (MSCs) to nanoscale cues (e.g. topography, chemistry and vibrations) will be considered. In particular, control of MSC self-renewal and differentiation. A focus will be on a new bioreactor that has been developed, the nanokick, that delivers precise nanovibrational cues to MSC cultures in 2D and 3D, driving the cells to turn into mineralizing osteoblasts. Mechanotransductive signalling will be considered looking at ion channel mediated differentiation

Mesenchymal stem cells (MSC) have potent immunomodulatory and regenerative effects via soluble factors. One approach to improve stem cell-based therapies is encapsulation of MSC in hydrogels based on natural proteins such as collagen and fibrin, which play critical roles in bone healing. In this work, we comparatively studied the influence of collagen and fibrin hydrogels of varying stiffness on the paracrine interactions established by MSC with macrophages and osteoblasts. Type I collagen and fibrin hydrogels in a similar stiffness range loaded with MSC from donants were prepared by modifying the protein concentration. Viability and morphology of MSC in hydrogels as well as cell migration rate from the matrices were determined. Paracrine actions of MSC in hydrogels were evaluated in co-cultures with human macrophages from healthy blood donors or with osteoblasts from bone explants of patients with osteonecrosis of the femoral head. Lower matrix stiffness resulted in higher MSC viability and migration. Cell migration rate from collagen hydrogels was higher than from fibrin matrices. The secretion of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E. 2. (PGE. 2. ) by MSC in both collagen and fibrin hydrogels increased with increasing matrix stiffness. Tumor necrosis factor-α (TNF-α) secretion by macrophages cultured on collagen hydrogels was lower than on fibrin matrices. Interestingly, higher collagen matrix stiffness resulted in lower secreted TNF-α while the trend was opposite on fibrin hydrogels. In all cases, TNF-α levels were lower when macrophages were cultured on hydrogels containing MSC than on empty gels, an effect partially mediated by PGE. 2. Finally, mineralization capacity of osteoblasts co-cultured with MSC in hydrogels increased with increasing matrix stiffness, although this effect was more notably for collagen hydrogels. Paracrine interactions established by MSC in hydrogels with macrophages and osteoblasts are regulated by matrix composition and stiffness

Summary Statement. Umbilical cord derived stem cell secretion could enhance the osteogenic differentiation of human bone marrow stem cells. It may promote bone, cartilage and tendon regeneration in rat models, but the effect was not significant up to now. Introduction. Mesenchymal stem cells (MSCs) are multipotent cells that have extensive proliferative capacity. MSCs synthesise various exosomes, growth factors and cytokines. Stem cell secretions were made from serum free conditioned medium of stem cells collected from different human tissues, such as adipose tissue and dental pulp. Our hypothesis is umbilical cord stem cell secretion could promote multiple proliferation and differentiation of MSCs, also enhance the regeneration of musculoskeletal tissues. Methods. In vitro: Human bone marrow mesenchymal stem cells (hBMSCs) were cultured in high glucose dulbecco's modified eagle medium with 10% serum. hBMSCs were treated by differential medium for osteogenic, tenogenic and chondrogenic differentiation. Alizarin red S staining, alcian blue staining and sirius red staining were used to test osteogenesis, chondrogenesis and tenogenesis of hBMSCs after treated by secretion. RNA expression level of hBMSCs were detected by real-time reverse transcriptase polymerase chain reaction. In vivo: 10 weeks male Sprague-Dawley rats were used in all the animal studies. Rat calvarial bone defect model, rat femoral closed fracture model with internal fixation, rat articular cartilage defect model and rat patella tendon window defect model were used in animal experiments. Radiography analysis, micro-computed tomography imaging analysis, mechanical test, ultrasound test and histology analysis were used to evaluate the regeneration of bone, cartilage and tendon. Results. Alizarin red S staining showed the minimal effective concentration of 20ug/ml umbilical cord stem cells secretion could promote strong osteogenesis of hBMSCs, with enhanced expression of osteogenic markers runx2 and ocn. 20ug/ml umbilical cord stem cells secretion could promote tenogenic differentiation. The bone defect healing study using rat calvarial defect model indicated no significant difference (p»0.05) between 0.5ug/1ug umbilical cord secretion treated group (agarose gel with secretion was implanted in defect) and control (PBS) in 4 weeks or 8 weeks time points. In the rat femoral closed fracture model, the difference of bone repair between 10ug umbilical cord secretion local injection group (injected 10ug in callus after surgery) and control (PBS injected) was not significant (p»0.05) in 4 weeks or 8 weeks. In the rat articular cartilage defect model, 1ug umbilical stem cell secretion with 20ul alginate gel group recovered better than alginate gel only group in 6 weeks(p<0.05), but the difference of cartilage healing was not significant (p»0.05) between other groups (alginate gel with BMSCs) in 6 weeks or 9 weeks. In the rat patella tendon window defect model, there were more compact collagen fibers in 1ug umbilical cord secretion group (secretion with fibrin glue), but the alignment of new tissue was not better than control (PBS with fibrin glue). Also the stress of defected area was not significantly different (p»0.05) between treated and control in 6 weeks and 9 weeks. Discussion/Conclusion. The umbilical cord stem cell secretion demonstrated osteogenic, and tenogenic effect in vitro, but the result in the healing of bone, cartilage and tendon was not significant. The optimal dosage and slow release method will be considered to improve the experiment. The mechanism of stem cell secretions will be studied in further research

Mesenchymal stem cells (MSCs) are believed to be immune-privileged due to lack of antigen-presenting-cell related markers, however, evidence suggests that MSCs are immunogenic and are attacked by the immune system. Our research investigates the hypothesis that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immune profile. Our goal is to discover immune-privileged stem cells, which can act as a universal allogenic mesenchymal stem cell donor to facilitate bone ingrowth for osteosarcoma patients status post tumor excision and prosthesis implantation. Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out in order to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. This procedure was repeated for another other 2 animals. The proliferation rate and cell doubling time of each clonal culture was measured. The proliferation rate of mixed clonal cultures was also measured. The tri-differentiation potential of the clonal cultures was compared and a comparison was also made with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. To measure the immune response a mixed leucocyte reaction was used but where leucocytes from a different individual were mixed with the clonal MSC cells. All isolates were able to differentiate into osteoblasts, chondrocytes and adipocytes. All clonal cultures revealed significantly different proliferation rates and doubling times when compared with each other and with mixed cultures. All clonal cultures showed different surface marker presentations, which included differences in the expression of MHC antigens. One clone isolated from ADMSCs showed lack of MHCI and MHCII. Our mixed leucocyte reaction and MHC staining showed variety of immune-modulation and this was related to the expression of the MHC antigens. All clones tri-differentiated and therefore show a degree of ‘stemness’. MSCs are generally are believed not to express MHC II and to be immune-privileged. However, this study shows that the expression of these antigens in clones isolated from bone marrow and from fat is variable. A heterogeneous result indicates individual differences between MSCs, even from same origin. The immune response elicited by MSCs is complicated. MSCs have been shown to release interleukin 10, which could inhibit the immune response but on the other hand interferon-gamma could enhance MHCII presentation in some MSCs. Our results confirmed our hypothesis because clonal cultures isolated from different sources of MSCs in the same animal showed significant differences in proliferation rate, morphology and surface marker presentation. Mesenchymal stem cells are not immunogenic or immune-privileged. Individual differences highlighted through single-cell clonal cultures may be the key to finding a universal immune-privileged MSCs for allogeneic transplantation

Abstract. Objective. Articular cartilage damaged through trauma or disease has a limited ability to repair. Untreated, these focal lesions progress to generalized changes including osteoarthritis. Musculoskeletal disorders including osteoarthritis are the most significant contributor to disability globally. There is increasing interest in the use of mesenchymal stem cells (MSCs) for the treatment of focal chondral lesions. There is some evidence to suggest that the tissue type from which MSCs are harvested play a role in determining their ability to regenerate cartilage in vitro and in vivo. In humans, MSCs derived from synovial tissue may have superior chondrogenic potential. Methods. We carried out a systematic literature review on the effectiveness of synovium-derived MSCs (sMSCs) in cartilage regeneration in in vivo studies in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol. Nineteen studies were included in our review; four examined the use of human sMSCs and the remainder were conducted using sMSCs harvested from animals. Results. Despite the variability of animals, cell harvesting techniques, methods of delivery, and outcome measures, all studies reported successful cartilage repair with sMSC transplantation. Conclusion. We conclude that sMSC transplantation holds promise as a treatment option for focal cartilage defects. We believe that defining the cell population being used, establishing standardized methods for MSC delivery, and the use of objective outcome measures should enable future high-quality studies such as randomized controlled clinical trials to provide the evidence needed to manage chondral lesions optimally. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Mesenchymal stem cells (MSCs) are tissue-resident stroma cells capable of modulating immune cells through the secretion of paracrine factors. However, the comparison of MSCs potential, from different sources and submitted to hypoxia within a 3D scaffold, in secreting pro-healing factors has never been investigated. With a chemical composition similar to type I collagen, a major component of connective tissues retrieved in dental pulp, bone and umbilical cord, Hemocollagene® haemostatic foam presented porous and interconnected structure (> 90%) and a relative low elastic modulus of around 60 kPa. All these criteria meet basic requirements for tissue engineering based material. Herein, we assessed and compared the effect of hypoxia (3% O. 2. ) on the regulation and release of pro-angiogenic factors (VEGF, b-FGF and IL-8) from bone marrow (BM), Wharton's jelly (WJ) and dental pulp (DP) derived MSCs cultured in Hemocollagene®. After 10 days of culture, qRT-PCR analysis showed an up-regulation of b-FGF and VEGF mRNA in BM- and WJ-derived MSCs, but not in DP-derived MSCs. Furthermore, hypoxia highly up-regulated IL-8 expression in WJ-derived MSCs and moderately in both BM and DP-derived MSCs. In contrast, ELISA analysis showed a higher amount of VEGF and IL-8 in supernatant provided from DP-derived MSCs culture compared to BM and WJ-derived MSCs. B-FGF was not detected whatever the experimental condition. In conclusion, MSCs derived from several tissues were able to release pro-angiogenic factors under hypoxic conditions. There was no clearly superior type of MSCs for therapeutic use, however DP-derived MSCs are likely to be more advantageous

Reconstruction of 10mm segmental bone defects in rat by mesenchymal stem cell derived chondrogenic cells (MSC-DC). Background. Mesenchymal stem cell derived condrogenic cells (MSC-DC) have excellent potential for healing 5 mm bone defect in rat femur. Purpose. To evaluate the effectiveness of MSC-DC on bone healing in 10 mm segmental bone defects in rat femur. Methods. 10 millimeter bone defects were produced in rat femur and fixed with external fixator. We divided this model into four groups according to the kind of graft for bone defects. These bone defects were grafted by MSC-DC seeded on a poly (DL-lactic acid-co-glycolic acid) (PLGA) scaffold in Group A, MSC seeded on a PLGA scaffold in Group B, PLGA scaffold only in Group C, and autologus bone graft in Group D. The healing processes were monitored radiographically and studied biomechanically and histologically. Results. All the bone defects in Group A healed radiographically with bridging callus formation at 4 weeks after the procedure, while none of Group B, C, and D had achieved bone union even at 8 weeks. Mechanical testing revealed that Group A showed approximately 40 % bending strength at 4 weeks compared with the contralateral side, and approximately 60 % at 8 weeks. In histology, Group A, maturation of bridging callus occurred from outside and enchondral ossification was prominent from inside. Conclusion. This study showed that MSC-DC with PLGA scaffold enhances bone healing even in large bone defects

Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects the whole joint. Mesenchymal stem cells ability to secrete anti-inflammatory and immuno-modulatory factors represents an attractive tool in the treatment of OA. Considering the risk of cell leakage and the massive cell death upon intra-articular injection, we developed a micromolding protocol of encapsulation that allows to obtain particles that (i) could be injected with a 26G needle into a mouse joint and (ii) could provide a 3D microenvironment supporting cell biological activity. Polydimethylsiloxane (PDMS) chips containing circular micromolds were manufactured and a solution of alginate (2% w/v) containing human adipose stem cells (3 millions/mL) was deposited on the chips. Cell loading into the micromolds was performed either by sedimentation or by centrifugation. Following Ca2+ crosslinking, alginate particles (diameter 150±0.7μm) were obtained. The number of cells per particle was 5 times higher when the micromolds were loaded by centrifugation. Cell number and metabolic activity remained stable for 7 days after encapsulation and injection through a 26G needle had no impact on cell viability. When cells were stimulated with TNF-alpha and INF-gamma, prostaglandin E2 (PGE2) concentration in the supernatant was multiplied by 13 and 7 and indoleamine2,3-dioxygenase (IDO) activity was 2 and 4 times higher when cell loading was performed by sedimentation or centrifugation, respectively. We have demonstrated that encapsulated cells were able to sense and respond to an inflammatory stimulus and their therapeutic potential will be evaluated in a murine model of osteoarthritis

Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model. Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function. Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs

An established rabbit model was used to preliminarily investigate the effect of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets on tendon-bone interface healing. Bone, fibrocartilage and tendon tissue were harvested from the rabbits and sectioned into a book-type scaffold. The scaffolds were decellularized and their characterization was presented. BMSCs were isolated and co-cultured with the scaffolds to verify their cytocompatibility. BMSCs sheets were fabricated and inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS complex. The complex was implated in the right knee of rabbits which operated standard partial patellectomy for TBI regeneration using Imaging, histological and biomechanical examinations. The bone, fibrocartilage and tendon tissue were sectioned into a book-type scaffold before decellularization. Then we decellularized the above tissue and mostly preserved their microstructure and composition of the natural extracellular matrix, including collagen and proteoglycan. After the physicochemical and biological properties of the book-type ATS were evaluated, autologous BMSCs sheets were inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS implants for TBI regeneration. In addition, the ATS has the advantages of non-toxicity, suitable for cell adhesion and growth as well as low immunogenicity while co-cultured with the BMSCs. At the same time, different scaffolds has the ability to induce the osteogenic, chondrogenic and tenogenic differentiation of BMSCs by immunofluorescence, reverse transcription-polymerase chain reaction and western blot analysis. To determine the efficacy of the tissue-engineered implants for TBI regeneration, we transplanted it into a rabbit patella-patellar tendon (PPT) injury model, and the rabbits were sacrificed at postoperative week 8 or 16 for the radiological, histological, and mechanical evaluation. Radiologically, Synchrotron radiation micro-computed tomography (SR-μCT) showed that BMSCs/ATS group significantly increased bone area, BV/TV, trabecular thickness and trabecular number at the healing interface as compared with other groups at postoperative week 8 or 16. Histologically, the BMSCs/ATS group showed more woven bone, and a more robust fibrocartilaginous junction with a characteristic matrix rich in proteoglycans was seen at the PPT healing interface in comparison with other groups after 8 weeks. At week 16, the healing interface in 3 groups displayed better remodeling with respect to postoperative week 8. Healing and remodeling at the PPT junction were almost complete, with a resemblance to a healthy BTI consisting of the characteristic 4 zones in all groups. At last, we used biomechanical test as functional parameters to evaluate the quality of tendon-bone healing. Biomechanical testing indicated that BMSCs/ATS group showed significantly higher failure load and stiffness than other groups at postoperative week 8 and 16. The complex composed of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets can simulate the gradient structure of tendon-bone interface, inducing stem cell directional differentiation, so as to promote patella-patellar tendon interface healing effectively after injury

Introduction. Mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. We have previously demonstrated that the infrapatellar synovial fat pad is a rich source of mesenchymal stem cells and these cells are able to undergo chondrogenic differentiation. Although synovial fat pad derived mesenchymal stem cells may represent a heterogenous population, clonal populations derived from the synovial fat pad have not previously been studied. Materials and Methods. Mesenchymal stem cells were isolated from the infrapatellar synovial fat pad of a patient undergoing total knee arthroplasty and expanded in culture. Six clonal populations were also isolated before initial plating using limiting dilution and expanded. The cells from the mixed parent population and the derived clonal populations were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium for 14 days. Gene expression analyses; glycosoaminoglycan and DNA assays; and immunohistochemical staining were determined to assess chondrogenic responses. Results. Cells from the mixed parent population and the derived clonal populations stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic marker CD34 and for the neural and myogenic marker CD56. Interestingly, a variable number of cells were also positive for the pericyte marker 3G5 both in the mixed parent and clonal populations. The clonal populations exhibited a variable chondrogenic response; one clonal cell population exhibited a significantly greater chondrogenic response when compared with the mixed parent population. Discussion. Pericytes are a candidate stem cell in many tissue and our results show that all six clonal populations derived from the heterogenous synovial fat pad population express the pericyte marker 3G5. The variable chondrogenic responses suggest inherent differences between these populations. The chondrogenic potential of the synovial fat pad could be optimised by the identification of clonal populations with a propensity to differentiate

Mesenchymal stem cells (MSCs) are usually believed to be immune-privileged. However, immunogenic MSCs were also reported. We hypothesize that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immunogenicity. Our goal is to discover immune-privileged stem cells for universal allogenic MSCs transplantation. Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. The proliferation rate of each clonal culture and mixed clonal culture were measured. The tri-differentiation potential of the clonal cultures was compared, as well as with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. Mixed leucocyte reaction (MLR) were also performed to investigate immunogenicity. Tri-differentiation was confirmed in all isolates. All clonal cultures revealed significant different morphology and proliferation rates, compared with each other and mixed cultures. All clonal cultures showed different surface markers, inclusive of MHC antigens. One clone from ADMSCs showed lack of MHC antigens. Our MLR and MHC staining disclosed variety of immune properties. All clones tri-differentiated which indicated a degree of ‘stemness’. MSCs are generally believed not to express MHC II, resulting in immune-privileged. Our results confirmed our hypothesis because clonal cultures isolated from different origins of same animal show differences in morphology, proliferation rate, and surface marker presentation. Individual immune differences highlighted through single-cell clonal cultures may be crucial to find universal immune-privileged MSCs as universal allogeneic donor

Objectives: Skeletal muscle trauma leads to severe functional deficits. Present therapeutic treatments are unsatisfying and insufficient posttraumatic regeneration is a problem in trauma and orthopaedic surgery. Mesenchymal stem cell (MSC) therapy is a promising tool in the regeneration of muscle function after severe trauma. Our group showed increased contraction forces compared to a non-treated control group 3 weeks after MSC transplantation (TX) into a skeletal muscle trauma. In addition we demonstrated a dose-response relationship of the amount of MSC and force enhancement. We furthermore investigated the fate of the transplanted MSC labelled with very small iron oxide particles using 7 Tesla-MRI. Histological analysis revealed fusion events between existing myofibers but only to a low amount. The increase of muscle force can not be explained by these events only. Before further steps are taken the impact of paracrine effects and the homing to the site of trauma of the MSC has to be evaluated. Experimental studies about the functional regeneration of traumatized skeletal muscule after systemic MSC-TX do not exist. Methods: 36 female SD-rats received open crush trauma of the left soleus muscle. One week after trauma 2.5 x 106 autologous MSC, harvested from tibial biopsies, were transplanted intraarterially (i.a., femoral arte-ria, group 1) or intravenously (i.v., tail vein, group 2) (n=18). Control animals received saline (i.a.: group 3; i.v.: group 4) (n=18). Histological analysis and biomechanical evaluation by in vivo muscle force measurement was performed 3 weeks after TX. Results: Twitch stimulation of the healthy right soleus muscles resulted in a contraction force of 0.52±0.14 N. Forces of tetanic contraction in the uninjured muscles reached 0.98±0.27 N. The i.a. MSC-TX improved the muscle force of the injured soleus significantly compared to control (twitch: 82,4%, p=0.02, tetany: 61.6%, p=0.02). Contraction forces of muscles treated i.v. (MSC vs. saline) showed no significant difference. The histological analysis showed no differences in the amount of fibrotic tissue. Conclusions: The presented study demonstrates the effect of systemic MSC-TX in the treatment of severe skeletal muscle injuries. Interestingly, the functional regeneration could only be increased by i.a. application. The entrapment of MSC in the lungs and the dilution effect in the circulation, when injecting the MSC i.v. could be the reason. For possible future therapeutic approaches a systemic application is considered to be favourable compared to local injections due to the better distribution of the cells in the target muscle

Purpose. The purpose of this study was to evaluate the effects of implantation of mesenchymal stem cell derived condrogenic cells (MSC-DC) on bone healing in segmental defects in rat femur. Methods. Five-millimeter segmental bone defects were produced in the mid-shaft of the femur of Fisher 344 rats and stabilized with external fixator. The Treatment Group received MSC-DC, seeded on a PLGA scaffold, locally at the site of the bone defect, and Control Group received scaffold only. The healing processes were monitored radiographically (Softex), and studied radiographically (Micro-CT) and histologically. Results. All the bone defects in the Treatment Group healed radiographically with bridging callus formation at 4 weeks after the procedure, while none of the Control Group had achieved bone union. Micro-CT showed that newly formed bone volume in the Treatment Group at 16 weeks was 1.5 times that of unaffected side. Histological examination showed that the implanted scaffold of the Treatment Group were covered with periosteum-derived bridging callus and filled with cancellous bone-like tissue derived from enchondral ossification. Conclusion. The results of this study suggest that implantation of MSC-DC surprisingly enhances bone healing in segmental bone defects in rat much better than previously reported similar therapy using MSC

The role of mesenchymal stem cells (MSCs) in enhancing healing process has been examined with allogeneic and xenogeneic cells in transplantation models. However, certain factors might limit the use of allogeneic cells in clinical practice, (e.g. disease transmission, ethical issues and patient acceptance). Adipose tissue represents an abundant source for autologous cells. The aim of this study was to evaluate adipose-derived autologous cells for preventing non-union. Adults male Wistar rats (n=5) underwent a previously published surgical procedure known to result in non-union if no treatment is given. This consisted of a mid-shaft tibial osteotomy with peri/endosteal stripping stabilised by intramedullary nail fixation with a 1mm gap maintained by a spacer. During the same operation, ipsilateral inguinal subcutaneous fat was harvested and processed for cell isolation. After three weeks in culture, the cell number reached 5×106 and were injected into the fracture site. At the end of the experiment, all tibias (injected with autologous fat-MSCs) developed union. These were compared with a control group injected with PBS (n=4) and with allogenic (n=5) and xenogeneic (n=6) cell transplantation groups. The amount of callus was noticeably large in the autologous cell group and the distal-callus index was significantly greater than that of the other groups, P-value =<0.05, unpaired t-test, corrected by Benjamini & Hochberg. We report a novel method for autologous MSCs implantation to stimulate fracture healing. Local injection of autologous fat-MSCs into the fracture site resulted in a solid union in all the tibias with statistically significantly greater amounts of callus

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

Background. Currently, there is a focus on the development of cell based therapies to treat intervertebral disc (IVD) degeneration, particularly for regenerating/repairing the central region, the nucleus pulposus (NP). Recently, we demonstrated that GDF6 promotes NP-like differentiation in mesenchymal stem cells (MSCs). However, bone marrow- (BM-MSCs) and adipose- (Ad-MSCs) showed differential responses to GDF6, with Ad-MSCs adopting a more NP-like phenotype. Here, we investigated GDF6 signalling in BM-MSCs and Ad-MSCs, with the aim to improve future IVD stem cell therapies. Methods. GDF6 receptor expression in patient-matched BM-MSCs and Ad-MSCs (N=6) was profiled through western blot and immunocytochemistry (ICC). GDF6 signal transduction was investigated through stimulation with 100 ng ml. −1. GDF6 for defined time periods. Subsequently smad1/5/9 phosphorylation and alternative non-smad pathway activation (phospho-p38; phospho-Erk1/2) was analysed (western blot, ELISA). Their role in inducing NP-like gene expression in Ad-MSCs was examined through pathway specific inhibitors. Results. Western blot and ICC established that BMPR profiles differed between MSC populations; specifically, BMPR2 (a GDF6 receptor) expression, was significantly higher in Ad-MSCs (p<0.05). ELISA and western blot analysis showed that smad1/5/9 phosphorylation was significantly higher in Ad-MSCs following GDF6 stimulation (p<0.05). GDF6 stimulation also phosphorylated p38 and Erk1/2 pathways. Blocking of both smad and non-smad pathways resulted in variation of GDF6 induced NP-like gene expression. Conclusions. The upregulation of BMPR2 in Ad-MSCs and corresponding differences in smad1/5/9 and non-smad pathway phosphorylation in response to GDF6 indicates an enhanced discogenic potential in Ad-MSCs, suggesting they may be more suitable for GDF6 mediated cellular IVD regeneration. Conflicts of interest. No conflicts of interest. Sources of funding. We would like to acknowledge UKRMP Acellular Hub, MRC, NIHR Musculoskeletal BRU and The Rosetrees Trust for funding this research

The current ‘gold’ standard surgical intervention for critical size bone defect repair involves autologous bone grafting, that risks inadequate graft containment and soft tissue invasion. Here, a new regenerative strategy was explored, that uses a barrier membrane to contain bone graft. The membrane is designed to prevent soft tissue ingrowth, whilst supporting periosteal regrowth, an important component to bone regeneration. This study shows the development of a collagen-based barrier membrane supportive of periosteal-derived mesenchymal stem cell (P-MSC) growth. P-MSC-homing barrier membranes were successfully obtained with nonaligned fibres, via free-surface electrospinning using type I collagen and poly(E-caprolactone) in 1,1,1,3,3,3-Hexafluoro-2-propanol. Introduction of collagen in the electrospinning mixture was correlated with decreased mean fibre diameter (d: 319 nm) and pore size (p: 0.2–0.6 μm), with respect to collagen-free membrane controls (d: 372 nm; p: 1–2 μm). Consequently, as the average MSC diameter is 20 μm, this provides convincing evidence of the creation of a MSC containment membrane. SEM-EDX confirmed Nitrogen and therefore collagen fibre localisation. Quantification of collagen content, using Picro Sirius Red dye, showed a 50% reduction after 24 hours (PBS, 37 °C), followed by a drop to 25% at week 3. The collagen-based membrane has a significantly higher elastic modulus compared to collagen-free control membranes. P-MSCs attached and proliferated when grown onto collagen-based membranes, imaged using confocal microscopy over 3 weeks. A modified transwell cell migration assay was developed, using MINUSHEET® tissue carriers to assess barrier functionality. In line with the matrix architecture, the collagen-based membrane proved to prevent cell migration (via confocal microscopy) in comparison to the migration facilitating positive control. The aforementioned results obtained at molecular, cellular and macroscopic scales, highlight the applicability of this barrier membrane in a new ‘hybrid graft’ regenerative approach for the surgical treatment of critical size bone defects