Synovial Stem Cells Research Articles

Lerociclib diminishes stemness in pediatric sarcoma cell lines.

e22010 Background: The outcomes for children with osteosarcoma and synovial sarcoma remain poor and are even worse for metastatic or relapsed disease. Those who do survive frequently suffer from long-term toxicities from current standard-of-care therapies. It is evident novel therapeutics are warranted to improve the outcomes for patients with these difficult tumors. Stem cell-like cancer cells (SCLCCs) are a subpopulation of tumor cells thought to be responsible for treatment resistance, development of metastases and tumor recurrence, making the targeting of this cell population critical. We sought to evaluate the effect of lerociclib, a CDK4/6 inhibitor, on pediatric sarcoma cell stemness. Methods: We investigated two established human osteosarcoma cells lines (U2-OS and MG-63), two metastatic synovial sarcoma patient-derived xenografts (PDXs) (COA-30 and COA-79) and a metastatic epithelioid sarcoma PDX (COA-171). Cells were treated with the CDK4/6 inhibitor, lerociclib, at concentrations below the known LD50. Synovial sarcoma stemness markers Octamer-binding transcription factor 4 (Oct4), homeobox protein Nanog, SOX 2 and nestin were evaluated by qPCR. CD117, a marker of stemness in osteosarcoma cells, was examined by flow cytometry. Finally, long-term passaged U2-OS and MG-63 cells were placed in low attachment serum-free conditions, and tumorsphere formation was evaluated using extreme limiting dilution assay (ELDA) in all cell lines. Results: Lerociclib treatment significantly decreased abundance of Oct4 (by 54%), Nanog (by 29%), SOX 2 (by 60%) and nestin (by 63%) mRNA in COA-30 synovial sarcoma cells. Cell surface expression of CD117 decreased from 17% to 11% in MG-63 cells when treated with lerociclib 2μM. Treatment of all cell types with lerociclib led to significantly decreased tumorsphere formation (Table). Conclusions: Treatment with lerociclib led to a decrease in mRNA abundance in known synovial sarcoma stem cell markers, a decrease in CD117 cell surface expression in osteosarcoma cells and decreased the ability of cells to form tumorspheres. These findings indicate lerociclib leads to decreased sarcoma cell stemness, which plays a key role in tumor progression and recurrence and should be further investigated for potential translation to the clinical setting.[Table: see text]

Stem cell-homing hydrogel-based miR-29b-5p delivery promotes cartilage regeneration by suppressing senescence in an osteoarthritis rat model.

Osteoarthritis (OA) is a common joint disease characterized by progressive loss of cartilage and reduction in lubricating synovial fluid, which lacks effective treatments currently. Here, we propose a hydrogel-based miRNA delivery strategy to rejuvenate impaired cartilage by creating a regenerative microenvironment to mitigate chondrocyte senescence that mainly contributes to cartilage breakdown during OA development. An aging-related miRNA, miR-29b-5p, was first found to be markedly down-regulated in OA cartilage, and their up-regulation suppressed the expression of matrix metalloproteinases and senescence-associated genes (P16INK4a/P21) via ten-eleven-translocation enzyme 1 (TET1). An injectable bioactive self-assembling peptide nanofiber hydrogel was applied to deliver agomir-29b-5p, which was functionalized by conjugating a stem cell–homing peptide SKPPGTSS for endogenous synovial stem cell recruitment simultaneously. Sustained miR-29b-5p delivery and recruitment of synovial stem cells and their subsequent differentiation into chondrocytes led to successful cartilage repair and chondrocyte rejuvenation. This strategy enables miRNA-based therapeutic modality to become a viable alternative for surgery in OA treatment.

Divergence in chondrogenic potential between in vitro and in vivo of adipose- and synovial-stem cells from mouse and human

BackgroundSomatic stem cell transplantation has been performed for cartilage injury, but the reparative mechanisms are still conflicting. The chondrogenic potential of stem cells are thought as promising features for cartilage therapy; however, the correlation between their potential for chondrogenesis in vitro and in vivo remains undefined. The purpose of this study was to investigate the intrinsic chondrogenic condition depends on cell types and explore an indicator to select useful stem cells for cartilage regeneration.MethodsThe chondrogenic potential of two different stem cell types derived from adipose tissue (ASCs) and synovium (SSCs) of mice and humans was assessed using bone morphogenic protein-2 (BMP2) and transforming growth factor-β1 (TGFβ1). Their in vivo chondrogenic potential was validated through transplantation into a mouse osteochondral defect model.ResultsAll cell types showed apparent chondrogenesis under the combination of BMP2 and TGFβ1 in vitro, as assessed by the formation of proteoglycan- and type 2 collagen (COL2)-rich tissues. However, our results vastly differed with those observed following single stimulation among species and cell types; apparent chondrogenesis of mouse SSCs was observed with supplementation of BMP2 or TGFβ1, whereas chondrogenesis of mouse ASCs and human SSCs was observed with supplementation of BMP2 not TGFβ1. Human ASCs showed no obvious chondrogenesis following single stimulation. Mouse SSCs showed the formation of hyaline-like cartilage which had less fibrous components (COL1/3) with supplementation of TGFβ1. However, human cells developed COL1/3+ tissues with all treatments. Transcriptomic analysis for TGFβ receptors and ligands of cells prior to chondrogenic induction did not indicate their distinct reactivity to the TGFβ1 or BMP2. In the transplanted site in vivo, mouse SSCs formed hyaline-like cartilage (proteoglycan+/COL2+/COL1−/COL3−) but other cell types mainly formed COL1/3-positive fibrous tissues in line with in vitro reactivity to TGFβ1.ConclusionOptimal chondrogenic factors driving chondrogenesis from somatic stem cells are intrinsically distinct among cell types and species. Among them, the response to TGFβ1 may possibly represent the fate of stem cells when locally transplanted into cartilage defects.

Alterations in cartilage quantification before and after injections of mesenchymal stem cells into osteoarthritic knees

Several studies have reported improvement in knee pain following mesenchymal stem cell (MSC) injections for knee osteoarthritis (OA). We developed a novel 3D magnetic resonance imaging (MRI) analysis software program that provides “projected cartilage area ratios” for automatic detection of changes in cartilage amounts. The primary objective of this prospective interventional study was to compare alterations in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral posteromedial region between 30 weeks before and 30 weeks after synovial MSC injections. Secondary objectives were to assess the clinical scores and safety of MSC injections. Patients with OA who complained of knee pain underwent autologous synovial MSC injections into the knee at time 0 and again 15 weeks later. MRI examinations were performed at − 30, − 15, − 1, and 30 weeks. Patients showing < 3% decreases in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral the posteromedial region from − 30 weeks to − 15 weeks were excluded from the study. The Lysholm Knee Score, Knee Injury and Osteoarthritis Outcome Scale (KOOS), and Numerical Rating Scale (NRS) scores were evaluated at − 30, − 15, − 5, − 2, 0, 5, 10, 15, 20, 25, and 30 weeks. Five patients were excluded because 3D MRI analysis showed no cartilage loss at − 15 weeks. Ultimately, eight OA patients underwent MSC injections. The projected cartilage area ratio significantly decreased by 0.07 in the 30 weeks before MSC injections (p = 0.01), but no further decreases occurred in the 30 weeks after MSC injections. The projected cartilage area ratio at the femoral posteromedial region showed a significant difference between 30 weeks before and 30 weeks after MSC injections. The Lysholm Knee Score, KOOS, and NRS values improved significantly after the injections. MSC injection could not be ruled out as the cause of two adverse events: transient knee pain and itching in both hands. Fully automatic 3D MRI analysis showed that synovial MSC injections suppressed cartilage loss in patients with progressive OA.Trial registration: Intraarticular injections of synovial stem cells for osteoarthritis of the knee (Number UMIN 000026732). Date of registration; June 1, 2017. https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000029967.

Paracrine Effect of Synovial-Derived Stem Cells on Induced Knee Osteoarthritis in Rats

Background: Osteoarthritis (OA) is a progressive degenerative disease. Previous treatment strategies have tried to slow the progress of OA. Objectives: The present study aimed to stop and reverse the progressive nature of OA by the paracrine effects of synovial stem cells. Methods: Fifty male Sprague Dawley rats were randomly allocated to five equal groups (n = 10). The control group received no treatment. The second group received an intra-articular Hyalgan injection. The third group received an intra-articular injection of secreta. The fourth group received an intra-articular injection of synovial-derived stem cells (5 × 106). The last group received an intra-articular injection of secreta combined with synovial-derived stem cells (5 × 106). Three months after treatment, the samples were harvested and evaluated by histopathological and radiological analyses. Results: Histopathological and radiological findings demonstrated significant differences between the synovial stem cell combined with the secreta group and the control and Hyalgan groups. Significant differences were observed in the subchondral bone and matrix scores between the secreta group and the synovial stem cell and Hyalgan groups (P-value = 0.042 and P-value = 0.0001, respectively). Both secreta and synovial stem cell groups showed better healing in terms of cell population viability index than the Hyalgan group (P-value = 0.015 and P-value = 0.005, respectively). The synovial stem cell combined with secreta group showed a significant difference from the synovia stem cell group in both medial femoral condyle and fabella osteophyte indices (P-value = 0.004 and P-value = 0.011, respectively). Conclusions: The group treated with synovial stem cells combined with secreta showed better outcomes than other groups in histopathological and radiological evaluations.

Abstract # 2078 Norepinephrine (NE) inhibits chondrogenic differentiation of synovial adipose-derived stem cells (ASCs)

Sympathetic nerve fibers and NE are present in healthy and osteoarthritic (OA) synovial tissue and fluid. The potential regenerative role of progenitors in cartilage has been confirmed, though, in OA, these cells exhibit reduced chondrogenic capacity. The aim of this study was to explore the role of NE on proliferation and chondrogenesis of ASCs. Synovium was obtained from OA patients undergoing total knee replacement. For proliferation and chondrogenesis experiments, cells were treated with NE (10–9 – 10–6 M) or specific alpha- and beta-adrenergic receptor (AR) agonists under physioxia (2% O2). Cell number, viability, AR profile, pellet size, sulfated glycosaminoglycan (sGAG) deposition, type II collagen synthesis, hypertrophic marker gene expression, and dsDNA content was analyzed. ASCs expressed alpha1b, alpha2a, alpha2b, alpha2c, and beta2AR. NE or specific agonists had no significant effect on proliferation. Chondrogenic pellets expressed alpha1b, alpha2a, alpha2b, alpha2c, and beta2AR. Pellets treated with 10–7 or 10–6 M NE or with specific beta2AR agonist were significantly smaller compared to untreated controls, while dsDNA was constant. NE in high concentrations or the beta2AR agonist decreased sGAG and type II collagen content. No differences were observed on hypertrophic genes (MMP13, Runx2). Our study confirms the important role of NE in progenitor cell chondrogenic function and new insights in OA pathophysiology. Future studies might unravel the potential of novel neuroendocrine-chondrogenic therapeutic options for OA treatment.

A co-culture system of rat synovial stem cells and meniscus cells promotes cell proliferation and differentiation as compared to mono-culture

A meniscus tear often happens during active sports. It needs to be repaired or replaced surgically to avoid further damage to the articular cartilage. To address the shortage of autologous meniscal cells, we designed a co-culture system of synovial stem cells (SMSCs) and meniscal cells (MCs) to produce a large cell number and to maintain characteristics of MCs. Different ratios of SMSCs and MCs at 3:1, 1:1, and 1:3 were tested. Mono-culture of SMSCs or MCs served as control groups. Proliferation and differentiation abilities were compared. The expression of extracellular matrix (ECM) genes in MCs was assessed using an ECM array to reveal the mechanism at the gene level. The co-culture system of SMSCs/MCs at the ratio of 1:3 showed better results than the control groups or those at other ratios. This co-culture system may be a promising strategy for meniscus repair with tissue engineering.

The Potential for Synovium-derived Stem Cells in Cartilage Repair.

Articular cartilage defects often result in pain, loss of function and finally osteoarthritis. Developing cell-based therapies for cartilage repair is a major goal of orthopaedic research. Autologous chondrocyte implantation is currently the gold standard cell-based surgical procedure for the treatment of large, isolated, full thickness cartilage defects. Several disadvantages such as the need for two surgical procedures or hypertrophic regenerative cartilage, underline the need for alternative cell sources. Mesenchymal stem cells, particularly synovium-derived mesenchymal stem cells, represent a promising cell source. Synovium-derived mesenchymal stem cells have attracted considerable attention since they display great chondrogenic potential and less hypertrophic differentiation than mesenchymal stem cells derived from bone marrow. The aim of this review was to summarize the current knowledge on the chondrogenic potential for synovial stem cells in regard to cartilage repair purposes. A literature search was carried out identifying 260 articles in the databases up to January 2017. Several in vitro and initial animal in vivo studies of cartilage repair using synovia stem cell application showed encouraging results. Since synvoium-derived stem cells are located in the direct vicinity of cartilage and cartilage lesions these cells might even contribute to natural cartilage regeneration. The only one published human in vivo study with 10 patients revealed good results concerning postoperative outcome, MRI, and histologic features after a two-stage implantation of synovial stem cells into an isolated cartilage defect of the femoral condyle. Synovium-derived stem cells possess great chondrogenic potential and showed encouraging results for cartilage repair purposes. Furthermore, synovial stem cells play an important role in joint homeostasis and possibly in natural cartilage repair. Further studies are needed to elucidate the interplay of synovial stem cells and chondrocytes, and the promising role of synovium-derived stem cells in cartilage tissue engineering.

The study of leucocyte and platelet-rich plasma combined with rabbit synovial stem cells for the repair of knee joint cartilage injury

Objective To investigate the effect of platelet-rich plasma (L-PRP) combined with rabbit synovium-derived mesenchymal stem cells (SMSCs) on cartilage repair in knee-joint injured. Methods 60 healthy New Zealand white rabbits were used to establish models of knee-joint cartilage defects, SMSCs were separated, purified and identified, and then L-PRP was prepared using two step centrifugation. The levels of platelet derived growth factor-BB (PDGF-BB), transforming growth factor-β1 (TGF-β1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) in whole blood and L-PRP were determined using enzyme linked immunosorbent assay (ELISA). After 12 weeks of modeling, the injured cartilage was grossly observed, and hematoxylin-eosin (HE) staining was used to observe cartilage repair under light microscope; histological examination was performed according to the O’Driscoll histologic standard. Results The levels of platelet in L-PRP increased to (135.77±50.56)×104/μl were more 6 times than whole blood [(22.38±3.20)×104/μl, P=0.001], and the levels of PDGF-BB, TGF-β1, bFGF, VEGF in L-PRP were increased as (32.50±5.10) ng/ml, (124.26±32.30) ng/ml, (87.14±12.50) pg/ml, (174.50±31.22) pg/ml significantly higher than whole blood as [(6.28±1.11) ng/ml, (21.72±3.70) ng/ml, (25.85±4.52) pg/ml, (37.87±5.33) pg/ml], the differences were statistically significant (P=0.005, 0.003, 0.008, 0.002). Post-operation 12 weeks, the results from grossly observation, HE staining and histological examination indicated that the repair effect in L-PRP group, SMSCs group, L-PRP combined with SMSCs group was significantly better than in normal saline group, L-PRP and SMSCs group, respectively. Conclusion Both L-PRP and SMSCs be able to promote cartilage repair of knee-joint injured, and L-PRP combined with SMSCs group was be superior to L-PRP and SMSCs group respectively. Key words: Knee-joint; Cartilage injury; Platelet-rich plasma; Rabbit synovium-derived mesenchymal stem cells; Stem cells

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation

BackgroundIn our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs.MethodsHuman synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer’s solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation.ResultsAfter preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer’s solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide– and annexin V– cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C.ConclusionThe viability and chondrogenic potential of synovial MSCs were maintained when the cells were suspended in human serum at 4 and 13 °C.

Stem cells sources for intervertebral disc regeneration.

Intervertebral disc regeneration field is rapidly growing since disc disorders represent a major health problem in industrialized countries with very few possible treatments. Indeed, current available therapies are symptomatic, and surgical procedures consist in disc removal and spinal fusion, which is not immune to regardable concerns about possible comorbidities, cost-effectiveness, secondary risks and long-lasting outcomes. This review paper aims to share recent advances in stem cell therapy for the treatment of intervertebral disc degeneration. In literature the potential use of different adult stem cells for intervertebral disc regeneration has already been reported. Bone marrow mesenchymal stromal/stem cells, adipose tissue derived stem cells, synovial stem cells, muscle-derived stem cells, olfactory neural stem cells, induced pluripotent stem cells, hematopoietic stem cells, disc stem cells, and embryonic stem cells have been studied for this purpose either in vitro or in vivo. Moreover, several engineered carriers (e.g., hydrogels), characterized by full biocompatibility and prompt biodegradation, have been designed and combined with different stem cell types in order to optimize the local and controlled delivery of cellular substrates in situ. The paper overviews the literature discussing the current status of our knowledge of the different stem cells types used as a cell-based therapy for disc regeneration.

Age-Independent Cartilage Generation for Synovium-Based Autologous Chondrocyte Implantation.

The articular cartilage layer of synovial joints is commonly lesioned by trauma or by a degenerative joint disease. Attempts to repair the damage frequently involve the performance of autologous chondrocyte implantation (ACI). Healthy cartilage must be first removed from the joint, and then, on a separate occasion, following the isolation of the chondrocytes and their expansion in vitro, implanted within the lesion. The disadvantages of this therapeutic approach include the destruction of healthy cartilage-which may predispose the joint to osteoarthritic degeneration-the necessarily restricted availability of healthy tissue, the limited proliferative capacity of the donor cells-which declines with age-and the need for two surgical interventions. We postulated that it should be possible to induce synovial stem cells, which are characterized by high, age-independent, proliferative and chondrogenic differentiation capacities, to lay down cartilage within the outer juxtasynovial space after the transcutaneous implantation of a carrier bearing BMP-2 in a slow-release system. The chondrocytes could be isolated on-site and immediately used for ACI. To test this hypothesis, Chinchilla rabbits were used as an experimental model. A collagenous patch bearing BMP-2 in a slow-delivery vehicle was sutured to the inner face of the synovial membrane. The neoformed tissue was excised 5, 8, 11 and 14 days postimplantation for histological and histomorphometric analyses. Neoformed tissue was observed within the outer juxtasynovial space already on the 5th postimplantation day. It contained connective and adipose tissues, and a central nugget of growing cartilage. Between days 5 and 14, the absolute volume of cartilage increased, attaining a value of 12 mm(3) at the latter juncture. Bone was deposited in measurable quantities from the 11th day onwards, but owing to resorption, the net volume did not exceed 1.5 mm(3) (14th day). The findings confirm our hypothesis. The quantity of neoformed cartilage that is deposited after only 1 week within the outer juxtasynovial space would yield sufficient cells for ACI. Since the BMP-2-bearing patches would be implanted transcutaneously in humans, only one surgical or arthroscopic intervention would be called for. Moreover, most importantly, sufficient numbers of cells could be generated in patients of all ages.

Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix

As a tissue-specific stem cell for chondrogenesis, synovium-derived stem cells (SDSCs) are a promising cell source for cartilage repair. However, a small biopsy can only provide a limited number of cells. Cell senescence from both in vitro expansion and donor age presents a big challenge for stem cell based cartilage regeneration. Here we found that expansion on decellularized extracellular matrix (dECM) full of three-dimensional nanostructured fibers provided SDSCs with unique surface profiles, low elasticity but large volume as well as a fibroblast-like shape. dECM expanded SDSCs yielded larger pellets with intensive staining of type II collagen and sulfated glycosaminoglycans compared to those grown on plastic flasks while SDSCs grown in ECM yielded 28-day pellets with minimal matrix as evidenced by pellet size and chondrogenic marker staining, which was confirmed by both biochemical data and real-time PCR data. Our results also found lower levels of inflammatory genes in dECM expanded SDSCs that might be responsible for enhanced chondrogenic differentiation. Despite an increase in type X collagen in chondrogenically induced cells, dECM expanded cells had significantly lower potential for endochondral bone formation. Wnt and MAPK signals were actively involved in both expansion and chondrogenic induction of dECM expanded cells. Since young and healthy people can be potential donors for this matrix expansion system and decellularization can minimize immune concerns, human SDSCs expanded on this future commercially available dECM could be a potential cell source for autologous cartilage repair.

Editor's Spotlight/Take 5: Mesenchymal Stem Cells in Synovial Fluid Increase After Meniscus Injury

Editor's Spotlight/Take 5: Mesenchymal Stem Cells in Synovial Fluid Increase After Meniscus Injury

Can Arthroscopically Harvested Synovial Stem Cells Be Preferentially Sorted Using Stage-Specific Embryonic Antigen 4 Antibody for Cartilage, Bone, and Adipose Regeneration?

The aim of this study was to investigate the relation between stage-specific embryonic antigen 4 (SSEA4) expression and synovium-derived stem cell (SDSC) lineage differentiation. Human SDSCs were collected during arthroscopic surgery from 4 young patients with anterior cruciate ligament injuries. Passage 2 SDSCs were sorted by fluorescence-activated cell sorting using phycoerythrin-conjugated monoclonal antibody against SSEA4 into 3 groups: SSEA4(+) cells, SSEA4(-) cells, and unsorted control cells. After 1 more passage, expanded cells from each group were evaluated for SSEA4 expression by use of flow cytometry as well as multilineage differentiation capacities, including chondrogenesis, adipogenesis, and osteogenesis, using biochemical analysis, histologic analysis, immunostaining, and real-time polymerase chain reaction. After cell sorting, 1 more passage expansion decreased SSEA4(+) cells from 99.8% to 79.2% and increased SSEA4(-) cells from 4.4% to 53.3% compared with 70.3% in the unsorted cell population. SSEA4(-) SDSCs with a lower cell proliferation exhibited higher chondrogenic potential (in terms of the ratio of glycosaminoglycan to DNA [P < .001] and COL2A1 [type II collagen] messenger RNA [mRNA] [P < .001]) and adipogenic potential (in terms of oil red O staining and quantitative assay [P= .007], LPL [lipoprotein lipase] mRNA [P= .005], and CEBP [CCAAT/enhancer-binding protein alpha] mRNA [P= .010]). In contrast, SSEA4(+) SDSCs retained cell expansion and enhanced osteogenic capacity, as evidenced by intense calcium deposition stained by alizarin red S and a significantly elevated expression of OPN (osteopontin) mRNA (P= .007). In this study, for the first time, we showed the benefit of using the surface marker SSEA4 in SDSCs to preferentially sort a mixed population of cells. SSEA4(+) SDSCs indicated a strong potential for osteogenesis rather than chondrogenesis and adipogenesis. SDSC-based mesenchymal tissue regeneration can be easily achieved by arthroscopic harvesting followed by quick cell sorting.

The pilot study of fibrin with temporomandibular joint derived synovial stem cells in repairing TMJ disc perforation.

TMJ disc related diseases are difficult to be cured due to the poor repair ability of the disc. TMJ-SDSCs were ideal cell sources for cartilage tissue engineering which have been widely used in hyaline cartilage regeneration. Fibrin gel has been demonstrated as a potential scaffold for neocartilage formation. The aim of this study was to repair the TMJ disc perforation using fibrin/chitosan hybrid scaffold combined with TMJ-SDSCs. Rat TMJ-SDSCs were cultured on hybrid scaffold or pure chitosan scaffolds. The cell seeding efficiency, distribution, proliferation, and chondrogenic differentiation capacity were investigated. To evaluate the in vivo repair ability of cell/scaffold construct, rat TMJ disc explants were punched with a defect to mimic TMJ disc perforation. Cell seeded scaffolds were inserted into the defect of TMJ disc explants and then were implanted subcutaneously in nude mice for 4 weeks. Results demonstrated that fibrin may improve cell seeding, proliferation, and chondrogenic induction in vitro. The in vivo experiments showed more cartilage ECM deposition in fibrin/chitosan scaffold, which suggested an enhanced reparative ability. This pilot study demonstrated that the regenerative ability of TMJ-SDSCs seeded in fibrin/chitosan scaffold could be applied for repairing TMJ disc perforation.

Synovial stem cells and their responses to the porosity of microfibrous scaffold

Tissue-specific stem cells can be coaxed or harvested for tissue regeneration. In this study, we identified and characterized a new type of stem cells from the synovial membrane of knee joint, named neural crest cell-like synovial stem cells (NCCL-SSCs). NCCL-SSCs showed the characteristics of neural crest stem cells: they expressed markers such as Sox10, Sox17 and S100β, were clonable, and could differentiate into neural lineages as well as mesenchymal lineages, although NCCL-SSCs were not derived from neural crest during the development. When treated with transforming growth factor β1 (TGF-β1), NCCL-SSCs differentiated into mesenchymal stem cells (MSCs), lost the expression of Sox17 and the differentiation potential into neural lineages, but retained the potential of differentiating into mesenchymal lineages. To determine the responses of NCCL-SSCs to microfibrous scaffolds for tissue engineering, electrospun composite scaffolds with various porosities were fabricated by co-electrospinning of structural and sacrificial microfibers. The increase in the porosity in microfibrous scaffolds enhanced cell infiltration in vitro and in vivo, but did not affect the morphology and the proliferation of NCCL-SSCs. Interestingly, microfibrous scaffolds with higher porosity increased the expression of chondrogenic and osteogenic genes but suppressed smooth muscle and adipogenic genes. These results suggest that the differentiation of NCCL-SSCs can be controlled by both soluble chemical factors and biophysical factors such as the porosity of the scaffold. Engineering both NCCL-SSCs and scaffolds will have tremendous potential for tissue regeneration.

Detection of abnormalities in the superficial zone of cartilage repaired using a tissue engineered construct derived from synovial stem cells

The present study investigated the surface structure and mechanical properties of repair cartilage generated from a tissue engineered construct (TEC) derived from synovial mesenchymal stem cells at six months post-implantation compared to those of uninjured cartilage. TEC-mediated repair tissue was cartilaginous with Safranin O staining, and had comparable macro-scale compressive properties with uninjured cartilage. However, morphological assessments revealed that the superficial zone of TEC-mediated tissue was more fibrocartilage-like, in contrast to the middle or deep zones that were more hyaline cartilage-like with Safranin O staining. Histological scoring of the TEC-mediated tissue was significantly lower in the superficial zone than in the middle and deep zones. Scanning electron microscopy showed a thick tangential bundle of collagen fibres at the most superficial layer of uninjured cartilage, while no corresponding structure was detected at the surface of TEC-mediated tissue. Immunohistochemical analysis revealed that PRG4 was localised in the superficial area of uninjured cartilage, as well as the TEC-mediated tissue. Friction testing showed that the lubrication properties of the two tissues was similar, however, micro-indentation analysis revealed that the surface stiffness of the TEC-repair tissue was significantly lower than that of uninjured cartilage. Permeability testing indicated that the TEC-mediated tissue exhibited lower water retaining capacity than did uninjured cartilage, specifically at the superficial zone. Thus, TEC-mediated tissue exhibited compromised mechanical properties at the superficial zone, properties which need improvement in the future for maintenance of long term repair cartilage integrity.

Identification of neural crest‐like synovial stem cells

Recent studies suggest that bioactive scaffolds can be used to regenerate cartilage by local cell homing. However, the specific cell types participating in regeneration are not fully understood. In this study, we identified a novel type of neural crest like synovial stem cell (NCL‐SSC) residing within the synovial membrane, which has several important characteristics distinct from previously identified stem cells. Protein marker screening revealed that NCL‐SSCs homogeneously expressed neural crest stem cell markers such as Sox1, Sox10, Snail and Vimentin. Furthermore, differentiation assays NCL‐SSCs can be induced into mesenchymal lineages (osteoblasts, adipocytes, and chondrocytes), peripheral neurons and Schwann cells. In addition, we found that the combination of chick embryo extract and basic fibroblast growth factor can maintain the multipotency and maker expression of NCL‐SSCs. Cloning assay showed that the average plating efficiency of NCL‐SSCs is about 10% and the cloned colonies retained the marker expression and multipotency. In summary, our study indentified a new type of local cells in synovial knee joint, which could be an important cell source for in situ tissue engineering.

Transcription Factor Rational Design Improves Directed Differentiation of Human Mesenchymal Stem Cells Into Skeletal Myocytes

There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.