Search

Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs.

Tendon/ligament injury is one of the most common impairments in sports medicine. The traditional treatments of damaged tissue repair are unsatisfactory, especially for athletes, due to lack of donor and immune rejection. The strategy of tissue engineering may break through these limitations, and bring new hopes to tendon/ligament repair, even regeneration. Silk is a kind of natural biomaterials, which has good biocompatibility, wide range of mechanical properties and tunable physical structures; so it could be applied as tendon/ligament tissue engineering scaffolds. The silk-based scaffold has robust mechanical properties; combined with other biological ingredients, it could increase the surface area, promote more cell adhesion and improve the biocompatibility. The potential clinical application of silk-based scaffold has been confirmed by in vivo studies on tendon/ligament repairing, such as anterior cruciate ligament, medial collateral ligament, achilles tendon and rotator cuff. To develop novel biomechanically stable and host integrated tissue engineered tendon/ligament needs more further micro and macro studies, combined with product development and clinical application, which will give new hope to patients with tendon/ligament injury.

Osteonecrosis of the femoral head (ONFH) is a common but intractable disease. In this study, we investigated the mechanisms regulating alterations in mesenchymal stem cell (MSC) differentiation in ONFH. Five patients who were diagnosed with ONFH were enrolled in this study. Bone MSCs (BMSCs) were isolated from the osteonecrotic zone in the femoral head (FH-pMSCs) and from the normal zone in the pelvis (hMSCs) of the same patient. Morphology, cell proliferation and expression of mediators of the Wnt signalling pathway were evaluated. There were significant differences in cell proliferation and expression of surface markers between the two populations of cells. FH-pMSCs exhibited significantly lower osteogenic differentiation compared with hMSCs (P < 0.0001). Dissection of the Wnt pathway showed that FH-pMSCs had significantly higher GSK3β expression compared with hMSCs (P < 0.001). Addition of LiCl, a GSK3β inhibitor, significantly increased osteogenic differentiation in FH-pMSCs, suggesting a relationship between the microenvironment and regulation of stem cell behaviour in ONFH. FH-pMSCs also exhibited significant downregulation of other mediators of the Wnt signalling pathway, including runx2 and β-catenin. Our data suggested that mediators of the Wnt signalling pathway, such as GSK3β could be important therapeutic targets for early-stage ONFH.

Poly (lactic-co-glycolic acid) (PLGA) and poly- l -lactate acid (PLLA) are biodegradable polymers widely utilized as scaffold materials for cartilage tissue engineering. Their acid degradation products have been widely recognized as being detrimental to cell function. However, the biological effects of lactate, rather than lactic acid, on chondrocytes have never been investigated. This is the major focus of this study. The amounts of lactate and the pH value (acid) of the PLGA and PLLA degradation medium were measured. The effects of PLGA and PLLA degradation medium, as well as different lactate concentrations and timing of exposure on chondrocytes proliferation and cartilage-specific matrix synthesis were investigated by various techniques including global gene expression profiling and gene knockdown experiments. It was shown that PLGA and PLLA degradation medium differentially regulated chondrocyte proliferation and matrix synthesis. Acidic pH caused by lactate inhibited chondrocyte proliferation and matrix synthesis. The effect of lactate on chondrocyte matrix synthesis was both time and dose dependent. A lactate concentration of 100 mM and exposure duration of 8 h significantly enhanced matrix synthesis. Lactate could also inhibit expression of cartilage matrix degradation genes in osteoarthritic chondrocytes, such as the major aggrecanase ADAMTS5, whilst promoting matrix synthesis simultaneously. Pulsed addition of lactate was shown to be more efficient in promoting COL2A1 expression. Global gene expression data and gene knock down experiments demonstrated that lactate promote matrix synthesis through up-regulation of HIF1A. These observed differential biological effects of lactate on chondrocytes would have implications for the future design of polymeric cartilage scaffolds. Statement of Significance Lactic acid is a widely used substrate for polymers synthesis, PLGA and PLLA in particular. Although physical and biological modifications have been made on these polymers to make them be better cartilage scaffolds, little concern has been given on the biological effect of lactic acid, the main degradation product of these polymers, on chondrocytes. Our finding illustrates the differential biological function of lactate and acid on chondrocytes matrix synthesis. These results can facilitate future design of lactate polymers-based cartilage scaffolds

This study aimed to develop a practical three-dimensional (3D) macroporous scaffold from aligned electrospun nanofibrous yarns for bone tissue engineering. A novel 3D unwoven macroporous nanofibrous (MNF) scaffold was manufactured with electrospun poly(L-lactic acid) and polycaprolactone (w/w 9:1) nanofibers through sequential yarns manufacture and honeycombing process at 65°C. The efficacy of 3D MNF scaffold for bone formation were evaluated using human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) differentiation model and rabbit tibia bone defect model. In vitro, more cell proliferation and cell ingrowth were observed in 3D MNF scaffold. Moreover, calcium deposit was obviously detected in vitro differentiation of hESC-MSCs. In vivo, histology and X-ray showed that 3D MNF scaffold treated bone defect had fine 3D bony tissue formation around the scaffold as well as inside the scaffold at 3 weeks and 6 weeks. This study demonstrated that 3D MNF scaffold provides a structural support for hESC-MSCs growth and guides bone formation suggesting that this novel strategy successfully makes use of electrospun fibers for bone tissue engineering, which may help realize the clinical translation of electrospun nanofibers for regenerative medicine in future.

To obtain the biomimetic scaffolding materials for bone tissue engineering, poly(lactide-co-glycolide) (PLGA) nanofibrous mesh (NFM) was mineralized in a 5× simulated body fluid (SBF) for different time after it was treated by air plasma for 15 min and subsequent collagen coating. The apatite particles were nucleated on the surface of individual nanofibers, gradually grew up, and finally covered the whole NFM surface. The mineral aggregates were mainly composed of tiny hydroxyapatite (HA) nanoparticles, whose content reached a constant value of 54 µg · cm−2 after 9 days. The collagen coating and apatite deposition enhanced the NFM strength pronouncedly too. In vitro cell culture demonstrated that the non- or less mineralized NFMs were more beneficial of cell spreading and proliferation than those highly mineralized NFMs, but the latter ones could strongly promote secretion of alkaline phosphatase (ALP) by osteoblasts after cultured for 14 days. Moreover, the highly mineralized NFMs also could significantly up-regulated ALP activity and calcium synthesis of bone marrow mesenchymal stem cells (BMSCs), demonstrating that these NFMs are more favorable of the osteoblast phenotype expression and osteogenic induction. Therefore, the biomimetic apatite deposited PLGA/collagen NFM could be a promising candidate scaffold for bone tissue engineering.

Tendinopathy of the Achilles tendon is a chronic degenerative condition that frequently does not respond to treatment. In the current study, we propose that autologous tenocytes therapy (ATT) is effective in treating tendon degeneration in a collagenase-induced rabbit Achilles tendinopathy model.Chronic tendinopathy was created in the left Achilles tendon of 44 rabbits by an intratendonous injection of type I collagenase. Forty-two rabbits were randomly allocated into three groups of 14 and received control treatment; autologous tenocytes digested from tendon tissue; and autologous tenocytes digested from epitendineum tissue. For cell tracking in vivo, the remaining two animals were injected with autologous tenocytes labeled with a nano-scale super-paramagnetic iron oxide (Feridex). Rabbits were sacrificed at 4 and 8 weeks after the therapeutic injection, and tendon tissue was analyzed by histology, immunostaining, and biomechanical testing to evaluate tissue repair.Autologous tenocyte treatment improved tendon remodeling, histological outcomes, collagen content, and tensile strength of tendinopathic Achilles tendons. Injected tenocytes were integrated into tendon matrix and could be tracked up to 8 weeks in vivo. Immunohistochemistry showed that ATT improved type I collagen expression in repaired tendon but did not affect type III collagen and secreted protein, acidic and rich in cysteine expression.ATT may be a useful treatment of chronic Achilles tendinopathy.

Poly(lactide-co-glycolide) (PLGA) sponge was filled with fibrin gel, bone marrow mesenchymal stem cells (BMSCs) and transforming growth factor-β1 (TGF-β1) to obtain a construct for cartilage restoration in vivo. The PLGA sponge lost its weight steadily in vitro, but degraded much faster in the construct of PLGA/fibrin gel/BMSCs implanted in the full-thickness cartilage defects. The in vivo degradation of the fibrin gel inside the construct was prolonged to 12 wk too. The CM-DiI labeled allogenic BMSCs were detectable after transplantation (implantation) into the defects for 12 wk by small animal in vivo fluorescence imaging and confocal laser scanning microscopy. In vivo repair experiments were firstly performed by implantation of the PLGA/fibrin gel/BMSCs and PLGA/BMSCs constructs into full-thickness cartilage defects (3 mm in diameter and 4 mm in depth) of New Zealand white rabbits for 12 wk. The defects implanted with the PLGA/fibrin gel/BMSCs constructs were filled with cartilage-like tissue containing collagen type II and glycosaminoglycans (GAGs), while those by the PLGA/BMSCs constructs were filled with fibrous-like tissues. To repair the defects of larger size (4 mm in diameter), addition of growth factors was mandatory as exemplified here by further loading of TGF-β1. Implantation of the PLGA/fibrin gel/BMSCs/TGF-β1 constructs into the full-thickness cartilage defects for 12 wk resulted in full restoration of the osteochondral tissue. The neo-cartilage integrated well with its surrounding cartilage and subchondral bone. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the cartilage special genes were significantly up-regulated compared with those of the TGF-β1 absent constructs.

This study developed a bioactive knitted silk-collagen sponge scaffold by incorporation of exogenous SDF-1 alpha, to enable selective migration and homing of cells for in situ tendon regeneration. With in vitro studies, it was observed that CXCR4 gene expression and migration of bone mesenchymal stromal cells and hypo-dermal fibroblasts were more sensitive to exogenous SDF-1 alpha, while expression of tendon repair gene markers by hypo-dermal fibroblasts and Achilles tendon fibroblasts were more sensitive to exogenous SDF-1 alpha. With a rat Achilles tendon injury model, exogenous SDF-1 alpha was shown to reduce infiltration of inflammatory cells and enhance migration of fibroblast-like cells into the scaffold at 4 days and 1 week post-surgery. After 4 weeks, SDF-1 alpha treated tendon had increased expression of tendon repair gene markers and endogenous SDF-1 alpha, exhibited more physiological microstructures with larger diameter collagen fibrils, and had better biomechanical properties than the control group. Hence, our bioactive scaffold improved efficacy of tendon regeneration by increasing the recruitment of fibroblast-like cells, enhancing local endogenous SDF-1 alpha and tendon extracellular matrix production, and decreasing accumulation of inflammatory cells. Incorporation of SDF-1 alpha within a knitted silk-collagen sponge scaffold can therefore be a practical application for tendon tissue engineering.

Numerous in vitro studies have indicated the potential of using electrospun nanofibrous scaffolds for tissue regeneration. However, few reports have demonstrated their utility in real tissue repair models. The present investigation tested the hypothesis that electrospun poly-L-lactic acid (PLLA) nanofibrous membrane leads to dense cortical bone regeneration and improves the efficacy of currently-used collagenous guided bone regeneration (GBR) membrane. In vitro, the function of bone marrow-derived mesenchymal stem cells (BMSCs) on nanofibrous scaffolds was evaluated. In an in vivo experiment, large bony defects were created in rabbit tibia and treated with a nanofiber-reinforced bilayer membrane, nanofibrous membrane, or collagenous membrane alone. Three and six weeks after operation, bone defect healing was assessed radiologically and histologically. In vitro differentiation studies showed that BMSCs had much higher expression of Runx2 and collagen type I, alpha 1 mRNAs, when cultured on nanofibrous scaffolds. The radiographic and histological data both showed that the group treated with bilayer membrane had more bony tissue formation at 3 weeks. Moreover, at 6 weeks, only the bilayer membrane-treated bone defects displayed better regeneration of cortical bone tissue, whereas in the other groups the defects were filled with spongy bone-like tissue. The results demonstrated that electrospun nanofibrous membrane improves the regeneration of cortical bone, suggesting that this type of membrane can be combined with current collagenous GBR membrane to improve guided bone regeneration technology.

As recent studies have suggested that bone morphogenetic protein-4 (BMP-4) and BMP-7 are promising cartilage differentiation factors, this study aimed to compare the efficacy of BMP-4 and BMP-7 for chondral-lineage differentiation in vitro as well as the efficacy of BMP-4 for articular cartilage repair in vivo.Rabbit mesenchymal stromal cells and articular chondrocytes were treated with 10 ng/mL human recombinant BMP-4 or BMP-7. The expression of cartilage-specific genes (col II, aggrecan, and Sox9) and fibroblast growth factor receptor genes was tested by real-time polymerase chain reaction in vitro. Also, full-thickness cartilage defects (diameter 4 mm, thickness 3 mm) were created in New Zealand white rabbits and untreated (group I), or treated with a bilayer collagen scaffold (group II) or BMP-4 with scaffold (group III) (n = 12/group). The repaired tissues were harvested for histology and mechanical testing after 6 or 12 weeks.Cartilage differentiation of mesenchymal stromal cells was more apparent after BMP-4 treatment, as evidenced by higher expression of type II collagen and aggrecan genes. Also, BMP-4 induced higher aggrecan and fibroblast growth factor receptor-2 gene expression in chondrocytes, whereas BMP-7 had no effect. In the in vivo experiments, group III treated with BMP-4 protein had the largest amounts of cartilage tissue, which restored a greater surface area of the defect and achieved higher International Cartilage Repair Society scores. Moreover, Young's modulus, which indicates the mechanical properties of the repaired tissue, was markedly higher in group III than in groups I and II (p0.05), but lower than in normal tissue.BMP-4 is more potent than BMP-7 for cartilage differentiation. The delivery of BMP-4 protein in a bilayer collagen scaffold stimulates the formation of cartilage tissue.

Tendon injuries are common especially in sports activities, but tendon is a unique connective tissue with poor self-repair capability. With advances in stem cell biology, tissue engineering is becoming increasingly powerful for tissue regeneration. Stem cells with capacity of multipotency and self-renewal are an ideal cell source for tissue engineering.This review focus on discussing the potential strategies including inductive growth factors, bio-scaffolds, mechanical stimulation, genetic modification and co-culture techniques to direct tendon-lineage differentiation of stem cells for complete tendon regeneration. Attempting to use embryonic stem cells as seed cells for tendon tissue engineering have achieved encouraging results. The combination of chemical and physical signals in stem cell microenvironment could be regulated to induce differentiation of the embryonic stem cells into tendon.We summarize fundamental questions, as well as future directions in tendon biology and tissue engineering.Multifaceted technologies are increasingly required to control stem cell differentiation, to develop novel stem cell-based therapy, and, ultimately, to achieve more effective repair or regeneration of injured tendons.

Tendon is a specific connective tissue composed of parallel collagen fibers. The effect of this tissue-specific matrix orientation on stem cell differentiation has not been investigated. This study aimed to determine the effects of nanotopography on the differentiation of human tendon stem/progenitor cells (hTSPCs) and develop a biomimetic scaffold for tendon tissue engineering. The immuno-phenotype of fetal hTSPCs was identified by flow cytometry. The multipotency of hTSPCs toward osteogenesis, adipogenesis, and chondrogenesis was confirmed. Then, the hTSPCs were seeded onto aligned or randomly-oriented poly (l-lactic acid) nanofibers. Scanning electron micrographs showed that hTSPCs were spindle-shaped and well orientated on the aligned nanofibers. The expression of tendon-specific genes was significantly higher in hTSPCs growing on aligned nanofibers than those on randomly-oriented nanofibers in both normal and osteogenic media. In addition, alkaline phosphatase activity and alizarin red staining showed that the randomly-oriented fibrous scaffold induced osteogenesis, while the aligned scaffold hindered the process. Moreover, aligned cells expressed significantly higher levels of integrin alpha1, alpha5 and beta1 subunits, and myosin II B. In in vivo experiments, the aligned nanofibers induced the formation of spindle-shaped cells and tendon-like tissue. In conclusion, the aligned electrospun nanofiber structure provides an instructive microenvironment for hTSPC differentiation and may lead to the development of desirable engineered tendons.

Mesenchymal stem cells (MSCs) hold great promise for bone regeneration. However, the power of mesenchymal stem cells has not been applied to structural bone allografts in clinical practice. This study designed a new strategy to enhance the efficiency of allografts for segmental bone regeneration. Isolated MSCs were cultured to form a cell sheet. The MSC sheet was then wrapped onto structural allografts. The assembled structures were cultured in vitro to evaluate the differentiation potential of MSC sheet. The assembled structures were implanted subcutaneously into nude mice as well as into the segmental radius defect of rabbits to investigate the efficiency of MSC sheets to repopulate allografts for bone repair. MSC sheets, upon assembling on bone grafts, showed similar differentiation properties to the in situ periosteum in vitro. After implantation the MSC sheets accelerated the repopulation of bone grafts in nude mice. Moreover, MSC sheets induced thicker cortical bone formation and more efficient graft-to-bone end fusion at the segmental bone defects in rabbits. This study thus presented a novel, more efficient, and practical strategy for large weight-bearing bone reconstruction by using MSC sheets to deliver large number of MSCs to repopulate the bone allografts.

Human embryonic stem cells (hESCs) are ideal seed cells for tissue regeneration, but no research has yet been reported concerning their potential for tendon regeneration. This study investigated the strategy and efficacy of using hESCs for tendon regeneration as well as the mechanism involved. hESCs were first induced to differentiate into mesenchymal stem cells (MSCs), which had the potential to differentiate into the three mesenchymal lineages and were positive for MSC surface markers. hESC-derived MSCs (hESC–MSCs) regenerated tendon tissues in both an in vitro tissue engineering model and an in vivo ectopic tendon regeneration model, as confirmed by the expression of tendon-specific genes and structure. In in-situ rat patellar tendon repair, tendon treated with hESC–MSCs had much better structural and mechanical properties than did controls. Furthermore, hESC–MSCs remained viable at the tendon wound site for at least 4 weeks and secreted human fetal tendon-specific matrix components and differentiation factors, which then activated the endogenous regeneration process in tendon. Moreover, no teratoma was found in any samples. These findings demonstrate a safe and practical strategy of applying ESCs for tendon regeneration and may assist in future strategies to treat tendon diseases. Disclosure of potential conflicts of interest is found at the end of this article.

In this study a 3-D alginate microbead platform was coated with cartilaginous extracellular matrix (ECM) components to emulate chondrogenic microenvironment in vivo for the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). BMSCs were seeded onto the microbead surface and the effect of the modified microbead on BMSC adhesion, proliferation and chondrogenic differentiation was studied, and compared to chondrogenesis in conventional pellet culture. Our results indicated that microbead system promoted BMSC proliferation and protein deposition resulting in the formation of bigger aggregates compared to conventional pellet culture. Analysis of the aggregates indicated that chondroitin sulfate (CS)- and Col2-coated microbeads enhanced the chondrogenic differentiation of hBMSCs, with increasing formation of glycosaminoglycan (GAG) and collagen II deposition in histology, immunohistochemistry and real time PCR analysis. In addition, Col2-coated microbeads resulted in hypertrophic maturation of the differentiated chondrocytes, similar to conventional pellet culture, while CS-coated microbeads were able to retain the pre-hypertrophy state of the differentiated cells. Our result suggested that provision of suitable cartilaginous microenvironment in a 3-D system can promote the chondrogenic differentiation of BMSC and influence the phenotype of resulting chondrocytes. Our microbead system provides an easy method of processing a 3-D alginate system that allows the possibility of scaling up chondrogenic pellet production for clinical application, while the modifiable microbeads also provide an adjustable 3-D platform for the study of co-interaction of ECM and differentiation factors during the stem cell differentiation.

Cartilage damage has been documented as one of the major problems leading to knee repair procedures worldwide. The low availability of cartilage that can be harvested without causing a negative health impact has led to the focus on the potential of stem cells, which have been transplanted into damaged areas and successfully grown into new healthy tissue. This study aims to compare the chondrogenic potential of two stem cell sources--adipose tissue and bone marrow. Stem cells were isolated from donor-matched adipose tissue and bone marrow, following established protocols. The cells were grown in a chondrogenic cocktail containing transforming growth factor-beta3 (TGF-beta3) up till 28 days, and assessed for expression changes of cartilage markers at the gene and protein level, using qualitative and quantitative methods. Controls were included for every time point. Real-time polymerase chain reaction (PCR) results showed increases in the gene expression of collagen II in both the cell types that received TGF-beta3 treatment. However, histological, immunohistochemical, and glycosaminoglycan (GAG) assay clearly showed that collagen II and proteoglycans (PG) were synthesized only in the growth factor-treated bone marrow stem cells (BMSCs). These findings support the results obtained in our in vivo comparative study done on an animal model, suggesting that BMSCs are more suitable than adipose-derived stem cells (ADSCs) for chondrogenesis.

Introduction Interleukin-1β (IL-1β) and nerve growth factor (NGF) are key regulators in the pathogenesis of inflammatory arthritis; specifically, IL-1β is involved in tissue degeneration and NGF is involved in joint pain. However, the cellular and molecular interactions between IL-1β and NGF in articular cartilage are not known. Cartilage stem/progenitor cells (CSPCs) have recently been identified in osteoarthritic (OA) cartilage on the basis of their migratory properties. Here we hypothesize that IL-1β/NGF signaling is involved in OA cartilage degeneration by targeting CSPCs. Method NGF and NGF receptor (NGFR: TrkA and p75NTR) expression in healthy and OA human articular cartilage and isolated chondrocytes was determined by immunostaining, qRT-PCR, flow cytometry and western blot. Articular cartilage derived stem/progenitor cells were collected and identified by stem/progenitor cell characteristics. 3D-cultured CSPC pellets and cartilage explants were treated with NGF and NGF neutralizing antibody, and extracellular matrix changes were examined by sulfated glycosaminoglycan (GAG) release and MMP expression and activity. Results Expression of NGF, TrkA and p75NTR was found to be elevated in human OA cartilage. Cellular changes upon IL-1β and/or NGF treatment were then examined. NGF mRNA and NGFR proteins levels were upregulated in cultured chondrocytes exposed to IL-1β. NGF was chemotactic for cells isolated from OA cartilage. Cells isolated on the basis of their chemotactic migration towards NGF demonstrated stem/progenitor cell characteristics, including colony-forming ability, multi-lineage differentiation potential, and stem cell surface markers. The effects of NGF perturbation in cartilage explants and 3D-cultured CSPCs were next analyzed. NGF treatment resulted in extracellular matrix catabolism indicated by increased sGAG release and MMP expression and activity; conversely, treatment with NGF neutralizing antibody inhibited increased MMP levels, and enhanced tissue inhibitor of matrix metalloprotease-1 (TIMP1) expression in OA cartilage explants. NGF blockade with neutralizing antibody also affected cartilage matrix remodeling in 3D-CSPC pellet cultures. Conclusion Our results strongly suggest that NGF signaling is a contributing factor in articular cartilage degeneration in OA, which likely targets a specific subpopulation of progenitor cells, the CSPCs, affecting their migratory and matrix remodeling activities. These findings provide novel cellular/signaling therapeutic targets in osteoarthritic cartilage. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0840-x) contains supplementary material, which is available to authorized users.

Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5'-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.

Tendon injuries are common and present a clinical challenge, as they often respond poorly to treatment and result in long-term functional impairment. Inferior tendon healing responses are mainly attributed to insufficient or failed tenogenesis. The main objective of this study was to establish an efficient approach to induce tenogenesis of bone marrow-derived mesenchymal stem cells (BMSCs), which are the most common seed cells in tendon tissue engineering. First, representative reported tenogenic growth factors were used as media supplementation to induce BMSC differentiation, and the expression of teno-lineage transcription factors and matrix proteins was compared. We found that transforming growth factor (TGF)-β1 significantly induced teno-lineage-specific gene scleraxis expression and collagen production. TGF-β1 combined with connective tissue growth factor (CTGF) elevated tenomodulin and Egr1 expression at day 7. Hence, a stepwise tenogenic differentiation approach was established by first using TGF-β1 stimulation, followed by combination with CTGF for another 7 days. Gene expression analysis showed that this stepwise protocol initiated and maintained highly efficient tenogenesis of BMSCs. Finally, regarding in situ rat patellar tendon repair, tendons treated with induced tenogenic BMSCs had better structural and mechanical properties than those of the control group, as evidenced by histological scoring, collagen I and tenomodulin immunohistochemical staining, and tendon mechanical testing. Collectively, these findings demonstrate a reliable and practical strategy of inducing tenogenesis of BMSCs for tendon regeneration and may enhance the effectiveness of cell therapy in treating tendon disorders. Significance The present study investigated the efficiency of representative tenogenic factors on mesenchymal stem cells' tenogenic differentiation and established an optimized stepwise tenogenic differentiation approach to commit tendon lineage differentiation for functional tissue regeneration. The reliable tenogenic differentiation approach for stem cells not only serves as a platform for further studies of underlying molecular mechanisms but also can be used to enhance cell therapy outcome in treating tendon disorders and develop novel therapeutics for tendon injury.

Multipotent mesenchymal stem cells (MSC) are reported to be immunoprivileged as well as immunosuppressive. Hence, they are ideal candidates for allogeneic transplantation to induce regeneration of diseased tissues and organs. However, it is not known whether MSC would retain their immunoprivileged and immunomodulatory properties after differentiating into the local cell types of the transplantation site. This study sought to investigate this question with a novel New Zealand White rabbit osteogenesis model. Results showed that osteogenic cells differentiated from MSC (DOC) in vitro did not express the MHC class II molecule, were incapable of inducing allogeneic lymphocyte proliferation in mixed lymphocyte culture or generating CTL, were inhibitory in ongoing lymphocyte proliferation, and secreted anti-inflammatory cytokines (IL-10 and TGF-β). There was a significantly higher secretion of IL-10 by DOC than that by MSC, while there was no significant difference between the TGF-β secretion of MSC and DOC in vitro. However, after IFN-γ treatment, TGF-β secretion by DOC significantly decreased despite the increased production by MSC. Four weeks after local DOC implantation, despite MHC class II expression, second-set allogeneic skin rejection showed similar survival to first-set allogeneic skin rejection and DOC appeared to function as osteoblasts. In conclusion, DOC retained their immunoprivileged and immunomodulatory properties in vitro, but the latter was lost following transplantation.

This study evaluates the ability of MSCs isolated from different origins--bone marrow, periosteum, or fat--to treat partial growth arrest in immature (6-week-old) New Zealand White rabbits. Up to 50% of the medial half of the proximal physis of the tibia was excised in these New Zealand White rabbits. Three weeks later, the bony bridge was excised, and fibrin glue with and without MSCs were transferred into the physeal defect of different rabbits. Contralateral tibias, without undergoing operation, served as self-control. Four groups of rabbits were involved in the study. Each group was injected separately with bone marrow-derived MSCs (group I), periosteum-derived MSCs (group II), fat-derived MSCs (group III), and fibrin glue alone (control, group IV). The rabbits were killed 8 and 16 weeks postoperatively. Clinical, radiological, and histological analyses were subsequently performed. Similar proliferative rates for three MSCs were demonstrated on days 4, 7, and 11 of primary culture. However, MSCs derived from bone-marrow and periosteum appeared to be more homogeneous than that from fat. All MSCs demonstrated chondrogenic and osteogenic differentiation potentials in vitro. The tibias in groups I and II showed significant correction of varus angulation at 16 weeks. However, the varus angulation in group III remained significantly obvious when compared with group I (p < 0.05). The length discrepancies between operated and normal tibiae in groups I, II, and III were significantly corrected compared with control (p < 0.01). In conclusion, bone-marrow and periosteum yielded more homogenous MSCs than fat, providing better correction of physeal arrest in rabbits. The source of MSCs itself could influence the success in the treatment of growth arrest.

Chondroitin sulfate is up-regulated in granulation tissue during wound healing. To investigate the role of chondroitin sulfate in the wound-healing process after surgical repair of cleft palate, we isolated and cultured rabbit palatal fibroblasts. Treatment with chondroitin-6-sulfate resulted in a dose-dependent increase in cell adhesion and cell proliferation, whereas the reverse effects were seen after chondroitinase degradation of chondroitin sulfate. The biological actions of chondroitin sulfate appeared to be dependent on the presence and position of sulfate groups. Inhibition of glycosaminoglycan sulfation by chlorate treatment led to reduced cell adhesion and cell proliferation and a slower rate of wound closure in vitro. Furthermore, exposure to chondroitin-4-sulfate resulted in a dose-dependent reduction in cell adhesion. Together, these results show that chondroitin sulfate is involved in palatal wound healing.

Bone marrow stromal cells are potentially useful for tendon repair and regeneration. To provide lasting benefits, the seeded cells must survive implantation at local tendon sites. Our objective was to determine the in vivo fate of allogeneic bone marrow stromal cells (bMSCs) at different time points after implantation into patella tendon defects (i.e., at 2, 3, 5, and 8 weeks). The protocol involved the labeling of bMSCs with green fluorescent protein (GFP) or carboxyfluorescein diacetate (CFDA) before implantation. A window defect (5 × 5 mm) was created at the central portion of rabbit patella tendon and subsequently treated with GFP- or CFDA-marked bMSCs. The marked bMSCs were loaded into the window defect with fibrin glue. Upon sacrifice of the rabbits at the different time points, the implant site of the patellar tendon was immediately retrieved and the viability of the labeled cells was assessed under confocal microscopy. The results showed that the seeded bMSCs remained viable within the tendon wound site for at least 8 weeks after implantation. The cell morphology was changed from a round shape at 2 weeks to a spindle shape at 5 weeks after implantation. This study demonstrated that the bMSCs remained viable for prolonged periods after implantation and therefore have the potential to influence the formation and remodeling of neotendon tissue after tendon repair.

In this article, ultrafine gelatin (Gt) fibers were successfully produced with the use of the electrical spinning or electrospinning technique. A fluorinated alcohol of 2,2,2-trifluoroethanol (TFE) was used as the dissolving solvent. The morphology of the electrospun gelatin fibers was found to be dependent on the alteration of gelatin concentration ranging from 2.5% w/v to 12.5% w/v at 2.5% increment intervals. Based on the electrospun gelatin fibers obtained, 10% w/v gelatin/TFE solution was selected and mixed with 10% w/v poly(epsilon-caprolactone) (PCL) in TFE at a ratio of 50:50 and co-electrospun to produce gelatin/PCL composite membranes. Contact-angle measurement and tensile tests indicated that the gelatin/PCL complex fibrous membrane exhibited improved mechanical properties as well as more favorable wettability than that obtained from either gelatin or PCL alone. The gelatin/PCL fibrous membranes were further investigated as a promising scaffold for bone-marrow stromal cell (BMSC) culture. Scanning electron microscopy (SEM) and laser confocal microscopy observations showed that the cells could not only favorably attach and grow well on the surface of these scaffolds, but were also able to migrate inside the scaffold up to 114 microm within 1 week of culture. These results suggest the potential of using composite gelatin/PCL fibrous scaffolds for engineering three-dimensional tissues.

The objectives of this study were to evaluate the morphology and biomechanical function of Achilles tendons regenerated using knitted poly-lactide-co-glycolide (PLGA) loaded with bone marrow stromal cells (bMSCs). The animal model used was that of an adult female New Zealand White rabbit with a 10-mm gap defect of the Achilles tendon. In group I, 19 hind legs with the created defects were treated with allogeneic bMSCs seeded on knitted PLGA scaffold. In group II, the Achilles tendon defects in 19 hind legs were repaired using the knitted PLGA scaffold alone, and in group III, 6 hind legs were used as normal control. The tendon-implant constructs of groups I and II were evaluated postoperatively at 2, 4, 8, and 12 weeks using macroscopic, histological, and immunohistochemical techniques. In addition, specimens from group I (n = 7), group II (n = 7), and group III (n = 6) were harvested for biomechanical test 12 weeks after surgery. Postoperatively, at 2 and 4 weeks, the histology of group I specimens exhibited a higher rate of tissue formation and remodeling as compared with group II, whereas at 8 and 12 weeks postoperation, the histology of both group I and group II was similar to that of native tendon tissue. The wound sites of group I healed well and there was no apparent lymphocyte infiltration. Immunohistochemical analysis showed that the regenerated tendons were composed of collagen types I and type III fibers. The tensile stiffness and modulus of group I were 87 and 62.6% of normal tendon, respectively, whereas those of group II were about 56.4 and 52.9% of normal tendon, respectively. These results suggest that the knitted PLGA biodegradable scaffold loaded with allogeneic bone marrow stromal cells has the potential to regenerate and repair gap defect of Achilles tendon and to effectively restore structure and function.

In this study, the adhesion, proliferation and morphology of rabbit anterior cruciate ligament (ACL) cells and bone marrow stromal cells (bMSCs) on synthetic biodegradable polymeric films were investigated. Tissue culture polystyrene (TCP) was used as control. Seven biodegradable polymers were used; they are as follows: poly(e-caprolactone) (PCL), poly(DL-lactide) (D-PLA), poly(L-lactide) (L-PLA), PLA/PCL (50:50), PLA/PCL (75:25), high molecular weight (HMW) poly(DL-lactide-co-glycolide (PLGA50:50) and HMW PLGA75:25. Polymeric film substrates were manufactured using solvent spin-casting technique. After 8 h of cell culture, a high percentage of ACL cells was found attached to PLGA50:50 (38.6 ± 8.4%) and TCP (39.3 ± 6.1%) as compared to the other six polymeric films (p ≤ 0.001). As for bMSCs, 76.4 ± 10%, 76.3 ± 16% and 76.1 ± 19% of seeded bMSCs were adhered to TCP, PLGA50:50 and PLGA75:25, respectively. These were significantly more than those of the other five polymeric films (p

Recent advances have demonstrated the power of small molecules in promoting cellular reprogramming. Yet, the full potential of such chemicals in cell fate manipulation and the underlying mechanisms require further characterization. Through functional screening assays, we find that mouse embryonic fibroblast cells can be induced to trans-differentiate into a wide range of somatic lineages simultaneously by treatment with a combination of four chemicals. Genomic analysis of the process indicates activation of multi-lineage modules and relaxation of epigenetic silencing programs. In addition, we identify Sox2 as an important regulator within the induced network. Single cell analysis uncovers a novel priming state that enables transition from fibroblast cells to diverse somatic lineages. Finally, we demonstrate that modification of the culture system enables directional trans-differentiation towards myocytic, glial or adipocytic lineages. Our study describes a cell fate control system that may be harnessed for regenerative medicine.

Osteoarthritis (OA) remains an intractable clinical challenge. Few drugs are available for reversing this degenerative disease, although some promising candidates have performed well in preclinical studies. Tumor necrosis factor α (TNFα) has been identified as a crucial effector modulating OA pathogenesis. This study aimed to investigate the therapeutic effects of Atsttrin, a novel TNFα blocker, on OA treatment. We developed genetically modified mesenchymal stem cells (MSCs) that expressed recombinant Atsttrin (named as MSC-Atsttrin). Expression levels of ADAMTS-5, MMP13, and iNOS of human chondrocytes were analyzed when cocultured with MSC-GFP/Atsttrin. OA animal models were induced by anterior cruciate ligament transection, and MSC-GFP/Atsttrin were injected into the articular cavity 1 week postsurgery. The results showed that MSC-Atsttrin significantly suppressed TNFα-driven up-regulation of matrix proteases and inflammatory factors. Intra-articular injection of MSC-Atsttrin prevented the progression of degenerative changes in the surgically induced OA mouse model. Additionally, levels of detrimental matrix hydrolases were significantly diminished. Compared with nontreated OA samples at 8 weeks postsurgery, the percentages of MMP13- and ADAMTS-5-positive cells were significantly reduced from 91.33% ± 9.87% to 24.33% ± 5.7% (p < .001) and from 91.33% ± 7.1% to 16.67% ± 3.1% (p < .001), respectively. Our results thus indicated that suppression of TNFα activity is an effective strategy for OA treatment and that intra-articular injection of MSCs-Atsttrin could be a promising therapeutic modality. Significance The main novelty of this study is the finding of Atsttrin modified mesenchymal stem cells (MSCs-Atsttrin) for blocking osteoarthritis (OA) development within an in vivo mouse surgically induced osteoarthritis model. Because MSCs have already been widely used in the treatment of patients and have demonstrated good efficacy and safety, MSC-based Atsttrin gene therapy could be a promising modality for the treatment of OA patients.

Physical topographic cues from various substrata have been shown to exert profound effects on the growth and differentiation of stem cells due to their niche-mimicking features. However, the biological function of different topographic materials utilized as bio-scaffolds in vivo have not been rigorously characterized. This study investigated the divergent differentiation pathways of mesenchymal stem cells (MSCs) and neo-tissue formation trigged by aligned and randomly-oriented fibrous scaffolds, both in vitro and in vivo. The aligned group was observed to form more mature tendon-like tissue in the Achilles tendon injury model, as evidenced by histological scoring and collagen I immunohistochemical staining data. In contrast, the randomly-oriented group exhibited much chondrogenesis and subsequent bone tissue formation through ossification. Additionally, X-ray imaging and osteocalcin immunohistochemical staining also demonstrated that osteogenesis in vivo is driven by randomly oriented topography. Furthermore, MSCs on the aligned substrate exhibited tenocyte-like morphology and enhanced tenogenic differentiation compared to cells grown on randomly-oriented scaffold. qRT-PCR analysis of osteogenic marker genes and alkaline phosphatase (ALP) staining demonstrated that MSCs cultured on randomly-oriented fiber scaffolds displayed enhanced osteogenic differentiation compared with cells cultured on aligned fiber scaffolds. Finally, it was demonstrated that cytoskeletal tension release abrogated the divergent differentiation pathways on different substrate topography. Collectively, these findings illustrate the relationship between topographic cues of the scaffold and their inductive role in tissue regeneration; thus providing an insight into future development of smart functionalized bio-scaffold design and its application in tissue engineering.

Injured adult tendons do not exhibit optimal healing through a regenerative process, whereas fetal tendons can heal in a regenerative fashion without scar formation. Hence, we compared FFs (mouse fetal fibroblasts) and AFs (mouse adult fibroblasts) as seed cells for the fabrication of scaffold-free engineered tendons. Our results demonstrated that FFs had more potential for tendon tissue engineering, as shown by higher levels of tendon-related gene expression. In the in situ AT injury model, the FFs group also demonstrated much better structural and functional properties after healing, with higher levels of collagen deposition and better microstructure repair. Moreover, fetal fibroblasts could increase the recruitment of fibroblast-like cells and reduce the infiltration of inflammatory cells to the injury site during the regeneration process. Our results suggest that the underlying mechanisms of better regeneration with FFs should be elucidated and be used to enhance adult tendon healing. This may assist in the development of future strategies to treat tendon injuries., Scientific Reports, 4, ISSN:2045-2322

The transcription factor Mohawk (Mkx) is expressed in developing tendons and is an important regulator of tenogenic differentiation. However, the exact roles of Mkx in tendinopathy and tendon repair remain unclear. Using gene expression Omnibus datasets and immunofluorescence assays, we found that Mkx expression level was dramatically lower in human tendinopathy tissue and it is activated at specific stages of tendon development. In mesenchymal stem cells (MSCs), ectopic Mkx expression strikingly promoted tenogenesis more efficiently than Scleraxis (Scx), a well-known master transcription factor of tendon. Significantly higher levels of tenogenic gene expression and collagen fibril growth were observed with Mkx versus Scx. Interestingly, it was observed that Mkx dramatically upregulated Scx through binding to the Tgfb2 promoter. Additionally, the transplantation of Mkx-expressing-MSC sheets promoted tendon repair in a mouse model of Achilles-tendon defect. Taken together, these data shed light on previously unrecognized roles of Mkx in tendinopathy, tenogenesis, and tendon repair as well as in regulating the TGFβ pathway. Stem Cells 2015;33:443–455

Anterior cruciate ligament (ACL) is difficult to heal after injury due to the dynamic fluid environment of joint. Previously, we have achieved satisfactory regeneration of subcutaneous tendon/ligament with knitted silk–collagen sponge scaffold due to its specific “internal-space-preservation” property. This study aims to investigate the long-term effects of knitted silk–collagen sponge scaffold on ACL regeneration and osteoarthritis prevention. The knitted silk–collagen sponge scaffold was fabricated and implanted into a rabbit ACL injury model. The knitted silk–collagen sponge scaffold was found to enhance migration and adhesion of spindle-shaped cells into the scaffold at 2 months post-surgery. After 6 months, ACL treated with the knitted silk–collagen sponge scaffold exhibited increased expression of ligament genes and better microstructural morphology. After 18 months, the knitted silk–collagen sponge scaffold-treated group had more mature ligament structure and direct ligament-to-bone healing. Implanted knitted silk–collagen sponge scaffolds degraded much more slowly compared to subcutaneous implantation. Furthermore, the knitted silk–collagen sponge scaffold effectively protected joint surface cartilage and preserved joint space for up to 18 months post-surgery. These findings thus demonstrated that the knitted silk–collagen sponge scaffold can regenerate functional ACL and prevent osteoarthritis in the long-term, suggesting its clinical use as a functional bioscaffold for ACL reconstruction.

Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into β-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering.

The cell-cycle inhibitor flavopiridol has been shown to improve recovery from spinal cord injury in animal models. However, the systemic dose of flavopiridol has side-effects and the mechanism of action is not clear. This study aimed to develop a strategy for the local delivery of flavopiridol and investigate its mechanisms of action. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were used for the sustained delivery of flavopiridol. The spinal cord was right-hemisectioned and NPs were delivered into the injury site. Transparent spinal cord technology was used for the three-dimensional observation of anterograde tracing. The results showed that flavopiridol NPs had a sustained release of up to 3 days in vitro. Flavopiridol NPs significantly decreased inflammatory factor synthesis by astrocytes, including TNF-α, IL-1β, and IL-6, while the IL-10 expression was elevated. In vivo study demonstrated that flavopiridol NPs decreased cell-cycle activation, inflammatory expression and glial scarring, and facilitated neuronal survival and regeneration. The cavitation volume was decreased by ~90%. Administration of flavopiridol NPs also improved the motor recovery of injured animals. These findings demonstrated that local delivery of flavopiridol in PLGA NPs improves recovery from spinal cord injury by inhibiting astrocyte growth and inflammatory factor synthesis.

Although cell transplantation therapy can effectively promote functional tendon repair, occasional ectopic ossification during tendon regeneration undermines its efficacy. The effect of transplanted cell types on ectopic ossification has not yet been systematically evaluated. This study compared the rate of ectopic ossification during tendon repair upon transplantation with mouse fetal fibroblasts (FFs) and their adult counterparts (adult fibroblasts [AFs]). Alkaline phosphatase (ALP) staining, immunofluorescence, and gene expression analysis were used to compare the spontaneous osteogenic differentiation of FFs and AFs in vitro. X-ray, histology, and gene expression analysis were used to investigate the ectopic ossification in a mouse Achilles tendon repair model in vivo. ALP staining and immunofluorescence data in vitro showed that FFs had less spontaneous osteogenic differentiation capacity, and lower expression of runt-related transcription factor 2 (runx2). For the in vivo study, the FFs transplant group displayed reduced ectopic ossification (2/7 vs. 7/7, Mann-Whitney test p

Terminal differentiation often occurs in the repair progress of osteochondral defects. It is reported that parathyroid hormone-related protein (PTHrP) could inhibit terminal differentiation but may elicit adverse effects on chondrogenesis when administrated at improper time. Here we apply PTHrP by optimal intra-articular injection together with implantation of a collagen-silk scaffold to treat osteochondral defect. The injection schedule was set at three different time windows: 4-6, 7-9 and 10-12 weeks post-operatively. Samples were obtained and evaluated macroscopically, histologically and immunohistochemically at 16 weeks post-injury. In vivo delivery of PTHrP at 4-6 weeks exhibited better repairing effects and less terminal differentiation compared with other time windows. These results showed that PTHrP would invoke best repair effects for osteochondral defects when intraarticularly injected at 4-6 weeks post-injury, which not only prevented terminal differentiation but also promoted cartilage regeneration in rabbit model. The findings offer insight into understanding the mechanisms involved and pave the way for clinical application of PTHrP for osteochondral defects repair.

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b‐estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose‐derived stem cells (hEASCs) in vitro with hydrogen peroxide (H2O2)‐induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H2O2‐induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI., Journal of Cellular and Molecular Medicine, 18 (2), ISSN:1582-1838, ISSN:1582-4934

Despite our previous study that demonstrates that human embryonic stem cells (hESCs) can be used as seed cells for tendon tissue engineering after stepwise induction, suboptimal tendon regeneration implies that a new strategy needs to be developed for tendon repair. We investigated whether overexpression of the tendon-specific transcription factor scleraxis (SCX) in hESC-derived mesenchymal stem cells (hESC-MSCs) together with knitted silk-collagen sponge scaffold could promote tendon regeneration.hESCs were initially differentiated into MSCs and then engineered with scleraxis (SCX+hESC-MSCs). Engineered tendons were constructed with SCX+hESC-MSCs and a knitted silk-collagen sponge scaffold and then mechanical stress was applied. SCX elevated tendon gene expression in hESC-MSCs and concomitantly attenuated their adipogenic and chondrogenic potential. Mechanical stress further augmented the expression of tendon-specific genes in SCX+hESC-MSC-engineered tendon. Moreover, in vivo mechanical stimulation promoted the alignment of cells and increased the diameter of collagen fibers after ectopic transplantation. In the in vivo tendon repair model, the SCX+hESC-MSC-engineered tendon enhanced the regeneration process as shown by histological scores and superior mechanical performance compared with control cells, especially at early stages.Our study offers new evidence concerning the roles of SCX in tendon differentiation and regeneration. We demonstrated a novel strategy of combining hESCs, genetic engineering, and tissue-engineering principles for tendon regeneration, which are important for the future application of hESCs and silk scaffolds for tendon repair.

Degradation of proteoglycan is the key early event in the development of osteoarthritis (OA). The aggrecanase ADAMTS-5 has been identified as the major enzyme responsible for the degradation and thus is an attractive therapeutic target for OA. However, currently there is no report on using an ADAMTS-5 inhibition strategy for OA treatment. The present study aimed to investigate the synergic effect of combining an ADAMTS-5 inhibitor (114810) with a hyaluronic acid hydrogel (HAX) for OA therapeutics. Two OA models were induced by surgically creating an osteochondral defect or removing the anterior cruciate ligament (ACL) in Sprague-Dawley rats. Human OA cartilage was obtained from total joint replacement patients. Both human and rat OA cartilage showed marked proteoglycan loss with significantly increased ADAMTS-5 expression. The effectiveness of ADAMTS-5 inhibition by 114810 was confirmed by a cartilage explants assay in vitro, which showed that the 114810 halted the aggrecanase-mediated (374)ARGS neoepitope released from aggrecan induced by IL-1β stimulation. The in vivo effect of ADAMTS-5 inhibition was assessed by the articular injection of HAX with 114810 into OA knee joints. Evaluated eight weeks after injection, 114810 with HAX significantly promoted the in vivo cartilage healing in the osteochondral defect model, and prevented the progression of degenerative changes in the ACL model. Our results confirmed that ADAMTS-5 is an effective target for OA treatment, and the intra-articular injection of an ADAMTS-5 inhibitor within HAX gel could be a promising strategy for OA treatment.

Meniscus injury is frequently encountered in clinical practice. Current surgical therapy involving partial or complete meniscectomy relieves pain in the short-term but often leads to osteoarthritis (OA) in the long-term. Here, this study aimed to identify and characterize a novel population of meniscus-derived stem cells (MeSCs) and develop a new strategy of articular cartilage protection by intra-articular injection of these cells. The "stemness" and immune properties of MeSCs were investigated in vitro, while the efficacy of intra-articular injection of MeSCs for meniscus regeneration and OA prevention were investigated in vivo at 4, 8, and 12 weeks postsurgery. MeSCs displayed typical stem cell characteristics such as low immunogenicity and even possessed immunosuppressive function. In a rabbit meniscus injury model, transplantation of allogenous MeSCs did not elicit immunological rejection, but promoted neo-tissue formation with better-defined shape and more matured extracellular matrix. In a rabbit experimental OA model, transplantation of MeSCs further protected joint surface cartilage and maintained joint space at 12 weeks postsurgery, whereas extensive joint surface irregularities and joint space stenosis were observed in the control group. This study thus evoked a new strategy for articular cartilage protection and meniscus regeneration by intra-articular injection of MeSCs for patients undergoing meniscectomy.

It is reported that decellularized collagen matrices derived from dermal skin and bone have been clinically used for tendon repair. However, the varying biological and physical properties of matrices originating from different tissues may influence the differentiation of tendon stem cells, which has not been systematically evaluated. In this study, the effects of collagenous matrices derived from different tissues (tendon, bone and dermis) on the cell differentiation of human tendon stem/progenitor cells (hTSPCs) were investigated, in the context of tendon repair. It was found that all three matrices supported the adhesion and proliferation of hTSPCs despite differences in topography. Interestingly, tendon-derived decellularized matrix promoted the tendinous phenotype in hTSPCs and inhibited their osteogenesis, even under osteogenic induction conditions, through modulation of the teno- and osteolineage-specific transcription factors Scleraxis and Runx2. Bone-derived decellularized matrix robustly induced osteogenic differentiation of hTSPCs, whereas dermal skin-derived collagen matrix had no apparent effect on hTSPC differentiation. Based on the specific biological function of the tendon-derived decellularized matrix, a tissue-engineered tendon comprising TSPCs and tendon-derived matrix was successfully fabricated for Achilles tendon reconstruction. Implantation of this cell-scaffold construct led to a more mature structure (histology score: 4.08 ± 0.61 vs. 8.51 ± 1.66), larger collagen fibrils (52.2 ± 1.6 nm vs. 47.5 ± 2.8 nm) and stronger mechanical properties (stiffness: 21.68 ± 7.1 Nm m(-1) vs.13.2 ± 5.9 Nm m(-1)) of repaired tendons compared to the control group. The results suggest that stem cells promote the rate of repair of Achilles tendon in the presence of a tendinous matrix. This study thus highlights the potential of decellularized matrix for future tissue engineering applications, as well as developing a practical strategy for functional tendon regeneration by utilizing TSPCs combined with tendon-derived decellularized matrix.

Injured tendons often heal with scar tissue formation, resulting in uniformly smaller collagen fibrils and poor mechanical properties. The small leucine-rich proteoglycan decorin is well known to regulate fusion of collagen fibrils. Rat patellar tendon cells were transfected with lentiviral-encoded shRNA that specifically targets decorin. Silencing of decorin expression resulted in decreased cell growth. Three types of scaffold-free engineered tendons with different mix ratios of anti-decorin shRNA-treated cells to untreated cells at 1:0 (DCN), 1:1 (MIX), and 0:1 (CON) were utilized for repair of injured patellar tendons. Four weeks after implantation in situ, the MIX group manifested the best results (best coordination of histology, more mature collagen deposition, and larger collagen fibril diameter). Although the DCN group exhibited the largest collagen fibril diameter, this was associated with abnormal shape. Hence, regulation of decorin expression to an appropriate level is crucial for tendon repair with gene therapy.

Some complex clinical conditions require regeneration of large bone sections, such as in the case of massive traumatic injury and atrophic nonunions. To date, regeneration of bone tissue has been an important focus for biological repair in the field of regenerative medicine. Bone tissue engineering – an interdisciplinary field at the intersection of engineering, biology and medicine, has emerged as one of the most promising approaches for developing biological bone substitutes. In the past half century, the potential use of MSCs in bone tissue engineering has been increasingly recognized because of its unique characteristics. Many studies have utilized bone marrow derived mesenchymal stem cells (BM-MSCs) as seed cells for cell sheets and biomaterial scaffolds. In the past decade, many researchers have sought to exploit their potential as seed cells for diverse applications in regenerative medicine. The availability of robust clinical and scientific data supports the use of MSC in bone repair.