RESUMEN

Equine adult bone marrow-derived MSCs (BM-MSCs) may be induced into the tenogenic lineage after exposure with bone morphogenetic protein-12 (BMP-12). Despite fetal BM-MSCs have showed a greater differentiation potential compared to adults, the tenogenic differentiation capacity of equine fetal BM-MSC have not been reported. Thus, the aim of the present study was to evaluate the in vitro tenogenic differentiation potential of equine fetal BM-MSCs under the effect of BMP-12. Equine fetal BM-MSCs were exposed to three concentrations of BMP-12 (25, 50 and 100 ng/mL) during a 21-day culture period. Levels of mRNA of tenogenic markers decorin (DCN), tenomodulin (TNMD), scleraxis (SCX), collagen 1α1 (COL1α1) and protein expression of Col1α1 were evaluated. Plastic adherent cells exhibited specific MSC profile including expression of CD73 and lack of expression of CD34. Gene expression levels of DCN, TNMD, SCX and COL1α1 were increased in equine fetal BM-MSC exposed to three different concentrations of BMP-12 during a 21-day culture period. Equine fetal BM-MSCs displayed specific expression profiles suggesting features of MSCs and multipotent capacity. Furthermore, up-regulation of tenogenic markers DCN, TNMD, COL1α1 and SCX after exposure to different concentrations of BMP-12 suggests that equine fetal BM-MSCs have potential to activate selected genes that control tenogenic differentiation.

Asunto(s)

Células Madre Fetales , Células Madre Mesenquimatosas , Animales , Proteínas Morfogenéticas Óseas , Diferenciación Celular , Colágeno , Caballos

RESUMEN

BACKGROUND: Achilles tendon (AT) defects often occur in traumatic and chronic injuries. Currently, no graft can satisfactorily regenerate parallel tendinous tissue at the defect site to completely restore AT function. PURPOSE: To develop a cell-free functional graft by tethering bone morphogenetic protein 12 (BMP-12) on a book-shaped decellularized tendon matrix (BDTM) and to determine whether this graft is more beneficial for AT defect healing than an autograft. STUDY DESIGN: Controlled laboratory study. METHODS: Canine patellar tendon was sectioned into a book shape and decellularized to fabricate a BDTM. The collagen-binding domain (CBD) was fused into the N-terminus of BMP-12 to synthesize a recombinant BMP-12 (CBD-BMP-12), which was tethered to the BDTM to prepare a cell-free functional graft (CBD-BMP-12/BDTM). After its tensile resistance, tenogenic inducibility, and BMP-12 release dynamics were evaluated, the efficacy of the graft for tendon regeneration was determined in a rat model. A total of 140 mature male Sprague-Dawley rats underwent AT tenotomy. The defect was reconstructed with reversed AT (autograft group), native BMP-12 tethered to an intact decellularized tendon matrix (IDTM; NAT-BMP-12/IDTM group), native BMP-12 tethered to a BDTM (NAT-BMP-12/BDTM group), CBD-BMP-12 tethered on an IDTM (CBD-BMP-12/IDTM group), and CBD-BMP-12 tethered on a BDTM (CBD-BMP-12/BDTM group). The rats were sacrificed 4 or 8 weeks after surgery to harvest AT specimens. Six specimens from each group at each time point were used for histological evaluation; the remaining 8 specimens were used for biomechanical testing. RESULTS: In vitro CBD-BMP-12/BDTM was noncytotoxic, showed high biomimetics with native tendons, was suitable for cell adhesion and growth, and had superior tenogenic inducibility. In vivo the defective AT in the CBD-BMP-12/BDTM group regenerated more naturally than in the other groups, as indicated by more spindle-shaped fibroblasts embedded in a matrix of parallel fibers. The biomechanical properties of the regenerated AT in the CBD-BMP-12/BDTM group also increased more significantly than in the other groups. CONCLUSION: CBD-BMP-12/BDTM is more beneficial than autograft for healing AT defects in a rat model. CLINICAL RELEVANCE: The findings of this study demonstrate that CBD-BMP-12/BDTM can serve as a practical graft for reconstructing AT defects.

Asunto(s)

Tendón Calcáneo , Cicatrización de Heridas , Animales , Fenómenos Biomecánicos , Proteínas Morfogenéticas Óseas , Preparaciones de Acción Retardada , Perros , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

The objective of this study was to investigate the curative effects of mesenchymal stem cells' tenogenic differentiation on linea alba incision healing induced by bone morphogenetic protein-12. Mesenchymal stem cells were isolated and induced by 10 ng/ml of bone morphogenetic protein-12 for 48 h. Expression of scleraxis, collagen I and collagen III were examined at 48 h, 5 and 7 days to investigate the tenogenic differentiation. The expression of scleraxis increases continually even in the absence of bone morphogenetic protein-12 for 5 days (P<0.01). The expression of collagen I and III requires persistent inducing. Then fifty Sprague-Dawley rats were randomly divided into five groups: negative control, positive control, sham group, native mesenchymal stem cells and tenogenically differentiated mesenchymal stem cells. Tensiometric testing and modified semiquantitative histological analysis were performed to explore the curative effects. The tension levels in the positive control, sham, native mesenchymal stem cells and tenogenically differentiated mesenchymal stem cells were 44, 41.8, 51.6 and 69.7%, respectively, compared with the negative control. Tenogenically differentiated mesenchymal stem cells exhibited a greater increase in tension compared with positive control, sham and native mesenchymal stem cell groups (P<0.05). From the sections stained with Masson's Trichrome, collagen organization and amount of tenogenically differentiated mesenchymal stem cells was better than the other three groups (P<0.05). In conclusion, mesenchymal stem cells' tenogenic differentiation induced by bone morphogenetic protein-12 can enhance linea alba incision healing.

RESUMEN

AIM: Adipose-derived stem cells (ASCs) have been deeply characterized for their usefulness in musculoskeletal tissue regeneration; recently, other mesenchymal stem cell (MSC) sources have also been proposed. This study compares for the first time human tendon stem/progenitor cells isolated from hamstring tendons with human ASCs. MATERIALS & METHODS: Human TSPCs and ASCs were isolated from hamstring tendon portions and adipose tissue of healthy donors undergoing ACL reconstruction or liposuction, respectively (n = 7). Clonogenic ability, immunophenotype and multi-differentiation potential were assessed and compared. RESULTS: Both populations showed similar proliferation and clonogenic ability and expressed embryonic stem cell genes and MSC surface markers. Tendon stem/progenitor cells showed lower adipogenic and osteogenic ability, but after the chondrogenic differentiation, they produced more abundant glycosaminoglycans and expressed higher levels of aggrecan with regards to ASCs. The tenogenic induction with BMP-12 upregulated SCX and DCN gene expression in both populations. CONCLUSION: Our results demonstrate that waste hamstring tendon fragments could represent a convenient MSC source for musculoskeletal regenerative medicine.

Asunto(s)

Tejido Adiposo/citología , Células Madre Mesenquimatosas/citología , Trasplante de Células Madre/métodos , Tendones/patología , Adipocitos/citología , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Proliferación Celular , Separación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Condrocitos/citología , Fibroblastos/citología , Citometría de Flujo , Regulación de la Expresión Génica , Factores de Diferenciación de Crecimiento/metabolismo , Humanos , Inmunofenotipificación , Músculo Esquelético/patología , Osteogénesis , Regeneración , Medicina Regenerativa , Regulación hacia Arriba

RESUMEN

Objective To investigate the feasibility of committed differentiation of rhesus bone marrow mesenchymal stem cells (MSCs) into tenocytes induced by BMP12. Methods MSCs were transfected with pTARGETTM bearing BMP12 gene by electroporation. The transfected cells were identified by morphological observation and molecular biological measure. Results Under the observation of light microscope, the morphological features of transfected cells changed significantly compared to parental MSCs. RT-PCR data showed the transfected cells had the mRNA expression of BMP12 and collagen Ⅰ, but without that of collagen Ⅲ. 98.39% of the transfected cells were CD44+ and negative for HLA-DR. Conclusion BMP12 could induce MSCs into tenocytes, and bone marrow MSCs might be the optional seed cells for tendon tissue engineering.