Abstract
BackgroundTendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Exogenous growth factors that activate TSPC proliferation have emerged as a promising approach for treatment. Here, we evaluated the pigment epithelial-derived factor (PEDF)-derived short peptide (PSP; 29-mer) for treating acute tendon injury and to determine the timing and anatomical features of CD146- and necleostemin-positive TSPC in the tendon healing process.MethodsTendon cells were isolated from rabbit Achilles tendons, stimulated by the 29-mer and analyzed for colony-forming capacity. The expression of the TSPC markers CD146, Oct4, and nestin, induced by the 29-mer, was examined by immunostaining and western blotting. Tendo-Achilles injury was induced in rats by full-thickness insertion of an 18-G needle and immediately treated topically with an alginate gel, loaded with 29-mer. The distribution of TSPC in the injured tendon and their proliferation were monitored using immunohistochemistry with antibodies to CD146 and nucleostemin and by BrdU labeling.ResultsTSPC markers were enriched among the primary tendon cells when stimulated by the 29-mer. The 29-mer also induced the clonogenicity of CD146+ TSPC, implying TSPC stemness was retained during TSPC expansion in culture. Correspondingly, the expanded TSPC differentiated readily into tenocyte-like cells after removal of the 29-mer from culture. 29-mer/alginate gel treatment caused extensive expansion of CD146+ TSPC in their niche on postoperative day 2, followed by infiltration of CD146+/BrdU− TSPC into the injured tendon on day 7. The nucleostemin+ TSPC were located predominantly in the healing region of the injured tendon in the later phase (day 7) and exhibited proliferative capacity. By 3 weeks, 29-mer-treated tendons showed more organized collagen fiber regeneration and higher tensile strength than control tendons. In culture, the mitogenic effect of the 29-mer was found to be mediated by the phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC.ConclusionsThe anatomical analysis of TSPC populations in the wound healing process supports the hypothesis that substantial expansion of resident TSPC by exogenous growth factor is beneficial for tendon healing. The study suggests that synthetic 29-mer peptide may be an innovative therapy for acute tendon rupture.
Highlights
Tendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes
It has been reported that hydrogel combinations of fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-1, and platelet-derived growth factor (PDGF)-BB can improve the survival of adipose-derived mesenchymal stem cells (ASCs) and aid tendon healing [6, 7]
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotic–antimicotic solutions, and trypsin were purchased from Invitrogen (Carlsbad, CA). 5-Bromo2′-deoxyuridine (BrdU), insulin–transferrin–sodium selenite (ITSE) media supplement, Hoechst 33258 dye, Trichrome Stain (Masson) Kit, and all chemicals were from Sigma-Aldrich
Summary
Tendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Tendons have limited ability for self-healing following severe injury, because of their avascularity and acellularity [1, 2]. Unlike another type of connective tissue, bone marrow mesenchymal stromal cells (BM-MSCs) are difficult to mobilize into the injured sites of tendons [1]. Growth factors injected into and around the injured site to stimulate tendon stem/ progenitor cells (TSPC) proliferation may offer an alternative option for promoting tendon repair. Connective tissue growth factor (CTGF) can promote tendon wound healing by stimulating proliferation of a TSPC population marked by CD146. The mitogenic signaling stimulating TSPC proliferation remains largely unknown
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