ObjectiveTo investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor γ (PPARγ), 1ipoprotein lipase (LPL), and fatty acid binding protein (aP2). ResultsThe final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P<0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P<0.05). ConclusionInhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.
Objective To evaluate the effectiveness of hamstring tendon and flexor hallucis longus (FHL) tendon autograft for Achilles tendon defects reconstruction. Methods Between February 2009 and October 2011, 9 patients (9 feet) with Achilles tendon defect were treated with hamstring tendon and FHL tendon autograft. Of 9 cases, 6 were male and 3 were female with an average age of 43 years (range, 21-65 years), including 5 cases of chronic Achilles tendon ruptures caused by sport injury and 4 cases of Achilles tendon defects caused by resection of tendon lesion (2 cases of hyaline degeneration with necrosis, 1 case of giant cell tumor, and 1 case of chronic inflammation with hyaline degeneration). The disease duration ranged from 31 to 387 days (mean, 137.6 days). The defect length was 5 to 18 cm (mean, 8.6 cm). Functional exercise of the ankle began at 6 weeks after plaster fixation. Results Dehiscence and effusion occurred in 2 cases and plantar pain caused by injury of tibial nerve in 1 case; primary healing of wound was obtained in the other patients without complication. Nine patients were followed up 19.7 months on average (range, 13-25 months); no re-rupture was observed. There was no significant difference in the dorsal extension between at preoperation and at 1 year and last follow-up after operation (P gt; 0.05); the ankle plantar flexion at 1 year and last follow-up after operation was significantly larger than that at preoperation (P lt; 0.05). The ankle plantar flexion and dorsal extension at 1 year and last follow-up after operation were significantly larger than those at 3 months after operation (P lt; 0.05), but no significant difference was found between at 1 year and last follow-up (P gt; 0.05). American Orthopaedic Foot and Ankle Society (AOFAS) and short-form 36 health survey scale (SF-36) scores were significantly increased at postoperation when compared with scores at preoperation (P lt; 0.05), and the scores at last follow-up were significantly higher than those at 3 months after operation (P lt; 0.05). The Achilles tendon total rupture score (ATRS) at last follow-up was significantly higher than that at 3 months after operation (t= — 7.982, P=0.000). Conclusion Combined hamstring tendon and FHL tendon autograft is one of the effect methods to reconstruction Achilles tendon defects.
Objective To investigate the effects of bone morphogenetic protein 2 (BMP-2) on the chondrogenic differentiation of human Achilles tendon-derived stem cells (hATDSCs) in vitro. Methods Achilles tendon was harvested from a voluntary donor with acute Achilles tendon rupture. And nucleated cells were obtained by digesting with collagenase and were cultured to the 3rd passage. The flow cytometry was used to measure the immunophenotyping; and Oil red O staining, alizarin red staining, and Safranin O/fast green staining were used to identify the adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation, respectively. The hATDSCs pellet was cultured in complete culture medium with (experimental group) or without recombinant human BMP-2 (rhBMP-2) (control grup) for 3 weeks. Chondrogenic differentiation of hATDSCs was evaluated by HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II; and the mRNA expressions of SOX9, collagen type II, and Aggrecan were detected by real-time fluorescence quantitative PCR. Results Primary hATDSCs cultured in vitro showed clonal growth; after cell passage, homogeneous spindle fibroblast-like cells were seen. The cells were positive for CD44, CD90, and CD105, while negative for CD34, CD45, and CD146. The results were positive for Oil red O staining at 3 weeks after adipogenic differentiation, for alizarin red staining at 4 weeks after osteogenic differentiation, and for Safranin O/fast green staining at 3 weeks after chondrogenic differentiation. After hATDSCs were induced with rhBMP-2 for 3 weeks, pellets formed in the experimental group, and the size of pellets was significantly larger than that in the control group; the results of HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II were all positive. The results of real-time fluorescence quantitative PCR showed that the mRNA expressions of SOX9, collagen type II, and Aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion BMP-2 can promote proteoglycan deposition and induce chondrogenic differentiation of hATDSCs in vitro. The effect of BMP-2 on hATDSCs might provide a possible explanation for histopathological changes of tendinopathy.
Objective To review the latest researches of Tenomodulin in tendon tissue engineering, to predict the progress of research and application of Tenomodulin. Methods The literature concerning Tenomodulin in tendon tissue engineering was collected and analyzed. Results Tenomodulin is a type II transmembrane glycoprotein that can regulate growth of tendon and contains a C-terminal anti-angiogenic domain. The human Tenomodulin gene spans approximately 1 360 bp and is mapped to Xq22.1. The expression of Tenomodulin is regulated by various biological factors, especially Scleraxis; and the nature and structure of scaffold material as well as the stain loading and cell passage, can modulate the expression of Tenomodulin. Conclusion Tenomodulin, as relatively specific molecule makers for tendon and containing a C-terminal anti-angiogenic domain, is expected to play a significant role in tendon tissue engineering.
Objective To investigate the effect of repeated freezing and thawing combining nuclease treatment on the decellularization of bovine tendons, and the morphology, structure, biochemical compositions, and mechanical properties of the decellularized tendons. Methods A total of 48 fresh 1-day-old bovine Achilles tendons were randomly divided into 3 groups (n=16): fresh normal tendons (group A), repeated freezing and thawing for 5 times (liquid nitrogen refrigeration/37℃ thawing, group B), and repeated freezing and thawing combining nuclease processing for 24 hours (group C). In each group, 2 tendons were used for scanning electron microscope (SEM), 3 tendons for histological and immunohistochemical observations, 3 tendons for DNA content detection, and 8 tendons for biomechanical testing. Results SEM observation indicated the intact, aligned, and densely packed collagen fibers with no disruption in groups A and B, and the slightly loose collagen fibers with little disruption in group C. The alcian blue staining, sirius red staining, and immunohistochemical staining showed that the most of glycosaminoglycan, collagen type I, collagen type III, and fibronectin in group C were retained after decellularization treatment. HE and DAPI staining showed that the cell nuclei between the collagen fibers were clearly visible in groups A and B; however, the cell nuclei between collagen fibers almost were invisible with a few residual nuclei on the endotendineum in group C. DNA quantitative detection confirmed that DNA content in group C [(0.05 ± 0.02) μg/mg] was significantly lower than those in group A [(0.24 ± 0.12) μg/mg] and group B [(0.16 ± 0.07) μg/mg] (P lt; 0.05). Biomechanical testing showed that the values of tensile strength, failure strain, stiffness, and elastic modulus were different among 3 groups, but no significant difference was found (P gt; 0.05). Conclusion Repeated freezing and thawing combining nuclease processing can effectively remove the component of cells, and simultaneously retain the original collagen fibrous structure, morphology, most of the extracellular matrix compositions, and mechanical properties of the bovine tendons.
Objective To review the research progress of cell-scaffold complex in the tendon tissue engineering. Methods Recent literature concerning cell-scaffold complex in the tendon tissue engineering was reviewed, the research situation of the cell-scaffold complex was elaborated in the aspects of seed cells, scaffolds, cell culture, and application. Results In tendon tissue engineering, a cell-scaffold complex is built by appropriate seed cells and engineered scaffolds. Experiments showed that modified seed cells had better therapeutic effects. Further, scaffold functionality could be improved through surface modification, growth factor cure, mechanical stimulation, and contact guidance. Among these methods, mechanical stimulation revealed the most significant results in promoting cell proliferation and function. Through a variety of defect models, it is demonstrated that the use of cell-scaffold complex could achieve satisfactory results for tendon regeneration. Conclusion The cell-scaffold complex for tendon tissue engineering is a popular research topic. Although it has not yet met the requirement of clinical use, it has broad application prospects.
Objective To investigate the feasibility and effect of human amniotic membrane in prevention of tendon adhension after tendon sheat defect repair. Methods The amniotic membrane in size of 1.5 cm × 1.0 cm was harvested from human placenta which was voluntary donated from maternal after cesarean. Forty healthy male Leghorn chicken (aged 3-6 months) were selected, weighing (1.86 ± 0.04) kg. The model of flexor digitorum profundus tendon and tendon sheath defects was established at the third toe. After repair of the flexor digitorum profundus tendon, the human amniotic membrane was used to repair the tendon sheath defect in the right foot (group A), but tendon sheath defect was not repaired in the left foot (group B) . At 1, 2, 4, and 6 weeks after operation, the gross and histological observations were done; the degree of tendon adhesions was graded according to Tang’s tendon adhesion general observation grading standards; and the biomechanical properties (tendon slip length and total flexion angle) were tested. Results All animals survived after operation and incisions healed. Gross and histological observations showed that the new tendon sheath formed with time passing after operation in groups A and B; new tendon sheath was more maturer and smoother in group A than in group B. The degree of tendon adhesions in group A was significantly less than that in group B (P lt; 0.05) at 1 and 6 weeks after operation. The biomechanical test results showed there was no significant difference in the tendon slip length between 2 groups at 1 and 2 weeks after operation (P gt; 0.05), but the tendon slip length of group A was significantly longer than that of group B at 4 and 6 weeks after operation (P lt; 0.05). The total flexion angle of group A was significantly smaller than that of group B at 1, 2, 4, and 6 weeks after operation (P lt; 0.05). Conclusion It is effective in the prevention of tendon adhesion to use the amniotic membrane for repairing the tendon sheath defect, which is beneficial to recovery of the tendon sliding function.
Objective To investigate the effectiveness of the terminal extensor tendon reconstrution by palmaris longus tendon graft for the treatment of old mallet finger deformity. Methods Between February 2009 and February 2011, 32 patients with old mallet finger deformity were treated with palmaris longus tendon graft. There were 28 males and 4 females with an average age of 32.5 years (range, 22-58 years). The injury causes included sports injury in 26 cases and punctured injury in 6 cases. The index finger was involved in 8 cases, the middle finger in 3 cases, the ring finger in 16 cases, and the little finger in 5 cases. According to the Rockwell’s classification, all patients were classified as type I. The time from injury to operation was 4-16 weeks (mean, 6 weeks). Results Primary healing was obtained in all incisions; no necrosis, infection, or nail bed injury occurred. All patients were followed up 12-20 months (mean, 14 months). The patients had no pain or paresthesia of volar finger. According to Patel’s functional assessment system, the results were excellent in 8 cases, good in 21 cases, fair in 2 cases, and poor in 1 case at last follow-up, with an excellent and good rate of 90.6%. Conclusion Reconstruction of the terminal extensor tendon by palmaris longus tendon graft is a reliable method to treat old mallet finger deformity.