Objective By culturing tendon sheath fibroblasts, epitenon tenocytes and endotenon tenocytes of rabbits’ tendon in vitro, to study the effects of mannose-6-phosphate on transforming growth factor β (TGF-β) peptide and receptor expression, and to provide the experimental basis for preventing the tendon heal ing adhesion by mannose- 6-phosphate. Methods Eight adult New Zealand white rabbits, regardless of their gender and weighing 4.0-4.5 kg, were selected. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All 3 cells were divided into 2 groups at random after cells were adjusted to a concentration of 4 × 104 per well and 1 × 104/mL. The first was the control group without supplementation. The experimental group was supplemented withmannose-6-phosphate. The expressions of TGF-β and TGF-β receptor were quantified with enzyme-l inked immunosorbent assay. The expression of TGF-β1 mRNA was also assessed with in situ hybridization and the expression of TGF-β1 was assessed with immunohistochemistry. Results The expressions of TGF-β and TGF-β receptor in experimental group were significantly lower than that in control group (P lt; 0.05). The expression levels of TGF-β1 and TGF-β2 decreased in descending order of tendon sheath fibroblasts (36.1%, 37.9%), epitenon tenocytes (31.0%, 32.1%), and endotenon tenocytes (31.2%, 27.0%). The expression levels of TGF-β3 decreased in descending order of endotenon tenocytes (42.5%), tendon sheath fibroblasts (41.2%), and epitenon tenocytes (33.3%). The expression levels of TGF-β receptor 1 and TGF-β receptor 2 decreased in descending order of epitenon tenocytes (29.9%, 26.2%), endotenon tenocytes (27.8%, 23.5%), and tendon sheath fibroblasts (23.1%, 20.0%). The expression levels of TGF-β receptor 3 decreased in descending order of endotenon tenocytes (26.1%), epitenon tenocytes (19.2%), and tendon sheath fibroblasts (15.8%). In experimental group, the positive expression of TGF-β1 mRNA and the expression level of intracellular TGF-β1 mRNA in all 3 tendon cells were significantly lower than those in the control group (P lt; 0.05). Immunohistochemical staining showed the expressions of TGF-β1 in all 3 tendon cells were significantly lower in theexperimental group than in the control group. Conclusion Mannose-6-phosphate can significantly decrease the expressions of TGF-β peptide, TGF-β receptor, and TGF-β1 mRNA. Modulation of mannose-6-phosphate levels may provide a mean of modulating the effects of TGF-β on adhesion formation in flexor tendon wound heal ing.
Objective To investigate the cellular compatibil ity of polyvinyl alcohol (PVA)/wild antheraea pernyisilk fibroin (WSF), and to explore the feasibil ity for tendon tissue engineering scaffold in vitro. Methods The solutions of WSF (11%), PVA (11%), and PVA/WSF (11%) were prepared with 98% formic acid (mass fraction) at a mass ratio of 9 : 1. The electrospinning membranes of WSF, PVA, and PVA/WSF were prepared by electrostatic spinning apparatus. The morphologies of scaffolds were evaluated using scanning electronic microscope (SEM). The tendon cells were isolated from tail tendon of 3-dayold Sprague Dawley rats in vitro. The experiment was performed using the 3rd generation cells. The tendon cells (1 × 106/mL) were cocultured with PVA and PVA/WSF electrospinning film, respectively, and MTT test was used to assess the cell adhesion rate 4, 12 hours after coculture. The tendon cells were cultured in PVA and PVA/WSF extraction medium of different concentration (1, 1/2, and 1/4), respectively; and the absorbance (A) values were detected at 1, 3, 5, and 7 days to evaluate the cytotoxicity. The composite of tendon cells and the PVA or PVA/WSF scaffold were observed by HE staining at 7 days and characterized by SEM at 1,3, 5, and 7 days. Results The solution of WSF could not be used to electrospin; and the solution of PVA and PVA/WSF could be electrospun. After coculture of tendon and PVA or PVA/WSF electrospinning membranes, the cell adhesion rates were 26.9% ±0.4% and 87.0% ± 1.0%, respectively for 4 hours, showing significant difference (t=100.400, P=0.000); the cell adhesion rates were 35.2% ± 0.6% and 110.0% ± 1.7%, respectively for 12 hours, showing significant difference (t=42.500, P=0.000). The cytotoxicity of PVA/WSF was less significantly than that of PVA (P lt; 0.05) and significant difference was observed between 1/2 PVA and 1/4PVA (P lt; 0.05). HE staining and SEM images showed that the tendon cells could adhere to PVA and PVA/WSF scaffolds, but that the cells grew better in PVA/WSF scaffold than in PVA scaffold in vitro. Conclusion PVA/WSF electrospinning membrane scaffold has good cell compatibility, and it is expected to be an ideal scaffold of tendon tissue engineering.
OBJECTIVE: To study the feasibility of calcium polyphosphate fiber (CPPF) as the scaffold material of tendon tissue engineering. METHODS: CPPF (15 microns in diameter) were woven to form pigtail of 3 mm x 2 mm transverse area; and the tensile strength, porous ratio and permeability ratio were evaluated in vitro. Tendon cells (5 x 10(4)/ml) derived from phalangeal flexor tendon of SD rats were co-culture with CPPF scaffold or CPPF scaffold resurfaced with collagen type-I within 1 week. The co-cultured specimens were examined under optical and electric scanning microscope. RESULTS: The tensile strength of CPPF scaffolds was (122.80 +/- 17.34) N; permeability ratio was 61.56% +/- 14.57%; and porous ratio was 50.29% +/- 8.16%. CPPF had no obvious adhesive interaction with tendon cells, while CPPF of surface modified with collagen type-I showed good adhesive interaction with tendon cells. CONCLUSION: The above results show that CPPF has some good physical characteristics as scaffold of tendon tissue engineering, but its surface should be modified with organic substance or even bioactive factors.