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 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 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.