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 investigate the possibility of repairing defected tendon with a tissue engineering tendon, combined culture of allogenous tenocyte and derived tendon. Methods Macaca tenocytes labelled by BrdU were seeded on the derived tendon. The flexor digitorum profundus of five fingers of left hand in 15 Macaca mulatta were resected and made 2.5cm defects as experimental model. They were divided into three groups according to repair methods (Group A: Combined culture of derived tendon materials and alloggenous tendon cells; Group B; Derived tendon materials; Group C; Autograft). In different stages, the labeled BrdU of tendon cells were observed. Results In Groupo A, after iin vivo implantation, the tenocytes could proliferate and synthesize collagen; the new tissue was white and glossy and the collagen fibers fused to form dense tendon structure as several weeks passed. Twelve weeks after implantation, the tenocytes still survived and synthesized collagen, the results of labelled cells were positive by immunothistochemical methods. By scanning electron microscopic observation, the tenocytes arraged regularly and evely among the derived tendon; the collagen fibers formed a network and its main direction was accord with that of the derived tendon. Normal nucleus, nucleolus, and cell organelles were seen under transmission electron microscope. Conclusion Combined culture of tenocytes with derived tendon is able to make tendon like tissue. The structure of tissue engineering tendon in similar to that of normal tendon.
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.
For the purpose of understanding the distribution of insulin-like growth factor-1 (IGF-1) receptor on the tendon cell, the continuous cultured tendon cell line was studied by following experiments. With the methods of immunohistochemical study and flow cytometric study, the density of IGF-1 receptor of the primary, 6th and 13th generation of tendon cell was analyzed. The results showed that there was no difference of the receptor density among those generations. However, in the cell cycle, the numbers of IGF-1 receptor in G2M phase tendon cells were more than that in G1 phase cells (P lt; 0.01). These works provided sufficient evident which suggested there were stable density of IGF-1 receptor on the tendon cell though out the life span of tendon cell. This may build some foundation in growth control of tendon cell by growth factor in the research of tendon tissue engineering.
This paper reviewed the main achievements in the research on tissue engineering tendon, focusing on major problems concerning the substitute for extracellular matrix (ECM) of tendon, biological characteristics of tendon cells, and tendon cells compounding with ECM substitute. It was concluded the important problems in the study of the tissue engineering having specific reparative functions could be: to prepare the ECM materials suitable for the tendon cells to attach, grow, and function; to establish the tendon cell line whose growth, proliferation, and immunological antigenicity could be modulated and controlled, and simulating the mechanical environment of tendon in vivo, to adopt three-dimensional tendon cell culture method.
The purpose of this study was to find some solutions to the problem of tendon cell proliferation control. Under the condition of in vitro culture, several materials including IGF-1 receptor antibody and mRNA antisense oligonucleotide were added to the culture medium to block the IGF-1-Receptor system. The effect of the material on the tendon cell proliferation was judged by cell count after incubation of 48 hours. The results showed that both IGF-1 Receptor antibody (IGF-1R alpha) and IGF-1 Receptor mRNA antisense oligonucleotide had negative effect on tendon cell proliferation (P lt; 0.01 and P lt; 0.05). These findings lead us to think that the above two materials could be used in the experiment of tendon adhesion preventing and living ready-made tendon producing.
In order to investigate the possibility of repairing injuried tendon with living artificial tendon, after combining culture, subcultured autogenous tendon cells with carbon fibers were implanted into the calcaneous tendon of rabbits. In different stages, the synthesis of type I collagen and their relevant morphological changes were observed. The results showed as follows: after implantation, tendon cells continued proliferating. Four weeks after implantation, tendon cells were detached from the carbon fibers and proliferated and produced collagen among the carbon fibers. The collagen fibrils were linked with each other to formed a dense structure. In the linkage site, the collagen fibrils originated from the implants joined to that from the ruptured end of the tendon, which meaned that the implant was healed with the recipient tendon. Observed under scanning electronic microscope, the tendon cells were lined among the carbon fibers evenly and in order, the collagen fibrils joined each other and formed an network, the fibrils were lined parallel to the carbon fibers. Under transparent electron microscope, the nucleolus were clear and organelle were abundant.
In order to investigate the compatibility and growth between the tendon cell or fibroblast of rabbit and artificial materials, the combined-culture of the two cells with the carbon fiber, terylene and chitin was observed respectively. Results showed as following: in vitro, the compatibility of carbon fiber with these two cells was well, cell-adhesion ability was good as well. Few cells grew on terylene. Chitin inhibited the growth of either cells. No matter the tendon cell or the fibroblast, the amount of cells adhering on the carbon fiber was far more than that on terylene or chitin. When the three materials were interlaced together, the collagen fibers produced by the cells were arranged in direction parallel to the carbon fibers. As the time elapsed, the cells on the carbon fiber distributed evenly and enveloped the material in network-like fashion, this suggested that carbon fiber was a good material for producing living artificial tendon and ligament.
In order to study the expression change of insulin-like growth factor-1 (IGF-1) and its receptor genes in different generations of tendon cell in culture, Dig-labeled synthesized oligonucleotide probes were used to detect the mRNA expression in primary, 6th and 13th generation of tendon cell. The results showed that IGF-1 receptor mRNA was expressed in all of the 3 above generation tendon cells. IGF-1 mRNA was expressed only in primary and 6th generation cells. Tendon cell of 13th generation did not express IGF-1 mRNA. It might suggest that the absence of IGF-1 mRNA expression be one of the causes which led to the decrease of reproductive ability of 13th generation tendon cell.