A Study On The Biological Effect Of Intermittent Cyclic Stretching Tension On Tissue Engineered Tendon In Vitro

Objective: To fabricate the tissue engineered tendon under the condition of intermittent dynamic stretch tension with tenocytes of rabbit and small intestinal submucosa (SIS) in vitro , and to primarily evaluate the role of cyclic stretchy tension on the proliferation of tenocytes and the gene expresion collagen typeⅠⅢ, and to primarily investigate its possible mechanism involved in.Methods:1. The rabbit tenocytes were isolated by the means of enzymic digestion, morphology of the cells were assayed with inverted microscope and Masson's trichrome stain, collagen typeⅠⅢand Vimentin of the tenocytes were charactered by immunofluorescence stain and confocal laser scanning microscope. The SIS was isolated from the porcine small intestines with machanical and chemical cleaning, its histological structure ,cellular remnants and collagen fibres were investigated by HE stain and optical microscope, the topography in its both surfaces were charactered by scan eletronic microscope, its cellular compatibility to the rabbit tenocytas was evaluated with MTT assay and co-cultuing with rabbit tenocytes. Its hydrophilisity was decided by its absorption rate.2. The tenocytes-SIS scaffold composites were composed of rabbit tenocytes and porcine SIS, and were cultured respectively under the condition of dynamic cyclic stretch tension of all stretching tension regime and static state. The tenocytes-SIS composites were sampled for the use in the following procedures.The cells-SIS composites cultivated under static state and dynamic cyclic stretching condition were rutinely paraffin embedded and sliced, then stained with hematoxylin and eosin(HE) stain and Masson's trichrome stain. The discrepancy of the histological structure and morphology of the specimens were compared under optical microscope. The specimens were collected at 1st ,2nd 4th and 9th day, and the MTT assay were porformed as the protocol of MTT kit, and optical density(OD) value were measured by enzymic linked immunocemetry, then relative quantity of the cells in the specimens were evaluated.The specimens were havested after cultivated for 3days. To investigate the effect of cyclic stretch tension on the cellular cycle of the tenocytes in the engineered tendons, the percentage of the tenocytes in all phases were surveyed with flow cytometry, and percentage of the cells in G2 and S phase were defined as the proliiferation ratio.Real time reverse transcription polymerse chain reaction(RT-PCR) were performed to investigate the change of gene expression of COLI, COLIII, TGF-β3, decorin and Biglycan in the specimens cultivated 3days. Extraction of total RNA and systhesize of complementary deoxyribonucleic acid(c-DNA) were performed as the protocol of reagent kit. The sequece of the target gene and housekeeping gene were listed as following table.The CT value of gene expression of the target gene were read in an automatic PCR metry and analyzed using the GAPDH as an internal standard, and the relative multiple of gene expression were reckoned. And the expression of COLI to COLIII ratio were defined as the ratio of reative value of them.Results:(1)Rabbit tenocytes emerged long fusiform shape or polygon shape under inverted microscope, with stronge refactile. the nucleus were stained red and the celluar plasm were dyed blue by Masson's trichrome stain. Immunofluorescence stain analysis showed the cells with positively stain of Vimentin and collagen typeⅠ, and with negative stain of collagen typeⅢ.(2)①The ioslated SIS were gray and white after lyophilized, hematoxylin and eosin staining(HE stain)analysis showed no cellular remnants as nucleus debris were seen in the SIS, and Masson's teichrome stain revealed the SIS with loose and porouse structure and stained blue. The scan electronic microscope confirmed the SIS possessed of two surface , the mucosa surface and the serosa surface, fibres in the mucosa surface were thick, the one in the other surface were ethin.②The absorption of the SIS reached to 279% at 0.25 hour time point, and hit its peak at point 4～6hour(490%).③MTT assay confirmed cytotoxity of SIS ranged from 1 grade to 2 grade, and tenocytes grown in the SIS flourished, the tenocytes overgrown in the surface of SIS cultureed the cell-SIS for 1 week.3.⑴Cell-SIS structures were gray and white ,the diameter grown small and the length become long, the formation was incompact yet. The paraffin slices of cell-SIS constructions cultivated under congdition of dynamic cyclic stretch tension with HE stain confirmed that the cellular density were large than that of control group's under the optical microscope, the seed cells combine with the scaffold firm, and part of the cells had grown into depth of the SIS scaffold in the transverse slices. And in the longitude section slice of the experimential groups, the cells arrayed in linage along the scaffold and distributed more uniformly than that of the control groups.⑵MTT assay found that proliferation of the seed cells in all the experimental groups were remarkably higher than that of the control group at other time points(P<0.05). Except at the point 1st day , cellular proliferation had no statistically diference(P>0.05) in all the groups. The frenquency of the stretch tension selected in the experimental groups promoted the seed cells'proliferation, which was the most markable especially at the frequency of 0.5Hz, and its growth-curve drift ahead and its growth peak period prolonged, and the peak value heightened. The magnitude of the stretch tension stimulated the multiplication, which was the most prominent especially at the magnitude of 4%. No interaction effect was found between the frequency and magnitude of the stretch tension. The effect on the proliferation of the frequency of stretch tension were greater than that of the magnitude at the other point except at 1st day. The growth curve were found to have a uniform trendence in the culture period, and the most obvious proliferation were found in the period from cultured 2nd to 5th day.⑶The stretch tension were found to noticeably reduce the pencentage of G1 period cells in the cell-SIS constructions (P<0.05), at the same time, the pencentage of the cells in G2 and in S period were found to noticeably increase in the structures (P=0.00024), and there were no statistical dicrepancy (P=0.136) between the experimental and control groups.⑷The frequency of the stretch tension were found to remarkably(P=0.00017)enhance the expresion of collagen typeⅠgene (COLⅠ), the stimulative effect on the COLⅠexpresion the frequency of 0.5Hz were the most remarkable at the same magnitude. The magnitude of the stretch tension were found to evidently(F=4.369, P=0.028) enchance the expresion of COLⅠof the cell in the cell-scaffold constructions, and the stimulative effect on the COLⅠexpresion of the stretch tension were found to be the remarkable. While no interaction effect were found between the frequency and the magnitude of the stretch tension yet(F=0.426,P=0.788).The respose of COLⅠexpresion to the frequency of stretch tension were found to more obvious than that of the magnitude of the stretch tension. The magnitude of the stretch tension were found to remarkably(F=13.87, P=0.0002) promote the expresion of COLⅢ, and 8% were the magnitude promote the gene expresion the most noticeablely. The frequency of the stretch were revealed to heighten the COLⅢexpresion observably(F=3.57, P=0.047), it was found that 1.0Hz was the frequency to heighten the gene expresion most observably. The frequency and the magnitude of the stretch tension were not found to have interaction effect on the expresion of COLⅢ(F=0.252, P=0.905).⑸There were a markable discrepancy(F=2.772,P=0.028) of expression of COLI to COL III ratio between the specimens cultivated under different tensional condition, the COL I to COL III rario in the group of 0.5Hz/4% was the highest (2.68±0.26) in them.⑹The stretch tension were observed to obviously(t=-3.665, P=0.043) down regulate the expresion of Decorin gene, at the same time to remarkably(t=6.308, P=0.024) up regulate the expresion of Biglycan gene. Conclusions:1. The 2nd passage tenocytes could be used as the seed cells in the tissue engineering tendon research.2. SIS,an acellular matrix, were suitable to be used as scaffold in tissue engineering tendon. The homemade SIS have a smooth surface facing the mucosa and a rough surface towards the serosa, no statistical difference between both of its surfaces in cellular adhesion to tenocytes(P > 0.05). It possessed of excellent hydrophilisity, and good cellular compatibility. After co-cultured with tenocytes for 2 weeks under the condition of cyclic stretch tension, the SIS scaffolsd remained intact.3. The stretch tension could effectively promote the tenocytes'proliferation in vitro, which might be relate to the tension's increasing the percentage of the G2 and S phase cells in the cellular cycle.4. The stretch tension could upregulate the expression of COL I and COL III and heighten the COL I to COL III ratio, tension's regulation of the expression of COL I and III might be relate to the change of Decorin's expression.5. The stretch tension at 0.5Hz/4% could effectively promote proliferation and heightening ratio of COL I to COL III, thereby it was forseeable to form more resemble tendon substitute.