Repair Of Bladder Defect In Rabbit With Hair Follicle Stem Cells Acellular Scaffold Complex

Objective:(1) To compare different methods of hair follicle stem cells(HFSCs) culture in vitro, and using current recognized markers and other means to identify;(2) To find the method of hair follicle stem cell markers, and using CM-Dil to label HFSCs and to investigate the tracing feasibility of CM-Dil. To observe the expression of fluorescence labeling in cell-scaffolds co-cultured in HFSCs and using bladder acellular matrix to ensure the tracing effect of labeled cell and quality of transplanted cells;(3) To induce and identify HFSCs differentiating into smooth muscle cells, and to observe the growth states of the cells after fluorescence labeled cell-scaffolds;(4) To evaluate the function of cell-scaffolds in repairing rabbit bladder defect model. To observe and identify the expression of the specific biomarkers in cell-scaffolds. Methods:(1) 2-month New Zealand rabbits, with strictly disinfected and excised tentacles parts, were cut tissue containing hair follicles. Hair follicle was isolated from hair follicle outer root sheath with microsurgical technique in the sterile environment. Hair follicle outer root sheath was digested by neutral protease and trypsin. Cultured cells for further propagation or frozen treatment could be used after differentiation of adherent screening adhered cells. Inverted microscope, electron microscope, Giemsa staining, immune-fluorescence staining and flow cytometry were used to identify cultivated cells. Integrin beta 1, CK15, CD34, CK7 and other cell surface markers were the basis for identification.(2) To isolate, culture and identify rabbit hair follicle stem cells. 5ul stock solution of CM-Dil was diluted with PBS into 1ml(5ug/m1, 5u M) working solution. The culture of adhered hair follicle stem cells(P3) in good condition(total number of cell: 5-6×106) was ended, and culture liquid was aspirated. The adhered cells were light washed twice by PBS, added 5ug/m1(5uM) CM-Dil working solution, incubated for 15 minutes at 37℃, incubated for 5minutes at-4℃, washed twice by PBS, added in 25 ml volume of culture flask, and cultured for second day liquid. Observation was conducted under the green fluorescence of the inverted phase contrast fluorescence microscope. The third generation of MFSCs was differentiate into smooth muscle cell under the action of transforming growth factor beta 1(TGF- beta 1) and bone morphogenetic protein 4(BMP4). The expression of specific markers of smooth muscle cell was detected by cellular immunohistochemistry;(3) HFSCs was labeled by CM-Dil, and co-cultured with rabbit bladder acellular matrix into cell-scaffolds. The fluorescence microscope was used to observe the labeling cells and cell-scaffolds, and to determine the labeling situation of fluorescence. The cell-scaffolds was re-implanted into rabbit bladder defect model by surgery, and observed for the growth. The experimental animal bladder was obtained. The location of repairing was then selected, and made of paraffin. The fluorescence expression of tissue slices was observed by using inverted fluorescence microscope. The tissue structural components and the expression of specific markers of smooth muscle cell were detected with immunohistochemical method. Results :(1) The third generation of HFSCs have showed cobblestone morphology under inverted microscope. HFSCs were well-cultured with two-step enzymatic method, microdissection combined withminiMACS, and differential adhesion method;(2) The stable fluorescence expression of the third generation of HFSCs labeled by CM-Dil also has a strong fluorescence expression of rabbit bladder acellular matrix co-cultured onto cell-scaffolds. 7 days after the differentiation of HFSCs into smooth muscle cells, there is a peak to trough growth characteristics that only appears on smooth muscle cell specific.a-SMA was the specific markers of smooth muscle cell expression;(3) Two experimental rabbits died in 7th day, and 16 th day respectively because of the leakage of urine. Although the expression of tissue fluorescence is weakened, the expression of tissue fluorescence still existed, which shows a clear boundary from the location of repairmen;(4) Masson staining found that the organizational structure is complete, and the muscle fiber was significant. Furthermore, a-SMA was the specific markers of immunohistochemical expression of smooth muscle cell. Conclusion:(1) After accessed, the primary of HFSCs grows well when cultured in vitro, and can maintain the characteristics of stem cells, which can be used as tissue engineering research and application for seed cells. Hair follicle stem cells can be well cultivated by two-step enzymatic method, and microdissection combined withminiMACS, and differential adhesion method;(2) HFSCs labeled by fluorescence can stabilized expression in vivo; TGF-beta 1 and BMP 4 can make HFSCs differentiate into smooth muscle cells;(3) after 8 weeks, bladder acellular matrix BAMG in experimental rabbits can be absorbed and degraded, which is good scaffold material. In addition, HFSCs can be differentiated into smooth muscle cells in vivo. The HFSCs and BAMG methods can be used to construct tissue engineering bladder in vitro, and to repair the defect of rabbit bladder with good effect, which is ideal material for repair of bladder.