Studies Of Hair Follicle Stem Cells In Co-culture With Xenografic Acellular Scaffold And Its Tissue Compatibility

Object:1. To establish a method to culture the hair follicle stem cells (Hair folliclestem cells, HFSCs) and identify these cultured cell with accepted markers and other ways.2. To construc rabbit bladder acellular matrix (bladder acellular matrix, BAM) scaffold,then hair follicle stem cells grown in the BAM to build cell-seeded-scaffold complexe andobserve the biological compatibility of the complex in vitro。3. To define hair follicle stemcells and to identify multipotent differentiation potential; simultaneously to mpare theefficiency of hair follicle stem cells cultured in vitro,which are the volume fraction ofrelatively free DMEM/F12+10%fetal bovine serum, serum-free keratinocyte culturemedium and serum-free keratinocyte base+volume fraction of10%FBS culture medium.Materials and Methods:1.To select7-9days neonatal SD rats, sterilize and after removalof tentacles, cut off the tissue containing hair follicles. Iisolate hair follicles and hairfollicle root sheath using microsurgical techniques in a sterile environment. With a neutralprotease and trypsin digestion follicle outer root sheath; choose the fast proliferations withthe differential adhesion after the adherent cells were cultured in10%serum-free fetalbovion filthy cells. Cultured cells can be frozen for further processing. Using an invertedmicroscope, electron microscope, Giemsa staining, immunofluorescence staining, flowcytometry to identify the cultured cells, of which the most important surface markers areCK15, β1integrins and CD34.2. In order to get more high-follicle stem cells, platelet-richplasma on P3generation of hair follicle stem cells in the hair follicle stem cellproliferation were tested; And cultured stem cell suspension level is divided into threegroups, each were cultured with a volume fraction of DMEM/F12culture+10%fetalbovine serum, keratinocyte keratinocyte serum-free medium and the volume fraction ofserum-free medium+10%FBS culture respectively to determine their viability, culture efficiency of three media were compared with growth curves, flow cytometry.3.NewZealand Rabbits aged from3to5month old were killed by air embolism, withmicrosurgery cut of bladder submucosa.Chemically washe the bladder for acellularmatrix, which were identified by scanning of electron microscopy morphology, BAMsurface makers to determine whether the presence of residual surface observation cells orcell debris; scaffold with Masson staining identified again;with GB/T168865.-2003vitrotoxicity and national standard cell biological evaluation of medical devices to determinetheir cytotoxicity. Finally,with the cultured hair follicle stem cells suspension seed thescaffold,,to clarify its biocompatibility through the results of the growth curve, scanningelectron microscopy and histology. Results:1.An observation of uniform, folding,lightand strong of cell were detected by inverted phase contrast microscope and showing atypical "cobblestone-like" cells; TEM observed that plasma cells characterized by a largeproportion of nuclear,organelles immature, small size, cells were thought being in originalcondition. Growth curves show: P3-P6cells have strong capability of proliferatin. Thethird generation HFSCs cells, The flow cytometry checked that four indications whichare stem cell marker CK15, CD34, β1integrin andCK7. markers CD34and β1integrin, CK15in the experimental group were higher (p <0.05), there was significantdifference between two groups. CK7expression between experimental groups and controlgroup had no significant difference after statistical analysis (P>0.05).2Test the twicecentrifuged obtained platelet(A group) and thrice centrifuged obtained platelet (group B)with the CCK-8Colorimetric.Showed the effects of cell proliferation were that2daysbefore culturing,cells absorbance A value of each group between each groups and tocontrol group had no significant difference after statistical analysis (P>0.05). However,compared A,B Platelet-rich plasma(PRP) to control group, there was significantdifferences(P <0.05) on fourth day. On the fourth,fifth sixth day, thrice centrifugedobtained and twice centrifuged obtained PRP(including concentration of2%PRP and4%PRP) compared each other displayed statistically significant (P <0.05). Indicate that theeffect of thrice centrifuged obtained PRP is more obvious to hair follicle stem cell onproliferation. While it is evidenced that the high-dose platelet-rich plasma(PRP) wassignificantly better than low-dose PRP group on hair follicle stem cell proliferation,underthe identical conditions. It was also showed that thrice centrifuged obtained concentrationof4%PRP is the best on hair follicle stem cell proliferation. Preparing K-SFM+10%FBS,K-SFM, DMEN/F12+10%FBS for culturing. On the sixth day of the third generationHFSCs cells, cell viability was calculated st atistically as DMEN+10%FBS group (96.62 ±2.00)%, K-SFM+10%FBS group (95.26±2.27)%, K-SFM group (93.95±2.54)%, while comparison each other among the three groups showed no statisticallysignificant (P>0.05) on cell viability.All three groups grew slowly at early2days, DMEN+10%FBS and K-SFM+10%FBS cell got into logarithmic growth phase after4days;but after5days for K-SFM group, K-SFM+10%FBS group got into plateau after7days;K-SFM group and DMEN+10%FBS group entered the plateau after8days.On the6thday of the third generation HFSCs cells, marked and tested the β1integrins (CD29),CK15, and CD34, indicated that β1integrins (CD29), CK15, and CD34in K-SFM andK-SFM+10%FBS group expressed more obviously than DMEN+10%FBS group, thedifference was statistically significant (P <0.05), but only CD34is higher betweenK-SFM group and K-SFM+10%FBS group (P <0.05), it was not statistically significant(P>0.05) among β1integrin (CD29) and CK15two cell surface markers K-SFM+10%FBS and K-SFM.3. Observation under scanning electron microscopic, prepared BAMwas a fiber network structure within visible irregular voids, and without residual cells onthe surface. Masson staining showed that it was clear anatomical structure of the bladderlayers but only see the state of homogeneous thin collagen fibers in blue, and withoutresidual cells. BAM extracts of different concentrations of cells for toxicity grading for0or1. Observed the cell scaffold inverting under the microscope, adhered cell of scaffoldsurface grew in fusiform, cells arranged in a direction consistently, dish bottom celladhered, proliferation in good condition, growing as cobblestone. On the fifth day ofcomposite culturing, dish bottom cell around material converged above80%, The numberof cells on surface increased obviously. Among the cell-scaffold culture, growth curve ofcontrol group and experimental group had no differences, equally got into the plateau on7-8th day. Proved that cells on surface grow well, the hair follicle stem cells and BAMmaterial has good biocompatibility. Conclusion: HFSCs can obtaining by microdissectionmethod, two step enzyme method and differential velocity adherent method. Combinedthe CD34, β1integrin, CK15can identify follicle stem cells. The differentiation of thefollicle stem cells to fat and bone cells, make it becomes seed cells of tissue engineering aswell.2. The type and volume of platelet-rich plasma has a correlation of cell proliferation;platelet-rich plasma and K-SFM+10%FBS improves cell proliferation and plasma has notinfluence to cell purity.3. Bladder BAM is not only less cytotoxic, but also has a goodbiocompatibility with rat HFSCs, thus it can be biological scaffolds of bladderconstructions. After7-8days in vitro, cells on the dish surface and scellular scaffold gotinto plateau which is the best time of transplanting the hair follicle stem cells-bladder acellular scaffold complexes into the rat. This study is an exploration of hair follicle stemcells in vitro, build BAM and Cell-BAM Complexes, In addition it provides importantexperimental basis for planting HFSCs-BAM complex into bodies and making bladdertissue engineering in clinical medicine.