Lab Research Of Tissue Engineered Oral Mucosa Lamina Propria In Vivo And Clinical Research Of Oral Mucosa Lamina Propria

Tissue engineering was the new method in the field of wound repair,it was possible to repair the huge tissue defects by a lots of cells of a piece of tissue. recently Apligraft and Dermagraft were used to repair skin wounds,oral mucosa fibroblasts(OMF)underwent contraction of fibroblast- populated collagen lat-tices(FPCL)group more strongly than that of skin fibroblasts in vitro,so that this study focused at setting up tissue Engineered Oral Mucosa Lamina Propria (OMLP)to repair oral buccal tissue round defects and skin round defects on rats" back by means of type 1 rat tail collagen,OMF of rat,chitosan,et al.oral maxillofacial wound healing was the issue for oral surgeon,no report now on his-tologic research of skin scar and mucosa scar of the deft lip,no report now on the grafting research of oral mucosa lamina propria on the lip skin wound,so that this study did the clinical research for his histologic structure and autograf-ted pieces of oral mucosa in skin incision,to study the wound healing results af-ter grafting.Materials and methodsMaterials;DMEM,trypsin,foetal serum,rat tall 1 collagen(1.8- 1.9 mg/ml),chitosan,antibiotics,monoclonal anti- BrdU,BrdU,et al.Specimens of scars were obtained from 7 re - inpatients,fixed in 10%for-malin,embedded in paraffin,sections,two scars were placed in 2.5%amyldi-aldehyde.OMLP for grafting were obtained from specimens of 21 inpatients(to- gether with cleft palates),the epidermal and fat of lip mucosa were peeled off, then the OMLP was minced and kept in N.S,robust Wistar rats,21 -25 week old.Methods;cell culture,cultures of OMF were obtained from specimens of rats'buccal,palate mucosa undergoing minor surgery,cultures were maintained in serum containing medium and at 37℃in a 5%CO₂ humidified atmosphere at 90 -95%confluence,OMF were split at a ratio of 1;3.Fabrication of FPCL was done as described previously(Zhiqiang Wu's paper 2000).Fabrication of FPCCL was done as described previously(Zhutang You 2003).OMF marked, on the third day,after FPCCL and FPCL were constructed,FPCCL and FPCL in the 24 -well plates were maintained in 10uM SCM confining 10uM BrdU for 48 hours,then the mediums were replaced by SCM.Buccal mucosa defect animal model,21 wistar rats were arranged into two groups,FPCCL group of 12 rats, control one of 9 rats.under general aneasthesia,and antiseptic condition,one side buccal mucosa of rats were performed by round knife(diameter of 0.8cm), then FPCCLs prepared for allografting were moved and placed on the round de-fects,and then covered by petrolatum gauge and common gauge,then sutured and fixed,buccal mucosa round defects were not treated.Newly growing mucosa in the round mucosa defects of rats were biopsied and placed in 10%formalin on the 7th,14th,21st days respectively and postoperatively,then embedded in par-aft'm,then 5urn sections.Histology staining,all sections(mentioned)were stained by H.E,sections on 7th,14th days were detected with immunohisto-chemical assays.Animal model of skin round defects,41 Wistar rats were ar-ranged into three groups,FPCL group of 14 rats,FPCCL group of 14 rats,con-trol group of 13 rats.according to similar methods,skin round defects were per-formed with round knife(diameter of 0.8cm)on the back of rats,Among two experimental groups the round Wounds were grafted by FPCLs,FPCCLs respec-tively and covered with petrolatum gauge and common gauge,then fixed by 6 -0 stitches,the round wounds of control group were covered only with petrolatum gauge,newly growing skin in the round skin defects of rats were biopsied on 4th,7th,14th,21st,26th days respectively and postoperatively,the specimens were placed in 10%formalin and embedded in paraffin.Histology staining,all sections were stained by H.E method,sections on 4th,7th,14th,21th days were detected with immunohistochemical asssays respectively.Patients' sections were stained by.H.E method,Weigert's resorcin- fuchsin method,and routing method of scanning electronic microscope.Surgery style for patients included tennison method and OLMP grafting method,follow- up when reinpatients for cleft palates.Observation for postop-eratively;wound repair procedure of round wounds of 5 rats" groups whether or not infection,blooding and coming off of grafts,wound healing situation of inci-sions of nostril sills.Measurement and statistics analysis of round wounds by Stephens'method with sliding gage,all the round wounds diameters on the backs (and buccal mucosas)were measured on the 4th,7th,14th,and 21st days re-spectively.All the wounds' diameters on the buccal mucosas were done on the 4th,7th,14th days respectively,data was showed by means and analysed by va-fiance of SPSS11.5.Study of specimens" histologic structure under light micro-scope;specimens of the mucosa wounds,skin wounds of upper lips;Study of specimens' OMF of upper lips under electronic microscope.ResultsObservation under macrography;the small bags of 9 rats'buccal wounds ex-isted well,on 4th day,postoperatively without evident redness and edema,the stitches were took off on 7th day post - operatively,recipient wounds were cov-ered with yellowish membrane,on 14th day,there was light on the surface of newly growing buccal mucosa,on 21st day,the color of newly growing buccal mucosa was similar to the normal mucosa.Skin defect animal group,the small bags of 26 rats" skin wounds existed without infection and blood on the first day postoperatively,all stitches were took off,on 7th day,and all wounds of three groups were covered by scab and gauge,but on 14th day,the gauze and scab on the wounds of FPCL group,wounds of FPCCL group(one on 17th day)and con-trol group(one on 17th day)were replaced by the new epidermis naturally.On 21 st day after grafting,all of new skin were smooth without hair,the color of them was similar to the normal one. There was a significant tendency that three groups'diameters became much smaller and smaller on the 4th,7th and 14th days(P＜0.01),then there was not significant difference after 14th day.On the 7th day,the wound diameter of FPCL group was significantly smaller than that of FPCCL group and that of con-trol group(P＜0.01),however,that of FPCCL group is similar to that of control group.On the 21st day after grafting,the new skin diameter of FPCCL was big-gest,that of control group was smallest,although the diameters of three groups wounds were not more significantly different.Observation under light microscope,buccal mucosa defects models,on 7th day postoperatively,the mucosa defect wounds were covered only partially by newly growing mucosa,the ganuletissue of lamina propria was rich in inflamma-tion cells,fibroblasts and vessels and of positive OMF grafted,on 14th,21st days the newly growing epidermal was the same as the control one,rich in big rete ridge,there were lots of inflammation cells and fibroblasts and vessels in the newly.growing lamina propria and some positive OMF grafted,skin defect animal models,on 4th day postoperatively,the decayed tissue on the surface of the re-cipient wounds of FPCL group and FPCCL group separated with lower granula tissue in which were lots of inflammation cells,fibroblasts,new vessels and some positive OMF marked,wound histologic structure of two experimental groups by H.E staining was similar to that of control group,on the 7th day,in the skin defects'wounds of two experimental groups were growing epidermis like front part of sword,and positive OMF,the controlgroup was the same as others. on 14th day,the recipient wounds keratinocyed thoroughly with rite ridges,in new dermis were some positive OMF,but in control group,there was not almost evident rete ridge,on 21st day,fibers in new dermis were small and tiny,many vessels with positive OMF,control group keratinocyed without rite ridge on the 26th.day,the new skin structures by H.E staining were similar to those on 21st day.Study to the scars of cleft lips under light microscope,the epidermises of upper lip incisions consisted of four kinds of cells,the membrane of the scars u-sually was parallel to the surface of the epidermises of new skin,epidermises of upper lip mucosa incisions consisted of 3 layers of keratinocyts,their rete ridges were long and large,fibers in skin scars were larger than that of mucosa scar of lips.there were elastic fibers in the skin scar,but not in mucosa scar,papillia dermis of mucosa scar was rich in vessels,the fibers in it were thin,short of e-lastic fibers,there were a few long fibroblasts,under scanning electron micro-scope,the fibroblast plasm in mucosa scar was not rich,two fibroblast plasm in skin scar were not rich.During 7th day postoperatively,incisions and skins of the 21 nostril sills mentioned above healed well without redness and evident ede-ma,the follow up investigations showed that 15 incision scars of nostril sills were flat and the similar color to the normal skin one 1.5 year later postoperatively.ConclusionOMF as the new seed cells joined in the repair and reconstruction of skin and buccal mucosa defects effectively and feasibly.Collagen httice and chitosan -collagen lattice had good histocompatibility and acted as extracellular matrix,chitosan - collagen lattices limited the con-traction of recipient area more significantly than the collagen lattices did.FPCLs and FPCCLs repaired and reconstructed the skin or mucosa defects effectively and feasibly,quality of new skin of two experimental groups was bet-ter than that of control one.Histologic character of lip,mucosa incision scar,long and large rete ridge, long papilla almost closed to the surface of the epidermis,dermal papilla was rich in blood vessels,fibers were thin,short of elastic fiber.OMLP was autografted effectively in skin incision of upper cleft lip without allergic reaction,OMLP could accelerate the healing of skin wound.