The Preliminary Study On Biological Transition Of Acellular Dermal Matrix

Skin damage is a common clinical problem in the field of surgery.As a new dermis substitutes, acellular dermal matrix(ADM)has been primitively used in Neurosurgery,Otolaryngology,Burns, Periodontology and so on.But there are many controversies among domestic and foreign researchers remoulding and transition of ADM in vivo.In this study,ADM manufactured by the Dispase-Triton method was used to implant beneath the back skin muscle of rats in order to observe its biological transition.Objective:Investigate the remoulding and transition of ADM in vivo.Method:Part one:preparation of ADM and allogenic dermis.8 healthy grown male Wistar rats were selected for this experiment.The entire level skin was cut from the rats' back with 4×4cm in size. Those skin pieces were incided to cortices with 0.6mm in thickness using the drum dermatome and then were sheared into skin pieces with 2×2cm in thickness,which number is 32.The skin pieces stochastically were divided into two groups with 16 in each group.Each group of skins piece were incubated respectively in 2.5U/ml Dispase at 4℃and 1M/L NaCl solution at normal temperature for 48hours to remove epidermis,then respectively thrown into 0.5%Triton X-100 solution for 48 hours and 0.5%SDS solution for 6 hours,which were kept on shaking to take off cell composition at normal temperature.The histology and the molecular biology methods were used to examine the quality of ADMs prepared by two methods after samples acquired.At end chose another 4 rats to prepare for allogenic dermis only removing epidermis by Dispase.Part two:establishment of animal model.16 healthy grown male Wistar rats were selected for this experiment.Straight incisions were fabricated with 2cm in length on the upper rats' back which were parallel with vertebral column and on the left of it.To make the incision becoming trapezoid,the two verges should extend to axifugal direction with the angle 45 and 0.5cm in length.The full-thickness of skin and cutaneous muscle were incided to get to the surface of fasciaprofunda,and demixed sluggishly in order to require enough space.The ADMs(2×2cm in size) were flatten and fixed to fascia with 0~# suture line after being filled in the space.The ADMs were covered by skin flaps and sutured intermittently in situ.The incisions were covered by petrolatum gauze and absorbent gauze and fixed safely with bandage and rubberized fabric.The samples were acquired at the time points of 3d,1,2,4w after operation to observe the stability of the model.Part three:study on transition of ADM and allogenic dermis.112 healthy grown male Wistar rats were selected for this experiment.14 rats were used to prepare ADM(applying the Dispase-Triton method),and 14 for allogenic dermis.The surplus animals(84 in number) were divided into two groups stochastically for ADM group 42 and allogenic dermis group 42.Then ADM and allogenic dermis were implanted using above animal model.The animals were killed to acquire samples at the time points of 1,2,3,4weeks,2,4,6 mouths after operation.Samples were processed with routine HE dyeing and the picric acid-Sirius red collagen special staining, simultaneously examined the collagen total quantity(hydroxyproline,OHP),and applied the immunohistochemical method to detect the content of matrix metalloproteinase-1.Through the above analysis,we could sum up the change rule of thickness,collogen total quantity,Ⅰ/Ⅲcollagen proportion,MMP-1 activeness and fibroblast counts in ADM and allogenic dermis at different time point.Result:Part one:ADM manufactured by the Dispase-Triton method had removed cell component completely,maintained the collagen structure and arrangement normally and theⅠ/Ⅲcollagen ratio in it hadn't statistical difference with normal skin.ADM by the NaCl-SDS method had much cell debris and theⅠ/Ⅲcollagen ratio was higher than that in normal skin.Part two:3 d,1,2,4w after being implanted,ADMs were seen flat in space and no wrinkle,no significant leakage,and inosculated with peripheral tissues gradually;the count of fibroblast increased piecemeally(peaked at 2w),decreased significantly at 4w in microscope and did not see any obvious inflammatory cells.3 d,1,2,4w after being implanted,there was significant leakage and allogeneic dermis was flat in space,also obvious inflammatory reaction in peripheral tissues. The effiusion disappeared feckly at 4w.In microscope there were many inflammatory cells all the time and fibroblast could be found.Part three:(1) fibroblast count:the cell counts in ADM and allogeneic dermis peaked at 2w (ADM more than allogeneic dermis),then trended to decrease all along;the decreasing range of count in allogeneic dermis was small,which made cell count more than that in ADM after 4w. There was statistical difference between the two materials at all time points(P＜0.05).(2)Ⅰ/Ⅲcollagen ratio:the ratio of the two material had been declining and there were no significant difference(P＞0.05).The ratio in allogeneic dermis increased gradually after 4w and ADM remain original lever;there was statistical difference between the two materials at those time points (P＜0.05).(3) OHP content:the OHP content of ADM peaked at 4w and declined gradually to the lever of null point;the OHP content of allogeneic dermis reduced at 1w firstly then rose to culminated point at 4w and then decreased persistently.There was statistical difference between the two materials at all time points(P＜0.05).(5) MMP-1 activity:MMP-1 expressed on endochylema of fibroblast.The MMP-1 activity in ADM arrived at tiptop at 3w then reduced to the lever of null point;the activity in allogeneic dermis peaked at 4w then declined slowly but higher that in ADM all along.There was statistical difference between the two materials at other time points except 4w(P＜0.05).Conclusion:1.ADMs manufactured by the Dispase-Triton method were excellent than ADMs made by NaCl -SDS method.2.The animal model of ADM grafted in vivo could guarantee ADM to participate in the organism rebuilding in the stable environment.3.ADM could inosculate with organism tissues to participate in normal rebuilding.Collagen synthesis and degradation in ADM achieved balance primitively after being implanted 4w. Then ADM could maintain long-dated stability and possess major clinical application value.The collagen fibers in allogeneic dermis degraded gradually to lead to resolution of collagen scaffold.4.Fibroblast played the foundational and main role in the biological transition of ADM.