| Part 1.The Construction of Laser Microporous Porcine Acellular Dermal Matrix(LPADM)In this part,LPADM material combined with chemical hypertonic saline acellular cells and cross-linking of glutaraldehyde were used to form LPADM,which was formed through 3D matrix formation by ultraviolet laser.Bacteriology,gross,histology,scanning electron microscopy and hyperspectral image smart identification were used to analyse the basic feature of LPADM.Objective:To construct laser microporous porcine acellular dermal matrix(LPADM).METHODS:One healthy white pig with body mass of about 25Kg was sacrificed and washed with soapy water.After stripping,the whole skin was stripped.After defatting,the skin was soaked in 0.1%benzalkonium bromide for 15mins,rinsed with saline,Knife take 0.2mm thick blade thick skin,remove the epidermis and basement membrane layer,then cut about 0.3mm thick layer of dermis,divided into two parts for the following treatment.(1)for one part of the pig leather,we usea unique laser template hanging hole technology.Fix the leather sheet suspended on the platform of the laser processing equipment to keep the upper and lower surfaces of the leather sheet smooth and moist.The parameters of the adjusting equipment are the minimum output power density 10000W/mm2,the hole clearance 1mm,the aperture 0.1-0.8mm(preferably 135um)The aperture and aperture gap laser template was used to guide the formation of 100 glitches in the 1.0 cm*1.0 cm area to rapidly breakdown the sample to obtain a laser microporous porcine dermal matrix.2%NaCL,0.5%glutaraldehyde to remove microporous porcine dermal cells and cross-link.Obtained LPADM,60Co irradiation sterilization made of 5.0 cm × 5.0 cm size,4 ? refrigerated storage.(2)the other part of the pig leather is not perforated,acellular and cross-linked as above to make the same size non-hole pig ADM,according to the same technical means acellular and cross-linked,4 ? cold preservation.The prepared LPADM was subjected to gross,histological,scanning electron microscopy,and hyperspectral analysis.Results:LPADM,non-porous PADM was porcelain white,soft,flexible,histological examination showed that dermal matrix decellularization was clean,no cellular components.In addition to the above morphological characteristics,LPADM has penetrating circular,uniform pore structure.Histological examination showed that LPADM microporous edge laser was lightly carbonized.Scanning electron microscopy showed collagen fibers arranged in an orderly pore.LPADM micropore laser processing,the spectral curve in the 20 band,showing the characteristics of deep "V" fingerprints,the organization showed an obvious difference in characteristics of the fingerprint in a great degree,suggesting that the material itself before and after management is of different traits.Conclusion:LPADM is a new type of acellular dermal matrix with innovative physical structureand structural characteristics.Part 2.LPADM in vitro experimentsThis part consists of two parts.The first part of the experiment:LPADM-Fb co-culture and early detection of a variety of cytokines in culture medium were used to verify the low/non-toxic LPADM,good cell compatibility,and demonstrated the necessity and rationality of changing LPADM in vitro into an active material of LPADM containing active fibroblasts in advance at the same time.There is no need for this in vitro operation.Part two:BMSC and ADSCs derived from mesenchymal stem cells were co-cultured with LPADM in vitro respectively.The activity of LPADM-BMSC was not significantly affected by the formation of LPADM-BMSC.It is demonstrated that LPADM has a good combination of multiple cell-compatible functions as a biological platform for cell culture.Lay the foundation for the next step in vivo test.OBJECTIVE:LPADM was co-cultured with fibroblasts,mesenchymal stem cells,BMSCs and ADSCs in vitro respectively to verify the impact of LPADM on the cytotoxicity,cell compatibility and cell biological activity of thethree kinds of cells above.Methods:Primary cultured fibroblasts and bone marrow mesenchymal stem cells were co-cultured with LPADM in six-well culture plates in vitro respectively,which were divided into the following five steps:(1)Prepare LPADM and nonporous PADM of 2.0cm x 2.0 cm x 0.3 mm,respectively.The cells were placed in 6-well culture dishes and cultured in Fb medium supplemented with 10%FBS in DMEM medium.One SD rat was isolated and cultured in its own skin Fb.The third generation of Fb was inoculated into the two kinds of ADM surfaces at a density of 1 x 10 5/mL,respectively,which were LPADM-FB group and nonporous PADM-FB group.(2)40 times inverted phase contrast microscope were used to observe the growth of Fb in each group.On the 1 st,3rd and 5th day after inoculation,the absorbance at 492 nm of Fb active factors IL-10,IL-6,TGF-?1,LN and VEGF in each group were measured by double antibody sandwich ABC-ELISA,6 samples at each time point in each group,the specific operation is according to the kit instructions.(3)Take the third generation of adherent cells of BMSCs to make cell suspension,adjust the cell concentration to 1 ×104 cells/mL,and inoculate 2 6-well plates according to 1 mL of cell suspension per well,and set up 3 holes for the osteogenic group and adipogenic group,the other three holes were set as negative control groups,and then identifying the differentiation.Osteoblasts were cultured in osteoblast-induced medium(high glucose DMEM containing 10%FBS,100 u/mL penicillin-streptomycin PBs,5 mmol/L glycerophosphate,g/mL ascorbic acid,2-phosphoric acid,1 x 10-7mmol/L dexamethasone).The adipocyte-induced medium(adipose-induced DMEM medium containing 10%10ug/mL insulin,1 x 10-3mmol/L dexamethasone,0.5mmol/L isobutylmethylxanthine,200umol/L indomethacin).The culture medium of negative control group was not changed,and the medium was changed every 2 days.After induction for 10 days,the 3-hole cells in the osteoblast group and the adipogenic group were covered with 4%formalin,lg/L alizarin red and 70%ethanol,respectively.Observing under the light microscope.(4)The LPADM was cut into a size of 2 cm x 2 cm in a clean bench environment.The microporous dermal matrix was placed on a 6-well plate with its epidermis side up,and infiltrated into low glucose DMEM medium containing 10%FBS.Placed in 37 5%CO2 saturated humidity incubator and stored for 4 h.The third generation of well-grown BMSCs were trypsinized into cell suspension and adjusted to a density of 4 × 10 5 cells/mL.One mL of the cell suspension was seeded onto the surface of the LPADM of the six-well plate and placed in a 37 ?.,5%CO2 saturated humidity incubator for 4 h and then change medium,cultured in 37 0?,5%CO2 saturated humidity incubator changed the medium in every 2 d,a total of 5 days in vitro culture.A similar batch of non-porous dermal matrix(PADIM)was seeded in the same manner.The microporous and nonporous dermal matrix materials containing BMSC-LPADM and BMSC-PADM were then constructed.(5)As above,the adipose-derived stem cells(ADSCs)were purchased and passaged for the third generation in vitro.The cells were co-cultured with ADSCs-LPADM in vitro to observe the proliferation,growth and migration of ADSCs.Results:(1)The fibroblasts were co-cultured with LPADM.When they reach the third generation,their morphology tended to be the same as Fb-like and grew rapidly;(2)There was statistical significance(F was 0.050?1.763,P>0.05)of absorbance values at 492 nm of Fb cytokines(IL-10,IL-6,TGF-?1,LN and VEGF)at 1,3 and 5 days of inoculation measured by double antibody sandwich ABC-ELISA;(3)The morphological,adipogenic and osteogenic differentiation and differentiation of BM-MSCs and the co-growth characteristics of BM-MSCs-BMSC-LPADM.Isolated mononuclear cells had strong refractivityin a round inverted phase contrast microscope.After culturing for 2 days,some cells grew adherently and appeared as fusiform.After culturing for 5 days,the cells had appeared to proliferate.When cultured for 12 days,the confluency rate of cells reached 80%,and the cells showed as Fbat this time.By the third generation,the morphology of the cells tended to be the same as that of the Fb-like cells,and the cells grew rapidly and were passaged once every 7 days.Osteoblast-induced culture medium cultured for 3 days,cell density increased,showing as short spindle.After culturing for 5 days,the cells were in different morphologies and some of the cells showed polygons.After culturing for 7 days,small calcifications were observed in the cells.Culturing for 10 days to form obvious calcium nodules;negative control group increased cell density,no calcium nodules appeared.Osteogenic cells scattered calcium nodules using Alizarin red staining,showing dark red under high magnification,the negative control group showed no significant change.Adipocytes were cultured in adipocyte medium for 3 days.Small lipid droplets were seen in the cells,and some cytoplasm reflections were enhanced.After 5 days,small lipid droplets appeared in the cytoplasm and gradually fused.After 10 days,obvious lipid droplets appeared and cells containing lipid droplets turned a round or polygon shape;negative control group cells did not change significantly.Adipogenic group see a large number of adipose cells containing red lipid droplets using Oil red O staining,negative control group showed no significant change.(4)Adipose-derived stem cells(ADSCs)-LPADM observed ADSCs cell proliferation activity,growth and cell climbing.1-2 days covered with 12-well plate base.CONCLUSION:After acellular and laser continuous porcine dermal tissue with microporous(gasification)protein-derived material,the biological activities of the three kinds of cells were co-cultured with fibroblasts,mesenchymal stem cells and ADSCs in vitro respectively.The three cells showed normal bioactivity.LPADM showed no obvious cytotoxicity;LPADM had good cell compatibilityas a dermal substitute,andcan provide 3D bio-derived platform for two kinds of terminal differentiated cells-fibroblasts,mesenchymal origined stem cells(bone marrow mesenchymal stem cells,Adipose-derived stem cells)of cell culture in vitro.Part 3.LPADM transplantation experiment in vivoThis part includes the following five parts.First step,the evaluation of histocompatibility and vascularization ability was demonstrated by subcutaneous burial experiment.In the second step,free medium and thick skin graft was used to test the degree of early vascularization in LPADM.The third step was continuous dynamic acquisition in vivo Histopathological HE histological images,hyperspectral analysis of LPADM spectral characteristics,optical characteristics of fingerprints to access its feasibility and characteristics as a process of vascular process of transplantation;The fourth step,LPADM-BMsc transplantation in vivo,the phenomenon of nascent differentiate into skin appendages of exogenous BMsc suggests that LPADM may have a potential role as a carrier of BMsc and continue to promote BMsc differentiation.The fifth step is to use another kind of human adipose-derived endogenous stem cell to demonstrate the stability and general applicability of LPADM as a "carrier" for stem cell transplantation in vivo.Objectives:1.To evaluate the cellular,biocompatibility and vascularization ability of LPADM in vivo.2.LPADM complex of bone marrow mesenchymal cells containing bone marrow-derived mesenchymal stem cells(BMsc)Annexe cell regeneration repair role;3,LPADM can provide more ideal mesenchymal stem cells 3D biological platform.method:(1)Subcutaneous burial test.Twenty-one rats were taken out and anesthetized by intraperitoneal injection of 30 g/L sodium pentobarbital 50 mg/kg after 24 h dehairing with sodium sulfide.The symmetrical "U" shape was cut off from the entire back of the spine to the deep fascia to make a 2.0 cm x 2.0 cm wound.The LPADM and the nonporous pig ADM were cut into corresponding sizes and transplanted on the fascia layers on the left and right respectively.The wounds were packed and fixed in a single cage.At 1,3 and 10 days after operation,the changes of body weight,mortality and skin allergy,infection signs and so on were observed.Meanwhile,histological examination of LPADM and non-porous pig ADM specimens was performed.(2)medium-thickness skin graft test.A total of 35 rats were used to establish a 2.0 cm x 2.0 cm "mouth" type of wound on the back of the rats.The LPADM was placed on the surface of the deep fascia.The entire thickness of the wound was thinned into medium Thick skin graft simultaneously transplanted on it,stitching,packing fixed,single cage feeding.The change of body weight,mortality,skin allergy,infection signs and so on were observed.At the same time,an examination of Graft survival criteria for the early are skin flap ruddy,flat,and the skin base closely attached.(3)LPADM specimens collected from 1-15 days in(2)in different time periods were made to HE pathological sections for hyperspectral imaging analysis.(4)BMsc-LPADM in vivo transplantation test.One SD rat was sacrificed.Femur and tibia were taken from bone marrow-derived bone marrow mesenchymal cells isolated,take the 3rd generation adherent cells osteogenic and adipocyte differentiation experiments.After that,they were inoculated on LPADM and nonporous PADM to construct bone marrow mesenchymal cells.LPADM and Bone Marrow Mesenchymal Cells-Nonporous PADM.Twenty-one healthy nude mice were randomly divided into 2 groups:BMSC-LPADM + non-porous pig ADM group(A group,6 rats),LPADM + blade thick skin graft group(B group,(P<0.05),and there was no significant difference between the two groups(P>0.05)2 cm x 2 cn,the full-thickness skin defect wound up to deep fascia,while taking the same size blade thickness flap spare,and respectively transplanting the corresponding material covering the wound on the 5th,7th and 14th day after transplantation,observe the operation area of the nude mouse Healing and adverse reactions were observed.All nude mice were sacrificed and all the implants were excised.HE staining was performed to observe the histological changes.The newborn skin appendages in the corresponding complexes were observed under transmission electron microscope 7 and 14 days after transplantation.(5)LPADM + adipose derived stem cells(ADSCs)live animal imaging test.LPADM+ ADSCs were constructed in vitro.The rats were sacrificed on the back of the transplanted mice.The experimental group on the left was LPADM + ADSCs.The control group was LPADM on the right.On the 5th,7th and 14th after surgery,observe the ex vivo activity of exogenous ADSCs and proliferation and migration were observed by small animal biopsy technique.Results:(1)LPADM subcutaneous burial experiment.HE examination:After 24 h of LPADM transplanted in the back of the SD rat wound,collagen deposition can be observed around the material,no new tissue and neovascularization;the new type of vascular endothelial cells under the transmission electron microscope to observe.At 3 days after operation,the LPADM material was significantly reddish and adhered tightly to the fascia and its surrounding tissues.HE staining showed that LPADM laser drilling was used to grow new tissue,in which the neovascularization was clearly visible and contained multiple signs of red blood cells with rare inflammatory cells.At 10 days after operation,the vascularization of LPADM was networked.Transmission electron microscopy did not see non-porous ADM newborn vascular endothelial cells.Postoperative non-invasive ADM histological observation of inflammatory cells in the wound,but no blood vessels.(2)LPADM combined with medium thickness skin graft experiment.1.The two groups of animals survived after transplantation,but the color and appearance of skin grafts were different at different time.The skin grafts of group LPADM and group PADM were pale no obvious difference in blood color,and the general difference was not obvious 24h after operation.72h after operation,LPADM group of skin grafts showed a white base color,its LPADM is visibly reddening,tight adhesion,,group PADM skin pale,its PADM was porcelain white,.It can pull down the natural light separation,apparent exudate,with different taste 5 days after operation.the LPADM group showed the basic pink skin,and the skin grafts in group PADM showed a different degree of black,some even dry or wet.Its smell is aggravating;10 days LPADM group showed pink skin.14 days after operation,the experimental group wound skin graft fusion,and had a good growth of skin,no scar formation.The skin necrosis in the control group was significant.2.The healing rate of the wounds 2 weeks after operation,the composite skin all survived in the experimental group,and most of the epithelium in the control group died of necrosis.The wound healing rate in the two groups was 99.40%± 0.24%compared with 25.41%± 1.32%(p<0.05)Sex).3,HE tissue examination results 24h after operation.It can be seen leukocyte infiltration and exudation of cellulose,no neovascularization,in LPADM pores.At 72h after operation,neovascularization was seen in the microwells,with multiple erythrocytes and few inflammatory cells.After 5 days of operation,LPADM showed a good inflammatory reaction,and a large number of naive capillaries flushed upward from the basal surface of the wound;At 10 days after operation,the blood vessels around LPADM were network-like,and the inflammatory cells were slightly dispersed.On the 15th day,the vascularization around LPADM was rich and the inflammation was mild.Nonporous PADM,postoperative HE observation showed a large number of inflammatory cells in the wound,no blood vessels.4.The migration of fibroblasts in the two groups of ADM collagen network and the comparison of the 1,3-day post-operation group showed that the growth of rubeosis tissue was observed at the bottom of the micropore structure.The fusion of LPADM with the basal tissue was found in the drawing process and the control group was relatively loose,Light easy to separate from the substrate.Three days after the operation,the experimental group showed that there were many new tissues in the micropore structure that grew from the basal to the inside of the pores,and more fibroblasts were migrated into the collagen around the pore structure.The control group of non-perforated collagen network can even see the migration of fibroblasts.5.Electron microscopy results.At 3 days after the scanning electron microscopy showed that the experimental group of basal tissue and nascent granulation tissue through the pore structure along the pore size perpendicular to the epidermis to the LPADM collagen network growth in small blood vessels within the growth of new tissue collagen network Fibroblasts in the growth,proliferation,division and secretion activities provide a good microenvironment.After 14 days of transmission electron microscopy showed that although the experimental group and a small amount of inflammatory cells in LPADM immigration,moving into the fibroblasts large irregular nuclei,split strong cellular fluid filled with rough endoplasmic reticulum,Fibroblasts play a role in the regeneration of the dermis,while the newly secreted collagen rebuilds the dermal matrix collagen network with the original dermal matrix as a template.The control group of PADM newborn tissue is difficult to grow into the PADM structure after decellularization antigen sites due to lack of adequate fibroblast repair and prolonged exposure,making the migration of PADM collagen structure of the cells to inflammatory cells.1,Generally observation.Autologous medium-thick skin survived 14 days after transplantation.30d to mention the operation area skin,skin healing good quality.Histological examination showed that LPADM had sufficient vascularization,and no apparent dissolution,absorption,reduction,or encapsulation of LPADM Non-observed.The rats were all survived by compound transplantation.No obvious local or systemic allergic reaction was found,and their daily behaviors were normal and their body weight increased normally.2,Partial observation of transplantation area.Animals were alive in both groups,but the grafted skin at different times the color appearance of significant differences.LPADM group and PADM group at 24h were pale,no color,no significant difference between the two groups;The group of 72h LPADM presented a basic pink skin,LPADM under its material was significantly red,and fascia and the surrounding tissue adhesion,PADM group of pale skin,which PADM was porcelain white,with the fascia and surrounding tissue adhesion in general,gently pull the natural separation,exudate obvious,accompanied by smell;5 days of LPADM group showed pink skin,PADM In the group of LPADM for 10 days and 15 days,the skin of the LPADM group was obviously pink,the skin of PADM group was completely black,dry or damp,and the pus,The difference is significant.3,HE tissue examination results.At 24h after operation,LPADM pores can be seen leukocyte infiltration and exudation of cellulose,no neovascularization;At 72h after operation,there is no new blood vessels within the pores,containing multiple red blood cells,rare inflammatory cells 5 days after operation.LPADM showed a good inflammatory reaction,a large number of naive capillaries from the basement of the wound surface upwards to the micropore;At 10 days after operation,the blood vessels around the LPADM were network-like,the inflammatory response was good,the inner wall of the laser microporosity ADM color and hole around the integration of blue denatured tissue weakened.On the 15th day,the vascularization around LPADM was rich and the inflammatory response was good.The color of the inner wall of the laser microporosity was faded and the ADM around the pore was more compatible.Nonporous PADM,postoperative HE observation showed a large number of inflammatory cells in the wound,no blood vessels.(4)Dynamic hyperspectral analysis of HE specimens.1?LPADM laser hyperspectral analysis showed that LPADM green curves in vivo were compared with LPADM full-band spectroscopy(0-day LPADM,black curve),which were not involved in in vivo experiments.The green curve of LPADM in vivo showed a deep "V" shape along the horizontal axis.However,with the passage of time from 1 day to 15 days,the spectral and vertical axis brightness of local structure of LPADM pore wall gradually increases from near 1500(0 days)of LPADM to near 3000(15 days),indicating that,with the compatibility of tissues,inflammation,necrosis of the inner wall of the hole after the improvement of absorption,transmittance also increased accordingly;2?After LPADM transplantation,hypersensitivity analysis of erythrocytes in the micropore.Compared with the full-band red blood cell curve(black curve)in normal skin,the red curve of LPADM showed the characteristic "V" in the bands around 10,25 and 40,which were different from the mature red blood cell curve,However,with the passage of time,the erythrocyte spectral curve in the neovasculature gradually coincides with the spectral curve of normal mature erythrocytes.For example,on the third day,the brightness value of the 10 band has reached 3600,which is close to the normal 3800;on the tenth day 10-band brightness value has reached the normal 3800;that on the 3rd day LPADM neovascularization vitality is close to normal mature skin blood vessels;3.After LPADM transplantation,hyperspectral analysis of inflammatory cells in and around the micropores showed that at the same time period,the ratio of the inflammatory cells in the micropores(green curve)and the surrounding inflammatory cells(blue curve)The curve shows the corresponding deep "V" shape in the band of 20 on the horizontal axis,but there is no obvious difference between them.With the increase of time,the luminosity value of the inflammatory cells did not show obvious ups and downs,indicating that after the laser penetrating,chemical decellularization None of the rats had any stress on the immune system and affected the normal wound repair in rats.(4)BMsc-LPADM in vivo transplantation test.1,LPADM,non-porous porcine ADM was porcelain white,soft,flexible.Histological examination showed no dermal matrix cell components;scanning electron microscopy showed pore collagen fibers arranged in order;LPADM microporous structure.2,After the cells passed to the third generation,the morphology tends to be consistent,Fb-like,rapid growth.3,Induced differentiation experiments show that cells can differentiate into osteoblasts and adipocytes.4,At 5 d after transplantation,A group of non-porous ADM local dry,dry skin necrosis in Group D,A,D group were not infected and inflammatory reactions;B,c graft skin graft survived.On the 7th and 14th day after transplantation,the partial color of the covering on the surface of group A was black,dry and hard;the skin of group D appeared completely necrotic under darkness,and the yellowish exudate was visible under the skin;Obvious purulent discharge.B,c skin appearance and the color of the skin around the close;5.At 5 and 7 days after transplantation,the vascularization was observed in the micro-pore structure of dermal matrix in group A,B and c,and the visible erythrocytes were seen in it.At 14 days after transplantation,the micro vesicular structures of the dermal matrix of group A,B,c were completely vascularized,and a large number of erythrocytes were seen in them.In group A,the microporous dermal matrix survived,Hog ADM did not bind tightly;Group B,c skin and dermal matrix Q asked closely,no skin appendages in the skin,c skin tissue dermis and the junction of the dermis can see special monolayer cells;6,D group skin graft failed to survive,and give up electron microscopy.At 7 days after transplantation,there was no significant difference in the TEM images of groups A,B and C:on day 14 after transplantation,no sebaceous-like and sweat-like cells were observed in the grafts of groups A and B,Fb immigration,C group wound surface thick leather and dermis matrix junction can be seen a large number of nascent capillaries hyperplasia,Fb rough endoplasmic reticulum proliferation proliferation,showing the new unmyelinated nerve endings;in the dermal matrix shallow,there is a single free Sebaceous and sweat gland-like cells.(5)LPADM combining with adipose derived stem cells(ADSCs)live animal imaging test.On the back of the transplanted mice,the left experimental group:LPADM combining with ADSCs,.the right control group:LPADM.The mice were housed in a single cage for 7 days and 14 days and then were taken to the living body imaging room.3.5%chloral hydrate intraperitoneal injection of anesthetized mice,each injection of 0.2mL.Imaging in a small animal imager(IVIS LuminaII)and recorded.Fluorescence was observed and proliferation and migration of the stem cell-free zone to the wound area was observed.Conclusions:LPADM is a new dermal substitute with low immunogenicity and early stabilizing vascularization.The characteristic fingerprint of LPADM can be quickly identified by vascularization and the fingerprint spectrum can be used as an identification marker by hyperspectral analysis.LPADM helps promote cell repair and even repair of skin appendages. |
PDF Full Text Request