| Background:Clinically,inflammation,full thickness burn,surface tumor resection,acute trauma,chronic skin ulcers and other diseases can cause full-thickness skin defect.At present,the prevalence of the impaired skin wound is higher,and the treatment for them is difficult,often disappointed,which gives rise to a negative impact on the society and economies.Skin wound healing is a complex process involving many cell types and processes,such as epidermal,fibroblastic,and endothelial cell proliferation,cell migration,extracellular matrix(ECM)synthesis,and wound contraction.In clinical,there are commonly used methods to repair full-thickness skin defects such as autologous free skin graft,skin flap transplantation,allograft skin/skin graft and artificial substitute.At present,the treatment of skin defect is mainly carried on autologous skin graft which has some drawbacks in color,texture,scar hyperplasia,donor site healing.Meanwhilie,the number of donor skin is limited.With the continuous development of tissue engineering,tissue engineering skin application has received extensive attention.At present,tissue engineering skin products of the clinical application mainly include artificial skin,artificial dermis and composite skin.Currently available skin substitutes for wound healing often suffer from a range of problems,such as wound contraction,scar formation,poor integration with host tissue,and early poor vascularization.The current clinical application of tissue engineering skin is limited,and the success rate of its transplantation is not high,mainly due to the lack of vascular structure in the early stage of transplantation,not be established in a timely manner.Particular concern is that scars have psychologically associated issues such as esthetic disfiguration and pain that significantly affect the life quality of patients,besides loss of tissue function in various degrees.Tissue engineering approaches,the combination of stem cells with a biomaterial-based matrix,have gained more and more attention for skin wound healing over the years.However,there exist many obstacles,especially how to introduce proper cues to induce skin regeneration while avoiding scarring and promoting neovascularization.Bone marrow mesenchymal stem cells(BMSCs)have been reported as attractive players in the improvement of wound healing,and they actively participate in different phases of wound healing.BMSCs participate in attenuating inflammation in the wound,enhancing the secretion of angiogenic factors and reprogramming the resident immune and wound healing cells to favor tissue regeneration and inhibit fibrotic tissue formation.As a result.BMSCs have been considered and tested as a potential candidate in cell-based therapy to promote wound healing while reducing scarring and improving the new blood vessel of the wound.Biomaterial scaffold is the bridge of' tissue engineering research,which provides the necessary space for cell proliferation,differentiation,nutrition,metabolism and other physiological activities,and plays an important role in the construction of the organization.Therefore,the pros and cons of biomaterial scaffold,not only affects the biological behavior and the effect of culture,but also determine whether the cells can be well adapted and combined with the host.so as to affect the final repair of damaged skin.Graphene foam(GF),a three-dimensional(3D)porous structure,has been recently utilized in the field of tissue engineering.3D-GFs exhibit a monolith of continuous and porous structure.There are reports and studies demonstrating that the porosity of the biomaterial is up to 99.5 ± 0.2%and has a pore size of 100-300 ?m that may possess high water retention capability to maintain wound's moisture.Therefore,the 3D-GF structure is a promising candidate for a novel cell scaffold as it may incorporate topographical,chemical,and electrical cues in the same scaffold to provide an environment for skin tissue regeneration that is superior to conventional inert biomaterials.The unique properties of two-dimensional(2D)graphene can induce stem cell to preferentially differentiate into specific lineages.The 3D-GFs were recently reported as a biocompatible and conductive scaffold,which directed the differentiation and proliferation of neural stem cells,and the differentiation of BMSCs into bone.Of clinical interest,the 3D-GFs may direct the differentiation and proliferation of cells cultured inside,and the porous 3D structure can provide 3D microenvironments in which cells are able to resemble their in vivo counterparts during skin wound healing.In addition,graphene materials demonstrate strong antibacterial and antifungal properties in wound infection.The above properties of GF provide a new way for the treatment as a tissue engineering cell scaffold.In this work,we hypothesized that bone marrow derived MSCs together with 3D-GF biomechanical and biochemical features synergize to provide a better wound healing environment,enhance early vascularization and reduce scarring.Based on the above background,we will isolate and culture BMSCs from rat bone marrow,then transplant them into damaged skin wound,tegether with 3D-GFs,and observe the effect of wound healing,neovascularization and anti-scarring.ObjectiveTo detect biocompatibility of the 3D-GF scaffold;to observe the effect of 3D-GFs and bone marrow mesenchymal stem cells in wound healing;and further to observe the effect of 3D-GFs and bone marrow mesenchymal stem cells in the neovascularization and reducing scarring;eventually to provide new ideas and methods for the treatment of skin wound,and lay the foundation for its application in tissue engineering.Methods1.The femoral and tibial bone marrow cells were taken from adult male SD rats and then used to isolate and culture MSCs by means of the whole bone marrow culture method.The third generation cells were used to detect stem cell surface antigen marker CD54,CD73,CD45 and CD34 expression by flow cytometry,and to detect osteogenic and adipogenic capacity simultaneously.Changes in cell morphology were observed under the inverted microscope,and the induced effect was further observed by oil red staining and alizarin red staining.2.The three-dimensional graphene foams were synthesized by chemical vapor deposition method,by scanning electron microscopy and metallurgical microscope to observe the surface morphology,energy dispersive spectrometer and Raman spectroscopy to analyze the composition of the samples.3.The rat BMSCs were respectively seeded on the 3D-GFs and tissue culture polystyrene(TCPS).The cell proliferation was detected by MTT method,LDH method and BrdU method.Meanwhile,the growth of rat BMSCs in the 3D-GF was detected by immunofluorescence assay.4.Animal experiment:The male SD rats,weighing 220-300g,were assigned randomly to three groups:(1)empty wound(control),(2)scaffold alone(GF).and(3)scaffold plus mesenchymal stem cells(GF+MSCs).The full-thickness skin defect wounds,perfomed in the dorsum of each animal,were covered with transparent dressings and fixed by elastic bandage.Animals were kept in individuals cages with food and water ad libitum,and observed daily during the total period of the experiment.At days 3,7,10,and 14 post-surgery,animals were euthanized by CO2 inhalation and the explants retrieved and processed for real time RT-PCR to detect the expression of vascular endothelial growth factor(VEGF).basic fibroblast growth factor(bFGF)and transforming growth factor-betal(TGF-?1),transforming growth factor-beta3(TGF-?3),alpha-smooth muscle actin(a-SMA).At day 14 post-surgery,the regenerated skins were stained for hematoxylin&eosin(H&E),and at days 7,14 post-surgery,the contents of soluble collagen in the regenerated skins were examined.After wound healing,the healing skins were used to detect hydration,skin moisture loss,tensile strength and elastic displacement.Results1.By means of whole bone marrow culture method,BMSCs were successfully isolated.On fifth day.cells grew in in a manner of colony,mainly with the forms of spindle cell,and growed vigorously.There was no obvious degeneration after continuous 4 generations.Flow cytometry analysis demonstrated that the cells isolated in the study were positive in MSC surface markers of CD54(98%)and CD73(97%)while negative in hematopoietic stem cell-associated markers CD45(1%)and CD34(1.5%).The isolated BMSCs were proved to be able to differentiate into both the adipogenic and osteogenic lineages by oil red staining and alizarin red staining.2.The 3D-GFs were successfully synthesized by chemical vapor deposition method.SEM and metallurgical microscope observation revealed a monolith of continuous and porous structure.The porosity of 3D-GFs was determined to be 99.7±0.3%and had a pore size of 100-350 ?m,while the width of the graphene skeleton was about 40-100?m.Raman spectroscopy proved that 3D-GFs contained single and few layer graphene,and energy dispersive spectrometer showed that carbon was the main element and nickel was absent on the surface of 3D-GFs.3.Both 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay assay and lactate dehydrogenase(LDH)assay demonstrated that there was no significant difference in cell viability when cultured on the 3D-GFs,compared to that cultured on the TCPS(P>0.05).BrdU(5-bromo-2'-deoxyuridine)incorporation assay analysis showed that the BrdU incorporation increased significantly from day5,when cultured on the 3D-GFs.compared to the control of TCPS(P<0.01).At the same time,immunofluorescence confocal microscopy showed that rat BMSCs had good cell adhesion in 3D-GFs.and formed three-dimensional network.4.Analysis of wound area and healing time showed that there was no significant difference between GF group and control group(P>0.05).However,a significantly different wound area was observed from day 3 post-transplantation in the GF + MSCs group,compared to the control(P<0.01)and GF groups(P<0.01),and the wound healing time was also significantly shortened(P<0.01).Compared to the control,the levels of VEGF and bFGF mRNA in the GF group demonstrated no significant differences(P>0.05).Significantly higher expressions of VEGF and bFGF mRNA were observed from day 3 to day 14 in the GF + MSCs group,compared to that in the control(P<0.01)and GF(P<0.01)groups.The TGF-?1 level was significantly lower in the GF + MSCs group.compared to the other 2 groups(P<0.01).An opposite trend was observed in the expression of TGF-?3 in the 3 groups,and its level in the GF + MSCs group was significantly higher than the other 2 groups(P<0.01).The trend of the a-SMA mRNA level over time followed the TGF-?1 expression level.Worthy of mention,there existed significant dif-ferences in the levels of TGF-(31(from day 10),TGF-?3(from day 10),and a-SMA(from day 7)between the control and the GF group(P<0.05).Skin thickness,soluble collagen content,tensile strength and elastic displacement in the GF + MSCs group were significantly better than the other 2 groups(P<0.01? P<0.05);compared to the control,the above indeies in the GF group demonstrated no significant differences(P>0.05).Also,the hydration effect and the water loss of the skin in the GF + MSCs group were significantly better than the other 2 groups(P<0.01?P<0.05);the above indeies in the GF group was significantly better than the control group(P<0.05).Conclusion1.BMSCs were successfully isolated and amplified by means of the whole bone marrow adherent culture method,and expressed mesenchymal stem cell surface antigen,and had a number of differentiation potential.2.The 3D-GFs were synthesized by chemical vapor deposition method and exhibited a monolith of continuous and porous structure,with few impurities,few defects and stable structure.3.The 3D-GFs had no cytotoxic effect on bone marrow mesenchymal stem cells,high compatibility,and could promote cell proliferation.4.The 3D-GFs scaffold loaded with BMSCs could upregulate VEGF and bFGF,which led to neo-vascularization,and downregulate TGF-?3,upregulate TGF-?1 and a-SMA.which had anti-scarring effect.5.The 3D-GFs scaffold loaded with BMSCs obviously facilitated wound closure and provided a better quality neo-skin.6.The 3D-GFs scaffold could improve the quality of neo-skin to a certain extent,and reduce scar formation of the wound. |
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