| BackgroundArtificial dermis synthesized with collagen sponge scaffold(CSS)has been widely used in the treatment of skin and soft tissue defects.However,its current function as a dermal matrix template is relatively simple,and it is still more difficult to improve re-epithelialization and graft ischemia.Adipose-derived stem cells(ASC)can differentiate into keratinocytes(KC),which are key cells for re-epithelization under certain conditions.However,most of the previous researches were two-dimensional(2D)induction culture,without considering the role of important components of stem cell niches such as extracellular matrix.It has been reported that 3D induction culture system with biomaterials can promote the differentiation of ASC to KC,suggesting that CSS is expected to promote ASC to better differentiate into KC.Platelet-rich plasma(PRP)can release growth factors to improve blood supply to the graft.However,the "burst effect" often affects its efficacy.The sustained-release drug delivery system can effectively delay the drug release rate,and polydopamine(pDA)can enhance the adhesion of the material surface.Studies have shown that collagen-based biomaterials can slowly release growth factors,suggesting that CSS is expected to be a sustained-release drug delivery system for PRP.Based on the CSS material,this research,on the one hand,explored whether it could function in the construction of stem cell niches to promote the differentiation of ASC to KC,in order to explore its potential to improve the re-epithelialization of skin and soft tissue defect;On the other hand,pDA-CSS was prepared to give it a powerful sustained-release drug delivery property,and construct a sustained-release drug delivery system to explore whether it can improve graft revascularization.Methods1.Preparation and characterization of collagen sponge scaffold,cultivation and identification of hASCCollagen stock solution was prepared from bovine Achilles tendon according to acid dissolving method and enzymatic hydrolysis method,and then CSS was fabricated.At the same time,its microscopic morphology,structure and mechanical properties were characterized.hASC was identified based on cell morphology,cell surface markers and multi-directional differentiation potential.2.Three-dimensional indirect co-culture induction strategy of collagen sponge scaffoldsIn order to better mimic the characteristics of stem cell niches,a 3D induction system was constructed with CSS,and the degree of hASC epithelial differentiation was evaluated from cell morphology and cell differentiation markers.At the same time,the preliminary mechanisms involved was also conducted.3.Surface modification and optimization of thin-layer CSS modified by polydopamineThe thin layer of CSS was prepared and its surface was modified with polydopamine(pDA).From the appearance,surface morphology,molecular structure,mechanical properties,adhesion ability,and cell proliferation toxicity,the CSS undergoing different modification times of pDA was evaluated and optimized continuously.4.In vitro biological evaluation of optimized pDA-CSS loaded with PRPThe sustained-release drug delivery property was evaluated by testing the drug loading of PRP and growth factor release experiments.Meanwhile,the effects of optimized pDA-CSS loaded with PRP on the proliferation,migration and growth of key cells in skin and soft tissue repair were evaluated.5.The effect of optimized pDA-CSS loaded PRP on the survival of ischemic flaps in nude miceThe thin layer of pDA-CSS-PRP was transplanted into ischemic skin flaps in nude mice,and the efficacy in vivo was evaluated based on the flap survival rate,neovascularization,tissue cell proliferation,and biocompatibility.Results1.Preparation and characterization of collagen sponge scaffoldsCompared with the collagen layer of the commercially available Lando? bi-layer artificial dermal scaffold products,the CSS prepared in this study is also a porous structure,and the microscopic morphology,structure and mechanical properties of them are basically the same.And the cells tested were identified to be hASC.2.Three-dimensional indirect co-culture induction strategy of collagen sponge scaffoldsConsistent with H&E staining,phalloidin staining observed that cells adhered and proliferated well on CSS.Inverted microscope and scanning electron microscope confirmed that hASC seeded on CSS showed more KC-like cells,and immunofluorescence staining and gene expression were also confirmed.3.Construction and optimization of thin-layer CSS modified by polydopamineThe thin-layer CSS with a thickness of about 200 μm was successfully prepared;In the process of modification of scaffolds with pDA,it was observed to increase its chemical cross-linking effect and affect its degradation property;pDA-CSS with an optimal modification time of 12 hours were considered to have both h better adhesion and proliferation promoting properties,which could be utilized in the subsequent experiments.4.In vitro biological evaluation of optimized pDA-CSS loaded with PRPThe optimized pDA-CSS could not only load more PRP and continuously release growth factors related,but also has certain safety and proliferation-promote to related cells,which meet the basic requirements as a skin and soft tissue repair and regeneration materials.5.The effect of optimized pDA-CSS loaded with PRP on the survival of ischemic flaps in nude miceAccording to the experiment of ischemic skin flap in nude mice,it was confirmed that the optimized pDA-CSS loaded with PRP can promote the survival of ischemic skin flaps,and its mechanism is related to the improvement of vascularization with excellent in vivo biocompatibility.ConclusionThis study preliminarily confirmed that the CSS can construct stem cell niche to facilitate the differentiation of hASC into KC,and construct a sustained-release drug delivery system for PRP to promote revascularization of ischemic flaps,indicating to be a new regenerative biomaterial,which will be of great significance to the regeneration of skin and soft tissue defects. |
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