The Research Of Tissue Engineered Tarsal Plate Substitutes In Eyelid Defect Repair

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Fabrication and biocompatibilities studies of novel branched polyethylene porous scaffoldsPurposeIn this study,the novel branched polyethylene(B-PE)elastomer was used as the basic material to construct a tissue engineering scaffold for soft tissue repair.We aimed to prepare an integrated B-PE scaffold with suitable porosity,interconnectivity and pore size,and study its physicochemical characteristics,biocompatibility in vitro and in vivo.We hope to provide a promising technique to produce a tarsus substitute with good biocompatibility and easily integrated by host tissues.MethodsB-PE scaffolds were prepared using a gelatin porogen-leaching method.The microstructure of the basic material and scaffolds were observed by scanning electron microscopy,and the porosity of the scaffold was determined by an ethanol displacement method.To investigate the cytotoxicity,extract of B-PE was used to culture NIH/3T3 fibroblasts and human vascular endothelial cells(ECs).FDA living cell staining was used to observe cell adhesion and proliferation on the material.The endothelialization of the scaffolds was observed under confocal microscopy.Histological evaluation and related gene expression of host tissue response was performed after subcutaneously implantation in rats.ResultsThe branched polyethylene was a foam-like elastomer in appearance,with a loose structure,and a hydrophobic surface.The B-PE scaffolds using gelatin-leaching method had high porosity and interconnectivity.CCK-8 tests were performed to determine cytotoxicity of B-PE extract to NIH3T3 fibroblasts and human vascular ECs in vitro.The extract did not induce significant changes in cell viability.Human vascular ECs adhered,spread and proliferated well on the surface of B-PE films,while the NIH3T3 fibroblasts showed a poorer initial adhesion on the B-PE films than on TCPS.After a 7-day culture,the human vascular ECs proliferated and formed an endothelium lining.The porous scaffolds implanted subcutaneously in rats exhibited mild inflammatory response,collagen deposition and fast fibro-vascularization,suggesting their good biocompatibility.Quantitative PCR analysis(q PCR)showed low expression of pro-inflammatory genes and up-regulated expressions of collagen deposition and vascularization-related genes,validating the results of historical evaluation in a molecular level.ConclusionB-PE elastomer had good processability and film-forming ability.The homesynthesized B-PE elastomer was also able to be made into a porous scaffold by a porogenleaching method.In vitro cytotoxicity tests by both extract test and direct contact test showed that the B-PE exhibited no obvious cytotoxicity.Furthermore,accompanied by a moderate inflammatory response,the rapid fibrovascular engraftment of scaffolds was observed in vivo,which is regarded as key factor for its successful tissue integration.Part ? A tarsus construct of novel branched polyethylene with good elasticity for eyelid defect repair in vivoPurposeThe purpose of this part was to study the mechanical properties and microstructure of natural tarsal plate and B-PE scaffold,and investigate its potential application as a tarsal construct.Lay an experimental basis and provide a new strategy for the application of B-PE scaffold in tissue engineering.MethodThe fresh rabbit tarsal plates were harvested for the evaluation of mechanical properties,composition and biological functions.The mechanical properties and microstructure of B-PE scaffolds and Medpor controls were characterized.The rabbit tarsal plate defect model was established,and the B-PE scaffold and Medpor controls were implanted in situ.The interaction between the material and eyelid tissue,such as inflammation,grafts location and tissue integration,was comprehensively evaluated by H&E and Masson staining.ResultsEmbedded within the plate are the regularly arranged Meibomian glands,around which is dense fibrous connective tissue.Of the tarsus samples tested,the mean elastic modulus was 0.9 ± 0.1 MPa with an extensibility of 50.8 ± 10.4%.The mean elastic modulus and elongation of B-PE scaffolds and Medpor materials were 0.8 ± 0.1 MPa,76.2 ± 2.8% and 30.2 ± 4.5 MPa,7.0 ± 1.1% respectively.The rabbit tarsal plate defect model was successfully established.After 4 weeks implantation,severe contraction of tarsal defects could be found in the blank control group.The operated eyes showed good wound healing in the B-PE group than the Medpor spacers group.Histologically,the tissue response to the B-PE scaffolds presented abundant connective tissue infiltration,while the Medpor group showed tissue infiltration deficiency.Masson staining showed that the thickness of fibrous encapsulation induced by B-PE scaffolds and Medpor was 134.4 ± 24.9 ?m and 271.6 ± 46.5 ?m.ConclusionThe B-PE scaffolds exhibited comparable mechanical properties with the natural eyelid tarsal plates,making them elastic enough to be mould to the shape of globe.The scaffolds could be rapidly integrated by host tissue in vivo,leading to batter stabilization.This study attempted to construct alternative materials that match the structure and mechanical properties of the tissue,providing new materials and ideas for eyelid reconstruction.Part ? A biomimetic tarso-conjunctival biphasic scaffold for eyelid defect repair in vivoPurposeBased on the composition and structure of the tarso-conjunctival tissue,we aimed to explore a construct of a two-layer composite material that could reconstruct the tarsoconjunctival tissue.The impact on cell behavior was analyzed in vitro and the application of repairing the defect of posterior lamina was further evaluated in situ.MethodThe soft collagen/chitosan(Col/CS)was composed to hard polypropylene fumarate/hydroxyethyl methacrylate(PPF-HEMA).The morphology and pore structure of the scaffolds were observed under scanning electron microscopy.The porosity of the scaffold was measured by ethanol displacement method.The mechanical properties were tested by a universal mechanical testing instrument.Degradation in vitro were analyzed according to the mass loss of the scaffolds in PBS.The adhesion,proliferation and migration of human conjunctival epithelial cells(Cj ECs)in the scaffold were observed.The model of tarso-conjunctival defect was constructed,and the repair effect was evaluated by in-situ implantation.ResultsThe biphasic scaffold consisting of the hard PPF–HEMA and soft Col/CS layers was fabricated successfully.The PPF–HEMA layer functioned as the skeleton as tarsal plate substitute,while Col/CS sponge mimicked the conjunctiva lining.The scaffolds had high porosity of ~ 90%,The average pore size of the Col/CS layer and PPF–HEMA layer was around 95 ?m and 235 ?m.The scaffolds were stable in vitro degradation study.After 12 weeks of degradation,the porous macrostructure was well maintained without notable collapse.With the combination of Col/CS sponge onto PPF–HEMA skeleton,the biphasic scaffolds showed improved cell adhesion and proliferation in vitro.The Cj ECs tended to migrate and confluent on the surface of the scaffolds.The in vivo results of rabbit posterior lamella full thickness defects demonstrated that PPF-COL biphasic scaffolds could promote wound closure,re-epithelialization.The regenerated conjunctiva was stained positively by CK4 and MUC5 AC immunofluorescence staining.ConclusionHierarchical 3D biphasic scaffolds were designed and constructed,which were mechanically improved and favorable for cell adhesion and proliferation.In a rabbit tarsoconjunctival defect model,the grafted biphasic scaffolds promoted re-epithelization with functional regenerated conjunctiva.The results of this study provide a new concept for the design and construction of an ideal substitute for eyelid defect reconstruction.