Fabrication Of Poly(Propylene Fumarate)-based Copolymer Materials For Tarsal Plate Repair And Eyelid Reconstruction

Part I Development and characterization of poly(propylene fumarate)-2-hydroxyethyl methacrylate copolymersPurposeThe purpose of this study was to investigate the feasibility of introducing 2-hydroxyethyl methacrylate(HEMA)to copolymerize with PPF to enhance the toughness of PPF gels.Different mass ratios of PPF and HEMA were fabricated,of which crosslinking density,hydrophilicity and mechanical properties were characterized.To provide an experimental basis on the studies of the tarsal plate damage repair materials and to lay the experimental foundation of further optimization of this kind of tarsal plate repair copolymer.MethodsBased on observation of the morphology and examination of biomechanical properties of rabbit tarsal plates,we used natural polymer materials of PPF and HEMA as the main raw materials to fabricate an elastic copolymer network.The mass ratio of PPF and HEMA were optimized.The chemical composition and changes in the copolymers was characterized by Fourier transform infrared(FTIR)spectroscopy.The thermal behavior of the polymer was characterized by a dynamic mechanical analyzer(DMA).The tensile strength,Young's modulus and the maximum elongation of the film were detected by the universal testing machine.Static water contact angle was tested to analyzed the film's hydrophobicity and hydrophilicity.The swelling ratio of the polymer was also studied.ResultsAverage Young's modulus of the rabbit tarsal plates was 25.30 � 6.97MPa.The strong absorbance at 3400-1 and 1740 cm-1 were the typical vibrations of hydroxyl and carbonyl groups,respectively.Because the hydroxyl groups are mainly derived from the PHEMA component,the increase in the absorbance of the hydroxyl groups and the decrease in the absorbance of the carbonyl groups from PPF-HEMA0.5 to PPF-HEMA2 reveal that the relative content of PHEMA in PPF-HEMA networks increased consistently.The water contact angle on the PPF-HEMA networks decreased with an increase of the PPF-HEMA copolymers.The swelling ratio of PPF-HEMA0.5,PPF-HEMA 1 and PPF-HEMA2 was 16%.20%and 30%,respectively.PPF-HEMA0.5,which has the lowest crosslinking density,had the largest elongation at break of 20%.For all the samples,the compression modulus decreased from 15-20 MPa to 1.5-2 MPa after swelling.According to 50 cycling compressive stress-strain curves,PPF-HEMA networks showed good shape stability and reproducibility.By dividing the stress(strain at 8%)of the 50th and 1st compression,the stress remaining for PPF-HEMA0.5,PPF-HEMA1,and PPF-HEMA2 was approximately 90.1%,82.5%,and 85.9%.Therefore,all the samples exhibit outstanding fatigue-resistant properties.ConclusionsIn this study,the hydrophilic HEMA monomer was successfully introduced into PPF by bulk polymerization.With the addition of HEMA,the obtained PPF-HEMA copolymer showed better swelling ability and outstanding toughness compared to the traditional PPF gels.Artificial simulation of the biomechanical properties of tarsal plate has been achieve.This will lay a reliable experimental basis for clinical tarsal plate repair and eyelid reconstruction.Part ? Preparation and characterization of porous PPF-HEMA tissue engineering scaffoldsPurposeThe purpose of this study was fabrication and characterization of the porous PPF-HEMA scaffolds.To explore the influence of chemical composition and porosity on the physical and chemical properties of the PPF-HEMA scaffolds,also on the cytocompatibility of the porous scaffolds as a tarsal plate substitute.And to prepare new methods and research ideas to the fabrication of tissue engineering porous scaffolds for tarsal plate repair.MethodPPF-HEMA tarsal substitutes were fabricated using a gelatin porogen-leaching method.The microstructure of PPF-HEMA scaffolds and ADM was observed under scanning electron microscopy(SEM).The porosity of PPF-HEMA scaffolds was assessed by Archimedes method,and that of ADM was quantified from the SEM images.In vitro degradation of PPF-HEMA scaffolds was evaluated by mass loss at 1,2,3,6,9,and 12 weeks.To investigate the cytotoxicity of scaffolds,extract liquid of PPF-HEMA scaffolds was used to culture HDFs.PrestoBlue assay was used to compare the adhesion and proliferation of human conjunctival epithelium cells and human conjunctival stroma cells cultured on different scaffolds.Immunofluorescent staining of specific markers of cells was used to observed the morphology of cells.ResultsIt is observed that the porous PPF-HEMA scaffolds porogen-leached with the 180-280p.m gelatin particles is one that has no cracks compared to scaffolds fabricated with 280-450?m and 480-600p?m gelatin porogen.Commercial ADM had thinner pore walls and higher porosity(91.5�3.2%),while PPF-HEMA scaffolds had porosities of 88.9�2.3%,82.3�1.6%and 84.6�3.1%for PP-HEMA0.5,PPF-HEMA1,and PPF-HEMA2 scaffolds,respectively.Both PPF-HEMA scaffolds and ADM were white in appearance and showed a porous structure.With an increase in PHEMA ratio,the degradation degree was reduced at equal incubation times.The PPF-HEMA2 scaffold exhibited the lowest rate of mass loss.After cultured with PPF-HEMA scaffolds 24 h extractant,MTT assay showed no statistical difference in the cell metabolic activities of HDFs.Cell adhesion and proliferation on PPF-HEMA 1 scaffold is higher than PPF-HEMA0.5 and PPF-HEMA2 scaffold,and CjECs can grow on the surface of CjSCs cultured with PPF-HEMA 1 scaffold.ConclusionPorous PPF-HEMA tissue engineering scaffolds with high porosity were successfully fabricated by gelatin porogen-leaching method.By varying the ratio of PPF and HEM A,the physical and chemical properties could be controlled.The porous scaffolds showed good biocompatibility and no cytotoxicity in vitro.Considering the outstanding toughness and biocompatibility,PPF-HEMA is a promising material for tarsal plate repair.Part ? In vivo biocompatibilities and tarsal plate repair of porous PPF-HEMA ScaffoldsPurposeThe purpose of this study was to analyze and evaluate the biocompatibility of porous PPF-HEMA scaffolds.Lay a theoretical and experimental basis to the clinical application and industrialization of the PPF-HEMA scaffolds in the eyelid reconstruction.To fundamentally promote the development PPF materials in tarsal plate repair.MethodThe rabbit tarsal plate repair model was established,hematoxylin-eosin staining was used to detect the tissue response to materials,and Masson staining was used to measure the thickness of the fibrous capsule around the scaffolds and interstice qualitatively.Histologic evaluations were carried out 8 weeks after implantation using a modified histological grading scale for the accurate quantitative and qualitative analysis of the soft-tissue response.Operability and in vivo biocompatibility of the scaffolds were evaluated,acellular dermal matrix was used as control.ResultsAfter 8 weeks,the operated eyes showed good wound healing and complete hair clothing of operation areas in the ADM group and PPF-HEMA2 group,while the contracture of tarsal defects was found mildly in the PPF-HEMA 1 group and severely in the blank control group.The overall degradation scores at the 8th week were very small with values of 2.8�1.0,2.3�0.5,and 2�0.8 for the ADM,PPF-HEMA1 and PPF-HEMA2 group.The thickness of the peri-implant capsules at the 8th week measured by Image J was 28.0�9.3?m,65.2�16.8?m,and 63.5�17.3 ?m for the ADM,PPF-HEMA 1,and PPF-HEMA2 groups,respectively.At the 4th and 8th week,the PPF-HEMA1 and PPF-HEMA2 groups showed a lower score in interstice qualitatively than the ADM control group.Moreover,no statistical significance was manifested in either group.ConclusionThe in vivo repair results of rabbit tarsal plate full-thickness defects demonstrated that the PPF-HEMA2 group had better eyelid reconstruction than the PPF-HEMA1 group.Moreover,the histologic study showed a mild inflammatory response and good compatibility with fibroblast growth and fibrous capsulation.Compared to PPF-HEMA2 and the ADM control group,PPF-HEMA1 produced a relatively higher inflammatory score in the 4th week after implantation.In summary,biodegradable PPF-HEMA2 scaffolds show great potential for the development of tarsal defect:repair in the rabbit experiment in vivo.