Based Poly (p-dioxanone) Composite With Small Intestinal Submucosa For Soft Tissue Repair And Reconstruction

Background and Objectives:Soft tissue defect is not particularly difficult clinical issue,but it is particularly common in clinic and it has been plagued by clinicians.According to the American society of plastic surgeons data in 2015,there are more than 5.8 million reconstruction operations have been performed.The traditional treatment is often"removing east to west",which causes the tissue deformity,infection and the survival of transplant tissue in the donor site.Tissue engineering is the most promising approach.One of the three essential elements of tissue engineering architecture is tissue engineering scaffolds,and the ideal scaffold materials should have a certain three-dimensional structure and function of extracellular matrix（ECM）.The purpose of this study is to explore and develop new tissue engineering scaffolds for the repair of soft tissue defects.Our team members have successfully conducted a variety of scaffolds,such as cryogel,double layer membrane material which were based a kind of extracellular matrix-porcine small intestinal submucosa（P-SIS）.P-SIS has good histocompatibility and biological activity.Moreover,it is easy to obtain and cost effectively.However,it is not conducive to space-occupying retained to satisfied repair soft tissue defect material demand,which need slow degradation duration and no residue of chemical crosslinking agent.With the development of materials and scaffolds research,more and more biodegradable polymers have been used in medical field,poly（p-dioxanone）PPDO is a kind of aliphatic polyester.Due to the PPDO molecular chain containing ether bond,it has showed good flexibility,strong tensile strength,etc.In the late 1970s,the United States food and drug administration（FDA）has approved PPDO for surgical suture material.In addition,it also has slow degradation time.However,its hydrophilicity leads to difficulties in processing and limits its development and application.Methods:Part Ⅰ:Preparation of decellular matrix,detection of materials and selection of right matrix for composite scaffold.1.Preparation of acellular porcine pericardial membrane（P-PC）and porcine small intestinal submucosa membrane（P-SIS）and tests for P-PC&P-SIS,including:DNA extraction for identification of cell residual,observation for characteristics of the materials,scanning electron microscope to observe the microstructure and mechanical properties analysis,etc.;2.According to the above experimental results and literature review,a comprehensive analysis was carried out,which was aiming to find out the suitable ECM for further use.Finally,p-sis was selected as the natural biological matrix material to process the composite scaffold;3.Preparation of P-SIS powder.Part Ⅱ:preparation of PPDO-SIS porous composite scaffold.1.PPDO dissolution experiment.PPDO is difficult to dissolve in general organic solvents.Therefore,it is necessary to find a suitable organic solvent for the preparation of composite materials;2.Forming PPDO film experiment;3.Preparation of PPDO-SIS composite scaffolds;4.The characterization and microstructure of PPDO-SIS composite scaffolds.PPDO membrane was as control group;5.The component characteristics of PPDO-SIS composite scaffolds（FTIR and X-ray diffraction patterns were analyzed by Fourier infrared spectroscopy and XRD experiment）.The results were compared with P-SIS membrane and PPDO film;6.Mechanical properties of PPDO-SIS composite scaffolds（the control group was the same as above）;7.Thermogravimetry analysis of PPSO-SIS composite scaffolds（the control group was the same as above）;8.Determination of degradation rate of PPDO-SIS composite scaffolds in 58℃protease K solution and 37℃phosphate buffer（PBS）（the control group was the same as above）;9.Water absorption experimental of PPDO-SIS composite scaffolds（the control group was the same as above）.Part Ⅲ:Biological observation of PPDO-SIS composite porous scaffolds in vitro.1.Isolation and culture of adipose derived stromal cells（ADSCs）from green fluorescent transgenic mice;2.Compound culture of PPDO-SIS scaffold materials and ADSCs in vitro;3.CCK-8 kit was used to detect cell activity and proliferation（P-SIS membrane and PPDO membrane were as control group）;4.Scanning electron microscope（SEM）to observe the cell state.Part Ⅳ:experimental observation of PPDO-SIS composite porous scaffolds in vivo.1.The production of soft tissue defect in mice model,comparing with the blank group,P-SIS membrane and PPDO membrane group as the control group,the PPDO-SIS composite scaffold embedded subcutaneously in mice,to observe each material for soft tissue defect repair.2.By tissue section of HE staining and immunofluorescence detection expression of CD34,immunohistochemical detection CD90 expression and Western blot detection CD31and VEGF expression,observation analysis PPDO-SIS the local promote vascularization of composite scaffolds after transplantation and cell proliferation.Results:1.P-PC membrane,P-SIS membrane and P-SIS powder were prepared.P-SIS powder is more conducive to the preparation of composite scaffolds.2.The PPDO-SIS composite scaffold was successfully prepared.The scaffold is observed by SEM and has a three-dimensional porous structure.The pore wall has micropores and can be intercommunicated within the material.PPDO-SIS composite scaffold materials are with good water absorption performance,compared with the P-SIS membrane and a slight higher than the biological matrix membrane.Compared with polymer PPDO membrane,the water absorption performance was significant higher than PPDO film（P<0.05）.FTIR display PPDO-SIS composite scaffold wave number 1736 cm^-1 C=O stretching vibration absorption peak,in 1221 cm^-1,1052 cm^-1,1126 cm^-1 shows a C-O stretching vibration peak,after that blend of PPDO-SIS compound still retain PPDO chemical structure characteristics of the support.XRD results show that the PPDO-SIS X-ray diffraction peaks of the composite scaffold mainly appear in 2 theta Angles are 21.92°,23.78°and 29.12°.The result was confirmed the P-SIS of the mix of PPDO-SIS composite material of crystal type and has no effect.It was showed the characteristic of PPDO diffraction peak.The strain rate of PPDO-SIS composite stent was increased compared with P-SIS membrane,which can meet the mechanical requirements of soft tissue defect repair.After 48 hours in proteinase K solution,the degradation of the P-SIS rate up to nearly 85%,PPDO-SIS composite scaffold is about 10%,the degradation of PPDO membrane rate less than 1%,by statistical analysis,PPDO-SIS composite scaffold and the degradation rate of P-SIS have ever had extremely significant difference（P<0.01%）,and the degradation of PPDO membrane at12 h rate exist significant differences（P<0.05%）.With the increase of degradation time,this tended to be more significant difference（P<0.01%）,and this PPDO-SIS might contain of P-SIS composite scaffold which had much to do so.PBS buffer in vitro degradation rate of the experimental results showed that although the PPDO-SIS composite scaffold with PPDO membrane in four time points（1w,2w,3w,4w）degradation rate were extremely significantly statistical differences（P<0.01%）,but the PPDO-SIS composite scaffolds in 4w were at the same time as the degradation rate of only 25%.The PPDO-SIS composite scaffold showed good water absorption performance,slightly higher than the P-SIS membrane.Compared with the PPDO membrane,its water absorption performance was much higher than that of the polymer PPDO film,and it showed statistical significance（P<0.05）.3.The fluorescence microscope and scanning electron microscope result showed ADSCs cells with green fluorescence in the P-SIS membrane,PPDO-SIS composite scaffold materials and PPDO membrane could widely distributed on the growth.By scanning electron microscope,clear cells could been seen form a spindle,good stretch,especially visible ADSCs grew in PPDO-SIS composite scaffold holes on the wall.There was a significant difference between the 24h PPDO-SIS and PPDO membrane tested by CCK-8 kit（P<0.05）.Measurement 1d,3d,5d,7d of CCK8 absorbency showed the cell proliferation on each material.P-SIS of the membrane was the best cell proliferation and PPDO-SIS composite scaffold was better,PPDO membrane was the worst.However,as time going on,the difference of cell proliferation and cell viability between PPDO-SIS composite scaffolds and P-SIS membrane was changed from the extremely significant difference（P<0.01）into a significant difference（P<0.05）,but the differences between PPDO membrane and PPDO-SIS composite scaffold was showed extremely significant in statistics at any point of time（P<0.01）.4.Different scaffolds were buried in the soft tissue defect sites of mice.The result was that P-SIS membrane,PPDO-SIS composite scaffolds,PPDO membrane in mice showed good biocompatibilities and cell proliferation.Additionally at 14d,P-SIS were absorbed to amorphous state of shape,and PPDO-SIS were still keeping original shape,and PPDO-SIS membrane and PPDO membrane group could still be observed on the 28d with respectively（0.57+0.06）cm and（0.55+0.07）cm,and there was no significant difference in statistics.In the control group at 14d and 28d,the back of the mice were still able to observe the soft tissue defect.At the time point of 2 weeks and 4 weeks,HE staining histopathological morphology showed all of the material groups had neutrophil infiltration.The P-SIS group was absorbed quickly.All materials were clearly observed new angiogenesis,but control group not.Immunofluorescence staining showed positive expression of CD34 in the P-SIS membrane group,the PPDO-SIS composite scaffold group and PPDO film group,and there were significant differences between these groups with the control group（P<0.05）.Immunohistochemical results showed that the CD90 expression of the PPDO-SIS composite scaffold was significantly higher than that in the control group（P<0.05）.The Western blot of CD31 and VEGF showed that the different materials were significantly higher than the control group（P<0.05）.Conclusion:1.The successful preparation of PPDO-SIS composite scaffolds has performed and a good three-dimensional porous structure with the physical and chemical properties meet the requirements of tissue engineering scaffolds.2.Compound culture of PPDO-SIS composite scaffolds and ADSCs in vitro confirmed that PPDO-SIS composite scaffolds showed good cell viability and proliferation,and could be used in vivo transplantation.3.The PPDO-SIS composite scaffold in vivo degradation was slower and could have space-occupying repair effect in the space.The bracket can effectively promote the vascularization of graft site,conducive to cell regeneration,which can effectively solve the soft tissue defect repair.