Small Intestinal Submucosa Cryogel As A Scaffold For Cell Transplantation

Background and ObjectivesThe pain,functional defect and disfiguration caused by chronic wounds and tissue absence always affect the ambulation and life quality of patients,even induce psychological and social problems.When autologous and allogeneic transplantation limitated by donor number,ethics,infectious diseases or other restrictions,tissue engineering become an inevitable choice.As a focal point of the tissue engineering,animal-derived scaffold are inexhaustible,safe and no ethical problem.As an ideal material for tissue reconstruction,the porcine small intestinal submucosa(SIS)has low immunogenicity,good histocompatibility and contains multiple growth factors.But there are still some deficiencies exist: Firstly,SIS is a film membrane material that lack of adequate three-dimensional structures;Secondly,SIS has compact structure that lack of pore size which is essential for cell migration and nutrient transportation.In this study,we found a new crosslink method for SIS modification and prepared a new type of tissue engineering scaffold:SIS cryogel which has elastic deformation and shape memory capabilities.The surface and internal structure was detected by scanning electron microscopy and pore size was measured.Water absorption,in vitro degradation,mechanical properties and cytotoxicity between different EDC concentration cryogels.SIS cryogel can adsorb the cell suspension automatically and cells grown on well on SIS cryogel in vitro cell co-culture.SIS cryogel has good biological and physical properties,the integrity can be still matained even under strong compression which is very useful for transplantation.These findings suggest that SIS cryogel could serve as a cell and drug deliver scaffold for tissue reconstruction,would repairing and Injection Filling.MethodsThere are four parts in this studyPart I: Preparation of acellular SIS film and scanning electron microscopy exam1.The procedure involved mechanical disassociation,methanol / chloroform degrease,trypsin digestion,detergent treatment.2.SEM exam the acellular SIS to see if there is any cell remains on the surface.Part II: Preparation of acellular SIS powder and optimize the SIS’s concentration in solution.1.The procedure involved pepsin digestion,filtration,salting out,centrifugation,dialysis and lyophilization to make the SIS powder.2.To optimize the SIS concentration by observing the solution’s properties,fluidity and the sponge’s appearance lyophilized from solution between different SIS concentrations.Part III: Preparation and Evaluation of SIS cryogel1.Preparing a new biomaterials:SIS cryogel that have elastic deformation and shape memory capabilities by using a self-made mixing devices and through a freeze-crosslink process.2.Observe the SIS cryogel’s microstructure and pore size by SEM.3.Detect the water absorption,in vitro degradation and mechanical properties between different concentrations of EDC cryogel.PartⅥ In vitro cell culture on SIS cryogel1.Isolation and culture of fibroblasts from GFP transgenic neonatal mice.2.Fluorescence microscopy and SEM.3.CCK-8 kit for different EDC concentration cryogel compare to the commercial collagen sponge.Results1.We have prepared the acellular SIS film successfully,SEM suggested that there is no significant residual cell on the surface.2.The white acelluar SIS powder is flocculent.2mg/ml,5mg/ml and 10 mg/ml concentration of SIS can be dissolved in 0.1% HAC solution.The morphology and physical properties of the materials became better when the concentration increased,but the solution becomes more viscous and less flowable.It’s difficult to dissolve when the concentration rised to 10mg/ml.Much more higher concentrations are unable to stirring and dissolve.So we choice 10mg/ml for the next experiment.3.With shape-memory properties and elastic deformation,SIS cryogel can rise up quickly to recover the volume and shape withstand reversible deformations at over 85% strain level.SEM obtained that SIS cryogel has porous structure.The pore size are between 50-200 um,and the average is(106.63 ± 28.9)um.As EDC concentration rised up,there is no significant change in water absorption.But the vitro degradation and mechanical properties are different from eachother.30mM-50 mM EDC concentration would be appropriate.4.The SIS cryogel can absorp cell suspension rapidly after been squeezed up by sterile gauze.Fibroblasts from GFP transgenic neonatal mice were seed on the scaffold.Fluorescence microscopy and SEM results showed that cells grows well on the surface of the pore walls.There are numerous fibroblasts adhernt on the scaffold with linear,spindle or film shape and abundant pseudopodia.CCK-8 kit prompted that there is no significant difference between different EDC concentration cryogels compared with commercial collagen sponge scaffold.Conclusion1.The SIS cryogel been prepered by freezing-crosslink method through self-made device and EDC has hape-memory properties and elastic deformation.It can be used as an injectable,biodegradable scaffold.There is no need to crosslink additional chemical groups on the scaffold which avoid potential toxicty of chemical reagents,and the preparation prograse is much easier than other cryogels been reported.2.With porous structure,SIS cryogel is friendly to cell migration and nutrients exchange.Different EDC cryogels have the same water absorption but different degradation rates and mechanical properties.30mM-50 mM EDC concentration would be appropriate.3.EDC concentration had no effect on the cytotoxicity of SIS cryogel.Cells grown well on the surface and pore wall.