Study On Liver Tissue Engineering Based On Acellular Matrix Scaffold

The liver is the largest organ of human body and the center of metabolism，playing an important role in glucose metabolism, lipid metabolism, protein synthesis,detoxification and so on. Liver diseases, such as hepatitis, liver cirrhosis, and livercancer, are being serious threat to human health. The mortality caused by liver diseasehas been reached up to2.5%all over the world. Liver failure is the most seriousdisease, of which mortality is as high as60-80%. Especially, hepatitis and liver cancerhappen frequently in China, causing the annual number of deaths up to50million.Each year, medical treatment and health care for the treatment of hepatitis and liverdisease cost as high as100billion. Acute liver failure has a poor prognosis, leading to70%-80%deaths. At present, liver transplantation is the most effective treatment forthe patients with acute and chronic liver failure, but most of the patients died due toshortage of liver transplantation. Therefore, it is necessary to look for new therapiesor alternative liver, and the liver reconstruction constructed based on tissueengineering method may provide a promising alternative strategy for liver failure.Tissue engineering is a multidisciplinary science combining engineering,material science, life sciences, which aims to construct and replace human tissue andorgan. Liver tissue engineering research has always been a focus in last three decadesand has made lots of great breakthroughs. Studies have shown that liverreconstruction in vitro could serve as a treatment for liver diseases, which hassuccessfully applied into clinical bioartificial liver. Nevertheless, because of thecomplexity of liver structure and function, constructing engineered liver tissue in vitrois still facing a number of challenges, such as poor biocompatibility, lack ofvasculature, limited liver volume. In addition, the existing scaffold structure andcomposition are relatively simple and cannot provide a suitable environment for stemcell growth and differentiation. For overcoming these difficulties, liver tissueengineering research based on acellular matrix biomaterial has attracted more andmore attention recently, which is rapidly developed and makes a number ofbreakthroughs.Decellularized materials are novel biological scaffolds, removing all cells fromextracellular matrix (ECM) while retaining the native composition and structure of theassociated matrix typically involves exposure of the tissue/organ to detergents,proteases and chemicals. The retention of main proteins, vasculature and ultrastructure makes it excellent scaffolds for cell growth and tissue regeneration. Therapid development of decellularization makes a hot trend about acellular matrix. In2008, Ott, H C and his colleagues first successfully prepared whole-organ heartacellular matrix,and then other kinds of organ decellularized matrix were preparedsuccessfully, such as liver, kidney and lung. As the largest organ of the body, liver hasbeen one focus of research to prepare decellularized material and has made numbersof breakthroughs. In present days, decellularized matrix based scaffolds are mainlybased on two sources—whole liver decellularized matrix and other tissues or organsderived acellular matrix.In the study of liver tissue engineering based on non-liver decelluarized matrix,liver decellularized scaffolds also facing the same difficult for limited sources, whichmake researchers all over the world explore other tissue derived acellular matrix. Forexample, researchers have successfully constructed engineered liver-like tissue basedon prepared decellularized porcine with preserved vascular structures. Results showedthat this acellular matrix from jejunal segment could support the normal morphologyand metabolic activity of the hepatocytes, which suggesting the feasibility of non-liverdecellularized scaffolds applied into liver tissue engineering. Above all, liver tissueengineering based on non-liver acellular matrix has become one of the most focusesfor cell growth and tissue regeneration.Greater omentum is kind of peritoneum, connecting the stomach bend withtransverse colon. It is full of strong elasticity and flexibility as well as highlyvascularized structure, which characteristics make it excellent natural materials in thefield of tissue engineering and regenerative medicine. At present, the greater omentumhas been applied into clinic for surgical reconstruction to repair the inflammatory ordefect tissues. In the field of tissue engineering research, greater omentum can beused as "natural biological reactor" to culture engineered constructs fortransplantation, which promotes the vascularization, strengthens the function ofconstructs. Based on above advantages, it has been successfully applied in themyocardial tissue engineering, cartilage tissue engineering, neural tissue engineeringand other fields. In recent years, with the rapid development of decellularization,researchers start to focus on greater omentum, and successfully prepared thedecellularized greater omentum matrix. However, weather greater omentum acellularmatrix can be applied to liver tissue engineering or not remains to further exploration.In the study of liver tissure engineering based on liver whole-organdecellularized matrix, researchers have constructed engineered liver tissue or organ like constructswith some degree of function successfully using fetal hepatocytes, primaryhepatocytes and hepatocytes lineage as cell sources. The previous studies showed thatthe liver decellularized matrix could offer more natural mimic microenvironment forseeding cells than other scaffold materials. Besides, due to the high conservation ofextracellular matrix among different species, the acellular matrix scaffolds preparedwith decellularization are endowed with the advantage of low immunogenicity,therefore, the acellular matrix could be used in liver tissue engineering as goodscaffolds.The preparation of decellularized liver matrix with ideal constructure andcompositions is the prerequisite of acellular matrix based liver tissue engineering.Nowadays, the solutions of preparation of liver acellular matrix mainly comprisephysical methods, chemical methods, enzymatic digestion and the combination ofabove methods.Although decellularization has been constantly developed, methodscome to be insufficiency because of long time for exposure, loss main proteins andgrowth factors of ECM. Therefore, ideal methods for preparation of liver whole-organdecellularized matrix remains to be further optimized.This study can be divided into three parts:Part1: Liver tissue engineering based on greater omentum decellularizedmatrixObjective: Explore the component and biocompatibility of greater omentumdecellularized matrix and its feasibility for liver tissue engineering application.Methods: Explore the component and surface structure of four greater omentumdecellularized matrices (No.1,2,3,4) respectively with immunohistochemicalstaining and the scan electron microscopy; In addition, we constructed engineeredliver-like tissue with primary hepatocytes.Itsviability was measured using Live/Deadstaining; Besides, the morphology and functions of hepatocytes were investigatedusing HE and immunohistochemical staining and enzyme linked immunosorbentassay (ELISA) assay. Results: The greater omentum decellularized matrix retainedmain proteins of natural ECM with typical reticular structure; In addition, No.1,3matrix had smaller pore size while No.2,4matrix had bigger pore size; Moreover,compared with matrix with smaller pore size, matrix with bigger pore size couldbetter support cell adhesion, immersion and metabolic function. Conclusions: Thegreater omentum decellularized matrix could serve as excellent scaffold material to maintain hepatocytes morphology and functions, which made it a potential matrix forliver tissue engineering application.Part2: Preparation and optimization of liver whole-organ decellularizedmatrixObjective: Comparison of two different decellularization methods in order toscreen more effective way and preparation of liver whole-organ decellularized matrix.Methods: Liver whole-organ decellularized matrix was prepared by two differentdecellularization methods respectively using sodium dodecylsulfate (SDS) and TritonX-100+SDS (Tx+SDS) detergents. The three dimensional morphology ofacellularized matrix the preservation of original intact vessel structure was observedafter decellularization. The obtained matrix was submitted to histological staining.Results: Liver whole-organ decellularized matrix was successfully developed usingtwo methods; However, SDS method impaired vessel structure and retains lesscollagen and growth factors; Tx+SDS method obtained integrated vascular networkand retained higer proteins and growth factors. Conclusions: This study successfullyprepared superior liver whole-organ decellularized matrix with Tx+SDS method,which could provide a more effective scaffold for tissue engineered liver andregenerative medicine application.Part3: Liver tissue engineering based on liver whole-organ decellularizedmatrixObjective: Explore biocompatibility of liver whole-organ decellularized matrixand its possible application in liver tissue engineering.Methods: Liver whole-organdecellularized matrixwasdeveloped with Tx+SDS method and cut into discs. Inaddition, we constructed engineered liver tissue with HepG2.Its morphology andbiological functions were investigated after culturing7d usingHEstaining,immunohistochemical staining and PAS staining. Results: Histological results showedthat the liver whole-organ decellularized matrix we developed with Tx+SDS couldsupport cell adhesion, growth and maintain cell morphology and proliferation. PASstaining result showed that the liver whole-organ decellularized matrix could supportmetabolic function of hepatocytes.Conclusions:Liver whole-organ decellularizedmatrix we developed with Tx+SDS method processed good biocompatibility andcould maintain morphology and biological functions of hepatocytes, indicating that itwas a potential matrix for liver tissue engineering application.Above all, in the study of liver tissue engineering based on non-liver decelluarized matrix,this thesis explored the component and biocompatibility ofgreater omentum decellularized matrix. Results showed that all this fourdecellularized matrices from omentum retained main proteins of natural ECM and hadgood biological properties; Compared with matrix with smaller pore size, matrix withbigger pore size could better support cell adhesion, immersion and metabolic function.This results demonstrated the feasibility of omentum acellular materials for livertissue engineering application and broadened the sources of decellularized matrix forapplication into liver tissue engineering. In the study of liver tissue engineering basedon liver whole-organ decelluarized matrix,thisthesis successfully screened an effectivemethod for preparation liver whole-organ decellularized matrix with Tx+SDSdetergents. The prepared acellular matrix reserved the three-dimensional networkmorphology, vascular structure and the basic components of natural ECM; Engineeredliver-like tissue based on acellular matrix could support cell appearance and functions,indicating it had good biocompatibility and could provide the excellent scaffoldmaterialfor liver tissue engineering. Above all, this study broadened the sources ofdecellularized matrix for application into liver tissue engineering and provided newstrategies for the clinical treatment of liver failure.