טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTamari Tal
SubjectACellular Collagen Scaffold :The Osteoinductive and
Vascularization Promotion
DepartmentDepartment of Medicine
Supervisors Clinical Professor Dror Aizenbud
Professor Abraham Reznick
Full Thesis textFull thesis text - English Version


Abstract

Although bone has a remarkable capacity to heal itself, disease or injury often results in a loss of significant amount of bone tissue for the body to be naturally replaced. Bone autograft is associated with severe pain and morbidity at the site of removal. Allogenic transplants are also used, but are often of poor quality, carrying the risk of rejection and the transmission of disease from donor to recipient. The use of bone substitutes has shown insufficient vascularization and osteointegration. Indeed, the insufficient vascularization is considered a major limitation in attaining tissue functionality. Due to these drawbacks, the development of new -low immunogenicity treatment methods with improved healing effects of autograft - is a pressing need.

Recently, the role of the extracellular matrix (ECM) microenvironment (‘‘niche’’) in facilitating and regulating stem cell behavior in vivo has been elucidated. As a result, investigators have shifted their efforts toward developing three dimensional scaffolds capable of functioning like the native tissue ECM. In this study, we hypothesize that, three dimensional based ECM, acellularized scaffolds, will induce osteogenesis and improve vascularization of implants and thereby a new bone tissue formation will be enhanced.  The ECM was synthesized by Mesenchymal Stem Cells (MSC) to reconstitute the tissue-specific 3D microenvironment in vitro. MSC were seeded on to the collagen scaffold cultured in differentiation medium.  In this way osteogenic differentiation of the MSC was induced facilitating the generation of a pro-osteogenic matrix. Following deposition of the ECM inside collagen scaffold layers, the construct was acellularized (i.e. acellular collagen scaffold). In the study we used an optimized acellularization protocol, using hypertonic solution combining extensive washing and DNase treatment, for eliminating living cells generated by MSC from the matrix. In order to identify the ECM composition, ECM was chemically isolated and scanned for protein quantification and identification analysis. We identified 1370 gene products, including proteins related to the extracellular matrix and ion transportation, as well as enzymes, cytoskeletal, and regulatory proteins. In addition, immunofluorescence method using antibodies against fibronectin and laminin revealed that these proteins had preserved their unique composition even after acellularization process. mCT scanning of the acellular enriched ECM collagen scaffold proved calcium remains.  The results showed that, MSC cultured over acellular collagen scaffold, had osteogenic differentiation capacity compare to untreated collagen scaffold. When scaffolds were subcutaneously implanted into immundeficient mice, it was found to induce bone formation even in ectopic location only in the acellular collagen scaffold.

During vasculogenesis, ECM has a key role in regulating the function of endothelial cells.  I tested the ECM influence on scaffolds’ vascularization Using two different techniques: dextran injection and ultrasonography. Both techniques revealed that acellular collagen scaffold extensively vascularized, compared to collagen scaffold, two weeks after implantation. The in vivo study further confirmed that the acellular collagen scaffold was suitable to enhance bone and vascularization formation.

In conclusions, this novel 3D, acellularized, MSC derived ECM collagen scaffold, has the potential to be developed into a biomedical platform for the regeneration of natural bone tissues.