Microarray and Proteomics Techniques for Analysis of Human Msc-Biomaterials Interactions

Mesenchymal stem cells (MSCs) isolated from the bone marrow of adult organisms were initially characterized as plastic adherent when transplanted in vivo. In recent years, MSCs have been shown to reside within the connective tissue of most organs, and their surface phenotype has been well described. However, new findings suggest that the ability of MSCs to alter the tissue microenvironment via secretion of soluble factors may contribute more significantly than their capacity for transdifferentiation in tissue repair. Success in tissue engineering requires an understanding of how cells integrate the signals presented from the microenvironment created by biomaterial scaffolds to alter their responses. The present PI Dr Chen investigated the osteoblastic differentiation of rabbit MSCs loaded in a carrier system of Pluronic F127 and Interpore and developed a PLGA and calcium phosphate collagen composite as a bone substitute for posterolateral spinal fusion. Our associate PI Dr. Wu investigated the adherent effect of mammalian cells cultured within the collagen and hydroxyapatite composed microcarriers. Our associate PI Dr. Wang investigated the functional analysis of signature gene expression in bone marrow MSCs. Our associate PI Dr. Pan investigated the spontaneous tolerance in rat liver transplantation using genomic and proteomic tools. The present study will use DNA microarray, proteomics techniques, and animal model to analysis genes and proteins expression during osteogenesis of MSCs which cultured in different combination of ECM matrix (collagen, fibrin glue, and Pul-F127) and osteoconductive materials (Interpore, PLGA, calcium sulfate, and HAP/β-TCP): In the first year (DNA microarray analysis): We will harvest the MSCs from 21 patients who receive posterolateral spine fusion with autogenous iliac bone graft. We focus MSCs-different biomaterials interactions on identifying gene expression in osteogenesis of MSCs from patients. Some of these gene transcripts are further analyzed by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) to validate data gathered from the DNA microarray analysis. In the second year (proteomics analysis): We will harvest the MSCs from 21 patients who receive posterolateral spine fusion with autogenous iliac bone graft. We focus MSCs-different biomaterials interactions on identifying proteins expression in osteogenesis of MSCs from patients. Compared to conventional molecular biological approaches conducted on a limited number of proteins and on a protein-by-protein basis, proteomics has emerged as a systematic approach to the performance of large-scale studies for the qualitative and quantitative mapping of the whole proteome, and is also a useful complement to high-throughput gene expression analyses at the RNA level In the third year (animal model): The third year in vivo animal experiment is designed to evaluate the effect of mesenchymal stem cell loaded scaffolds as a bone substitute for posterolateral spinal fusion. 1. PLA/PGA-Interpore copolymer and collagen as a carrier matrix 2. PLA/PGA-Interpore copolymer and fibrin glue as a carrier matrix 3. PLA/PGA-Interpore copolymer and Pul-F127 as a carrier matrix 4. PLA/PGA- calcium sulfate copolymer and collagen as a carrier matrix 5. PLA/PGA- calcium sulfate copolymer and fibrin glue as a carrier matrix 6. PLA/PGA- calcium sulfate copolymer and Pul-F127 as a carrier matrix 7. PLA/PGA- HAP/β-TCP copolymer and collagen as a carrier matrix 8. PLA/PGA- HAP/β-TCP copolymer and fibrin glue as a carrier matrix 9. PLA/PGA- HAP/β-TCP copolymer and Pul-F127 as a carrier matrix 10 Autograft 50 rabbits will be evenly killed at 12 weeks for radiographic examination, manual testing, histological study and torsional loading (MTS) to evaluate the results of spinal fusion.

Project ID:PC9709-0109
External Project ID:NSC97-2314-B182-002-MY3