Functional And Molecular Characterization Of Human Mastoid-Derived Periosteal Cell For Tissue Engineering

Background:Tissue loss or end-stage organ failure caused by injury or other types of damage is one of the most devastating and costly problems in human health care. Tissue engineering is "an interdisciplinary field that applies the principles of engineering and of life science towards the development of biological substitutes that restore, maintain, or improve tissue or organ function." The sources of cells used in tissue engineering play a crucial role to tissue regeneration and repair. Adult stem cells, which are capable of self-renewal and differentiation into various cell lineages, hold great promise for treating affected tissue in which the source of cells for repair is including bone, cartilage, fat, tendon, muscle, and marrow stroma, without any immunogenic and ethical problem.To date, periosteum-derived progenitor cells(PC) were isolated and could be induced to differentiate into osteocytic and chondrocytic lineages. Although PC display a stable phenotype in terms of therapeutic approaches, many limitations and unsolved issues remain: isolation technique, antigen phenotype, multi-lineage differentiation potential, biocompatible with scaffolds, especially as for human PC. Therefore, the issues regarding isolation, proliferation, and differentiation of human PC to form a complex tissue or whole organ must be extensively studied.Aims:The aim of this study was: to study in vitro expansion capacity of PC using allogeneic human serum;to investigate their stem cell-related surface marker expression using FACS and genome-wide microarray technology, and their osteogenic, chondrogenic and, for the first time, adipogenic developmental potential;to characterize the osteogenic differentiation of PC in 3-dimensional (3D) Poly-lactic-co-glycolic acid (PGLA) fleeces cultured in medium containing allogeneichuman serum;to study the better understanding of the global gene expression profile of PC grown in these constructs.Methods:Periosteal tissues were harvested from human mastoid of three patients undergoing mastoidectomy. PC were isolated according to the modifications of an enzymatic digestion method by use of collagenase II. The resulting PC were subsequently harvested, resuspended in PC medium, placed into cell culture flasks and cultured at 37°C with 5% CO2 and 95% humidified air. Adherent growing PC were cultured and sub-cultured until P7 under standard cell culture conditions. Dividing the number of PC obtained in passage Pn through those obtained in Pn_i demonstrates the proliferative activity during culture expansion.We studied their antigen phenotype with flow cytometry (FACS) using monoclonal antibodies(CD73-PE, CD34-PE, CD44-FITC, CD45-FITC, CD166-PE and CD 105) against antigens associated with mesenchymal stem cells. The multilineage potential of human PC (P3) was demonstrated by culturing these cells under standard conditions that promote mesenchymal stem cell differentiation. During osteogenesis, express of alkaline phosphatase and deposition of mineralized matrix were visualized by staining with fast BCIP/NBT and with von Kossa staining. Chondrogenesis was documented by histological staining of alcian blue 8GS as well as by immunohistochemical methods. Adipocytes were identified using vital staining with oil red O.Expanded cells of P3 were seeded into PGLA constructs and cultured in osteogenic medium for a maximum period of 28 days. Morphological, histological and cell viability analyses of three-dimensionally cultured PC were performed to elucidate osseous synthesis and deposition of a calcified matrix.Furthermore, Affymetrix oligonucleotide microarrays HG-U133 Plus 2.0 were used to monitor gene expression of PC during osteogenic process in one of these tissue-engineered bone transplants. Hierarchical clustering and k-means clustering in Genesis Software were used to systematically search for osteogenic differentiation-related genes.Results:PC grew in distinct colonies by day 7-10 and displayed a typical fibroblast-like morphology which remained stable during prolonged culture and subculture in monolayer. In higher cell passages, PC appeared slightly larger and more stretched in a homogeneous cell compartment.Although donor dependent, single primary cultures starting with less than 100 adherent cells and could be expanded to well over 13 X 106 cells in P3 (about PI X 33), and 2.5 X 109 cells in P7 (about PI X 6000). Analysis of PC(P3) by FACS showed a predominant cell population within the forward and sideward scatter in the density plot. PC of this cell population were negative for CD34. In contrast, PC showed a uniformly high expression of CD 166, CD 105. CD73> CD44 and CD45.Microarray analysis was performed to investigate whether PC express stem cell-related markers on the mRNA level. Summarized, PC do not express most of the early markers described for embryonic stem cells like the transcription factor Oct4/POU5F1, Nanog, SSEA-4/SIAT6, SOX1 and hTERT. PC showed no expression of typical hematopoietic stem cell surface antigens, like CD11, CD 14 and the early hematopoietic stem cell marker CD34. In contrast, they showed a similar but not identical expression of markers also expressed by mesenchymal stem cells, like CD44, CD71, SH-3, CD90, CD105 and ALCAM.Human PC (P3) were induced to develop into distinct mesenchymal lineages. Osteogenesis was documented by visualization of alkaline phosphatase (AP) activity, by matrix deposition of calcium demonstrated by von Kossa staining. Chondrogenesis was documented by alcian blue staining of cartilage proteoglycans, and by immunohistochemistry using type II collagen specific antibodies. To our knowledge for the first time, adipogenesis of PC was verified by cell morphology and by oil red O staining.Seeding of the expanded PC was technically feasible. The fibrin gel immobilization technique provided homogeneous cell distribution without significant cell loss during the seeding process. FDA/PI live-dead staining showed the vital PC inside the PGLA scaffolds. PGLA fibers remained intact by day 7. On day 28, theviability rate of PC was nevertheless estimated at approx. 90%, while PGLA fibers came into degradation phase indicated by red fluorescence.Two weeks after osteogenic induction, the constructs displayed a transparent appearance. HE sections of cell/PGLA constructs confirmed a homogeneous distribution of cells within the scaffolds as well. The pores of the PGLA fleece matrix were completely filled with fibrin gel. Cell clusters were seen within the deep constructs. Some were attached to the fleece matrix fibers. Von Kossa staining showed osteogenic structures (matrix mineralization) on the surface of the PLGA constructs. After 28 days in culture, the architecture of constructs showed bony appearance. A change in distribution occurred with a decrease of cell density inside the constructs. Von Kossa staining showed bridges between the fibers of PLGA fleeces and osseous nodule formation.Among about 47,000 genes analyzed, 8526 were changed more than two-fold at one or more time points in cells that developed bone matrix, and 1391 were changed more than 5-fold of their normal level of expression. A set of differentially expressed genes were discovered, including Osteoprotegerin, Osteomodulin, Stanniocalcin, Thrombospondin, IL-8, VEGF, Angiopoietin, JAK1, Hypoxia induced factor, Integrin, and so on. Quantitative RT-PCR of Selected GAPDH, type I collagen, Osteocalcin and Osteonectin genes verify the results of the microarray analysis.Conclusions:Successful harvest and isolation of PC can be achieved by enzymatic digestion method and in culture medium containing allogeneic human serum. PC show a similar but not identical expression profile of antigen markers also expressed by mesenchymal stem cells. PC have the osteogenic, chondrogenic and adipogenic multilineage developmental capacity. Stem cell-like PC are promising candidates for advanced cell therapies of complex skeletal tissue defects.The model of 3D PLGA seeded with the expanded PC was technically feasible. This model apply a promising method for creation of new tissue-engineered tissue in vitro. PC-PLGA constructs displayed a bone-like appearance, implicated a relativelyconstant level of matrix deposition, osteoblastic phenotype of the PC grown in 3D cell-seeded constructs under osteogenic differentiation conditions.Microarray results displayed a complex pattern of differentiation markers on stimulation. Our identification of a set of genes that may be associated with osteogenesis in tissue-engineered bone provides a better information toward individual treatment.