In VitroChondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells (original) (raw)
Related papers
Chondrogenic potential of blood-acquired mesenchymal progenitor cells
Introduction: The associated morbidity from the acquisition of mesenchymal stem cells (MSC) from the bone marrow has led to the investigation of alternative stem cell sources. We propose that such cells may be isolated from non-mobilised blood and demonstrate their differentiation into a chondrocytic lineage. This safe and abundant source of cells may be useful for tissue engineering cartilage. Method: Peripheral blood mononuclear cells (PBMC) were isolated from healthy adults and cultured in RPMI medium supplemented with serum. The non-adherent and adherent cells were analysed for cell surface marker expression of CD14, CD34, CD133, CD105 and CD45 by flow cytometry. Adherent cells were also cultured on glass slides in chondrogenic media and analysed for the expression of collagen I and II on day 14 of culture. Results: The adherent cells were fibroblastic in morphology and were confluent on day 14. The non-adherent and adherent cell populations were shown to have distinct profiles by flow cytometry. The adherent cells were positive for CD105 and CD14 and also expressed collagen I and II precursors when cultured in chondrogenic media. Conclusion: Blood-acquired mesenchymal progenitor cells (BMPCs) can be isolated from non-mobilised blood. These unique cells are CD105 þ and CD14 þ and have chondrogenic differentiation capacity. BMPC may provide a potential source of MPC for tissue engineering applications.
Hypertrophic chondrocytes undergo further differentiation in culture
Journal of Cell Biology, 1992
Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102 :2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain oNG bone organogenesis occurs in the embryo by endochondral ossification from undifferentiated mesenchyme. During the early stages of development, mesenchymal cells in the limb buds condense to form a core of differentiated chondrocytes ; osteogenesis starts at the periphery ofthe cartilage core, which is subsequently invaded by blood vessels and replaced by bone marrow and trabecular bone. After birth, similar events take place in the long bone growth plate and at the bone fracture sites. Bone formation and remodeling have been extensively investigated, starting from pioneering work describing the morphological and biochemical changes occurring during early bone formation to more recent studies aimed at the elucidations of the cellular and molecular mechanisms involved (7, 22, 34). It is widely agreed that cells present in a continuous collar surrounding, but separated from the cartilage rudiment, give rise to osteoblasts, i.e., cells responsible for the synthesis and mineralization of the osteoid extracellular matrix. In the past, occasionally and recently more frequently, it has been postulated thatgrowth platehypertrophic chondrocytes might also contribute to the formation of a bone matrix, since in some organ culturesthese cells start to express bone markers. During culture of mouse mandibular condyles, the expression of type I collagen, osteonectin, alkaline phosphatase, osteopontin, and osteocalcin by mature chondrocytes was detected by in situ hybridization (38). A morphological study
In tissue engineering fields, recent interest has been focused on stem cell therapy to replace or repair damaged tissues. In particular, the repair of articular cartilage degeneration by stem cell-based tissue engineering could be of potential therapeutic. Bone marrow mesenchymal stem cells (BM-MSCs) therapy has offered new treatment for cartilage damaged; however, we must first understand the interaction of growth factor and receptors that initiate chondrogenic differentiation and the exact expression required for this differentiation. In this research, we focused on identifying the best combination of MSCs and functional extracellular matrices that provides essential elements for successful chondrogenesis. We experimentation mixture of growth factor, transforming growth factor beta1 (TGF-ß1), insulin like growth factor 1 (IGF-1), fibroblast growth factor, addition to dexamethasone, and ascorbic acid. We assessed the chondrogenic capacity by histology using H&E, alcian blue staining and immunohisto chemical analysis with chondrocyte markers which were positive for Collagen I, II proteins responsible for the chondrogenesis while advantageous cartilage acidic protein 1 (CRTAC1),exclusive proteins contribute to differentiation more than to proliferation. And use 3D (matrix) culture system prove to create cartilage like tissue that may use for damaged cartilage treatments
Molecular medicine reports, 2013
The objective of the current study was to investigate the ability of human umbilical cord blood‑derived mesenchymal stem cells (HUCB‑MSCs) to undergo chondrogenic differentiation, by co‑culture with rabbit chondrocytes. The aim was to obtain more seed cells for tissue engineering research and lay the foundation for the clinical repair of cartilage defects. The studies were performed using isolated rabbit cartilage cells and HUCB‑MSCs in vitro, which were co‑cultured in a 2:1 or 3:1 ratio with or without insulin‑like growth factor‑1 (IGF‑1). Following 7 and 14 days in culture, cell morphology was observed in each group. RNA and protein were extracted to assess the expression levels of aggrecan (ACAN) and collagen type II (COL2A) using quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Groups of cells that were co‑cultured exhibited significantly higher expression levels of ACAN and COL2A mRNA and protein, compared with the reduced effect of IGF‑1 at day...
Journal of Orthopaedic Research, 2008
Microfracture is frequently used to repair articular cartilage defects and allows mesenchymal progenitors to migrate from subchondral bone into the defect and form cartilaginous repair tissue. The aim of our study was to analyze the cell surface antigen pattern and the differentiation capacity of cells derived from human subchondral bone. Human progenitor cells were derived from subchondral corticospongious bone and grown in the presence of human serum. Stem cell-related cell surface antigens were analyzed by flowcytometry. Corticospongious progenitor (CSP) cells showed presence of CD73, CD90, CD105, and STRO-1. Multilineage differentiation potential of CSP cells was documented by histological staining and by gene expression analysis of osteogenic, adipogenic, and chondrogenic marker genes. CSP cells formed a mineralized matrix as demonstrated by von Kossa staining and showed induction of osteocalcin, independent of osteogenic stimulation. During adipogenic differentiation, the adipogenic marker genes fatty acid binding protein 4 and peroxisome proliferative activated receptor g were induced. Immunohistochemical staining of cartilage-specific type II collagen and induction of the chondrocytic marker genes cartilage oligomeric matrix protein, aggrecan, and types II and IX collagen confirmed TGFb3-mediated chondrogenic lineage development. CSP cells from subchondral bone, as known from microfracture, are multipotent stem cell-like mesenchymal progenitors with a high chondrogenic differentiation potential. ß
Biochemical and Biophysical Research Communications, 2010
Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed a difference in the temporal expression of chondrogenic markers which were up regulated in chondrogenic medium compared to levels in basal medium. Of the three cell types studied, adult chondrocytes offer a more promising cell source for cartilage tissue engineering. This comparative study revealed differences between the microenvironment of all three cell types and provides useful information to inform cell-based therapies for cartilage regeneration.
Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells
Arthritis & Rheumatism, 2008
Objective-Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers, e.g., collagen type X, by MSCs undergoing chondrogenesis raises concern for tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biological basis of MSC hypertrophy, we examine the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy-associated markers. Methods-Human MSC pellet cultures are pre-differentiated for two weeks in a chondrogenic medium, and hypertrophy induced with TGF-β withdrawal, dexamethasone reduction, and addition of thyroid hormone (T3). Cultures are characterized by histological, immunohistochemical, and biochemical methods, and for gene expression using quantitative RT-PCR. Results-A combination of TGF-β withdrawal, dexamethasone reduction, and T3 addition is essential for hypertrophy induction. Cyto-morphological changes are accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various hypertrophy markers, including collagen type X, receptors (FGFR1-3, PTHrPR, RARγ), MMP-13, Indian hedgehog, osteocalcin, and the pro-apoptotic gene, p53. However, hypertrophy is not induced uniformly throughout the pellet culture, with distinct regions of dedifferentiation seen. Conclusions-Chondrogenically differentiating MSCs behave functionally similar to growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC-derived chondrocytes undergo a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophyinhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering.
An immunohistochemical study of extracellular matrix formation during chondrogenesis
Developmental Biology, 1978
The accumulation of chondroitin sulfate proteoglycan was correlated with chick limb bud chondrogenesis by immunohistochemical methods. Antisera directed against chondroitin sulfate proteoglycan subunit were used in indirect immunohistochemical reactions which also utilized goat anti-rabbit IgG coupled to fluorescein isothiocyanate, peroxidase, or hemocyanin. Immunohistochemical reaction products were localized to alcian green staining regions in developing limb bud cultures. Developing cartilage nodules reacted with the immunohistochemical probes by 3-4 days and showed a progressive increase in reaction with time. Noncartilage cells in the same cultures did not react. Scanning electron microscopy revealed extensive binding of hemocyanin-antibody complexes to the chondrogenic matrix network. In contrast, the matrix synthesized by limb bud cultures blocked from differentiating by 5bromo-2'-deoxyuridine showed no reaction.
Developmental control of chondrogenesis and osteogenesis
2000
During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.
Biotechnology and Bioengineering, 2006
Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) β1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative “Real Time” RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF β1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF β1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF β1 to enhance the differentiation. © 2006 Wiley Periodicals, Inc.