Expression Of Chondrogenic Marker Genes Research Articles

Osteochondral Interface Tissue Engineering Using Macroscopic Gradients of Bioactive Signals

Continuous gradients exist at osteochondral interfaces, which may be engineered by applying spatially patterned gradients of biological cues. In the present study, a protein-loaded microsphere-based scaffold fabrication strategy was applied to achieve spatially and temporally controlled delivery of bioactive signals in three-dimensional (3D) tissue engineering scaffolds. Bone morphogenetic protein-2 and transforming growth factor-beta(1)-loaded poly(D,L-lactic-co-glycolic acid) microspheres were utilized with a gradient scaffold fabrication technology to produce microsphere-based scaffolds containing opposing gradients of these signals. Constructs were then seeded with human bone marrow stromal cells (hBMSCs) or human umbilical cord mesenchymal stromal cells (hUCMSCs), and osteochondral tissue regeneration was assessed in gradient scaffolds and compared to multiple control groups. Following a 6-week cell culture, the gradient scaffolds produced regionalized extracellular matrix, and outperformed the blank control scaffolds in cell number, glycosaminoglycan production, collagen content, alkaline phosphatase activity, and in some instances, gene expression of major osteogenic and chondrogenic markers. These results suggest that engineered signal gradients may be beneficial for osteochondral tissue engineering.

Animal serum-free expansion and differentiation of human mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo

BackgroundPattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis.ResultsIn this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II.ConclusionOur results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm.

Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Although pellet culture and encapsulation of chondrocytes into gel-like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate-fibrin, alginate-chitosan) and how do the cells behave in comparison to biomaterial-free (pellet) three-dimensional culturing? Monolayer-expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate-chitosan, alginate-fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate-fibrin-encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate- and alginate-chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D-cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate-cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D-environment.

Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro

Mustn1 encodes a small nuclear protein expressed specifically in the musculoskeletal system that was originally identified as a strongly up-regulated gene during bone regeneration, especially in fracture callus proliferating chondrocytes. Further experiments were undertaken to investigate its expression and role during chondrogenesis. Initially, whole mount mouse in situ hybridization was carried out and revealed Mustn1 expression in areas of active chondrogenesis that included limb buds, branchial arches and tail bud. To elucidate its function, experiments were carried out to perturb Mustn1 by overexpression and silencing in the pre-chondrocytic RCJ3.1C5.18 (RCJ) cell line. In these cells, Mustn1 is normally differentially regulated, with a spike in expression 2 days after induction of differentiation. Further, Mustn1 was successfully overexpressed in multiple RCJ cell lines by ∼2–6 fold, and reduced to ∼32–52% in silenced cell lines as compared to parental Mustn1 levels. Overexpressing, silenced, control, and parental RCJ cell lines were assayed for proliferation and differentiation. No statistically significant changes were observed in either proliferation or proteoglycan production when Mustn1 overexpressing lines were compared to parental and control. By contrast, both proliferation rate and differentiation were significantly reduced in Mustn1 silenced cell lines. Specifically, RNAi silenced cell lines showed reductions in populations of ∼55–75%, and also ∼34–40% less matrix (proteoglycan) production as compared to parental and random control lines. Further, this reduction in matrix production was accompanied by significant downregulation of chondrogenic marker genes, such as Sox9, Collagen type II (Col II), and Collagen type X (Col X). Lastly, reintroduction of Mustn1 into a silenced cell line rescued this phenotype, returning proliferation rate, matrix production, and chondrogenic marker gene expression back to parental levels. Taken together these data suggest that Mustn1 is a necessary regulator of chondrocyte function.

In Vitro Generation of a Scaffold-Free Tissue-Engineered Construct (TEC) Derived from Human Synovial Mesenchymal Stem Cells: Biological and Mechanical Properties and Further Chondrogenic Potential

The purpose of this study was to characterize a tissue-engineered construct (TEC) generated with human synovial mesenchymal stem cells (MSCs). MSCs were cultured in medium with ascorbic acid 2-phosphate (Asc-2P) and were subsequently detached from the substratum. The detached cell/matrix complex spontaneously contracted to develop a basic TEC. The volume of the TEC assessed by varying initial cell density showed that it was proportional to initial cell densities up to 4 x 10(5) cells/cm(2). Assessment of the mechanical properties of TEC using a custom device showed that the load at failure and stiffness of the constructs significantly increased with time of culture in the presence of Asc-2P, while in the absence of Asc-2P, the constructs were mechanically weak. Thus, the basic TEC possesses sufficiently self-supporting mechanical properties in spite of not containing artificial scaffolding. TEC further cultured in chondrogenic media exhibited positive alcian blue staining with elevated expression of chondrogenic marker genes. Based on these findings, such human TEC may be a promising method to promote cartilage repair for future clinical application.

Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells

The objective was to in vitro generate a mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC) to facilitate in vivo repair in a porcine chondral defect model. Porcine synovial MSCs were cultured in monolayer at high density and were subsequently detached from the substratum. The cell/matrix complex spontaneously contracted to develop a basic TEC. Immunohistochemical analysis showed that the basic TEC contained collagen I and III, fibronectin, and vitronectin. The basic TEC exhibited stable adhesion to the surface of a porcine cartilage matrix in an explant culture system. The TEC cultured in chondrogenic media exhibited elevated expression of glycosaminoglycan and chondrogenic marker genes. The TEC were implanted in vivo into chondral defects in the medial femoral condyle of 4-month-old pigs, followed by sacrifice after 6 months. Implantation of a TEC into chondral defects initiated repair with a chondrogenic-like tissue, as well as secure biological integration to the adjacent cartilage. Histologically, the repair tissue stained positively with Safranin O and for collagen II. Biomechanical evaluation revealed that repair tissue exhibited mechanical properties similar to those of normal porcine cartilage in static compression and friction tests. This technology is a unique and promising method for stem cell-based cartilage repair.

CD45-positive cells of haematopoietic origin enhance chondrogenic marker gene expression in rat marrow stromal cells

Adult mesenchymal stem cells (MSCs) can be readily isolated from bone marrow, expanded in culture and subsequently subjected to differentiation into various connective tissue lineages. In general, for animal studies separation of MSCs from other bone marrow-derived cells is achieved by sole adherence to plastic surface of tissue culture flasks; however, this procedure produces a heterogeneous cell population containing CD45-positive haematopoietic cells (HCs) and haematopoietic stem cells (HSCs). It is known, that mixed cell cultures consisting of cocultures of differentiated somatic cells with adult stem cells promote differentiation towards specific cell lineages. For determining the effect of the CD45-positive cell population on the differentiation potential of MSCs, we sorted out the bone marrow-derived adherent cells by immunomagnetic technique (MACS) to attain a subpopulation of CD45-depleted cells. Herein, we show that the presence of adherent CD45-positive HCs not only promote expression of the chondrogenic marker genes Col2a1, COMP and Sox9, but also of Col1a1, Col10a1 and to a certain degree Cbfa1 in MSCs when cultured in an appropriate three-dimensional environment. These observations constitute a step towards unravelling the influence of haematopoietic cells on chondrogenic differentiation of MSCs.

Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound enhances the chondrogenic phenotype in vitro

This study examined the effects of low intensity pulsed ultrasound (LIPUS) on human bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation. Aggregates of mesenchymal stem cells and mesenchymal stem cells seeded in three dimensional matrices were cultured in a defined chondrogenic medium and subjected to LIPUS for the first 7 days of culture. At 1, 7, 14 and 21 days, samples were harvested for histology, immunohistochemistry, RT-PCR, and quantitative DNA and matrix macromolecule analysis. Cell aggregates with daily treatment for 20 minutes showed no significant differences for proteoglycan and collagen content during chondrogenic differentiation. However ultrasound application for 40 minutes daily resulted in a statistically significant increase of the proteoglycan and collagen content after 21 days in culture. Aggregates treated for 20 minutes daily showed decreased expression of chondrogenic genes at all time points. In contrast, 40 minutes of daily treatment of aggregates resulted in a significant increase of chondrogenic marker genes after an initial decrease at day 7 with time in culture. Ultrasound treated cell-scaffold constructs showed a significant increase of chondrogenic marker gene expression and extracellular matrix deposition. This study indicates that LIPUS can be used to enhance the chondrogenesis of mesenchymal stem cells in cell aggregates and cell-scaffold constructs. We have found a dependency on the specific treatment parameters. We hypothesize that LIPUS can be used for an improved in vitro preparation of optimized tissue engineering implants for cartilage repair. Furthermore this non-invasive method could also be of potential use in vivo for regenerative therapy of cartilage in the future.

Gene Expression Analysis of Ectopic Bone Formation Induced by Electroporatic Gene Transfer of BMP4

Implantation of bone morphogenetic protein (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce bone formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and bone formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic bone formation by using semi-quantitative RT-PCR.A plasmid vector containing mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the process of endochondral bone formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day 10. As osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) gene expressions were clearly detected from day 10 and then increased by day 14. In conclusion, the present study proved that ectopic bone formation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix.

Influence of three-dimensional culture in a type II collagen sponge on primary cultured and dedifferentiated chondrocytes

Background. Once articular cartilage is destroyed, the intrinsic reparative ability is poor. Therefore, various techniques have been developed to repair articular defects. Many kinds of scaffolds have been used for cultured chondrocyte transplantation. In this study, we developed a sponge consisting of type II collagen. We investigated the influence of threedimensional culture on the maintenance of the chondrocyte phenotype and on the redifferentiation of dedifferentiated chondrocytes. Methods. Chondrocytes were isolated from the rib cartilage of rats and were cultured in plastic dishes for a week (P0). The cells were then dissociated with trypsin and subcultured for another 2weeks (P1). Primary isolated chondrocytes were cultured in the type II collagen sponges for 3weeks (S1). We compared the gene expression of S1 for chondrogenic markers with the expression of P0 and P1 by reverse transcription- polymerase chain reaction (RT-PCR). The cells were then dissociated with trypsin and subcultured for another 2weeks (P1) and then another 6weeks (P3). Cells of P1 were subsequently cultured in type II collagen sponges for 4weeks (P1r). At each time point, gene expression of chondrogenic markers was examined by RT-PCR. Results. Gene expression of COL2A1, COL10A1, and aggrecan in S1 was the same as in P0. Gene expression of COL10A1 and aggrecan in P1r was higher than in P1 and P3. Gene expression of COL1A1, COL2A1, and SOX9 in P1r was lower than in P1 and P3. Gene expression of ALP and osteocalcin in P1r was detected. Conclusions. These results show that culture in type II collagen sponges could maintain the chondrocyte phenotype; however, dedifferentiated chondrocytes differentiated to hypertrophic chondrocytes. These finding suggest that the complex of cells and scaffolds with primary cells was more useful than that with dedifferentiated chondrocytes in laboratory and clinical application.