Expression Of Chondrogenic Markers Research Articles

Comparison of Potentials of Stem Cells Isolated from Tendon and Bone Marrow for Musculoskeletal Tissue Engineering

The use of tendon-derived stem cells (TDSCs) as a cell source for musculoskeletal tissue engineering has not been compared with that of bone marrow stromal cells (BMSC). This study compared the mesenchymal stem cell (MSC) and embryonic stem cells (ESC) markers, clonogenicity, proliferative capacity, and multilineage differentiation potential of rat TDSC and BMSC in vitro. The MSC and ESC marker profiles of paired TDSC and BMSC were compared using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Their clonogenicity and proliferative capacity were compared using colony-forming and 5-bromo-2'-deoxyuridine assays, respectively. The expression of tenogenic, osteogenic, and chondrogenic markers at basal state were examined using qRT-PCR. Their osteogenic, chondrogenic, and adipogenic differentiation potentials were compared using standard assays. TDSC and BMSC showed similar expression of CD90 and CD73. TDSC expressed higher levels of Oct4 than BMSC. TDSC exhibited higher clonogenicity, proliferated faster, and expressed higher tenomodulin, scleraxis, collagen 1 α 1 (Col1A1), decorin, alkaline phosphatase, Col2A1, and biglycan messenger RNA levels than BMSC. There was higher calcium nodule formation and osteogenic marker expression in TDSC than BMSC upon osteogenic induction. More chondrocyte-like cells and higher glycosaminoglycan deposition and chondrogenic marker expression were observed in TDSC than BMSC upon chondrogenic induction. There were more oil droplets and expression of an adipogenic marker in TDSC than BMSC upon adipogenic induction. TDSC expressed higher Oct4 levels, which was reported to positively regulate mesendodermal lineage differentiation, showed higher clonogenicity and proliferative capacity, and had greater tenogenic, osteogenic, chondrogenic, and adipogenic markers and differentiation potential than BMSC. TDSC might be a better cell source than BMSC for musculoskeletal tissue regeneration.

The Effect of Two- and Three-Dimensional Cell Culture on the Chondrogenic Potential of Human Adipose-Derived Mesenchymal Stem Cells after Subcutaneous Transplantation with an Injectable Hydrogel

Articular cartilage is an avascular tissue composed of chondrocytes, a unique cell type responsible for abundant matrix synthesis and maintenance. When damaged, it never heals spontaneously under physiological circumstances. Therefore, the delivery of mesenchymal stem cells using hydrogel has been considered for cartilage repair. This study aims at investigating the influence of in vitro chondrogenic differentiation of human adipose tissue-derived stem cells (hATSCs) on in vivo cartilage formation when associated with a cellulose-based self-setting hydrogel (Si-HPMC). hATSCs were characterized for their proliferation, surface marker expression, and multipotency. The in vitro chondrogenic potential of hATSCs cultured within Si-HPMC in control or chondrogenic medium was evaluated by measuring COL2A1, ACAN, SOX9, and COMP expression by real-time PCR. Alcian blue and type II collagen staining were also performed. To determine whether in vitro chondrogenically differentiated hATSCs may give rise to cartilage in vivo, cells differentiated as a monolayer or in pellets were finally associated with Si-HPMC and implanted subcutaneously into nude mice. Cartilage formation was assessed histologically by alcian blue and type II collagen staining. Our data demonstrate that hATSCs exhibited proliferation and self-renewal. hATSCs also expressed typical stem cell surface markers and were able to differentiate towards the adipogenic, osteogenic, and chondrogenic lineages. Real-time PCR and histological analysis indicated that Si-HPMC enabled chondrogenic differentiation of hATSCs in inductive medium, as demonstrated by increased expression of chondrogenic markers. In addition, histological analysis of implants showed that chondrogenically differentiated hATSCs (monolayers or pellets) have the ability to form cartilaginous tissue, as indicated by the presence of sulphated glycosaminoglycans and type II collagen. This study therefore suggests that an in vitro induction of hATSCs in 2D was sufficient to obtain cartilaginous tissue formation in vivo. Si-HPMC associated with autologous hATSCs could thus be a significant tool for regenerative medicine in the context of cartilage damage.

Electroporation-Mediated Transfer ofSOX TrioGenes (SOX-5, SOX-6, andSOX-9) to Enhance the Chondrogenesis of Mesenchymal Stem Cells

The purpose of this study was to test the hypothesis that the SOX trio genes (SOX-5, SOX-6, and SOX-9) have a lower level of expression during the chondrogenic differentiation of mesenchymal stem cells (MSCs) compared with chondrocytes and that the electroporation-mediated gene transfer of SOX trio promotes chondrogenesis from human MSCs. An in vitro pellet culture was carried out using MSCs or chondrocytes at passage 3 and analyzed after 7 and 21 days. Then, MSCs were transfected with SOX trio genes and analyzed for the expression of chondrogenic markers after 21 days of in vitro culture. Without transforming growth factor-β1, the untransfected MSCs had a lower level of SOX trio gene and protein expression than chondrocytes. However, the level of SOX-9 gene expression increased in MSCs when treated with transforming growth factor-β1. GAG level significantly increased 7-fold in MSCs co-transfected with SOX trio, which was corroborated by Safranin-O staining. SOX trio co-transfection significantly increased COL2A1 gene and protein and decreased COL10A1 protein in MSCs. It is concluded that the SOX trio have a significantly lower expression in human MSCs than in chondrocytes and that the electroporation-mediated co-transfection of SOX trio enhances chondrogenesis and suppresses hypertrophy of human MSCs.

A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies

To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide) scaffold. By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water-dioxane-PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT-PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O'Driscoll score. In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells. The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide scaffold with cells.

Chondrogenic potential of electrospun nanofibres for cartilage tissue engineering

Articular cartilage has a heterogeneous structure, comprising elongated cells at the articulating surface and rounded cells elsewhere. This feature poses a complex challenge when fabricating 3D tissue engineering scaffolds able to mimic the native extracellular matrix (ECM) of cartilage for tissue repair and regeneration. Nanofibre scaffolds can provide an ECM-like structure, but are mechanically weak and typically have subcellular pore geometries. In this study, the use of poly(L,D-lactide) (PLDLA) nanofibre coatings on PLDLA microfibres or films (nanofibre composites) to influence bovine chondrocyte behaviour was investigated. It was demonstrated that electrospun nanofibres facilitated the adhesion of chondrocytes and helped to maintain smaller projected cell areas and a rounded cell phenotype, when compared to PLDLA films or microfibres. Random nanofibre composites were associated with the smallest and most rounded cells and aligned nanofibre composites also demonstrated a similar tendency. Quantitative PCR revealed that nanofibres promoted the expression of chondrogenic markers, such as collagen type IIaI and aggrecan, while maintaining low levels of collagen IaI. It was also found, by water contact angle measurement, that nanofibres were significantly more hydrophobic than cast films. The lower wettability of polymeric nanofibres favoured the maintenance of rounded chondrocyte morphology. To our knowledge this is the first study to confirm the positive influence on preserving chondrogenic phenotype and gene expression at the interface of true nano-microfibrous composites by using individual microfibres coated with aligned nanofibres. Such composites can potentially be fabricated into mechanically durable 3D scaffolds with better cell infiltration throughout the scaffolds.

Gene expression and cell differentiation in matrix-associated chondrocyte transplantation grafts: a comparative study

Although scaffold composition and architecture are considered to be important parameters for tissue engineering, their influence on gene expression and cell differentiation is rarely investigated in scaffolds used for matrix-associated autologous chondrocyte transplantation (MACT). In this study we have therefore comparatively analyzed the gene expression of important chondrogenic markers in four clinical applied cell-graft systems with very different scaffold characteristics. Residuals (n=165) of four different transplant types (MACI®, Hyalograft®C, CaReS® and Novocart®3D) were collected during surgery and analyzed for Col1, Col2, aggrecan, versican, melanoma inhibitory activity (MIA) and IL-1β by real-time PCR. Scaffold and cell morphology were evaluated by histology and electron microscopy. Despite the cultivation on 3D scaffolds, the cell differentiation on all transplant types didn't reach the levels of native cartilage. Gene expression highly differed between the transplant types. The highest differentiation of cells (Col2/Col1 ratio) was found in CaReS®, followed by Novocart®3D, Hyalograft®C and MACI®. IL-1β expression also exhibited high differences between the scaffolds showing low expression levels in Novocart®3D and CaReS® and higher expression levels in MACI® and Hyalograft®C. Our data indicate that scaffold characteristics as well as culture conditions highly influence gene expression in cartilage transplants and that these parameters may have profound impact on the tissue regeneration after MACT.

Enhanced chondrogenic marker expression of human mesenchymal stem cells by interaction with both TGF‐β3 and hyaluronic acid

This study was designed to evaluate the additive effects of transforming growth factor-beta3 (TGF-β3) and hyaluronic acid (HA) on chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The hMSCs were cultured on collagen type I-, HA-, or fibronectin-coated cell culture dishes with or without TGF-β3 added to the culture medium. Four weeks after cell culture, chondrogenic differentiation of hMSCs was determined by evaluating the expression of cartilage-specific markers using real-time polymerase chain reaction, immunocytochemistry, and Western blot analysis. hMSCs cultured on HA-coated dishes with TGF-β3 supplementation revealed a prominent increase in collagen type II, aggrecan, and Sox9. When hMSCs were cultured without TGF-β3 supplementation, only hMSCs cultured on HA-coated dishes showed prominent expression of the cartilage-specific markers. This study shows that chondrogenic differentiation of hMSCs can be enhanced additively by interactions with both a specific cell-adhesion matrix and a soluble growth factor.

Identification and characterization of GRP in articular cartilage and synovial fluid using a proteomic-based approach

Identification and characterization of GRP in articular cartilage and synovial fluid using a proteomic-based approach

Effects of oxygen tension on the chondrogenic potential of differentially cultured human adipose-derived stem cells

Effects of oxygen tension on the chondrogenic potential of differentially cultured human adipose-derived stem cells

Chondrogenesis of Rabbit Mesenchymal Stem Cells in Fibrin/Hyaluronan Composite ScaffoldIn Vitro

Scaffold material is expected to play a crucial role in induction of chondrogenic differentiation of mesenchymal stem cells (MSCs) for cartilage tissue engineering. Here we demonstrated the feasibility of a fibrin/hyaluronan (HA) composite hydrogel as a potent scaffold for support of chondrogenesis of rabbit MSCs (rMSCs). rMSCs were prepared in three-dimensional cultures of pellet, alginate layer, and fibrin/HA gel. Specimens in each group were cultured in chondrogenic defined media for 4 weeks in the absence or presence of transforming growth factor β1 (TGF-β1) treatment. Viability of rMSCs was somewhat reduced until 4 weeks, which was less significant in fibrin/HA gels than in the alginate layer (*p < 0.05). The fibrin/HA group showed transient size reduction by about 35% at 1 week, but showed significantly higher mechanical strength than the alginate group. In safranin-O and alcian blue stains, accumulation of sulfated glycosaminoglycans (GAGs) was observed clearly from 1 week, and homogenously in the entire area at 4 weeks in the fibrin/HA group. Of note, TGF-β1 treatment showed no additional effect on GAGs accumulation in the fibrin/HA group. The alginate and pellet groups, however, showed much lower levels of GAGs accumulation only in the presence of TGF-β1. Biochemical assays for GAGs and collagen, and expression of chondrogenic markers also showed much better results in the fibrin/HA group, even without TGF-β treatment than the other groups. These results demonstrated that fibrin/HA composite gel efficiently promoted chondrogenic differentiation of rMSCs, even without TGF-β treatment, and that it could be a useful tool for use in cartilage tissue engineering.

The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.

Adipose tissue-derived stem cells show both immunogenic and immunosuppressive properties after chondrogenic differentiation

The chondrogenic differentiation potential of mesenchymal stromal cells (MSC), as well as their immunosuppressive properties, have been studied extensively. So far, only a few studies have addressed the question of whether MSC still retain their immunosuppressive qualities after transdifferentiation. In particular, the expression of immunogenic markers, such as human leukocyte antigen (HLA)-DR, after differentiation has never been investigated. Chondrogenic transdifferentiation was induced in human adipose tissue-derived stem cell (ADSC) pellet cultures derived from 10 different patients, using 10 ng/mL transforming growth factor (TGF)-β3. Samples were harvested over a time-course of 28 days and analyzed by immunohistochemistry and reverse transcription (RT)-polymerase chain reaction (PCR). The cytokine levels in the supernatants of the samples were measured semi-quantitatively by dot-blots and quantitatively by enzyme-linked immunosorbant assays (ELISA). Undifferentiated ADSC were negative for chondrogenic markers, as well as HLA-ABC and HLA-DR epitopes in immunofluorescence. In contrast, TGF-β3-induced pellet cultures showed both expression of chondrogenic differentiation markers, such as transcription factor 9 (Sox 9), collagen type IIa and aggrecan, and an up-regulation of HLA-DR, beginning at day 7 after induction. Interferon-γ (INF-γ) is known to up-regulate HLA-DR. Therefore we measured INF-γ levels in the supernatants of TGF-β3-induced pellets and, indeed, INF-γ was up-regulated during chondrogenesis in ADSC pellet cultures. However, both undifferentiated and TGF-β3-induced ADSC also showed expression of immunosuppressive HLA-G and interleukin (IL)-10 up-regulation. These results suggest that the immunogenicity of adult stem cell-derived tissue should be tested in animal models before clinical trials for allogeneic engineered tissue are considered.

Arterial injury promotes medial chondrogenesis in Sm22 knockout mice

Expression of SM22 (also known as SM22alpha and transgelin), a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis. Sm22 knockout (Sm22(-/-)) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9). This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22(-/-) VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation. These findings suggest that disruption of SM22 alters the actin cytoskeleton and promotes chondrogenic conversion of VSMCs.

Microscale Versus Nanoscale Scaffold Architecture for Mesenchymal Stem Cell Chondrogenesis

Nanofiber scaffolds, produced by the electrospinning technique, have gained widespread attention in tissue engineering due to their morphological similarities to the native extracellular matrix. For cartilage repair, studies have examined their feasibility; however these studies have been limited, excluding the influence of other scaffold design features. This study evaluated the effect of scaffold design, specifically examining a range of nano to micron-sized fibers and resulting pore size and mechanical properties, on human mesenchymal stem cells (MSCs) derived from the adult bone marrow during chondrogenesis. MSC differentiation was examined on these scaffolds with an emphasis on temporal gene expression of chondrogenic markers and the pluripotent gene, Sox2, which has yet to be explored for MSCs during chondrogenesis and in combination with tissue engineering scaffolds. Chondrogenic markers of aggrecan, chondroadherin, sox9, and collagen type II were highest for cells on micron-sized fibers (5 and 9 μm) with pore sizes of 27 and 29 μm, respectively, in comparison to cells on nano-sized fibers (300 nm and 600 to 1400 nm) having pore sizes of 2 and 3 μm, respectively. Undifferentiated MSCs expressed high levels of the Sox2 gene but displayed negligible levels on all scaffolds with or without the presence of inductive factors, suggesting that the physical features of the scaffold play an important role in differentiation. Micron-sized fibers with large pore structures and mechanical properties comparable to the cartilage ECM enhanced chondrogenesis, demonstrating architectural features as well as mechanical properties of electrospun fibrous scaffolds enhance differentiation.

Effect of flow perfusion conditions in the chondrogenic differentiation of bone marrow stromal cells cultured onto starch based biodegradable scaffolds

Cartilage tissue engineering (TE) typically involves the combination of a 3-D biodegradable polymeric support material, with primary chondrocytes or other cell types able to differentiate into chondrocytes. The culture environment in which cell–material constructs are created and stored is an important factor. Various bioreactors have been introduced in TE approaches to provide specific culturing environments that might promote and accelerate cells’ potential for chondrogenic differentiation and enhance the production of cartilage extracellular matrix. The aim of the present study was to investigate the chondrogenic differentiation of goat bone marrow cells (GBMCs) under flow perfusion culture conditions. For that purpose, GBMCs were seeded into starch–polycaprolactone fiber mesh scaffolds and cultured in a flow perfusion bioreactor for up to 28days using culture medium supplemented with transforming growth factor-β1. The tissue-engineered constructs were characterized after several end points (7, 14, 21 and 28days) by histological staining and immunocytochemistry analysis, as well as by glycosaminoglycan and alkaline phosphatase quantification assays. In addition, the expression of typical chondrogenic markers was assessed by real-time reverse-transcription polymerase chain reaction analysis. In general, the results obtained suggest that a flow perfusion microenvironment favors the chondrogenic potential of GBMCs.

Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

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.

Effect of bone graft substitute on marrow stromal cell proliferation and differentiation

Marrow stromal cells (MSCs) are ideally suited for tissue engineered bone grafts since they have the potential to regenerate bone, but may also maintain the homeostasis of the repaired tissue through their ability for self-renewal. An ideal bone graft substitute should support MSC self-renewal as well as differentiation to ensure complete bone defect regeneration and maintenance. The purpose of this investigation was to determine the effect of different substrate materials on MSC expansion and differentiation. Calcium polyphosphate (CPP), bone and hydroxyapatite/tricalcium phosphate (HA/TCP) were seeded with rat MSCs and maintained in culture conditions that promote cell expansion. At 0, 3, 7, 14, and 21 days cell numbers were determined by measuring their metabolic activity using a MTT assay and the frequency of cycling cells by 24 hr BrdU incorporation. Osteogenic, chondrogenic, and adipogenic marker expression in these cultures was measured by qRT-PCR. An initial drop in cell numbers was observed on all substrates. CPP and bone, but not HA/TCP supported an increase in proliferating cells at day 14 and 21. In addition, no upregulation of mature bone markers was observed in cells cultured on CPP and bone, which suggests that these substrates support the expansion of undifferentiated MSCs. In contrast, cell numbers on HA/TCP decreased with time and only rare BrdU positive cells were observed. This decrease in proliferation correlated with the down regulation of osteogenic progenitor markers and the substantial increase in mature osteocyte markers, indicating that HA/TCP favors MSC differentiation and maturation along the osteogenic lineage.

Biophysical and chemical effects of fibrin on mesenchymal stromal cell gene expression

Mesenchymal stromal cells (MSCs) are multipotent cells that have high expansion yields and fibrin is a native extracellular matrix (ECM) material widely used for cell delivery and surgery. MSCs and fibrin have tremendous potential for tissue engineering applications, but the effect of fibrin on MSCs is not well characterized. The purpose of this study was to analyze the role of fibrin in modulating MSC phenotype by gene expression analysis. The results demonstrate that fibrin up-regulated MSC gene expression of vasculogenic (FLK1, ACTA2, VECAD, SM22 and CNN1), myogenic (MYF5 and MYH13), neurogenic (TH and GFAP) and chondrogenic (COL2A1) markers after 5 days incubation. These gene expression results were supported by induction of expression on the protein level for early lineage-specific markers such as ACTA2, FLK1 and MYF5. The ability of fibrin to modulate MSC gene expression was not affected by matrix pore size (80-110 microm diameter) or Young's modulus (5-25k Pa) and the differential expression of some phenotypic markers could be partially mimicked by other ECM proteins, such as fibronectin and collagen I. In some cases the inductive effect of fibrin on gene expression could be further augmented by the treatment with growth factors such as nerve growth factor. However, the effect of fibrin appeared to be limited, as MSCs did not differentiate into fully mature cells based on immunofluorescence staining after 12 days. This body of work provides a rational approach for studying the interactions of MSC with fibrin, which has important therapeutic implications for the delivery of stem cells.

Effects of Low-Intensity Pulsed Ultrasound, Dexamethasone/TGF-β1 and/or BMP-2 on the Transcriptional Expression of Genes in Human Mesenchymal Stem Cells: Chondrogenic vs. Osteogenic Differentiation

The effects of low-intensity pulsed ultrasound (LIPUS) on the differentiation of human mesenchymal stem cells (hMSCs) were investigated in this study. hMSCs were subjected to LIPUS with or without dexamethasone/transforming growth factor-β1 (TD) or bone morphogenetic protein-2 (BMP-2) and the effects of this treatment were assessed. TD-treated hMSCs exhibited characteristic chondrogenic morphology and increased messenger RNA (mRNA) expression of chondrogenic markers and LIPUS enhanced the chondrogenic differentiation of hMSCs treated with TD. The expression of Runx2, an osteogenic transcription factor was not altered in either TD treatment group; however, a significant increase was detected in the LIPUS only group. The osteogenic appearance exhibited 3 days after LIPUS and/or BMP-2 treatment. Increases in the mRNA expression levels of osteogenic markers, Runx2 and ALP were also detected. There was no additive or altered effect with combined LIPUS and BMP-2 treatment. LIPUS alone can increase osteogenic differentiation of hMSCs and LIPUS enhances TD-mediated chondrogenic differentiation of hMSCs. Clinically, LIPUS may differentially influence bone vs. cartilage repair. (E-mail: whchang@cycu.edu.tw or laitw@tmu.edu.tw or tlai@mclean.harvard.edu)

Gene expression profiling of primary human articular chondrocytes in high-density micromasses reveals patterns of recovery, maintenance, re- and dedifferentiation

The high-density micromass culture has been widely applied to study chondrocyte cell physiology and pathophysiological mechanisms. Since an integrated image has not been established so far, we analyzed the phenotypic alterations of human articular chondrocytes in this model on the broad molecular level. Freshly isolated chondrocytes were assembled as micromasses and maintained up to 6 weeks in medium containing human serum. Formation of cartilaginous extracellular matrix (ECM) was evaluated by histological and immunohistochemical staining. At 0, 3 and 6 weeks, chondrocyte micromasses were subjected to gene expression analysis using oligonucleotide microarrays and real-time RT-PCR. Micromasses developed a cartilaginous ECM rich in proteoglycans and type II collagen. On gene expression level, time-dependent expression patterns was observed. The induction of genes associated with cartilage-specific ECM ( COL2A1 and COL11A1) and developmental signaling ( GDF5, GDF10, ID1, ID4 and FGFR1 - 3) indicated redifferentiation within the first 3 weeks. The repression of genes related to stress response ( HSPA1A and HSPA4), apoptotic events ( HYOU1, NFKBIA and TRAF1), and degradation ( MMP1, MMP10 and MMP12) suggested a recovery of chondrocytes. Constant expression of other chondrogenic ( ACAN, FN1 and MGP) and hypertrophic markers ( COL10A1, ALPL, PTHR1 and PTHR2) indicated a pattern of phenotypic maintenance. Simultaneously, the expression of chondrogenic growth ( BMP6, TGFA, FGF1 and FGF2) and transcription factors ( SOX9, EGR1, HES1 and TGIF1), and other cartilage ECM-related genes ( COMP and PRG4) was consistently repressed and expression of collagens related to dedifferentiation ( COL1A1 and COL3A1) was steadily induced indicating a progressing loss of cartilage phenotype. Likewise, a steady increase of genes associated with proliferation ( GAS6, SERPINF1, VEGFB and VEGFC) and apoptosis ( DRAM, DPAK1, HSPB, GPX1, NGFRAP1 and TIA1) was observed. Sequence and interplay of identified expression patterns suggest that chondrocyte micromass cultures maintain a differentiated phenotype up to 3 weeks in vitro and might be useful for studying chondrocyte biology, pathophysiology and differentiation. Cultivation longer than 6 weeks leads to progressing dedifferentiation of chondrocytes that should be considered on long-term evaluations.