Chondrogenic Process Research Articles

Preliminary Characterization of the Epigenetic Modulation in the Human Mesenchymal Stem Cells during Chondrogenic Process.

Regenerative medicine represents a growing hot topic in biomedical sciences, aiming at setting out novel therapeutic strategies to repair or regenerate damaged tissues and organs. For this perspective, human mesenchymal stem cells (hMSCs) play a key role in tissue regeneration, having the potential to differentiate into many cell types, including chondrocytes. Accordingly, in the last few years, researchers have focused on several in vitro strategies to optimize hMSC differentiation protocols, including those relying on epigenetic manipulations that, in turn, lead to the modulation of gene expression patterns. Therefore, in the present study, we investigated the role of the class II histone deacetylase (HDAC) inhibitor, MC1568, in the hMSCs-derived chondrogenesis. The hMSCs we used for this work were the hMSCs obtained from the amniotic fluid, given their greater differentiation capacity. Our preliminary data documented that MC1568 drove both the improvement and acceleration of hMSCs chondrogenic differentiation in vitro, since the differentiation process in MC1568-treated cells took place in about seven days, much less than that normally observed, namely 21 days. Collectively, these preliminary data might shed light on the validity of such a new differentiative protocol, in order to better assess the potential role of the epigenetic modulation in the process of the hypertrophic cartilage formation, which represents the starting point for endochondral ossification.

The effect of matrix stiffness on the chondrogenic differentiation of mesenchymal stem cells.

Articular cartilage is one of the most important weight-bearing components in human body, thus the chondrogenesis of stem cells is reactive to many intracellular and extracellular mechanical signals. As a unique physical cue, matrix stiffness plays an integral role in commitment of stem cell fate. However, when examining the downstream effects of matrix stiffness, most studies used different soluble factors to assist physical inducing process, which may mask the chondrogenic effects of matrix stiffness. Here we fabricated polyacrylamide (PAAm) hydrogels with gradient stiffness to unravel the role of matrix stiffness in chondrogenic process of mesenchymal stem cells (MSCs), with or without TGF-β3 as induction factor. The results showed that with micromass culture mimicking relatively high cell density in vivo, the chondrogenic differentiation of MSCs can be promoted by soft substrates (about 0.5kPa) independently with assembled cytoskeleton. Further analysis indicated that addition of TGF-β3 generally increased expression level of cartilage-related markers and masked the stiffness-derived expression pattern of hypertrophic markers. These results demonstrate how mechanical cues experienced in developmental context regulate commitment of stem cell fate and have significant impact on the design of tissue regeneration materials.

The effect of mitochondrial fusion on chondrogenic differentiation of cartilage progenitor/stem cells via Notch2 signal pathway

BackgroundOsteoarthritis (OA) is a debilitating disease that inflicts intractable pain, a major problem that humanity faces, especially in aging populations. Stem cells have been used in the treatment of many chronic diseases, including OA. Cartilage progenitor/stem cells (CPSCs) are a type of stem cells with the ability to self- renew and differentiate. They hold a promising future for the understanding of the progression of OA and for its treatment. Previous studies have reported the relationship between mitochondrial dynamics and mesenchymal stem cell (MSC) proliferation, differentiation and aging. Mitochondrial dynamic and morphology change during stem cell differentiation.MethodsThis study was performed to access the relationship between mitochondrial dynamics and chondrogenic differentiation of CPSCs. Mitochondrial fusion and fission levels were measured during the chondrogenic differentiation process of CPSCs. After that, we used mitochondrial fusion promoter to induce fusion in CPSCs and then the chondrogenic markers were measured. Transmission electron microscopy (TEM) and confocal microscopy were used to capture the mass and fusion status of mitochondria. Lentiviruses were used to detect the role of mitofusin 2 (Mfn2) in CPSC chondrogenic differentiation. In vivo, Mfn2 was over-expressed in sheets of rat CPSCs, which were then injected intra-articularly into the knees of rats.ResultsMitochondrial fusion markers were upregulated during the chondrogenic induction process of CPSCs. The mass of mitochondria was higher in differentiated CPSC, and the fusion status was obvious relative to un-differentiated CPSC. Chondrogenesis of CPSCs was upregulated with the induction by mitochondrial fusion promoter. Mfn2 over-expression significantly increased chondrocyte-specific gene expression and reversed OA through NOTCH2 signal pathway.ConclusionsOur study demonstrated that the mitochondrial fusion promotes chondrogenesis differentiation of CPSCs. Mfn2 accelerates the chondrogenesis differentiation of CPSCs through Notch2. In vivo, Mfn2-OE in sheets of rCPSCs ameliorated OA in the rat model.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

SummaryBone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

Beneficial roles of the AhR ligand FICZ on the regenerative potentials of BMSCs and primed cartilage templates.

Bone marrow-derived mesenchymal stem cells (BMSCs) are commonly used seed cells, and BMSC-derived primed cartilage templates have been shown to achieve bone regeneration in bone tissue engineering. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in various cellular processes such as osteogenesis and immune regulation. This study investigated the effects of the AhR endogenous ligand 6-formyl (3,2-b) carbazole (FICZ) on the behavior of BMSCs and cartilage templates as well as the possible underlying molecular mechanisms. AhR expressions in rat bone marrow and isolated BMSCs were detected via immunohistochemistry (IHC) and immunofluorescent staining. Alkaline phosphatase staining and alizarin red staining showed that FICZ treatment enhanced the osteogenic potential of BMSCs without influencing their proliferation. FICZ was shown to alleviate the LPS-induced inflammatory cytokines IL-1β, 6 and TNF-α via the quantitative polymerase chain reaction (qPCR). In the chondrogenic process from BMSCs to primed cartilage templates, the expressions of AhR and its target gene cytochrome P450 subfamily B member 1 (CYP1B1) were inhibited. However, IHC staining demonstrated that AhR was still involved in the subcutaneous ossification of cartilage templates. Then, the effects of FICZ on cartilage templates were investigated. The osteogenic markers were upregulated by FICZ administration. The RAW 264.7 treated by condition medium of FICZ-treated cartilage templates exhibited an anti-inflammatory phenotype. Finally, high-throughput sequencing was applied to analyze the differentially expressed genes (DEGs) in the FICZ-treated cartilage templates. The upregulation of cytochrome P450 subfamily A member 1 (CYP1A1) and sphingomyelin phosphodiesterase 3 (Smpd3) were verified by qPCR, which might be the downstream targets of AhR in the cartilage templates promoting osteogenesis and macrophage polarization. These data implied a beneficial role of FICZ in the regenerative potentials of both BMSCs and primed cartilage templates. The FICZ/AhR axis might be a practical target to achieve optimal bone regeneration.

PNaSS-Grafted PCL Film-Guided rAAV TGF-β Gene Therapy Activates the Chondrogenic Activities in Human Bone Marrow Aspirates.

Scaffold-guided viral gene therapy is a novel, powerful tool to enhance the processes of tissue repair in articular cartilage lesions by the delivery and overexpression of therapeutic genes in a noninvasive, controlled release manner based on a procedure that may protect the gene vehicles from undesirable host immune responses. In this study, we examined the potential of transferring a recombinant adeno-associated virus (rAAV) vector carrying a sequence for the highly chondroregenerative transforming growth factor beta (TGF-β), using poly(ɛ-caprolactone) (PCL) films functionalized by the grafting of poly(sodium styrene sulfonate) (pNaSS) in chondrogenically competent bone marrow aspirates as future targets for therapy in cartilage lesions. Effective overexpression of TGF-β in the aspirates by rAAV was achieved upon delivery using pNaSS-grafted and ungrafted PCL films for up to 21 days (the longest time point evaluated), with superior levels using the grafted films, compared with respective conditions without vector coating. The production of rAAV-mediated TGF-β by pNaSS-grafted and ungrafted PCL films significantly triggered the biological activities and chondrogenic processes in the samples (proteoglycan and type-II collagen deposition and cell proliferation), while containing premature mineralization and hypertrophy relative to the other conditions, with overall superior effects supported by the pNaSS-grafted films. These observations demonstrate the potential of PCL film-assisted rAAV TGF-β gene transfer as a convenient, off-the-shelf technique to enhance the reparative potential of the bone marrow in patients in future approaches for improved cartilage repair.

Kartogenin Enhances Chondrogenic Differentiation of MSCs in 3D Tri-Copolymer Scaffolds and the Self-Designed Bioreactor System.

Human cartilage has relatively slow metabolism compared to other normal tissues. Cartilage damage is of great clinical consequence since cartilage has limited intrinsic healing potential. Cartilage tissue engineering is a rapidly emerging field that holds great promise for tissue function repair and artificial/engineered tissue substitutes. However, current clinical therapies for cartilage repair are less than satisfactory and rarely recover full function or return the diseased tissue to its native healthy state. Kartogenin (KGN), a small molecule, can promote chondrocyte differentiation both in vitro and in vivo. The purpose of this research is to optimize the chondrogenic process in mesenchymal stem cell (MSC)-based chondrogenic constructs with KGN for potential use in cartilage tissue engineering. In this study, we demonstrate that KGN treatment can promote MSC condensation and cell cluster formation within a tri-copolymer scaffold. Expression of Acan, Sox9, and Col2a1 was significantly up-regulated in three-dimensional (3D) culture conditions. The lacuna-like structure showed active deposition of type II collagen and aggrecan deposition. We expect these results will open new avenues for the use of small molecules in chondrogenic differentiation protocols in combination with scaffolds, which may yield better strategies for cartilage tissue engineering.

The involvement of cytokine-like 1 (Cytl1) in chondrogenesis and cartilage metabolism

The Cytokine-like 1 (Cytl1) is first identified in bone marrow cells and preferentially expressed in cartilaginous tissue, and showed chondrogenic effects in mesenchymal cells, not essential for cartilage or bone development as in Cytl1 knock-out mice but associated with cartilage inflammatory and destruction. Here, we show the involvement of Cytl1 in chondrogenesis. Using specified chondrogenic embryonic skeleton and adult cartilage, the Cytl1 gene expression was investigated with associated chondrogenic factors by quantitative RT-PCR. The effect of Cytl1 protein (rCytl1) on cultured chondrocytes to regulate expression of key factors and phenotypic markers was studied. The results revealed that Cytl1 was highly expressed in chondrogenic process in embryos and adult cartilage. The rCytl1 increased the expression of Sox9 and Col2α1 with stabilized Col1α1 in cultured chondrocytes (redifferentiation). The Cytl1 was expressed and involved at all stages of cartilage development. Furthermore, Cytl1 expression shared similar patterns as other chondrogenic factors, implying interactions with other factors in chondrogenic process. Cytl1 is involved in cartilage development and matrix homeostasis, which defines the dedifferentiation phenotype of chondrocytes, essential to forming of functional cartilage in both physiologic remodeling and pathologic regeneration.

The Role of MicroRNAs in Early Chondrogenesis of Human Induced Pluripotent Stem Cells (hiPSCs).

Human induced pluripotent stem cells (hiPSCs) play an important role in research regarding regenerative medicine. Particularly, chondrocytes differentiated from hiPSCs seems to be a promising solution for patients suffering from osteoarthritis. We decided to perform chondrogenesis in a three-week monolayer culture. Based on transcriptome analysis, hiPSC-derived chondrocytes (ChiPS) demonstrate the gene expression profile of cells from early chondrogenesis. Chondrogenic progenitors obtained by our group are characterized by significantly high expression of Hox genes, strongly upregulated during limb formation and morphogenesis. There are scanty literature data concerning the role of microRNAs in early chondrogenesis, especially in chondrogenic differentiation of hiPSCs. The main aim of this study was to investigate the microRNA expression profile and to select microRNAs (miRNAs) taking part in early chondrogenesis. Our findings allowed for selection crucial miRNAs engaged in both diminishing pluripotency state and chondrogenic process (inter alia hsa-miR-525-5p, hsa-miR-520c-3p, hsa-miR-628-3p, hsa-miR-196b-star, hsa-miR-629-star, hsa-miR-517b, has-miR-187). These miRNAs regulate early chondrogenic genes such as: HOXD10, HOXA11, RARB, SEMA3C. These results were confirmed by RT-qPCR analysis. This work contributes to a better understanding of the role of miRNAs directly involved in chondrogenic differentiation of hiPSCs. These data may result in the establishment of a more efficient protocol of obtaining chondrocyte-like cells from hiPSCs.

Melatonin rescued interleukin 1\u03b2-impaired chondrogenesis of human mesenchymal stem cells

BackgroundOsteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1β (IL-1β) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1β-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1β-induced inhibition of chondrogenesis and further study should be conducted.MethodsHuman bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1β and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs.ResultsThe current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs in different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein levels, and also regulate the expression levels of other chondrogenic markers like ACAN, SOX9, and COL10A1 in the presence of IL-1β. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing expression of catabolic genes, such as MMP, MMP13, and ADAMTS4. Furthermore, MLT can subdue IL-1β-induced cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65 and IκBα, which were elevated by IL-1β treatment, indicating that MLT can attenuate the IL-1β-induced activation of NF-κB signaling.ConclusionThe current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs by restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis. Our study also showed that MLT can attenuate the IL-1β-induced activation of the NF-κB signaling pathway, which is the most important pathway downstream of IL-1β, and plays a crucial role in inflammation, apoptosis, and metabolism. Thus, MLT has prospects for treating OA due to its multifaceted functions, such as mitigating inflammation, maintaining metabolic balance, and mitigating apoptosis.

Osteogenic and Chondrogenic Master Genes Expression Is Dependent on the Kir2.1 Potassium Channel Through the Bone Morphogenetic Protein Pathway.

Andersen's syndrome is a rare disorder affecting muscle, heart, and bone that is associated with mutations leading to a loss of function of the inwardly rectifying K+ channel Kir2.1. Although the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome-induced pluripotent stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the downregulation of master gene expression. This downregulation is the result of an impairment of the bone morphogenetic proteins signaling pathway through dephosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression and restored normal osteoblast and chondrocyte behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. © 2018 American Society for Bone and Mineral Research.

Impact of mechanical stimulation on the chondrogenic processes in human bone marrow aspirates modified to overexpress sox9 via rAAV vectors

BackgroundEvaluation of gene-based approaches to target human bone marrow aspirates in conditions of mechanical stimulation that aim at reproducing the natural joint environment may allow to develop improved treatments for articular cartilage injuries. In the present study, we investigated the potential of rAAV-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under established hydrodynamic conditions compared with the more commonly employed static culture conditions.MethodsFresh human bone marrow aspirates were transduced with rAAV-FLAG-hsox9 (40 μl) and maintained for up to 28 days in chondrogenic medium under mechanically-induced conditions in dynamic flow rotating bioreactors that permit tissue growth and matrix deposition relative to static culture conditions. The samples were then processed to examine the potential effects of sox9 overexpression on the cellular activities (matrix synthesis, proliferation) and on the chondrogenic differentiation potency compared with control treatments (absence of rAAV vector; reporter rAAV-lacZ, rAAV-RFP, and rAAV-luc gene transfer).ResultsProlonged, significant sox9 overexpression via rAAV was achieved in the aspirates for at least 28 days when applying the rAAV-FLAG-hsox9 construct, leading to higher, prolonged levels of matrix biosynthesis and to enhanced chondrogenic activities relative to control treatments especially when maintaining the samples under mechanical stimulation. Administration of sox9 however did not impact the indices of proliferation in the aspirates. Remarkably, sox9 gene transfer also durably delayed hypertrophic and osteogenic differentiation in the samples regardless of the conditions of culture applied versus control treatments.ConclusionsThe current observations show the value of genetically modifying human bone marrow aspirates upon mechanical stimulation by rAAV sox9 as a promising strategy for future treatments to improve cartilage repair by implantation in lesions where the tissue is submitted to natural mechanical forces.

S100A1 and S100B: Calcium Sensors at the Cross-Roads of Multiple Chondrogenic Pathways.

The S100 protein family comprises more than 20 members of small calcium binding proteins operating as Ca2 +-activated switches that interact and modulate the activity of a large number of targets. S100A1 and S100B, two members of this family, have been recently associated with the differentiation status of human articular chondrocytes. Both proteins are homogeneously expressed in all cartilage zones, their expression decreases during chondrocyte dedifferentiation, and can be induced under conditions promoting redifferentiation. Although S100 proteins have a broad range of extra- and intracellular roles, functional studies of S100 proteins expressed in chondrocytes have focused on their extracellular roles linked to catabolic processes. The intracellular roles of S100A1 and S100B in chondrocytes remain largely unexplored, yet the few studies addressing their intracellular activity point toward potentially important functions in chondrocyte biology. This review summarizes reported intracellular S100A1 and S100B regulatory functions described in other cell types that could be also involved in the regulation of chondrogenic processes in cartilage. Potential roles of S100A1 and S100B in the TGF-β-SMAD, the cAMP-PKA-CREB, and the PI3K-AKT pathways, Ca2+ homeostasis, cytoskeleton dynamics, the calcineurin-NFAT pathway, interactions with the p53 family, and the Hippo pathway are examined in the context of chondrocyte biology. Based on the plethora of interactions of S100A1 and S100B with different molecular partners playing essential roles in chondrocyte biology, and the staggering complexity and ubiquity of cross-talk among these partners, we hypothesize that these S100 proteins play fundamental roles in the spatial and temporal regulation of chondrogenesis. J. Cell. Physiol. 232: 1979-1987, 2017. © 2016 Wiley Periodicals, Inc.

Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation in vitro, part A.

Human induced pluripotent stem cells (hiPSCs) offer promise in regenerative medicine, however more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from the mesoderm and thus share a wide range of properties with chondrocytes, which also originate from the mesenchyme. Thus, the exclusion of dedifferentiation instead of chondrogenic differentiation is crucial. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in a chondrogenic medium supplemented with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on immunofluorescence and reverse transcription-quantiative polymerase chain reaction analysis, the results indicated that hiPSCs have the capacity for effective chondrogenic differentiation, in particular cells differentiated in the HC-402-05a-conditioned medium, which present morphological features and markers that are characteristic of mature human chondrocytes. By contrast, cells differentiated in the presence of TGF-β3 may demonstrate hypertrophic characteristics. Several genes [paired box 9, sex determining region Y-box (SOX) 5, SOX6, SOX9 and cartilage oligomeric matrix protein] were demonstrated to be good markers of early hiPSC chondrogenic differentiation: Insulin-like growth factor 1, Tenascin-C, and β-catenin were less valuable. These observations provide valuable data on the use of hiPSCs in cartilage tissue regeneration.

Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation invitro, part B.

The development of human induced pluripotent stem cells (hiPSCs) is considered a turning point in tissue engineering. However, more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly-used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from mesoderm and thus share a wide range of properties with chondrocytes, which originate from the mesenchyme. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in the presence of a chondrogenic medium with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on reverse transcription-quantitative polymerase chain reaction analysis, the results demonstrated that hiPSCs are capable of effective chondrogenic differentiation, with the cells obtained in the HC-402-05a medium presenting with morphological features and markers characteristic of mature human chondrocytes. In contrast, cells differentiated in the presence of TGF-β3 presented with certain undesirable hypertrophic characteristics. Several genes, most notably runt-related transcription factor 2, transforming growth factor β2 and transforming growth factor β3, were good markers of advanced and late hiPSC chondrogenic differentiation, whereas transforming growth factor β3I, II, III receptors and bone morphogenetic protein-2, bone morphogenetic protein-4 and growth differentiation factor 5 were less valuable. These findings provide valuable data on the use of stem cells in cartilage tissue regeneration.

RAAV-mediated overexpression of sox9, TGF-β and IGF-I in minipig bone marrow aspirates to enhance the chondrogenic processes for cartilage repair

Administration of therapeutic gene sequences coding for chondrogenic and chondroreparative factors in bone marrow aspirates using the clinically adapted recombinant adeno-associated virus (rAAV) vector may provide convenient, single-step approaches to improve cartilage repair. Here, we tested the ability of distinct rAAV constructs coding for the potent SOX9, transforming growth factor beta (TGF-β) and insulin-like growth factor I (IGF-I) candidate factors to modify marrow aspirates from minipigs to offer a preclinical large animal model system adapted for a translational evaluation of cartilage repair upon transplantation in sites of injury. Our results demonstrate that high, prolonged rAAV gene transfer efficiencies were achieved in the aspirates (up to 100% for at least 21 days) allowing to produce elevated amounts of the transcription factor SOX9 that led to increased levels of matrix synthesis and chondrogenic differentiation and of the growth factors TGF-β and IGF-I that both increased cell proliferation, matrix synthesis and chondrogenic differentiation (although to a lower level than SOX9) compared with control (lacZ) condition. Remarkably, application of the candidate SOX9 vector also led to reduced levels of hypertrophic differentiation in the aspirates, possibly by modulating the β-catenin, Indian hedgehog and PTHrP pathways. The present findings show the benefits of modifying minipig marrow concentrates via rAAV gene transfer as a future means to develop practical strategies to promote cartilage repair in a large animal model.

Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis

Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4’,6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.

Current Progress in Stem Cell-Based Gene Therapy for Articular Cartilage Repair

Administration of mesenchymal stem cells (MSCs) that have a reliable potential for chondrogenic differentiation is a promising approach currently employed to treat articular cartilage lesions (focal defects and osteoarthritis) in patients as a mean to enhance the poor intrinsic capabilities of this specialized tissue for self-repair. However, there is still a critical need for improved designs, as reproduction of a native structural and functional unit in sites of cartilage damage is not occurring upon implantation of such cells. With the availability of optimized gene transfer systems, gene therapy offers powerful tools to stimulate the chondrogenic process in MSCs via the effective, safe, and durable delivery of candidate sequences with chondroprotective and/or chondroregenerative properties, both in vitro and in experimental models of cartilage lesions in vivo. In the present article, we provide an overview of the current advances in gene- and stem cell-based treatments employed to promote cartilage repair in focal defects and for osteoarthritis, and discuss the challenges that remain to be addressed for a safe translation of such procedures into the clinics.

Determination of effective rAAV-mediated gene transfer conditions to support chondrogenic differentiation processes in human primary bone marrow aspirates.

The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of cartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the ability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in primary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results demonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes were achieved in transduced aspirates at very high efficiencies (90-94%) and over extended periods of time (up to 125 days) upon treatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of their targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the cellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of 0.5 mg ml(-1) hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting hypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify bone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage defects.

AB0769 Lipid Characterization of Chondrogenic Differentiation Using Maldi Mass Spectrometry Imaging

BackgroundRecent investigations have argued that there is a close relationship between altered lipid metabolism and Osteoarthritis (OA) disease. Mesenchymal stem cells (MSC) are an interesting alternative for cell-based therapy of...