MSC Chondrogenesis Research Articles

An injectable and 3D printable pro-chondrogenic hyaluronic acid and collagen type II composite hydrogel for the repair of articular cartilage defects

Current treatments for repairing articular cartilage defects are limited. However, pro-chondrogenic hydrogels formulated using articular cartilage matrix components (such as hyaluronic acid (HA) and collagen type II (Col II)), offer a potential solution if they could be injected into the defect via minimally invasive arthroscopic procedures, or used as bioinks to 3D print patient-specific customised regenerative scaffolds—potentially combined with cells. However, HA and Col II are difficult to incorporate into injectable/3D printable hydrogels due to poor physicochemical properties. This study aimed to overcome this by developing an articular cartilage matrix-inspired pro-chondrogenic hydrogel with improved physicochemical properties for both injectable and 3D printing (3DP) applications. To achieve this, HA was methacrylated to improve mechanical properties and mixed in a 1:1 ratio with Col I, a Col I/Col II blend or Col II. Col I possesses superior mechanical properties to Col II and so was hypothesised to enhance hydrogel mechanical properties. Rheological analysis showed that the pre-gels had viscoelastic and shear thinning properties. Subsequent physicochemical analysis of the crosslinked hydrogels showed that Col II inclusion resulted in a more swollen and softer polymer network, without affecting degradation time. While all hydrogels exhibited exemplary injectability, only the Col I-containing hydrogels had sufficient mechanical stability for 3DP applications. To facilitate 3DP of multi-layered scaffolds using methacrylated HA (MeHA)-Col I and MeHA-Col I/Col II, additional mechanical support in the form of a gelatin slurry support bath freeform reversible embedding of suspended hydrogels was utilised. Biological analysis revealed that Col II inclusion enhanced hydrogel-embedded MSC chondrogenesis, thus MeHA-Col II was selected as the optimal injectable hydrogel, and MeHA-Col I/Col II as the preferred bioink. In summary, this study demonstrates how tailoring biomaterial composition and physicochemical properties enables development of pro-chondrogenic hydrogels with potential for minimally invasive delivery to injured articular joints or 3DP of customised regenerative implants for cartilage repair.

Time-Dependent Anabolic Response of hMSC-Derived Cartilage Grafts to Hydrostatic Pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions.

Evaluation of Phosphate Treatment and Long-Term RUNX2 Suppression on Adult Human MSC Chondrogenesis and Neo-Cartilage Formation

Evaluation of Phosphate Treatment and Long-Term RUNX2 Suppression on Adult Human MSC Chondrogenesis and Neo-Cartilage Formation

Влияние комбинации ростовых факторов TGF и IGF на хондрогенный потенциал мезенхимальных стволовых клеток костного мозга

The authors studied the influence of the combination of TGF? and IGF growth factors, as well as the differentiation time, on the induction of MSC chondrogenesis in vitro. It is proved that MSCs located in 2D and 3D systems, when exposed to TGF?/ IGF, showed the signs of early chondroblast-like cells in 7 days. The TGF?/ IGF used for the induction of MSCs is more preferred, because it results in a more pronounced hypertrophic-suppression effect. The absence of significant differences in gene expression (excepting Sox9) on the 7th and 21st days of chondrogenic differentiation allows the process to be reduced in vitro to 7 days.

Predicting and Promoting Human Bone Marrow MSC Chondrogenesis by Way of TGFβ Receptor Profiles: Toward Personalized Medicine.

The use of human mesenchymal stromal cells (hMSCs) for cartilage regeneration has been hampered by the inherent donor variation of primary monolayer expanded cells. Although CD markers are typically used to characterize cell populations, there is no correlation between CD marker profile and functional outcomes. Therefore, we aimed to discover novel predictive MSC chondrogenesis markers. The chondrogenic potential of primary human bone marrow MSCs (hBMSCs) over multiple passages was assessed by standard pellet culture. We confirmed that the ratio of TGFβ-RI/TGFβ-RII at the time of cell recovery from the tissue culture plastic reliably predicted chondrogenic potential. Furthermore, it is possible to prospectively characterize any human BMSC cell population as responders or non-responders with respect to chondrogenic differentiation potential. Transient increase of the ratio with siRNA knockdown of TGFβ-RII reproducibly recovered the chondrogenic differentiation ability of non-responsive MSCs. Together this offers an opportunity to produce a more functionally characterized cell population for use in autologous cartilage repair therapies.

Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) is a lipid-lowering drug that also affects both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis can be initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM can augment the HAM-induced chondrogenesis but not osteogenesis of ADSCs and that this synergism can be applied for articular cartilage regeneration. ADSCs were treated with SIM in a HAM in HA-coated wells or an HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs and formation of cartilage were evaluated. Treatment with SIM in a HAM (SIM plus HAM) induced the expression of BMP-2 and transcription factors for chondrogenesis (SOX-9) and osteogenesis (RUNX-2) in ADSCs, and these effects were reversed by CD44 blockade. Furthermore, ADSCs exposed to SIM plus HAM exhibited cell aggregation, chondrogenic gene (collagen type II and aggrecan) expression and sulfated glycosaminoglycan formation. By contrast, SIM-induced osteogenesis in ADSCs was reduced in a HAM, as determined by measuring osteogenic gene (osteocalcin and alkaline phosphatase) expression and calcium deposition. These inhibitory effects were not altered by changes in the HA molecular weight (80~2000 kDa). In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. These results indicate that a HAM may be used as a chondroinductive agent to enhance SIM-induced MSC chondrogenesis but prevent osteogenesis.

TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation.

Background and ObjectivesMesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertrophy inducing medium. We investigated how intrinsic TGF-β signaling is altered in pro-hypertrophic conditions.Methods and ResultsHuman bone marrow derived MSC were chondrogenically differentiated in 3D culture. At day 14 medium conditions were changed to 1. pro-hypertrophic by addition of T3 and withdrawal of TGF-β and dexamethasone 2. pro-hypertrophic by addition of BMP 4 and withdrawal of TGF-β and dexamethasone and 3. kept in prochondrogenic medium conditions. All groups were treated with and without TGFβ-type-1-receptor inhibitor SB431542 from day 14 on. Aggregates were harvested for histo- and immunohistological analysis at d14 and d28, for gene expression analysis (rt-PCR) on d1, d3, d7, d14, d17, d21 and d28 and for Western blot analysis on d21 and d28. Induction of hypertrophy was achieved in the pro-hypertrophic groups while expression of TGFβ-type-1- and 2-receptor and Sox 9 were significantly downregulated compared to pro-chondrogenic conditions. Western blotting showed reduced phosphorylation of Smad 2 and 3 in hypertrophic samples, reduced TGF-β-1 receptor proteins and reduced SOX 9. Addition of SB431542 did not initiate hypertrophy under pro-chondrogenic conditions, but was capable of enhancing hypertrophy when applied simultaneously with BMP-4.ConclusionsOur results suggest that the enhancement of hypertrophy in this model is a result of both activation of pro-hypertrophic BMP signaling and reduction of anti-hypertrophic TGFβ signaling.

Functional Profiling of Chondrogenically Induced Multipotent Stromal Cell Aggregates Reveals Transcriptomic and Emergent Morphological Phenotypes Predictive of Differentiation Capacity.

Multipotent stromal cells (MSCs) are an attractive cell source for bone and cartilage tissue repair strategies. However, the functional heterogeneity of MSCs derived from different donors and manufacturing conditions has limited clinical translation, emphasizing the need for improved methods to assess MSC chondrogenic capacity. We used functionally relevant morphological profiling to dynamically monitor emergent morphological phenotypes of chondrogenically induced MSC aggregates to identify morphological features indicative of MSC chondrogenesis. Toward this goal, we characterized the morphology of chondrogenically stimulated MSC aggregates from eight different human cell‐lines at multiple passages and demonstrated that MSC aggregates exhibited unique morphological dynamics that were both cell line‐ and passage‐dependent. This variation in 3D morphology was shown to be informative of long‐term MSC chondrogenesis based on multiple quantitative functional assays. We found that the specific morphological features of spheroid area, radius, minimum feret diameter, and minor axis length to be strongly correlated with MSC chondrogenic synthetic activity but not gene expression as early as day 4 in 3D culture. Our high‐throughput, nondestructive approach could potentially serve as a tool to identify MSC lines with desired chondrogenic capacity toward improving manufacturing strategies for MSC‐based cellular products for cartilage tissue repair. Stem Cells Translational Medicine 2018;1–12

The osmolyte type affects cartilage associated pathologic marker expression during in vitro mesenchymal stem cell chondrogenesis under hypertonic conditions.

Stem cells' fate during in vitro differentiation is influenced by biophysicochemical cues. Osmotic stress has proved to enhance chondrocyte marker expression, however its potent negative impacts had never been surveyed. We questioned whether specific osmotic conditions, regarding the osmolyte agent, could benefit chondrogenesis while dampening undesired concomitant hypertrophy and inflammatory responses. To examine the potential side effects of hypertonicity, we assessed cell proliferation as well as chondrogenic and hypertrophic marker expression of human Adipose Derived-MSC after a two week induction in chondrogenic media with either NaCl or Sorbitol, as the osmolyte agent to reach a +100 mOsm hypertonic condition. Calcium deposition and TNF-α secretion as markers associated with hypertrophy and inflammation were then assayed. While both hyperosmotic conditions upregulated chondrogenic markers, sorbitol had a nearly three times higher chondro-promotive effect and a lesser hypertrophic effect compared to NaCl. Also, a significantly lesser calcium deposition was observed in sorbitol hypertonic group. NaCl showed an anti-proinflammatory effect while sorbitol had no effect on inflammatory markers. The ossification potential and cartilage associated pathologic markers were affected differentially by the type of the osmolyte. Thus, a vigilant application of the osmotic agent is inevitable in order to avoid or reduce undesired hypertrophic and inflammatory phenotype acquisition by MSC during chondrogenic differentiation. Our findings are a step towards developing a more reliable chondrogenic regimen using external hypertonic cues for MSC chondrogenesis with potential applications in chondral lesions cell therapy.

Expression of miR-145-5p During Chondrogenesis of Mesenchymal Stem Cells

Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.

A comparative study of chondroitin sulfate and heparan sulfate for directing three-dimensional chondrogenesis of mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) hold great promise for cartilage repair given their relative abundance, ease of isolation, and chondrogenic potential. To enhance MSC chondrogenesis, extracellular matrix components can be incorporated into three-dimensional (3D) scaffolds as an artificial cell niche. Chondroitin sulfate (CS)-containing hydrogels have been shown to support 3D chondrogenesis, but the effects of varying CS concentration and hydrogel stiffness on 3D MSC chondrogenesis remains elusive. Heparan sulfate (HS) is commonly used as a growth factor reservoir due to its ability to sequester growth factors; however, how it compares to CS in supporting 3D MSC chondrogenesis remains unknown.MethodsWe fabricated photocrosslinkable hydrogels containing physiologically relevant concentrations (0–10%) of CS or HS with two stiffnesses (~7.5 kPa and ~ 36 kPa) as a 3D niche for MSC chondrogenesis.ResultsCS is a more potent factor in enhancing MSC chondrogenesis, especially in soft hydrogels (~ 7.5 kPa). A moderate dosage of CS (5%) led to the highest amount of neocartilage deposition. Stiff hydrogels (~ 36 kPa) generally inhibited neocartilage formation regardless of the biochemical cues.ConclusionsTaken together, the results from this study demonstrated that CS-containing hydrogels at low mechanical stiffness can provide a promising scaffold for enhancing MSC-based cartilage tissue regeneration.

Osmolyte Type and the Osmolarity Level Affect Chondrogenesis of Mesenchymal Stem Cells.

The inductive effects of increased osmolarity on chondrogenesis are well approved. However, the effects of the osmolyte agent invoked to induce hyperosmolarity are largely neglected. Herein, we scrutinized how hyperosmotic conditions acquired by addition of different osmolytes would impact chondrogenesis. We briefly assessed whether such conditions would differentially affect hypertrophy and angiogenesis during MSC chondrogenesis. Chondrogenic and hypertrophic marker expression along with VEGF secretion during adipose-derived (AD)-MSC chondrogenesis under three osmolarity levels (350, 450, and 550mOsm) using three different osmolytes (NaCl, sorbitol, and PEG) were assessed. MTT assay, qRT-PCR, immunocytochemistry, Alcian Blue staining, ELISA, and ALP assays proved osmolyte-type dependent effects of hyperosmolarity on chondrogenesis, hypertrophy, and angiogenesis. At same osmolarity level, PEG had least cytotoxic/cytostatic effect and most prohibitive effects on angiogenesis. As expected, all hyperosmolar conditions led to enhanced chondrogenesis with slightly varying degrees. PEG and sorbitol had higher chondro-promotive and hypertrophy-suppressive effects compared to NaCl, while NaCl had exacerbated hypertrophy. We observed that TonEBP was involved in osmoadaptation of all treatments in varying degrees. Of importance, we highlighted differential effects of hyperosmolarity obtained by different osmolytes on the efficacy of chondrogenesis and more remarkably on the induction/suppression of cartilage pathologic markers. Our study underlies the need for a more vigilant exploitation of physicobiochemical inducers in order to maximize chondrogenesis while restraining unwanted hypertrophy and angiogenesis.

Scaffolds Derived from ECM Produced by Chondrogenically Induced Human MSC Condensates Support Human MSC Chondrogenesis.

Osteoarthritis is a leading cause of disability affecting an increasing number of individuals. However, cartilage replacement therapies are inadequate, and better cartilage regeneration products must be developed. In this work, we describe a human mesenchymal stem cell (hMSC)-based approach for fabricating extracellular matrix (ECM) scaffolds from tissue-engineered cartilage sheets and then for inducing chondrogenesis of reseeded hMSCs within the ECM scaffolds. Two types of ECM scaffolds were fabricated: one from high-density hMSC sheets cultured with media-supplemented transforming growth factor beta-1 (TGF-β1; -MS) and the other from high-density hMSC sheets incorporated with TGF-β1-laden gelatin microspheres (+MS), which significantly enhance chondrogenesis within the sheet system. Interestingly, when scaffolds were reseeded with hMSCs, -MS scaffolds lead to significantly more glycosaminoglycan (GAG) accumulation than +MS scaffolds. Importantly, ECM scaffolds could be soak loaded with TGF-β1 to produce cartilage of similar quality as that of constructs cultured with TGF-β1 in the media, thereby removing the need for supplementing the media with the growth factor. Lastly, tissues formed with the scaffolds were larger with more uniform cartilage matrix elaboration compared to scaffold-free groups making this strategy a clinically promising auto- or allogeneic therapy.

The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells.

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA.

Randomized double-blind study of a single intra-articular injection of allogeneic mesenchymal precursor cells + hyaluronan compared to hyaluronan alone to explore clinical and structural outcomes in patients post anterior cruciate ligament reconstruction

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S16 differentiation. lncRNAs can be derived from both intergenic and overlapping protein-coding gene regions, are expressed in a highly tissue-specific manner and are emerging as key regulators of gene expression. In this study we establish a role for lncRNAs in chondrogenesis and cartilage. Methods: Paired end sequencing was performed by Illumina Genome Analyzer IIx on total RNA isolated from human articular cartilage and human MSCs undergoing chondrogenic differentiation. lncRNAs and novel transcripts were identified by aligning sequence reads to the transcriptome and genome respectively. Novel transcripts were confirmed by rapid amplification of cDNA ends (RACE) and expression validated by real-time RT-PCR in a panel of human tissues. Expression was assessed in the tri-lineage differentiation of MSCs into chondrocytes, osteoblasts and adipocytes. lncRNA expression was depleted with specific siRNAs to establish their role during differentiation. Overexpression of lncRNAs was performed by cloning and lentiviral transduction as well as CRISPR/Cas9-VP16 mediated endogenous promoter activation. Chondrocyte and cartilage development was assessed by gene and protein expression measured with standard molecular biology, biochemical and histological techniques. lncRNA-regulated genes were identified by microarray and lncRNA-regulated signalling pathways in chondrogenesis assessed by Western blotting and reporter activation. Results: RNA sequencing indicates that human adult cartilage contains upwards of 3000 lncRNAs (FPKM >1) and that 188 of these are induced during MSC chondrogenic differentiation (>2fold). Of particular interest are a group of lncRNAs upstream of the SOX9 locus, found in a gene desert on chromosome 17, which includes SOX9-AS1, LINC01152 and a novel 4 exon version of the predicted 3 exon RNA, ENS0430908. This novel lncRNA is chondrocyte specific and upregulated during chondrogenic differentiation of MSCs. RNAi targeting novel ENS0430908 prevented MSC chondrogenesis with a complete loss of cartilage matrix formation and significant reductions in chondrocyte gene expression. Therefore we named this novel lncRNA ROCR (Regulator Of Chondrogenesis RNA). Depletion of ROCR totally blocked the induction of SOX9 during MSC chondrogenesis, consistent with the reduced cartilage-specific gene expression. Microarray analysis indicates that loss of ROCR phenocopies the loss of SOX9 on gene expression changes during the early stages of chondrogenesis. Loss of ROCR may also disrupt chondrogenic cell signalling, in particular activation of the TGF-b pathway. In contrast to the effect during chondrogenesis loss of ROCR enhances osteoblastogenic differentiation and gene expression. Conclusions: The cartilage transcriptome contains numerous noncoding transcripts many of which may have essential functions in cartilage biology. ROCR is essential for cartilage development and appears to promote a chondrocyte cell fate for MSCs over an osteoblast cell fate. Because SOX9-expressing cells are progenitors for numerous tissues identifying chondrocyte-specific regulatory elements might aid understanding of differentiation of chondrocytes from MSCs, potentially useful in chondrocyte tissue engineering applications. 15 SPECIFIC SYNOVIAL JOINT PROGENITOR CELL POPULATIONS UNIQUELY CONTRIBUTE TO ARTICULAR CARTILAGE GROWTH AND REPAIR R.S. Decker y, H.-B. Um y, N.A. Dyment z, N. Cottingham y, E. Koyama y, M. Kronenberg z, P. Maye z, D.W. Rowe z, M. Pacifici y. yChildren's Hosp. of Philadelphia, Philadelphia, PA, USA; zUniv. of Connecticut, Farmington,

Specific synovial joint progenitor cell populations uniquely contribute to articular cartilage growth and repair

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S16 differentiation. lncRNAs can be derived from both intergenic and overlapping protein-coding gene regions, are expressed in a highly tissue-specific manner and are emerging as key regulators of gene expression. In this study we establish a role for lncRNAs in chondrogenesis and cartilage. Methods: Paired end sequencing was performed by Illumina Genome Analyzer IIx on total RNA isolated from human articular cartilage and human MSCs undergoing chondrogenic differentiation. lncRNAs and novel transcripts were identified by aligning sequence reads to the transcriptome and genome respectively. Novel transcripts were confirmed by rapid amplification of cDNA ends (RACE) and expression validated by real-time RT-PCR in a panel of human tissues. Expression was assessed in the tri-lineage differentiation of MSCs into chondrocytes, osteoblasts and adipocytes. lncRNA expression was depleted with specific siRNAs to establish their role during differentiation. Overexpression of lncRNAs was performed by cloning and lentiviral transduction as well as CRISPR/Cas9-VP16 mediated endogenous promoter activation. Chondrocyte and cartilage development was assessed by gene and protein expression measured with standard molecular biology, biochemical and histological techniques. lncRNA-regulated genes were identified by microarray and lncRNA-regulated signalling pathways in chondrogenesis assessed by Western blotting and reporter activation. Results: RNA sequencing indicates that human adult cartilage contains upwards of 3000 lncRNAs (FPKM >1) and that 188 of these are induced during MSC chondrogenic differentiation (>2fold). Of particular interest are a group of lncRNAs upstream of the SOX9 locus, found in a gene desert on chromosome 17, which includes SOX9-AS1, LINC01152 and a novel 4 exon version of the predicted 3 exon RNA, ENS0430908. This novel lncRNA is chondrocyte specific and upregulated during chondrogenic differentiation of MSCs. RNAi targeting novel ENS0430908 prevented MSC chondrogenesis with a complete loss of cartilage matrix formation and significant reductions in chondrocyte gene expression. Therefore we named this novel lncRNA ROCR (Regulator Of Chondrogenesis RNA). Depletion of ROCR totally blocked the induction of SOX9 during MSC chondrogenesis, consistent with the reduced cartilage-specific gene expression. Microarray analysis indicates that loss of ROCR phenocopies the loss of SOX9 on gene expression changes during the early stages of chondrogenesis. Loss of ROCR may also disrupt chondrogenic cell signalling, in particular activation of the TGF-b pathway. In contrast to the effect during chondrogenesis loss of ROCR enhances osteoblastogenic differentiation and gene expression. Conclusions: The cartilage transcriptome contains numerous noncoding transcripts many of which may have essential functions in cartilage biology. ROCR is essential for cartilage development and appears to promote a chondrocyte cell fate for MSCs over an osteoblast cell fate. Because SOX9-expressing cells are progenitors for numerous tissues identifying chondrocyte-specific regulatory elements might aid understanding of differentiation of chondrocytes from MSCs, potentially useful in chondrocyte tissue engineering applications. 15 SPECIFIC SYNOVIAL JOINT PROGENITOR CELL POPULATIONS UNIQUELY CONTRIBUTE TO ARTICULAR CARTILAGE GROWTH AND REPAIR R.S. Decker y, H.-B. Um y, N.A. Dyment z, N. Cottingham y, E. Koyama y, M. Kronenberg z, P. Maye z, D.W. Rowe z, M. Pacifici y. yChildren's Hosp. of Philadelphia, Philadelphia, PA, USA; zUniv. of Connecticut, Farmington,

Differential effect of hypoxia on human mesenchymal stem cell chondrogenesis and hypertrophy in hyaluronic acid hydrogels

Photocrosslinked hyaluronic acid (HA) hydrogels provide a conducive 3-D environment that supports the chondrogenesis of human mesenchymal stem cells (hMSCs). The HA macromer concentration in the hydrogels has a significant impact on the chondrogenesis of the encapsulated MSCs due to changes in the physical properties of the hydrogels. Meanwhile, hypoxia has been shown to promote MSC chondrogenesis and suppress subsequent hypertrophy. This study investigates the combinatorial effect of tuning HA macromer concentration (1.5–5%w/v) and hypoxia on MSC chondrogenesis and hypertrophy. To decouple the effect of HA concentration from that of crosslinking density, the HA hydrogel crosslinking density was adjusted by varying the extent of the reaction through the light exposure time while keeping the HA concentration constant (5%w/v at 5 or 15min). It was found that hypoxia had no significant effect on the chondrogenesis and cartilaginous matrix synthesis of hMSCs under all hydrogel conditions. In contrast, the hypoxia-mediated positive or negative regulation of hMSC hypertrophy in HA hydrogels is dependent on the HA concentration but independent of the crosslinking density. Specifically, hypoxia significantly suppressed hMSC hypertrophy and neocartilage calcification in low HA concentration hydrogels, whereas hypoxia substantially enhanced hMSC hypertrophy, leading to elevated tissue calcification in high HA concentration hydrogels irrespective of their crosslinking density. In addition, at a constant high HA concentration, increasing hydrogel crosslinking density promoted hMSC hypertrophy and matrix calcification. To conclude, the findings from this study demonstrate that the effect of hypoxia on hMSC chondrogenesis and hypertrophy is differentially influenced by the encapsulating HA hydrogel properties.

Fibrous hyaluronic acid hydrogels that direct MSC chondrogenesis through mechanical and adhesive cues

Electrospinning has recently gained much interest due to its ability to form scaffolds that mimic the nanofibrous nature of the extracellular matrix, such as the size and depth-dependent alignment of collagen fibers within hyaline cartilage. While much progress has been made in developing bulk, isotropic hydrogels for tissue engineering and understanding how the microenvironment of such scaffolds affects cell response, these effects have not been extensively studied in a nanofibrous system. Here, we show that the mechanics (through intrafiber crosslink density) and adhesivity (through RGD density) of electrospun hyaluronic acid (HA) fibers significantly affect human mesenchymal stem cell (hMSC) interactions and gene expression. Specifically, hMSC spreading, proliferation, and focal adhesion formation were dependent on RGD density, but not on the range of fiber mechanics investigated. Moreover, traction-mediated fiber displacements generally increased with more adhesive fibers. The expression of chondrogenic markers, unlike trends in cell spreading and cytoskeletal organization, was influenced by both fiber mechanics and adhesivity, in which softer fibers and lower RGD densities generally enhanced chondrogenesis. This work not only reveals concurrent effects of mechanics and adhesivity in a fibrous context, but also highlights fibrous HA hydrogels as a promising scaffold for future cartilage repair strategies.

Interplay Between Local Versus Soluble Transforming Growth Factor-Beta and Fibrin Scaffolds: Role of Cells and Impact on Human Mesenchymal Stem Cell Chondrogenesis

Structural extracellular matrix molecules gain increasing attention as scaffolds for cartilage tissue engineering owing to their natural role as a growth factor repository. We recently observed that a collagen-type I/III (Col-I/III) matrix, human recombinant transforming growth factor-beta (TGF-β) protein, and fibrin hydrogel (FG) combined to a biphasic construct provided sufficient long-term TGF-β support to drive in vitro chondrogenesis of human mesenchymal stem cells (hMSC). Here we ask whether FG and Col-I/III can both retain TGF-β, describe the influence of cell seeding on TGF-β release, and compare the molecular path of hMSC chondrogenic differentiation under soluble versus local TGF-β supply. Release of growth factor from scaffolds augmented with increasing amounts of TGF-β was analyzed over 7 days and chondrogenesis was assessed over 42 days. Low TGF-β release rates from Col-I/III as opposed to higher release from FG indicated that both molecules retained TGF-β, with Col-I/III being the superior storage component. Cell seeding enhanced TGF-β retention in FG by about threefold and almost stopped release beyond 24 h. TGF-β remained bioactive and supported MSC chondrogenesis without impairing the amount of proteoglycan and collagen-type II deposition per cell and per construct compared to standard scaffold-free MSC pellets supplied with soluble TGF-β. Local TGF-β, however, mediated lower cell content, less collagen-type X relative to collagen-type II deposition and no matrix metalloproteinase-13 up-regulation. In conclusion, cells quickly halted release of local TGF-β from FG, turning FG and Col-I/III into attractive TGF-β repositories capable to drive full hMSC chondrogenesis, but via a modulated differentiation pathway. Since only part of the changes was reproduced by transient soluble TGF-β supply, release kinetics alone could not explain the molecular differences, suggesting that local TGF-β acts distinct from its soluble counterpart.

Histone deacetylase 4 promotes TGF-β1-induced synovium-derived stem cell chondrogenesis but inhibits chondrogenically differentiated stem cell hypertrophy

The transforming growth factor-beta (TGF-β) superfamily members play diverse roles in cartilage development and maintenance. TGF-β up-regulates chondrogenic gene expression by enhancing transcription factor SRY (sex determining region Y)-box 9 (Sox9) and inhibits osteoblast differentiation by repressing runt-related transcription factor 2 (Runx2). Recently, histone deacetylases (HDACs) were reported to act as negative regulators of chondrocyte hypertrophy. It was speculated that HDAC4 may promote TGF-β1-induced MSC chondrogenesis. In this study, the adenovirus-mediated HDAC4 gene (Ad.HDAC4) was utilized to infect synovium-derived stem cells (SDSCs). Adenovirus-mediated LacZ (Ad.LacZ) served as a control. The infected cells were centrifuged to form SDSC pellets followed by incubation in a serum-free chondrogenic medium for 15 days with or without 10 ng/mL TGF-β1. Transfection efficiency was determined in SDSCs using Ad.LacZ. Cytotoxicity was measured using lactate dehydrogenase assay. Histology, immunostaining, biochemical analysis, and real-time polymerase chain reaction were performed to assess chondrogenesis at protein and mRNA levels in infected SDSCs. Our data demonstrated that supplementation with TGF-β1 could initiate and promote SDSC chondrogenesis; however, TGF-β1 alone was insufficient to fully differentiate SDSCs into chondrocytes. Ad.HDAC4 could be efficiently transfected into SDSCs. Without TGF-β1 treatment, HDAC4 had no effect on SDSC chondrogenesis; however, in the presence of TGF-β1, HDAC4 could speed up and maintain a high level of chondrogenesis while down-regulating the hypertrophic marker – type X collagen expression. This study is the first report showing that HDAC4 overexpression promotes TGF-β1-induced SDSC chondrogenesis but inhibits chondrogenically differentiated stem cell hypertrophy. The mechanism underlying this process needs further investigation.