Differentiation Of Articular Cartilage Research Articles

Induced regeneration of articular cartilage: Identification of a dormant regeneration program for a non-regenerative tissue.

An organoid culture model was developed to regenerate articular cartilage by sequential treatment with BMP2 and BMP9 that parallels induced joint regeneration at digit amputation wounds in vivo. BMP9 induced chondrogenesis was used to identify clonal cell lines of articular chondrocyte and hypertrophic chondrocyte progenitor cells from digit fibroblasts. A protocol that includes cell aggregation enhanced by BMP2 followed by BMP9 induced chondrogenesis resulted in the differentiation of organized layers of articular chondrocytes similar to the organization of middle and deep zones of articular cartilage in situ and retained a differentiated phenotype following transplantation. In addition, the differentiation of a non-chondrogenic connective tissue layer containing articular chondrocyte progenitor cells demonstrates that progenitor cell sequestration is coupled with articular cartilage differentiation at a clonal level. The studies identify a dormant endogenous regenerative program for a non-regenerative tissue in which fibroblast-derived progenitor cells can be induced to initiate morphogenetic and differentiative programs that include progenitor cell sequestration. The identification of dormant regenerative programs in non-regenerative tissues such as articular cartilage represents a novel strategy that integrates regeneration biology with regenerative medicine.

Synovial cells secrete a temperature-stable protein that inhibits hypertrophic differentiation and induces articular cartilage differentiation of chondrocytes in vitro

Synovial cells secrete a temperature-stable protein that inhibits hypertrophic differentiation and induces articular cartilage differentiation of chondrocytes in vitro

Activation of the SOX-5, SOX-6, and SOX-9 Trio of Transcription Factors Using a Gene-Activated Scaffold Stimulates Mesenchymal Stromal Cell Chondrogenesis and Inhibits Endochondral Ossification.

Current treatments for articular cartilage defects relieve symptoms but often only delay cartilage degeneration. Mesenchymal stem cells (MSCs) have shown chondrogenic potential but tend to undergo endochondral ossification when implanted in vivo. Harnessing factors governing joint development to functionalize biomaterial scaffolds, termed developmental engineering, might allow to prime host MSCs to regenerate mature articular cartilage in situ without requiring cell isolation or ex vivo expansion. Therefore, the aim of this study is to develop a gene-activated scaffold capable of delivering developmental cues to host MSCs, thus priming MSCs for articular cartilage differentiation and inhibiting endochondral ossification. It is shown that delivery of the SOX-Trio induced MSCs to over-express COL2A1 and ACAN and deposit a sulfated and collagen type II rich extracellular matrix while hypertrophic gene expression and collagen type X deposition is inhibited. When cell-free SOX-Trio-activated scaffolds are implanted ectopically in vivo, they induced spontaneous chondrogenesis without evidence of hypertrophy. MSCs pre-cultured on SOX-Trio-activated scaffolds prior to implantation differentiate into phenotypically stable chondrocytes as evidenced by a lack of collagen X expression or vascular invasion. This SOX-trio-activated scaffold represents a potent, single treatment, developmentally inspired strategy to prime MSCs in situ for articular cartilage defect repair.

Distinct Articular and Endochondral Differentiation Pathways in Bone Marrow Chondrogenic Progenitor Cells

Abstract Bone marrow contains skeletal progenitor cells (mesenchymal stem cells) which are important actors in skeletal repair. Nevertheless, the therapeutic use of mesenchymal stem cells for articular cartilage repair remains a challenge with a limited efficacy on both clinical outcomes and cartilage regeneration. In rabbit periosteal graft models, we show that such skeletal progenitor cells are recruited and undergo differentiation through different skeletal tissue pathways (bone, cartilage, skeletal muscle) in bone marrow areas that are under periosteal graft influence. Moreover, histological observations show among cartilage differentiated cells two different cartilage pathways, corresponding respectively to articular cartilage formations and to endochondral cartilage formations. This underline the presence in bone marrow of cartilage progenitor cells that escape from the intrinsic endochondral differentiation program resulting in transient rather than permanent cartilage, and follow a specific articular cartilage differentiation pathway. With respect to tissue engineering, and while extremely complex techniques are being developed to artificially generate functionally integrated, stratified articular cartilage like structures, our observations urge the development of protocols that select the proper population of articular cartilage progenitor cells before its use as cartilage building units. Keywords Articular cartilage repair; Tissue engineering; Skeletal progenitors; Mesenchymal stem cells; Articular cartilage pathway; Endochondral cartilage pathway; Periosteum

NFIA and GATA3 are crucial regulators of embryonic articular cartilage differentiation

During appendicular skeletal development, the bi-potential cartilage anlagen gives rise to transient cartilage, which is eventually replaced by bone, and to articular cartilage that caps the ends of individual skeletal elements. While the molecular mechanism that regulates transient cartilage differentiation is relatively well understood, the mechanism of articular cartilage differentiation has only begun to be unraveled. Furthermore, the molecules that coordinate the articular and transient cartilage differentiation processes are poorly understood. Here, we have characterized in chick the regulatory roles of two transcription factors, NFIA and GATA3, in articular cartilage differentiation, maintenance and the coordinated differentiation of articular and transient cartilage. Both NFIA and GATA3 block hypertrophic differentiation. Our results suggest that NFIA is not sufficient but necessary for articular cartilage differentiation. Ectopic activation of GATA3 promotes articular cartilage differentiation, whereas inhibition of GATA3 activity promotes transient cartilage differentiation at the expense of articular cartilage. We propose a novel transcriptional circuitry involved in embryonic articular cartilage differentiation, maintenance and its crosstalk with the transient cartilage differentiation program.

Identifying the downstream target genes of Wnt signalling during articular cartilage differentiation

Identifying the downstream target genes of Wnt signalling during articular cartilage differentiation

Elucidating role of silk-gelatin bioink to recapitulate articular cartilage differentiation in 3D bioprinted constructs

Tissue engineered cartilage has never been evaluated with an aim to distinguish between transient and articular cartilage. A major drawback of existing state-of-the art engineered cartilage is cellular hypertrophy, leading to development of transient cartilage which ultimately undergoes endochondral ossification to form bone trabeculae. As a paradigm shift, using 3D bioprinting, we have evaluated six different conditions for best outcome vis-à-vis articular cartilage differentiation as assessed by expression of a constellation of markers (like Autotaxin, lubricin etc). Our study strongly suggests that BMSCs undergo hypertrophic differentiation in the presence of TGF-β1, while in the absence of TGF-β1 BMSCs encapsulated in 3D bioprinted silk-gelatin bioink matrix undergo articular cartilage differentiation. Our study provides novel insights into direct regulatory role of silk-gelatin bioink on IHH and Wnt signaling pathways in controlling hypertrophy during chondrogenic differentiation of BMSCs. 3D bioprinted silk-gelatin constructs enabled adequate cellular attachment, proliferation and most importantly, articular cartilage differentiation. Interestingly, we observed close similarities between the signaling pathways associated with the 3D bioprinted constructs with respect to the signaling pathways with embryonic cartilage development suggesting our engineered cartilage tissue to be a prospective tissue equivalent with potential of providing the essential instructive elements for activating pathways of organogenesis in patient-specific manner.

A comprehensive mRNA expression analysis of developing chicken articular cartilage

Articular cartilage present at the ends of appendicular skeletal elements provides friction-less movement to the synovial joints and any damage to this tissue can lead to a degenerative disease of joint called osteoarthritis. During past two decades although many genes e.g.,Gdf5, Wnt9a, Noggin etc. have been identified and characterized in joint development, still a comprehensive understanding of molecular network(s) operational in articular cartilage morphogenesis is far from being drawn. Here we report identification of 36 genes (19 from literature survey and 17 from microarray analysis) that are expressed in developing chicken phalangeal joints in a spatiotemporally dynamic manner. For both these set of genes across the time window investigated we observed three kinds of expression patterns: early, late and constant. The early expressed genes are invariably expressed in a domain broader than the interzone while the late expressed genes are expressed in restricted spatial domains. The comprehensive expression analysis presented in this report provides a candidate list of molecular players involved in articular cartilage differentiation and/or maintenance.

Precise spatial restriction of BMP signaling is essential for articular cartilage differentiation.

The articular cartilage, which lines the joints of the limb skeleton, is distinct from the adjoining transient cartilage, and yet, it differentiates as a unique population within a contiguous cartilage element. Current literature suggests that articular cartilage and transient cartilage originate from different cell populations. Using a combination of lineage tracing and pulse-chase of actively proliferating chondrocytes, we here demonstrate that, similar to transient cartilage, embryonic articular cartilage cells also originate from the proliferating chondrocytes situated near the distal ends of skeletal anlagen. We show that nascent cartilage cells are capable of differentiating as articular or transient cartilage, depending on exposure to Wnt or BMP signaling, respectively. The spatial organization of the articular cartilage results from a band of Nog-expressing cells, which insulates these proliferating chondrocytes from BMP signaling and allows them to differentiate as articular cartilage under the influence of Wnt signaling emanating from the interzone. Through experiments conducted in both chick and mouse embryos we have developed a model explaining simultaneous growth and differentiation of transient and articular cartilage in juxtaposed domains.

An echo in biology: Validating the executable chondrocyte

Purpose: Computational modeling of biological networks permits comprehensive analysis of cells and tissues to define molecular phenotypes and novel hypotheses. We recently presented ANIMO (Analysis of Networks with Interactive Modeling), an intuitive software tool for modeling molecular networks for use by biologists. We used ANIMO to generate a computational model of articular cartilage, ECHO. Over 1.5 million people in the Netherlands suffer from osteoarthritis (OA) in one or more joints. OA is a painful, disabling disease and currently cannot be cured. In a subset of OA patients, joint cartilage is replaced by bone via endochondral ossification. This is a natural process in growing long bones, where transient growth plate cartilage is replaced by bone. In contrast, healthy joint cartilage is permanent as it is protected against bone formation. Stable joint cartilage is under control of master transcription factor SOX9, whereas bone formation is controlled by RUNX2. The processes that regulate the switch between a SOX9+ state and a RUNX+ state are poorly understood, which greatly hampers the development of successful therapies. Methods: Based on a large-scale literature study and our own experiments, we recently developed ECHO (Executable Chondrocyte), a computational model of the key processes that regulate expression and activity of SOX9 and RUNX2. Simulations in ECHO were performed to investigate the robustness of the chondrocyte network. To validate ECHO predictions, we used FRAP to measure mobility of SOX9 and RUNX2, which we have shown to be a faithful readout of their activity. Primary chondrocytes of 2 OA donors were tested at passage 2 after isolation. In these donors we observed little interdonor variation in SOX9 mobility. Using lipofectamine LTX we obtained 40-70% transfection efficiencies in primary human chondrocytes even at low passages. Results: In its unperturbed form, ECHO displays two stable states in which activities of SOX9 and RUNX2 are mutually exclusive. We tested the hypothesis that addition of WNT (performed with a few mouse clicks) will change permanent into transient cartilage by inducing hypertrophy. Indeed, when we add WNT, a known regulator of bone formation, the permanent or SOX9+ state changes to a transient or RUNX2+ state. However, it is known that healthy articular cartilage is resistant to hypertrophic differentiation. Our group has previously found that this was probably due to the secretion of DKK1, FRZB and GREM1. We therefore added nodes to ECHO representing DKK1, FRZB and GREM1 (figure 1). GREM1 and DKK1 are able to stabilize the permanent cartilage or SOX9+ state even after addition of WNT to ECHO. We observed that in our model activation of WNT leads to a switch from a SOX9+ state to a RUNX2+ state. To prove that WNT/β-catenin signaling can directly regulate SOX9 function, we investigated the response of SOX9 mobility to WNT3A in live primary chondrocytes. Addition of WNT3A to human chondrocytes transfected with SOX9-GFP resulted in a significant decrease of the immobile SOX9 fraction from 53% to 34% within 15 minutes after addition. A direct correlation between the elevated levels of β-catenin after WNT addition and the activity of SOX9 indicates that β-catenin can directly change the mobility by complex formation with SOX9. Conclusions: Using ECHO we predicted the stimuli that prevents hypertrophic differentiation differentiation of articular cartilage, and tested this experimentally with FRAP using SOX9 and RUNX2 mobility as a read-out.

Quantitative zonal differentiation of articular cartilage by microscopic magnetic resonance imaging, polarized light microscopy, and Fourier‐transform infrared imaging

This study aimed to synchronize the zonal differentiation of the full-thickness articular cartilage by three micro-imaging techniques, namely microscopic magnetic resonance imaging (µMRI), polarized light microscopy (PLM), and Fourier-transform infrared imaging (FTIRI). Eighteen cartilage-bone blocks from three canine humeral joints were imaged by: (a) µMRI T2 relaxation at 0° and 55° orientations in a 7 T magnetic field, (b) PLM optical retardation and azimuthal angle, and (c) FTIRI amide I and amide II anisotropies at 0° and 90° polarizations relative to the articular surface. In addition, µMRI T1 relaxation was imaged before and after the tissue being immersed in gadolinium (contrast agent) solution, to calculate the proteoglycan concentration. A set of previously established criteria in cartilage imaging was revised. The new criteria could simultaneously correlate the thicknesses of the three consecutive subtissue zones in articular cartilage among these imaging techniques.

Gremlin 1, Frizzled‐related protein, and Dkk‐1 are key regulators of human articular cartilage homeostasis

The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation. Whole-genome gene-expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA. Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled-related protein (FRP), and Dkk-1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long-bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single-nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population-based cohorts. Taken together, our study identified Gremlin 1, FRP, and Dkk-1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

Chondrogenesis and Developments in Our Understanding

Conditions affecting cartilage through damage or age-related degeneration pose significant challenges to individual patients and their healthcare systems. The disease burden will rise in the future as life expectancy increases. This has resulted in vigorous efforts to develop novel therapies to meet current and future needs. Due to the limited regenerative capacity of cartilage, in vitro tissue engineering techniques have emerged as the favoured technique by which to develop replacements. Tissue engineering is mainly concerned with developing cartilage replacements in the form of chondrocyte suspensions and three-dimensional scaffolds seeded with chondrocytes. One major limiting factor in the development of clinically useful cartilage constructs is our understanding of the process by which cartilage is formed, chondrogenesis. For example, techniques of culturing chondrocytes in vitro have been used for decades, resulting in chondrocyte-like cells which produce an extracellular matrix of similar composition to native cartilage, but with inferior physical properties. It has now been realised that one aspect of chondrogenesis which had been ignored was the physical context in which cartilage exists in vivo. This has resulted in the development of bioreactor systems which aim to introduce various physical stresses to engineered cartilage in a controlled environment. This has resulted in some improvements in the quality of tissue engineered cartilage. This is but one example of how the knowledge of chondrogenesis has been translated into research practice. This paper aims to review what is currently known about the process of chondrogenesis and discusses how this knowledge can be applied to tissue engineering.

Distribution and expression of cartilage oligomeric matrix protein and bone sialoprotein show marked changes during rat femoral head development.

Distribution and sites of synthesis of a cartilage extracellular matrix protein, cartilage oligomeric matrix protein (COMP), and of a bone extracellular matrix protein, bone sialoprotein (BSP), were studied in the femoral head of growing Wistar rats from day 14 to day 60 by immunocytochemistry and in situ hybridization. This period includes formation of the secondary ossification center and differentiation of articular cartilage. At early stages, immunoreactivity for COMP was pronounced throughout the cartilage. The localization of COMP was predominantly territorial in the center of the immature femoral head and in the growth plate at all ages studied. In the superficial parts, a shift from a uniform extracellular matrix staining at day 14 to an interterritorial localization at day 33 to day 60 was seen, apparently concurrent with formation of articular cartilage. COMP staining, representing cartilage remnants, also extended into the center of the trabecular bone in the primary spongiosa. In the secondary ossification center, the staining for COMP decreased at the onset of calcification. The protein was only synthesized by chondrocytes, as shown by in situ hybridization. The highest level of COMP mRNA was detected in chondrocytes in the central region of the growth plate. In the layer corresponding to the articular cartilage of the femoral head, mRNA levels for COMP were low from day 14 to day 33 but were increased on day 60. This shows substantial synthesis in the developing articular cartilage. Immunoreactivity for BSP was detected in bone trabeculae of primary spongiosa. In situ hybridization showed the highest levels of BSP mRNA in regions of newly formed bone. BSP mRNA was detected in hypertrophic chondrocytes in the secondary ossification center as early as day 18, well before the appearance of immunochemically detectable BSP. Interestingly, simultaneous expression of COMP and BSP mRNA was seen after day 18 in hypertrophic chondrocytes of the growth plate and later also in hypertrophic chondrocytes close to the mineralization zone of the articular cartilage.

Expression of parathyroid hormone-related protein in rat articular cartilage

Expression and localization of parathyroid hormone-related protein (PTHrP) in rat articular cartilage during fetal and postnatal periods were investigated by immunohistochemistry and in situ hybridization. PHTrP displayed distinct distribution and intensity of staining at different ages. In fetal (18-day-old) and young (3-week-old) rats, articular chondrocytes expressed abundant PTHrP throughout the entire thickness of cartilage. In contrast, in 60-week-old rats, PTHrP was expressed in a few articular chondrocytes of superficial and middle layers. Regulation of PTHrP and PTH/PTHrP receptor mRNA was also studied in cultured rat articular chondrocytes. Northern blot analysis revealed that both transforming growth factor-beta (TGF-beta), an important stimulator for chondrocyte proliferation and differentiation, and 10% fetal bovine serum (FBS) stimulated the expression of PTHrP mRNA with down-regulation of its receptor mRNA. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulated the expression of receptor without changes of PTHrP mRNA level. These results suggest that the changes in abundance and localization of PTHrP and its receptor may be directly involved in the cell growth and differentiation of articular cartilage.

The effect of brain fraction with fibroblast growth factor activity on regeneration and differentiation of articular cartilage

From bovine brains a fraction with fibroblast growth factor activity was prepared according to the method given by Gospodarowicz et al. (1978) and tested regarding to its biological effectiveness on articular cartilage regeneration in rabbits the cartilage in one knee-joint of those had been injured in a defined manner. The intraarticular administration of the brain fraction supported the healing process of the intrachondral lesions by conspicuously increasing the chondrocyte proliferation starting from preexisting cartilage tissue. In the case of subchondral lesions the neodifferentiation of chondrocytes out of the simple connective tissue that primarily filled the defect was promoted significantly.