Deep Zone Cartilage Research Articles

Ankle distraction as a treatment for end stage osteoarthritis: follow-up at 10 years

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S273 spin sequence. A semi-automatic region-of-interest analysis was performed for specific areas (healthy cartilage, area of OA, region of MFX). To allow stratification with regards to the anatomical (collagen) structure, subregional analysis was carried out (deep superficial cartilage layer). Statistical analysis-of-variance was performed. Results: In healthy cartilage values were 56,76 ms (SD 14,44) in the superficial layer and 41,21 ms (SD 18,35) in the deep layer. The knees with OA showed results of 56,51ms (SD 14,52) in the superficial layer and 49,05 ms (SD 18,38) in the deep layer. After MFX, results for T2 averaged at 61,33 ms (SD 21,18) in the superficial layer and 53,35 (SD 21,89) in the deep layer. Comparison between layers within each group and (statistical) comparison between the groups showed that the superficial layer does not vary significantly between the groups, whereas the deep cartilage zone varies in between the groups. Most important, the increase of T2 values between deep and superficial zone showed a clearly significant difference in between the different groups and was able to differentiate in between healthy cartilage, induced OA and cartilage repair tissue after MFX (Table 1). Conclusions: Comparing ultra-high fieldMR T2 values between healthy cartilage, OA and cartilage repair sites at 7 T underlines the known fact that healthy cartilage is defined by a significant increase of T2 values between deep and superficial zone. With the used sheep model using T2 mapping, differences between healthy cartilage, OA and cartilage repair sites are detectable based on their zonal appearance. Hence, its application can help to evaluate cartilage repair procedures or therapeutical approaches in OA at the one hand and prove the value of ultrahigh field cartilage T2 mapping for diagnosis and follow-up imaging in the future on the other hand. This evaluation is supposed to be a step towards a more detailed understanding of high resolution zonal

Subchondral bone plate thickening precedes chondrocyte apoptosis and cartilage degradation in spontaneous animal models of osteoarthritis.

Osteoarthritis (OA) is the most common joint disorder characterised by bone remodelling and cartilage degradation and associated with chondrocyte apoptosis. These processes were investigated at 10, 16, 24, and 30 weeks in Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs that develop OA spontaneously. Both strains had a more pronounced chondrocyte apoptosis, cartilage degradation, and subchondral bone changes in the medial than the lateral side of the tibia, and between strains, the changes were always greater and faster in DH than BS2. In the medial side, a significant increase of chondrocyte apoptosis and cartilage degradation was observed in DH between 24 and 30 weeks of age preceded by a progressive thickening and stiffening of subchondral bone plate (Sbp). The Sbp thickness consistently increased over the 30-week study period but the bone mineral density (BMD) of the Sbp gradually decreased after 16 weeks. The absence of these changes in the medial side of BS2 may indicate that the Sbp of DH was undergoing remodelling. Chondrocyte apoptosis was largely confined to the deep zone of articular cartilage and correlated with thickness of the subchondral bone plate suggesting that cartilage degradation and chondrocyte apoptosis may be a consequence of continuous bone remodelling during the development of OA in these animal models of OA.

Increased Chondrocyte Apoptosis Is Associated with Progression of Osteoarthritis in Spontaneous Guinea Pig Models of the Disease

Osteoarthritis (OA) is the most common joint disease characterised by degradation of articular cartilage and bone remodelling. For almost a decade chondrocyte apoptosis has been investigated as a possible mechanism of cartilage damage in OA, but its precise role in initiation and/or progression of OA remains to the determined. The aim of this study is to determine the role of chondrocyte apoptosis in spontaneous animal models of OA. Right tibias from six male Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs were collected at 10, 16, 24 and 30 weeks of age. Fresh-frozen sections of tibial epiphysis were microscopically scored for OA, and immunostained with caspase-3 and TUNEL for apoptotic chondrocytes. The DH strain had more pronounced cartilage damage than BS2, especially at 30 weeks. At this time point, the apoptotic chondrocytes were largely confined to the deep zone of articular cartilage (AC) with a greater percentage in the medial side of DH than BS2 (DH: 5.7%, 95% CI: 4.2–7.2), BS2: 4.8%, 95% CI: 3.8–5.8), p > 0.05). DH had a significant progression of chondrocyte death between 24 to 30 weeks during which time significant changes were observed in AC fibrillation, proteoglycan depletion and overall microscopic OA score. A strong correlation (p ≤ 0.01) was found between chondrocyte apoptosis and AC fibrillation (r = 0.3), cellularity (r = 0.4) and overall microscopic OA scores (r = 0.4). Overall, the rate of progression in OA and apoptosis over the study period was greater in the DH (versus BS2) and the medial AC (versus lateral). Chondrocyte apoptosis was higher at the later stage of OA development when the cartilage matrix was hypocellular and highly fibrillated, suggesting that chondrocyte apoptosis is a late event in OA.

AB0136 A full depth cartilage explant model for the assessment of hypertrophic chondrocytes in osteoarthritis

BackgroundChondrocytes resist proliferation and terminal differentiation in healthy articular cartilage. Under disease condition, the chondrocytes change phenotype. Moreover, cartilage degradation, vascularization and calcification of the cartilage are initiated. Prehypertrophic chondrocytes...

OP0140 Circulating Carboxy-Terminal Type X Collagen Fragments (C-Col10), a Measure of Skeletal Hypertrophy, are Elevated in Patients with Osteoarthritis and Ankylosing Spondylitis

BackgroundAnkylosing Spondylitis (AS) is a chronic inflammatory disease of the spine and sacroiliac joints, whereas osteoarthritis (OA) is generally considered as a non-inflammatory condition of the synovial joints, predominantly knee...

Accumulation of Exogenous Activated TGF-β in the Superficial Zone of Articular Cartilage

It was recently demonstrated that mechanical shearing of synovial fluid (SF), induced during joint motion, rapidly activates latent transforming growth factor β (TGF-β). This discovery raised the possibility of a physiological process consisting of latent TGF-β supply to SF, activation via shearing, and transport of TGF-β into the cartilage matrix. Therefore, the two primary objectives of this investigation were to characterize the secretion rate of latent TGF-β into SF, and the transport of active TGF-β across the articular surface and into the cartilage layer. Experiments on tissue explants demonstrate that high levels of latent TGF-β1 are secreted from both the synovium and all three articular cartilage zones (superficial, middle, and deep), suggesting that these tissues are capable of continuously replenishing latent TGF-β to SF. Furthermore, upon exposure of cartilage to active TGF-β1, the peptide accumulates in the superficial zone (SZ) due to the presence of an overwhelming concentration of nonspecific TGF-β binding sites in the extracellular matrix. Although this response leads to high levels of active TGF-β in the SZ, the active peptide is unable to penetrate deeper into the middle and deep zones of cartilage. These results provide strong evidence for a sequential physiologic mechanism through which SZ chondrocytes gain access to active TGF-β: the synovium and articular cartilage secrete latent TGF-β into the SF and, upon activation, TGF-β transports back into the cartilage layer, binding exclusively to the SZ.

The importance of superficial collagen fibrils for the function of articular cartilage

It has been proposed that the superficial tangential zone (STZ) of articular cartilage is essential to the tissue's load-distributing function. However, the exact mechanism by which the STZ fulfills this function has not yet been revealed. Using a channel-indentation experiment, it was recently shown that compared to intact tissue, cartilage without STZ behaves slightly stiffer and deforms significantly different in regions adjacent to mechanically compressed areas (Bevill et al. in Osteoarthr Cartil 18:1310-1318, 2010). We aim to further explore the role of STZ in the load-transfer mechanism of AC by thorough biomechanical analysis of these experiments. Using our previously validated fibril-reinforced swelling model of articular cartilage, which accounts for the depth-dependent collagen structure and biochemical composition of articular cartilage, we simulated the above-mentioned channel-indenter compression experiments for both intact and STZ-removed cartilage. First, we show that the composition of the deep zone in cartilage is most effective in carrying cartilage compression, which explains the apparent tissue stiffening after STZ removal. Second, we show that tangential fibrils in the STZ are responsible for transferring compressive loads from directly loaded regions to adjacent tissue. Cartilage with an intact STZ has superior load-bearing properties compared to cartilage in which the STZ is compromised, because the STZ is able to recruit a larger area of deep zone cartilage to carry compressive loads.

Zone‐specific gene expression patterns in articular cartilage

To identify novel genes and pathways specific to the superficial zone (SZ), middle zone (MZ), and deep zone (DZ) of normal articular cartilage. Articular cartilage was obtained from the knees of 4 normal human donors. The cartilage zones were dissected on a microtome. RNA was analyzed on human genome arrays. The zone-specific DNA array data obtained from human tissue were compared to array data obtained from bovine cartilage. Genes differentially expressed between zones were evaluated using direct annotation for structural or functional features, and by enrichment analysis for integrated pathways or functions. The greatest differences in genome-wide RNA expression data were between the SZ and DZ in both human and bovine cartilage. The MZ, being a transitional zone between the SZ and DZ, thereby shared some of the same pathways as well as structural/functional features of the adjacent zones. Cellular functions and biologic processes that were enriched in the SZ relative to the DZ included, most prominently, extracellular matrix-receptor interactions, cell adhesion molecule functions, regulation of actin cytoskeleton, ribosome-related functions, and signaling aspects such as the IFN, IL4, Cdc42/Rac, and JAK/STAT signaling pathways. Two pathways were enriched in the DZ relative to the SZ, including PPARG and EGFR/SMRTE. These differences in cartilage zonal gene expression identify new markers and pathways that govern the unique differentiation status of chondrocyte subpopulations.

In vivo MRI of fresh stored osteochondral allograft transplantation with delayed gadolinium‐enhanced MRI of cartilage: Protocol considerations and recommendations

The protocol for delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) was adapted for the evaluation of transplanted osteochondral allograft cartilage. Eight patients with focal grade 4 cartilage defects of the femoral condyle were treated with single cylindrical osteochondral allografts. At 1 and 2 years, dGEMRIC image sequences were acquired and regions of interest (ROIs) were drawn in repair and native control cartilage. Mean T1 values of region of interest were used to calculate established dGEMRIC metrics. The correlation was measured between the ΔR1 and R1 -Post metrics for repair and native cartilage. T1 times were measured in deep and superficial zones of cartilage. A strong correlation was identified between full-thickness, deep, and superficial ΔR1 and R1 -Post values for native cartilage and repair cartilage for all years (range: 0.893-1.0). The mean T1 times and ΔR1 rate between deep and superficial regions of articular cartilage were statistically different for all regions of the distal femora analyzed at 1 year and 2 years after osteochondral allograft transplantation (P<0.05). The dGEMRIC pre-Gadolinium scan is unnecessary when evaluating transplanted osteochondral allograft cartilage. The observation of stratified T1 and ΔR1 values indicates a need to re-evaluate the methodology behind the placement of region of interest in dGEMRIC.

Distinct patterns of gene expression in the superficial, middle and deep zones of bovine articular cartilage

Hyaline articular cartilage will not heal spontaneously, and lesions in hyaline articular cartilage often result in degenerative joint disease. Considerable progress has been made with respect to the responsive stem cells, inductive signals and extracellular scaffolding required for the optimal regeneration of cartilage. However, many challenges remain, such as topographic differences in the functional zones of articular cartilage. We hypothesized that a distinct set of differentially expressed genes define the surface, middle and deep zones of hyaline articular cartilage. Microarray analysis of bovine articular cartilage from the superficial and middle zones revealed 52 genes differentially expressed ≥ 10-fold and 114 additional genes differentially expressed ≥ five-fold. However, no genes were identified with a ≥ five-fold difference in expression when comparing articular cartilage from the middle and deep zones. There are distinct, differential gene expression patterns in the superficial and middle zones of hyaline articular cartilage that highlight the functional differences between these zones. This investigation has implications for the tissue engineering and regeneration of hyaline articular cartilage.

Cross-talk between subchondral bone and articular cartilage in osteoarthritis

The interest in the relationship between articular cartilage and the structural and functional properties of peri-articular bone relates to the intimate contact that exists between these tissues in diarthrodial joints. Much of the interest and continuing controversy regarding the relationship between these tissues dates back to the original suggestion by Radin and Rose that alterations in the mechanical properties of subchondral bone could adversely affect the functional state of chondrocytes and the integrity of the overlying cartilage. They hypothesized that the deterioration in articular cartilage associated with the osteoarthritic process was secondary to an initial increase in subchondral bone stiffness induced by repetitive mechanical loading. Numerous approaches have been used to establish that the changes in periarticular bone occur very early in the development of OA. Although chondrocytes also have the capacity to modulate their functional state in response to loading, the capacity of these cells to repair and modify their surrounding extracellular matrix is relatively limited in comparison to the adjacent subchondral bone. This differential adaptive capacity likely underlies the more rapid appearance of detectable skeletal changes in OA in comparison to the articular cartilage. Given the intimate mechanical and biological interaction between the articular cartilage and subchondral bone it is likely that alterations in the composition and/or structural organization of either tissue will modulate the properties and function of the other joint component. An additional factor that affects the interaction between these tissues is the gradual expansion of the zone of calcified cartilage and advancement of the tidemark that occurs with aging and progression of OA and contributes to overall thinning of the articular cartilage. The precise mechanisms involved in this process have not been definitively established and could include the release of pro-angiogenic factors from chondrocytes in the deep zones of the articular cartilage that have undergone hypertrophy and/or the influences of microcracks that have initiated focal remodeling in the calcified cartilage in an attempt to repair the microdamage. In addition, this process markedly increases the mechanical stresses in the deep zones of the cartilage matrix, which likely contributes to the acceleration in OA cartilage deterioration. In summary, there is the need for further studies to define the pathophysiological mechanisms involved in the interaction between subchondral bone and articular cartilage and for applying this information to the development of therapeutic interventions to improve the outcomes in patients with OA.

Transport Phenomena in Articular Cartilage Cryopreservation as Predicted by the Modified Triphasic Model and the Effect of Natural Inhomogeneities

Knowledge of the spatial and temporal distribution of cryoprotective agent (CPA) is necessary for the cryopreservation of articular cartilage. Cartilage dehydration and shrinkage, as well as the change in extracellular osmolality, may have a significant impact on chondrocyte survival during and after CPA loading, freezing, and thawing, and during CPA unloading. In the literature, Fick's law of diffusion is commonly used to predict the spatial distribution and overall concentration of the CPA in the cartilage matrix, and the shrinkage and stress-strain in the cartilage matrix during CPA loading are neglected. In this study, we used a previously described biomechanical model to predict the spatial and temporal distributions of CPA during loading. We measured the intrinsic inhomogeneities in initial water and fixed charge densities in the cartilage using magnetic resonance imaging and introduced them into the model as initial conditions. We then compared the prediction results with the results obtained using uniform initial conditions. The simulation results in this study demonstrate the presence of a significant mechanical strain in the matrix of the cartilage, within all layers, during CPA loading. The osmotic response of the chondrocytes to the cartilage dehydration during CPA loading was also simulated. The results reveal that a transient shrinking occurs to different levels, and the chondrocytes experience a significant decrease in volume, particularly in the middle and deep zones of articular cartilage, during CPA loading.

Oxidant damage in Kashin‐Beck disease and a rat Kashin‐Beck disease model by employing T‐2 toxin treatment under selenium deficient conditions

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy, but the mechanisms underlying its pathogenesis remains unclear. This study compares antioxidant capacity and lipid peroxidation using a novel model, in which rats were administered a selenium-deficient diet for 4 weeks prior to their exposure to T-2 toxin for 4 weeks. Changes in cell morphology and empty chondrocyte lacunae indicative of cell death, as well as cartilage proteoglycan loss in the deep zone of articular cartilage of knee joints were observed in rats with selenium-deficient diet plus T-2 toxin treatment. These changes were similar to those observed previously in KBD. The levels of thiobarbituric acid reactive substances (TBARS), indicative of lipid peroxidation in serum and cartilage, were significantly increased in all experimental groups compared to the normal diet group, while the levels of antioxidants, measured as total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidases (GPX), in serum and cartilage were significantly lower than that in the normal diet group. The mRNA expression of those antioxidants in cartilage tissue was significantly reduced by T-2 toxin alone or by selenium-deficient diet plus T-2 toxin treatment. These results indicate that increasing TBARS and decreasing antioxidants in serum and cartilage by T-2 toxin treatment with a selenium-deficient nutritional status may alter oxidative stress in joint tissues and contribute to the pathological process of cartilage damage in KBD.

Histopathology of chondronecrosis development in knee articular cartilage in a rat model of Kashin–Beck disease using T-2 toxin and selenium deficiency conditions

The objective of this study is to observe pathogenic lesions of joint cartilages in rats fed with T-2 toxin under a selenium deficiency nutrition status in order to determine possible etiological factors causing Kashin-Beck disease (KBD). Sprague-Dawley rats were fed selenium-deficient or control diets for 4 weeks prior to their being exposed to T-2 toxin. Six dietary groups were formed and studied 4 weeks later, i.e., controls, selenium-deficient, low T-2 toxin, high T-2 toxin, selenium-deficient diet plus low T-2 toxin, and selenium-deficient diet plus high T-2 toxin. Selenium deficiencies were confirmed by the determination of glutathione peroxidase activity and selenium levels in serum. The morphology and pathology (chondronecrosis) of knee joint cartilage of experimental rats were observed using light microscopy and the expression of proteoglycans was determined by histochemical staining. Chondronecrosis in deep zone of articular cartilage of knee joints was seen in both the low and high T-2 toxin plus selenium-deficient diet groups, these chondronecrotic lesions being very similar to chondronecrosis observed in human KBD. However, the chondronecrosis observed in the rat epiphyseal growth plates of animals treated with T-2 toxin alone or T-2 toxin plus selenium-deficient diets were not similar to that found in human KBD. Our results indicate that the rat can be used as a suitable animal model for studying etiological factors contributing to the pathogenesis (chondronecrosis) observed in human KBD. However, those changes seen in epiphyseal growth plate differ from those seen in human KBD probably because of the absence of growth plate closure in the rat.

Response of Chondrocytes to Local Mechanical Injury in an Ex Vivo Model.

Background:Our goal was to set up an ex vivo culture system to assess whether cartilage wounding (partial-thickness defects) can induce morphological changes in neighboring chondrocytes and whether these cells can translocate to the surface of the defect.Methods:Two-millimeter partial-depth defects were created in human osteochondral explants followed by culture for up to 4 weeks. Frozen sections of defects and defect-free regions were labeled using immunofluorescence for a plasma membrane protein, CD44, and actin with TRITC-phalloidin. Viable nuclei were detected with Hoechst 33342. Differential interference contrast (DIC), confocal, and transmission electron microscopy (TEM) were used to examine process extension.Results:Significant changes in cell morphology occurred in response to wounding in the superficial and deep cartilage zones. These included cell flattening, polarization of the actin cytoskeleton, extension of pseudopods projecting towards the edge of the defect, and interactions of these filopodia with collagen fibers. Cell density decreased progressively in the 300-µm zone adjacent to the defect to an average of approximately 25% to 35% after 3 weeks. Concomitant increases in cell density in the defect margin were observed. By contrast, minimal changes were seen in the middle cartilage zone.Conclusions:These novel observations strongly suggest active cartilage cell responses and movements in response to wounding. It is proposed that cartilage cells use contact guidance on fibrillated collagen to move into and populate defect areas in the superficial and deep zones.

Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death

In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent HexabrixTM. Samples were prepared from bovine patellae and stored in Dulbecco’s Modified Eagle’s Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1MPa to 10MPa and from 0min to 15min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix™, stress-relaxation tests were conducted for the samples immersed in Hexabrix™ and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix™ were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8–10MPa contact stresses, the samples immersed in full strength Hexabrix™ showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.

Engineering articular cartilage with spatially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel

Despite significant advances in stem cell differentiation and tissue engineering, directing progenitor cells into three-dimensionally (3D) organized, native-like complex structures with spatially-varying mechanical properties and extra-cellular matrix (ECM) composition has not yet been achieved. The key innovations needed to achieve this would involve methods for directing a single stem cell population into multiple, spatially distinct phenotypes or lineages within a 3D scaffold structure. We have previously shown that specific combinations of natural and synthetic biomaterials can direct marrow-derived stem cells (MSC) into varying phenotypes of chondrocytes that resemble cells from the superficial, transitional, and deep zones of articular cartilage. In this current study, we demonstrate that layer-by-layer organization of these specific biomaterial compositions creates 3D niches that allow a single MSC population to differentiate into zone-specific chondrocytes and organize into a complex tissue structure. Our results indicate that a three-layer polyethylene glycol (PEG)-based hydrogel with chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) incorporated into the top layer (superficial zone, PEG:CS:MMP-pep), CS incorporated into the middle layer (transitional zone, PEG:CS) and hyaluronic acid incorporated in the bottom layer (deep zone, PEG:HA), creates native-like articular cartilage with spatially-varying mechanical and biochemical properties. Specifically, collagen II levels decreased gradually from the superficial to the deep zone, while collagen X and proteoglycan levels increased, leading to an increasing gradient of compressive modulus from the superficial to the deep zone. We conclude that spatially-varying biomaterial compositions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population.

Gene expression of alginate-embedded chondrocyte subpopulations and their response to exogenous IGF-1 delivery

The delivery of growth factors to aid in cartilage engineering has received considerable attention. However, phenotypical differences between chondrocyte cell populations and their distinct responses to growth factors are not fully understood. To address this issue, we have investigated the gene expression of chondrocytes isolated from the superficial, middle, and deep zones of bovine articular cartilage. A three-dimensional (3D) alginate bead model was used to encapsulate zonal chondrocytes and culture with or without exogenous insulin-like growth factor-1(IFG-1) delivery. Following culture, mRNA expression of type I collagen, type II collagen, aggregan, IGF-1 and IGF-1 binding protein (IGF-BP3) were analysed at 1, 4 and 8 days. To the best of our knowledge, this is the first study to investigate gene expression of IGF-1 and IGF-BP3 by zone, and among the first studies to investigate growth factor delivery to chondrocytes in a 3D culture environment. Histological images and cell count data confirm the isolation of chondrocyte subpopulations, and gene expression data show distinct profiles for each zone, both with and without IGF-1 delivery. The data also show similar gene expression for the middle and deep zone cells, while the superficial zone group displays unique activity. Deep zone cells appear the most robust in their phenotype retention and most responsive to IGF-1 delivery. The results highlight differences in metabolic activity and varying responses to delivered growth factors between zonal chondrocyte populations.

Differential response of cartilage oligomeric matrix protein (COMP) to morphogens of bone morphogenetic protein/transforming growth factor-β family in the surface, middle and deep zones of articular cartilage

Cartilage oligomeric matrix protein (COMP) is a prominent non-collagenous component of the extracellular matrix (ECM) in articular cartilage. The regulation of COMP synthesis and secretion is critical for the understanding of cartilage homeostasis in health and disease. We therefore investigated the role of bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) superfamily members on COMP. Articular chondrocytes were isolated from three distinct zones (surface, middle and deep) and cultured as monolayers in serum-free chemically defined medium. Protein levels of COMP were determined in the medium by enzyme-linked immunosorbent assay. The mRNA expression was determined by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). TGFβ1 significantly stimulated the expression of COMP at the mRNA and protein levels in the superficial zone in a time-dependent manner. An unexpected discovery was that surface chondrocytes were more responsive to TGFβ isoforms than those in the deep layer. However, BMP-7 and growth differentiation factor-5 (GDF-5) also upregulate COMP expression; the effects were not as potent as those of TGFβ1. Activins A, B and AB demonstrated no effects on COMP in any of the zones. In conclusion, COMP synthesis is differentially regulated by TGFβ1 in the surface and middle zones of bovine articular cartilage.

Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilage

Numerous studies have reported generation of cartilage-like tissue from chondrocytes and stem cells, using pellet cultures, bioreactors and various biomaterials, especially hydrogels. However, one of the primary unsolved challenges in the field has been the inability to produce tissue that mimics the highly organized zonal architecture of articular cartilage; specifically its spatially varying mechanical properties and extra-cellular matrix (ECM) composition. Here we show that different combinations of synthetic and natural biopolymers create unique niches that can “direct” a single marrow stem cell (MSC) population to differentiate into the superficial, transitional, or deep zones of articular cartilage. Specifically, incorporating chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) into PEG hydrogels (PEG:CS:MMP-pep) induced high levels of collagen II and low levels of proteoglycan expression resulting in a low compressive modulus, similar to the superficial zone. PEG:CS hydrogels produced intermediate-levels of both collagen II and proteoglycans, like the transitional zone, while PEG:hyaluronic acid (HA) hydrogels induced high proteoglycan and low collagen II levels leading to high compressive modulus, similar to the deep zone. Additionally, the compressive moduli of these zone-specific matrices following cartilage generation showed similar trend as the corresponding zones of articular cartilage, with PEG:CS:MMP-pep having the lowest compressive modulus, followed by PEG:CS while PEG:HA had the highest modulus. These results underscore the potential for composite scaffold structures incorporating these biomaterial compositions such that a single stem-progenitor cell population can give rise to zonally-organized, functional articular cartilage-like tissue.