Zone Of Articular Cartilage Research Articles

Profiling microRNA expression in bovine articular cartilage and implications for mechanotransduction

Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: the surface, middle, and deep zones. Each zone has a different gene expression pattern that plays a specific role in articular cartilage development and maintenance. MicroRNA (miRNA) are small noncoding gene products that play an important regulatory role in determining cell differentiation and function. The purpose of this study was to test our hypothesis that miRNA expression profiles in the different articular cartilage zones as well as between regions subjected to different levels of weight-bearing stresses are unique. Using an miRNA microarray approach in conjunction with quantitative reverse transcription-polymerase chain reaction, we identified miRNA in bovine articular cartilage that were differentially expressed in the different functional zones and in the anterior weight-bearing and posterior non-weight-bearing regions of the medial femoral condyle (M1 and M4, respectively). We identified miRNA-221 and miR-222 as part of a subset of differentially expressed miRNA that were up-regulated in articular cartilage in the anterior, M1, greater weight-bearing location. Additionally, miR-126, miR-145, and miR-335 were down-regulated in monolayers of tissue-cultured chondrocytes as compared with levels determined directly from intact native cartilage. In conclusion, miR-222 expression patterns in articular cartilage are higher in the weight-bearing anterior medial condyle as compared with the posterior non-weight-bearing medial condyle. Thus, miR-222 might be a potential regulator of an articular cartilage mechanotransduction pathway. These data implicate miRNA in the maintenance of articular cartilage homeostasis and are therefore targets for articular cartilage tissue engineering and regenerative medicine.

The collagen fibril structure in the superficial zone of articular cartilage by μMRI

To investigate the fibril architecture of the collagen matrix in the superficial zone (SZ) of articular cartilage non-destructively by microscopic magnetic resonance imaging (microMRI) T(2) anisotropy. Six specimens of canine humeral cartilage were rotated in such a way that the normal axis of the articular surface of the cartilage specimen remained stationary and perpendicular to the static magnetic field, over a range of 180 degrees and at a step of 15 degrees. At each rotation angle, a quantitative T(2) image was constructed at 13 microm pixel resolution. A set of complex and depth-dependent patterns was found in the microMRI T(2) anisotropy along the depth of the tissue. In the SZ, the T(2) anisotropy is clearly periodic, which demonstrates that the distribution of the collagen fibrils in the SZ is not random. In the transitional zone, the periodicity of the T(2) anisotropy approximately doubles with respect to that in the SZ. In the initial part of the radial zone (RZ), the T(2) anisotropy is also periodic but inverse to that in the SZ. In the deep part of the RZ, the T(2) anisotropy becomes increasingly weaker and eventually disappears. There exists a certain degree of collagen anisotropy in all zones of articular cartilage. The anisotropic imaging data can be interpreted with the aid of a collagen architecture model.

Chondrocyte death in mechanically injured articular cartilage—the influence of extracellular calcium

Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium-free media (approximately 0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium-rich media (2-20 mM, ANOVA at 2.5 h, p = 0.002). In calcium-rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium-free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium-free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium-free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells on Oriented Nanofibrous Scaffolds: Engineering the Superficial Zone of Articular Cartilage

Cell differentiation, adhesion, and orientation are known to influence the functionality of both natural and engineered tissues, such as articular cartilage. Several attempts have been devised to regulate these important cellular behaviors, including application of inexpensive but efficient electrospinning that can produce patterned extracellular matrix (ECM) features. Electrospun and oriented polycaprolactone (PCL) scaffolds (500 or 3000 nm fiber diameter) were created, and human mesenchymal stem cells (hMSCs) were cultured on these scaffolds. Cell viability, morphology, and orientation on the fibrous scaffolds were quantitatively determined as a function of time. While the fiber-guided initial cell orientation was maintained even after 5 weeks, cells cultured in the chondrogenic media proliferated and differentiated into the chondrogenic lineage, suggesting that cell orientation is controlled by the physical cues and minimally influenced by the soluble factors. Based on assessment by the chondrogenic markers, use of the nanofibrous scaffold (500 nm) appears to enhance the chondrogenic differentiation. These findings indicate that hMSCs seeded on a controllable PCL scaffold may lead to an alternate methodology to mimic the cell and ECM organization that is found, for example, in the superficial zone of articular cartilage.

Roles of adsorbed film and gel layer in hydration lubrication for articular cartilage

To maintain low friction and low wear in natural synovial joints, adsorbed film formation on articular cartilage surface appears to play an important role in mixed or boundary lubrication regime where local direct contact occurs. Furthermore, the proteoglycan gel layer at the uppermost superficial zone in articular cartilage appears to play an important role in preserving low friction and low wear by the hydration lubrication mechanism even after removal of adsorbed film. However, the interaction or synergistic action between adsorbed film and hydrated film/surface has not yet been clarified. To examine the roles of adsorbed film and gel layer on articular cartilage surface in hydration lubrication, the changes in friction were observed in the reciprocating test of articular cartilage against a glass plate, at repeated rubbing including restarting after interrupting—unloading process. It is noticed that at restarting immediately after loading, hydration lubrication is expected to become effective. The lubricating roles of protein-adsorbed film in hydration lubrication are discussed on the basis of experimental results in both cases with and without surface gel layer. The comparison of albumin and γ -globulin is described in connection with the existence of a gel layer.

Superficial Zone Protein (Lubricin) in the Different Tissue Compartments of the Knee Joint: Modulation by Transforming Growth Factor Beta 1 and Interleukin-1 Beta

Superficial-zone protein (SZP), also known as lubricin, is a key mediator of boundary lubrication and plays an important role in the functional integrity of the diarthrodial joint. The aim of this investigation was to examine the role of transforming growth factor beta (TGF-beta) and interleukin-1 beta (IL-1beta) on the expression of SZP in various compartments of the bovine knee joint: the superficial zone of articular cartilage, synovium, meniscus, and anterior and posterior cruciate ligaments. The effects of TGF-beta1 and IL-1beta on SZP expression were examined in explants and cells from the different tissue compartments. TGF-beta1 up-regulated the expression of SZP in cultured explants, but IL-1beta down-regulated it. Quantitative analysis of secreted proteins in the medium of the cells demonstrated significant stimulation by TGF-beta1 and inhibition by IL1-beta of the accumulation of SZP protein in all four tissues. Real-time polymerase chain reaction analysis revealed that TGF-beta1 significantly up-regulated SZP expression and that IL-1beta down-regulated it. These results revealed the modulation of SZP expression in various compartments of the knee joint by TGF-beta1 and IL-1beta. In addition, SZP was found to be immunolocalized at the surface layer of cells in histological sections of all four tissue compartments. Collectively, results of the current study on regulation of SZP expression by TGF-beta and IL-1 help provide new insights, into tissue engineering strategies to repair and regenerate the different tissue compartments in the articular joint with optimal lubrication.

Dependence of Zonal Chondrocyte Water Transport Properties on Osmotic Environment

OBJECTIVE: The increasing concentration of proteoglycans from the surface to the deep zone of articular cartilage produces a depth-dependent gradient in fixed charge density, and therefore extracellular osmolarity, which may vary with loading conditions, growth and development, or disease. In this study we examine the relationship between in situ variations in osmolarity on chondrocyte water transport properties. Chondrocytes from the depth-dependent zones of cartilage, effectively preconditioned in varying osmolarities, were used to probe this relationship. DESIGN: First, depth variation in osmolarity of juvenile bovine cartilage under resting and loaded conditions was characterized using a combined experimental/theoretical approach. Zonal chondrocytes were isolated into two representative "baseline" osmolarities chosen from this analysis to reflect in situ conditions. Osmotic challenge was then used as a tool for determination of water transport properties at each of these baselines. Cell calcium signaling was monitored simultaneously as a preliminary examination of osmotic baseline effects on cell signaling pathways. RESULTS: Osmotic baseline exhibits a significant effect on the cell membrane hydraulic permeability of certain zonal subpopulations but not on cell water content or incidence of calcium signaling. CONCLUSIONS: Chondrocyte properties can be sensitive to changes in baseline osmolarity, such as those occurring during OA progression (decrease) and de novo tissue synthesis (increase). Care should be taken in comparing chondrocyte properties across zones when cells are tested in vitro in non-physiologic culture media.

Induction of chondrogenesis and expression of superficial zone protein (SZP)/lubricin by mesenchymal progenitors in the infrapatellar fat pad of the knee joint treated with TGF-β1 and BMP-7

Superficial zone protein (SZP) is a key mediator of boundary lubrication of articular cartilage in joints. In this investigation, we made the unexpected discovery that SZP was expressed in infrapatellar fat pad (IFP) from bovine knee. Quantitative analysis of secreted proteins in the medium of the IFP stromal cells demonstrated a significant stimulation by TGF-β1 and BMP-7. Real-time PCR analysis revealed the SZP expression was up-regulated by TGF-β1 and BMP-7. Chondrogenically differentiated IFP progenitor cells were stimulated by TGF-β1 and BMP-7 to synthesize and secrete SZP. SZP mRNA was significantly up-regulated by chondrogenic induction for 21 days. These findings indicate that the stimulation of SZP expression by TGF-β and BMP-7 may lead to functional improvement of damaged intraarticular tissues and that IFP progenitor cells may be a potential useful source for inducing superficial zone of articular cartilage by tissue engineering for regeneration of damaged articular cartilage due to osteoarthritis

Cryopreservation and in Vitro Expansion of Chondroprogenitor Cells Isolated from the Superficial Zone of Articular Cartilage

Understanding the proliferation mechanisms of chondroprogenitor cells and their influence on cell differentiation is crucial in order to develop large-scale expansion processes for tissue engineering applications. Proliferation control mechanisms were mainly attributed to substrate limitation and cell-cell contact inhibition. The limiting substrates were found to be components of the FCS, with an optimal proliferation rate achieved in the presence of 40% FCS. In addition, the medium supply rate was found to be essential in reducing substrate limitation. In terms of FCS, 10 microL FCS cm(-2) h(-1) was the threshold feed rate required to prevent substrate limitation. Above this rate, maximum cell densities of 5.3 x 10(5) cells/cm2 were achieved, representing a 53-fold expansion. To reduce the need for high supply rates, the effect of specific growth factors was also investigated. Cell densities of 3.3 x 10(5) cells/cm2 were achieved in batch cultures using 40% FCS and 1 ng/mL TGF-beta1. Chondroprogenitor cells were expanded in this medium up to three passages without compromising their ability to differentiate and produce cartilage-like matrix in pellet cultures. In addition to substrate limitation, cell-cell contact, even at very sparse subconfluent densities, appeared capable of exerting some degree of growth inhibition. The cells exhibited deceleratory growth kinetics, characterized by a decrease of specific growth rates over time.

Glycosaminoglycans in the pericellular matrix of chondrons and chondrocytes

This is the first study to quantitate and profile the glycosaminoglycan (GAG) composition of the pericellular matrix (PCM) of chondrons and chondrocytes using the highly sensitive technique; fluorophore-assisted carbohydrate electrophoresis (FACE). Bovine articular chondrocytes and chondrons were isolated enzymatically. High cell yield and viability were obtained for both preparations. Chondrons had strong immunofluorescent labeling for keratan sulphate and chondroitin-6 sulphate but no labeling for hyaluronan. We compared the immunofluorescent data with FACE. The quantities of total keratan sulphate were determined to be 0.013 +/- 0.002 pg cell(-1) and 0.032 +/- 0.003 pg cell(-1) in the chondrocyte and chondron preparations, respectively. Four internal keratan sulphate sugars were detected (gal beta 1,4glcNAc6S, gal6S beta 1,4glcNAc6S, glcNAc beta 1,3gal and glcNAc6S beta 1,3gal) for both preparations but they were present at significantly higher concentrations in chondron preparations (P < 0.01). Total chondroitin sulphate (CS) was determined to be 0.054 +/- 0.004 pg cell(-1) and 0.077 +/- 0.005 pg cell(-1) for chondrocyte and chondron preparations, respectively. Unsulphated CS disaccharide levels were similar but chondrons had significantly more chondroitin-4 sulphated disaccharides and chondroitin-6 sulphated disaccharides (P < 0.05). Hyaluronan acid was present at low concentrations (0.010 +/- 0.001 pg cell(-1)) in both chondrocytes and chondrons. In this study, enzyme digestion coupled with FACE separation revealed new information about the differences in GAGs from isolated chondrocyte and chondron preparations. Further investigation of the differences in GAGs from chondrocytes and chondrons from different zones of articular cartilage may be useful for tissue engineering approaches.

Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs

Objective To investigate the pathogenesis of late phase osteoarthritic (OA) synovial fluid (SF) on normal articular cartilage in vivo and provide an understanding of degenerative cartilage extending in OA joint. Methods A random knee, each of 8 beagle dogs, received anterior cruciate ligament transection (ACLT) and was confirmed to have late phase OA degenerative changes at 24 weeks after operation. Thereafter, one random elbow of each canine was injected with autologous late phase OA knee SF. The contralateral elbow was injected with normal saline (NS) of the same volume as SF aspirated from ACLT knee. These two groups of elbows were labeled “SF” and “NS”. 8 other beagle dogs were left intact and placed in Group Control. After aseptic arthrocentesis was performed weekly on both elbows for 24 weeks, morphological changes were observed in the cartilage of the elbows, and expressions of 7 biological etiological factors of chondrocytes of the elbows were determined in Group SF, Group NS and Group Control, respectively. Results Morphological changes were observed in articular cartilage of the elbows in Group SF. Levels of unit area of collagen type I in the noncalcified, calcified and full zones of articular cartilage of the elbows in Group SF increased significantly. Level of unit area of collagen type III in the calcified zone of articular cartilage of the elbows in Group SF remained unchanged. Meanwhile, expressions of MMP-1 and MMP-3 of chondrocytes of the elbows in Group SF increased significantly. There was almost no difference between articular cartilage in Group NS and Group Control. Conclusion Based on these results, we conclude that OA degeneration of normal articular cartilage can be independently induced by late phase OA SF. Endogenous OA biological etiological factor may be one of the reasons causing degenerative cartilage extending in OA joint.

Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

IntroductionThe collagen structure in the superficial zone of articular cartilage is critical to the tissue's durability. Early osteoarthritis is often characterized with fissures on the articular surface. This is closely related to the disruption of the collagen network. However, the traditional histology can not offer visualization of the collagen structure in articular cartilage because it uses conventional optical microscopy that does not have insufficient imaging resolution to resolve collagen from proteoglycans in hyaline articular cartilage. This study examines the 3D collagen network of articular cartilage scored from 0 to 2 in the scoring system of International Cartilage Repair Society, and aims to develop a 3D histology for assessing early osteoarthritis.MethodsArticular cartilage was visually classified into five physiological groups: normal cartilage, aged cartilage, cartilage with artificial and natural surface disruption, and fibrillated. The 3D collagen matrix of the cartilage was acquired using a 3D imaging technique developed previously. Traditional histology was followed to grade the physiological status of the cartilage in the scoring system of International Cartilage Repair Society.ResultsNormal articular cartilage contains interwoven collagen bundles near the articular surface, approximately within the lamina splendens. However, its collagen fibres in the superficial zone orient predominantly in a direction spatially oblique to the articular surface. With age and disruption of the articular surface, the interwoven collagen bundles are gradually disappeared, and obliquely oriented collagen fibres change to align predominantly in a direction spatially perpendicular to the articular surface. Disruption of the articular surface is well related to the disappearance of the interwoven collagen bundles.ConclusionA 3D histology has been developed to supplement the traditional histology and study the subtle changes in the collagen network in the superficial zone during early physiological alteration of articular cartilage. The fibre confocal imaging technology used in this study has allowed developing confocal arthroscopy for in vivo studying the chondrocytes in different depth of articular cartilage. Therefore, the current study has potential to develop an in vivo 3D histology for diagnosis of early osteoarthritis.

Articular chondrocytes derived from distinct tissue zones differentially respond to in vitro oscillatory tensile loading

The cell morphology, gene expression, and matrix synthesis of articular chondrocytes are known to vary with depth from the tissue surface. The objective of this study was to investigate if chondrocytes from different zones respond to in vitro oscillatory tensile loading in distinct ways and whether tensile strain, which is most prevalent near the articular surface, would preferentially stimulate superficial zone chondrocytes. Chondrocytes were separately isolated from the superficial, middle, and deep zones of articular cartilage and seeded into three-dimensional fibrin hydrogel constructs. An intermittent protocol of oscillatory tensile loading was applied for 3 days, and the effects on extracellular matrix (ECM) synthesis were assessed by measuring the incorporation of radiolabed precursors, size exclusion gel chromatography, and western blotting. Tensile loading was found to be a potent stimulus for proteoglycan synthesis only in superficial zone chondrocytes. Although overall biosynthesis rates by deep zone chondrocytes were unaffected by tensile loading, the molecular characteristics of proteins and proteoglycans released to the culture medium were significantly altered so as to resemble those of superficial zone chondrocytes. Oscillatory tensile loading differentially affected subpopulations of articular chondrocytes in three-dimensional fibrin hydrogel constructs. Cells isolated from deeper regions of the tissue developed some characteristics of superficial zone chondrocytes after exposure to tensile loading, which may indicate an adaptive response to the new mechanical environment. Understanding how exogenous mechanical stimuli can differentially influence chondrocytes from distinct tissue zones will yield important insights into mechanobiological processes involved in cartilage tissue development, maintenance, disease, and repair.

The immunohistochemical localization of notch receptors and ligands in human articular cartilage, chondroprogenitor culture and ultrastructural characteristics of these progenitor cells

The presence of progenitor/stem cells in human articular cartilage remains controversial. Therefore, we attempted to isolate and culture progenitor/stem cells and to investigate their phenotypic characteristics. Biopsies were obtained (with consent) from patients undergoing arthroscopic surgery. Full depth explants were fixed and cryosectioned or enzymatically digested and the resulting cells cultured and plated on fibronectin-coated dishes. Chondrocytes were cultured until colonies of >32 cells were present. Colonies were trypsinized and expanded in monolayer for pellet culture. Immunolocalization of Notch and its ligands were detected in vivo and in vitro using immunocytochemistry. In vitro studies investigated differences in immunolocalization of Notch and its associated ligands in colony-forming cells and small clusters of non-colony-forming cells. The ultrastructure of the chondroprogenitors was examined by scanning and transmission electron microscopy. Results revealed that the immunolocalization of Notch-1 and its ligand Delta were concentrated in regions closest to the articular surface. Notch-1 was also densely localized in the deeper zone of articular cartilage. Notch-2 immunolabeling was densely localized in all zones of articular cartilage. Jagged-1 was concentrated in the deeper regions of articular cartilage. Notch-1, Delta and Jagged-1 were more abundant in colony-forming cells than non-colony-forming chondrocytes in vitro. Notch-3, Notch-4 and Jagged-2 were absent from all regions of the articular cartilage tissues and cultured cartilage cells in vitro. Ultrastructurally, chondrocytes cultured in monolayer dedifferentiated to fibroblast-like cells with cell surface processes of varying lengths, pellet cultured cells varied in morphology, as flattened and rounded. In conclusion, we propose that adult human articular cartilage may contain cells having progenitor cell features.

Distinct horizontal patterns in the spatial organization of superficial zone chondrocytes of human joints

A better understanding of the unique cellular and functional properties of the superficial zone of articular cartilage may aid current strategies in tissue engineering which attempts a layered design for the repair of cartilage lesions to avert or postpone the onset of osteoarthritis. However, data pertaining to the cellular organization of non-degenerated superficial zone of articular cartilage is not available for most human joints. The present study analyzed the arrangement of chondrocytes of non-degenerated human joints (shoulder, elbow, knee, and ankle) by using fluorescence microscopy of the superficial zone in a top-down view. The resulting horizontal chondrocyte arrangements were tested for randomness, homogeneity or a significant grouping via point pattern analysis and were correlated with the joint type in which they occurred. The present study demonstrated that human superficial chondrocytes occurred in four distinct patterns of strings, clusters, pairs or single chondrocytes. Those patterns represented a significant grouping (p < 0.0001) with horizontal alignment. Each articular joint surface was dominated by only one of these four patterns (p < 0.001). Specific patterns correlated with specific diarthrodial joint types (p < 0.001). Further studies need to establish whether these organizational patterns are a consequence of their surrounding environment or whether they are linked to a functional purpose.

Solute transport in the deep and calcified zones of articular cartilage

(1) To establish whether the tidemark and calcified cartilage are permeable to low molecular weight solutes, thereby providing a potential pathway for nutrition of cells in the deep cartilage. (2) To investigate transport from the subchondral microcirculation into calcified cartilage in an intact perfused joint and the effects on transport of static loading. The permeability of the tidemark and calcified cartilage was investigated in plugs of cartilage and subchondral bone which formed the membrane of a diffusion cell. Transport from the subchondral microcirculation and the effects of load were studied in an intact perfused joint. Both preparations used the metacarpophalangeal joints of mature horses and fluorescein and rhodamine (m.w. approximately 400 Da) were employed as tracers, assayed by quantitative fluorescence microscopy on histological sections. Calcified cartilage was permeable to both solutes, both from the superficial and the subchondral sides. The effective diffusivity of both solutes was of the order of 9 x 10(-9) cm(2) s(-1), fivefold less than in the uncalcified cartilage. The calcified zone was heterogeneous, with high uptake of both tracers in the vicinity of the tidemark. The distribution volume of rhodamine B was higher than for fluorescein, consistent with a significant anionic charge in the calcified matrix. Static loading of the intact joint did not affect the transport of rhodamine B but caused a significant decrease in concentration of fluorescein both in the surface and deep zones of the tissue. Calcified cartilage is permeable to small solutes and the subchondral circulation may make a significant contribution to the nutrition of deep cartilage in the mature horse. Static loading reduces the uptake of small anionic solutes in the intact joint.

Response of zonal chondrocytes to extracellular matrix-hydrogels

We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.

Interaction between zonal populations of articular chondrocytes suppresses chondrocyte mineralization and this process is mediated by PTHrP

Articular cartilage is separated from subchondral bone by the tidemark and a calcified cartilage zone. Advancement of the calcified region and tidemark duplication are both hallmarks of osteoarthritis (OA). Currently the mechanisms controlling post-natal articular cartilage mineralization are poorly understood. The objective of this study is to test the hypothesis that cellular communication between different cartilage layers regulates articular chondrocyte mineralization. Co-culture models were established to evaluate the interaction of chondrocytes derived from the surface, middle and deep zones of articular cartilage. The cultures were stimulated with triiodothyronine (T3) to promote chondrocyte hypertrophy. The effects of zonal chondrocyte interactions on chondrocyte mineralization were examined over time. Co-culture of deep zone chondrocytes (DZCs) with surface zone chondrocytes (SZCs) suppressed the T3-induced increase in alkaline phosphatase (ALP) activity and related mineralization. Moreover, SZC-DZC co-culture was associated with a significantly higher parathyroid hormone-related peptide (PTHrP) expression when compared to controls. When PTHrP(1-40) was added to the DZC-only culture, it suppressed DZC ALP activity similar to the inhibition observed in co-culture with SZC. In addition, treatment with PTHrP reversed the effect of T3 stimulation on the expression of hypertrophic markers (Indian hedgehog, ALP, matrix metalloproteinases-13, Type X collagen) in the DZC cultures. Moreover, blocking the action of PTHrP significantly increased ALP activity in SZC+DZC co-culture. Our findings demonstrate the role of zonal chondrocyte interactions in regulating cell mineralization and provide a plausible mechanism for the post-natal regulation of articular cartilage matrix organization. These findings also have significant implications in understanding the pathology of articular cartilage as well as devising strategies for functional cartilage repair.

Increased collagen and aggrecan degradation with age in the joints of Timp3−/− mice

To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage. Hind joints of Timp3(-/-) and wild-type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (... VDIPEN and ... NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme-linked immunosorbent assay. Articular cartilage from Timp3-knockout animals (ages > or =6 months) showed reduced Safranin O staining and an increase in ...VDIPEN content compared with cartilage from heterozygous and wild-type animals. There was also a slight increase in ... NVTEGE content in articular cartilage and menisci of Timp3(-/-) animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3(-/-) mice compared with wild-type animals. This increase was significant in 15-month-old animals. These results indicate that TIMP-3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP-3 may play a pathophysiologic role in the development of this disease.

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth ( p < 0.0 5 ) by a factor of 4–5 from the top 0.1 mm (28±13 kPa, 141±10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p < 0.0 0 1 ), and correlated with the modulus (both p < 0.0 1 ). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues.