Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1.

We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:1603-1615, 1999)     

Biology and pathology of Rho GTPase,PI‐3 kinase‐Akt,and MAP kinase signaling pathways in chondrocytes

Chondrocytes provide the framework for the developing skeleton and regulate long‐bone growth through the activity of the growth plate. Chondrocytes in the articular cartilage, found at the ends of bones in diarthroidial joints, are responsible for maintenance of the tissue through synthesis and degradation of the extracellular matrix. The processes of growth, differentiation, cell death and matrix remodeling are regulated by a network of cell signaling pathways in response to a variety of extracellular stimuli. These stimuli consist of soluble ligands, including growth factors and cytokines, extracellular matrix proteins, and mechanical factors that act in concert to regulate chondrocyte function through a variety of canonical and non‐canonical signaling pathways. Key chondrocyte signaling pathways include, but are not limited to, the p38, JNK and ERK MAP kinases, the PI‐3 kinase‐Akt pathway, the Jak‐STAT pathway, Rho GTPases and Wnt‐β‐catenin and Smad pathways. Modulation of the activity of any of these pathways has been associated with various pathological states in cartilage. This review focuses on the Rho GTPases, the PI‐3 kinase‐Akt pathway, and some selected aspects of MAP kinase signaling. Most studies to date have examined these pathways in isolation but it is becoming clear that there is significant cross‐talk among the pathways and that the overall effects on chondrocyte function depend on the balance in activity of multiple signaling proteins. J. Cell. Biochem. 110: 573–580, 2010. © 2010 Wiley‐Liss, Inc.     

Distribution of cartilage molecules in the developing mouse joint.

This study describes the precise spatial and temporal patterns of protein distribution for aggrecan, fibromodulin, cartilage oligomeric matrix protein (COMP) and cartilage matrix protein (CMP) in the developing mouse limb with particular attention to those cells destined to form articular chondrocytes in comparison to those cells destined to form a mineralized tissue and become replaced by bone. Mouse glenohumeral joints from fetal mice (12-18 days post coitus (dpc) to the young adult (37 days after birth) were immunostained with antibodies specific for these molecules. Aggrecan staining defined the general chondrocytic phenotype, whether articular or transient. Fibromodulin was associated with prechondrocytic mesenchymal cells in the interzone prior to joint cavitation and with the mesenchymal cells of the perichondrium or the periosteum encapsulating the joint elements of the maturing and young adult limb. Staining was most intense around developing articular chondrocytes and much less abundant or absent in those differentiating cells along the anlage. CMP showed an almost reciprocal staining pattern to fibromodulin and was not detected in the matrix surrounding articular chondrocytes. COMP was not detected in the cells at the articular surface prior to cavitation but by 18 dpc, as coordinated movement of the mouse forelimb intensifies, staining for COMP was most intense around the maturing articular chondrocytes. These results show that the cells that differentiate into articular chondrocytes elaborate an extracellular matrix distinct from those cells that are destined to form bone. Fibromodulin may function in the early genesis of articular cartilage and COMP may be associated with elaboration of a weight-bearing chondrocyte matrix.     

Chondroitin sulphate and heparan sulphate sulphation motifs and their proteoglycans are involved in articular cartilage formation during human foetal knee joint development

Novel sulphation motifs within the glycosaminoglycan chain structure of chondroitin sulphate (CS) containing proteoglycans (PGs) are associated with sites of growth, differentiation and repair in many biological systems and there is compelling evidence that they function as molecular recognition sites that are involved in the binding, sequestration or presentation of soluble signalling molecules (e.g. morphogens, growth factors and cytokines). Here, using monoclonal antibodies 3B3(-), 4C3 and 7D4, we examine the distribution of native CS sulphation motifs within the developing connective tissues of the human foetal knee joint, both during and after joint cavitation. We show that the CS motifs have broad, overlapping distributions within the differentiating connective tissues before the joint has fully cavitated; however, after cavitation, they all localise very specifically to the presumptive articular cartilage tissue. Comparisons with the labelling patterns of heparan sulphate (HS), HS-PGs (perlecan, syndecan-4 and glypican-6) and FGF-2, molecules with known signalling roles in development, indicate that these also become localised to the future articular cartilage tissue after joint cavitation. Furthermore, they display interesting, overlapping distributions with the CS motifs, reflective of early tissue zonation. The overlapping expression patterns of these molecules at this site suggests they are involved, or co-participate, in early morphogenetic events underlying articular cartilage formation; thus having potential clinical relevance to mechanisms involved in its repair/regeneration. We propose that these CS sulphation motifs are involved in modulating the signalling gradients responsible for the cellular behaviours (proliferation, differentiation, matrix turnover) that shape the zonal tissue architecture present in mature articular cartilage.     

Toward regeneration of articular cartilage

Articular cartilage is classified as permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in the epiphyseal growth plate. In the process of synovial joint development, articular cartilage originates from the interzone, developing at the edge of the cartilaginous anlagen, and establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators, such as Wnts, GDF5, Erg, and PTHLH, coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracellular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier's groove, the intra‐articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Furthermore, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site. Birth Defects Research (Part C) 99:192–202, 2013 . © 2013 Wiley Periodicals, Inc .     

CCN3-mediated promotion of sulfated proteoglycan synthesis in rat chondrocytes from developing joint heads

Chondrocytes forming articular cartilage are embedded in a vast amount of extracellular matrix having physical stiffness and elasticity, properties that support the mechanical load from bones and enable the flexible movement of synovial joints. Unlike chondrocytes that conduct the growth of long bones by forming the growth plate, articular chondrocytes show suppressed cell proliferation, unless these cells are exposed to pathological conditions such as mechanical overload. In the present study, we found that one of the members of the CCN family, CCN3, was significantly expressed in chondrocytes isolated from the epiphyseal head in developing rat synovial joints. Evaluation of the effect of recombinant CCN3 on those chondrocytes revealed that CCN3 promoted proteoglycan synthesis, whereas this factor repressed the proliferation of the same cells. These results suggest a critical role for CCN3 in the regulation of the biological properties of articular chondrocytes.     

Versican expression during synovial joint morphogenesis

The extracellular matrix (ECM) plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs) are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34). By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.     

Articular cartilage cells immortalized by a temperature sensitive mutant of SV40 large T antigen survive and form cartilage tissue in articular cartilage environment.

A chondrogenic cell line, TC6, was established by using cells derived from articular cartilage of transgenic mice harboring a temperature-sensitive mutant simian virus (SV) 40 large T-antigen gene. TC6 cells express genes encoding proteins related to cartilage phenotypes such as type II collagen. To examine the in vivo behavior of the TC6 cells, these cells were implanted into cavity-shaped full-thickness defects made in the articular cartilage of the central part of the patellar grooves of mouse femora. One week after implantation, the morphology of the cells was still fibroblastic but these cells were just about to start to form a cartilage-like matrix. By 6 weeks after implantation, the cells had produced abundant cartilaginous matrix and their morphology became closer to that of authentic chondrocytes. This was in sharp contrast to the fibroblastic morphology of these cells in an in vitro environment even after long-term culture. These observations indicate that a cartilage-matrix environment provides a scaffold for the TC6 cells to form cartilage tissues. Our data show that the genetically engineered chondrocytic cell line, TC6, can form a cartilage-like matrix in vivo.     

Dynamic 3D culture: Models of chondrogenesis and endochondral ossification

The formation of cartilage from stem cells during development is a complex process which is regulated by both local growth factors and biomechanical cues, and results in the differentiation of chondrocytes into a range of subtypes in specific regions of the tissue. In fetal development cartilage also acts as a precursor scaffold for many bones, and mineralization of this cartilaginous bone precursor occurs through the process of endochondral ossification. In the endochondral formation of bones during fetal development the interplay between cell signalling, growth factors, and biomechanics regulates the formation of load bearing bone, in addition to the joint capsule containing articular cartilage and synovium, generating complex, functional joints from a single precursor anlagen. These joint tissues are subsequently prone to degeneration in adult life and have poor regenerative capabilities, and so understanding how they are created during development may provide useful insights into therapies for diseases, such as osteoarthritis, and restoring bone and cartilage lost in adulthood. Of particular interest is how these tissues regenerate in the mechanically dynamic environment of a living joint, and so experiments performed using 3D models of cartilage development and endochondral ossification are proving insightful. In this review, we discuss some of the interesting models of cartilage development, such as the chick femur which can be observed in ovo, or isolated at a specific developmental stage and cultured organotypically in vitro. Biomaterial and hydrogel‐based strategies which have emerged from regenerative medicine are also covered, allowing researchers to make informed choices on the characteristics of the materials used for both original research and clinical translation. In all of these models, we illustrate the essential importance of mechanical forces and mechanotransduction as a regulator of cell behavior and ultimate structural function in cartilage. Birth Defects Research (Part C) 105:19–33, 2015. © 2015 Wiley Periodicals, Inc.     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Influence of stress rate on water loss, matrix deformation and chondrocyte viability in impacted articular cartilage

The biomechanical response of articular cartilage to a wide range of impact loading rates was investigated for stress magnitudes that exist during joint trauma. Viable, intact bovine cartilage explants were impacted in confined compression with stress rates of 25, 50, 130 and 1000 MPa/s and stress magnitudes of 10, 20, 30 and 40 MPa. Water loss, cell viability, dynamic impact modulus (DIM) and matrix deformation were measured. Under all loading conditions the water loss was small (approximately 15%); water loss increased linearly with increasing peak stress and decreased exponentially with increasing stress rate. Cell death was localized within the superficial zone (< or =12% of total tissue thickness); the depth of cell death from the articular surface increased with peak stress and decreased with increasing stress rate. The DIM increased (200-700 MPa) and matrix deformation decreased with increasing stress rate. Initial water and proteoglycan (PG) content had a weak, yet significant influence on water loss, cell death and DIM. However, the significance of the inhomogeneous structure and composition of the cartilage matrix was accentuated when explants impacted on the deep zone had less water loss and matrix deformation, higher DIM, and no cell death compared to explants impacted on the articular surface. The mechano-biological response of articular cartilage depended on magnitude and rate of impact loading.     

WARP is a novel multimeric component of the chondrocyte pericellular matrix that interacts with perlecan

WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with K(D) values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of "permanent" cartilage structures during development and in mature cartilages.     

Cell death activation during cavitation of embryoid bodies is mediated by hydrogen peroxide

The formation of the proamniotic cavity is the first indication of programmed cell death associated to a morphogenetic process in mammals. Although some growth factors have been implicated in proamniotic cavitation, very little is known about the intracellular mechanisms that control the cell death process itself. Reactive oxygen species (ROS) are potent activators of cell death, thus, in the present work we evaluated the role of ROS during the cavitation of embryoid bodies (EBs), a common model to study proamniotic cavitation. During cavitation, ROS concentration increases in the inner cells of EBs, and this ROS accumulation appears to be associated with the mitochondrial respiratory activity. In agreement with a role of ROS in cavitation, EBs derived from ES cells that overproduce catalase, an enzyme that specifically degrades hydrogen peroxide, do not cavitate, and caspase activation and cell death is markedly decreased. Notably, cell death, but not the rise in ROS, during EB cavitation is caspase-dependent. The apoptosis-inducing factor (Aif) is released from the mitochondria during cavitation, but EBs derived from Aif^−/y ES cells cavitate and ROS levels in the inner cells remain high. We conclude that hydrogen peroxide is a cell death activating signal essential for EB cavitation, suggesting that cell death during proamniotic cavitation is mediated by ROS.     

Permanence of proliferating cells in developing,juvenile and adult knee epiphyses of lizards in relation to bone growth and regeneration

The distribution of long‐labelling‐retaining cells, putative progenitor or stem cells, in the developing knees of embryo, juvenile and adult lizards has been analysed using H3‐thymidine autoradiography and 5BrdU immunohistochemistry. Proliferating cells are present in developing cartilaginous femur and tibia, especially in the epiphyses where a higher cell multiplication likely determines their typical enlarged shape in comparison with the diaphyses where chondroblast proliferation is low to absent. Sparse 5BrdU‐labelled cells remain in the articular and growth plate cartilages of the epiphyses in older stages of development and are still detected in developing epiphyses 13 days after injection of 5BrdU. This indicates they are slow‐cycling cells, a typical characteristic for progenitor or stem cells. Long retaining 5BrdU‐labelled cells remain in the articular surface also during adult life where they likely sustain the growth of long bones. Adult epiphyses show secondary ossification centres where the articular cartilage is partially or largely replaced by bone trabeculae. The damage in the epiphysis of lizards stimulates the proliferation of progenitor cells for the regeneration of new cartilaginous epiphyses. The localization of cells capable of proliferation in the epiphyses of adult femur and tibia pre‐adapts these lizards to cartilage regeneration in case of injury.     

Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar,growth plate,and articular cartilage in rats

Summary Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. Therefore, we concluded that TIMP-1 and TIMP-2 were general inhibitors of MMP-2 and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix.     

Chondroitin sulfate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells.

Osteoarthritis is a chronic, debilitating joint disease characterized by progressive destruction of articular cartilage. Recently, a number of studies have identified a chondroprogenitor cell population within articular cartilage with significant potential for repair/regeneration. As yet, there are few robust biomarkers of these cells. In this study, we show that monoclonal antibodies recognizing novel chondroitin sulfate sulfation motif epitopes in glycosaminoglycans on proteoglycans can be used to identify metabolically distinct subpopulations of cells specifically within the superficial zone of the tissue and that flow cytometric analysis can recognize these cell subpopulations. Fluorochrome co-localization analysis suggests that the chondroitin sulfate sulphation motifs are associated with a range of cell and extracellular matrix proteoglycans within the stem cell niche that include perlecan and aggrecan but not versican. The unique distributions of these sulphation motifs within the microenvironment of superficial zone chondrocytes, seems to designate early stages of stem/progenitor cell differentiation and is consistent with these molecules playing a functional role in regulating aspects of chondrogenesis. The isolation and further characterization of these cells will lead to an improved understanding of the role novel chondroitin sulfate sulfation plays in articular cartilage development and may contribute significantly to the field of articular cartilage repair.     

Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis

Two members of the fibroblast growth factor (FGF) family, basic FGF (bFGF) and FGF-18, have been implicated in the regulation of articular and intervertebral disc (IVD) cartilage homeostasis. Studies on bFGF from a variety of species have yielded contradictory results with regards to its precise role in cartilage matrix synthesis and degradation. In contrast, FGF-18 is a well-known anabolic growth factor involved in chondrogenesis and articular cartilage repair. In this review, we examined the biological actions of bFGF and FGF-18 in articular and IVD cartilage, the specific cell surface receptors bound by each factor, and the unique signaling cascades and molecular pathways utilized to exert their biological effects. Evidence suggests that bFGF selectively activates FGF receptor 1 (FGFR1) to exert degradative effects in both human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme activity, inhibition of matrix production, and increased cell proliferation resulting in clustering of cells seen in arthritic states. FGF-18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating FGFR3, increasing matrix formation and cell differentiation while inhibiting cell proliferation, leading to dispersed cells surrounded by abundant matrix. The results from in vitro and in vivo studies suggest the potential usefulness of bFGF and FGFR1 antagonists, as well as FGF-18 and FGFR3 agonists, as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future.     

Effect of Compressive Strain on Cell Viability in Statically Loaded Articular Cartilage

Physiological loading of articulating joints is necessary for normal cartilage function. However, conditions of excessive overloading or trauma can cause cartilage injury resulting in matrix damage and cell death. The objective of this study was to evaluate chondrocyte viability within mechanically compressed articular cartilage removed from immature and mature bovine knees. Twenty-three mature and 68 immature cartilage specimens were subjected to static uniaxial confined-creep compressions of 0–70% and the extent of cell death was measured using fluorescent microscopic imaging. In both age groups, cell death was always initiated at the articular surface and increased linearly in depth with increasing strain magnitude. However, most of the cell death was localized within the superficial zone (SZ) of the cartilage matrix with the depth never greater than ~ 500 μm or 25% of the thickness of the test specimen. The immature cartilage was found to have a significantly greater (> 2 times) amount (depth) of cell death compared to the mature cartilage, especially at the higher strains. This finding was attributed to the lower compressive modulus of the immature cartilage in the SZ compared to that of the mature cartilage, resulting in a greater local matrix strain and concomitant cell surface membrane strain in this zone when the matrix was compressed. These results provide further insight into the capacity of articular cartilage in different age groups to resist the severity of traumatic injury from compressive loads.