At birth, articular cartilage is relatively homogeneous. That is, the cellular and molecular structures do not vary from site to site topologically across the joint 123456. During postnatal maturation in mammals, articular cartilage undergoes architectural reorganization associated with growth and changes in biomechanical demands on the tissue 1235. For equine articular chondrocytes, the process is characterized by altered matrix protein production patterns with exercise during the first five months of postnatal life 27. This adaptation affects the tissue longitudinally from the joint surface to deeper layers, as well as topologically across a joint between weight-bearing and non-weight-bearing areas 128. Sites that receive low-level constant weight-bearing load have higher amounts of glycosaminoglycan and proteoglycan, providing resistance to compressive stresses 18. Sites that receive high sheer stress through intermittent peak loading and movement have high levels of type II collagen for tensile strength 18. As noted, these changes in architectural heterogeneity develop in the articular cartilage of foals during the first 5 months of life 23. However, if exercise is restricted, articular cartilage fails to fully develop these characteristics of maturation. In addition, re-establishing normal levels of activity in a previously exercise-restricted foal is ineffective at promoting proper articular cartilage adaptation 23. In this same population of horses, van den Hoogen et al. found that proteoglycan synthesis increased with free pasture exercise over the first 5 months followed by a light exercise regimen 9. These results suggest that the architecture of articular cartilage changes with age and mechanical stimulation. If normal exercise is severely restricted in a horse during the first 5 months of postnatal life, then adaptation of matrix and anchorage proteins in articular cartilage to weight- and shear-stresses can be permanently compromised.

Postnatal articular cartilage changes have been characterized in several mammals. The transition from isotropic to anisotropic architecture was demonstrated in the articular cartilage of the proximal tibia of rats by Sasano et al. and in the medial femoral condyle of rabbits by Hunziker et al. 510. Increased postnatal matrix deposition of type II collagen occurs in the rabbit and rat 1011. Changes and topological differences in cross-linking of collagens with age in postnatal horses as well as in humans have been established 7812. Moreover, moderate exercise in young adult dogs, guinea pigs, and humans seem to cause zonal changes in proteoglycan and collagen content that allow for adaptation and strengthening of articular cartilage 13. While past studies have described biochemical and histological changes to articular cartilage during the postnatal period, much remains unexplained with regards to the cellular mechanisms that achieve growth and matrix re-organization. The current study utilizes microarray-based transcriptional profiling to assess differential gene expression between neonatal and mature articular cartilage across a large subset of genes in the equine genome. In addition to individual genes, including those with established functional annotation in cartilage, the approach enables expression differences between gene ontology categories to be compared. The hypothesis tested was that changes in chondrocyte gene expression occur during postnatal maturation and reflect a functional transition from growth and matrix synthesis to cartilage homeostasis.

Transcriptional profiling results indicate that maturation-dependent differences in cellular activity exist between neonatal and adult equine articular chondrocytes. These differences likely reflect age-dependent adaptation that occurs as chondrocytes are acting to meet changing functional needs. Neonatal articular cartilage is in a state of growth and expansion. The chondrocytes are secreting large amounts of extracellular matrix (ECM) in response to the expanding articular surface. This can be seen with the upregulation of collagen types II and IX in neonatal chondrocytes. Microarray hybridization data showing increased abundance of type II collagen transcripts in neonatal articular cartilage relative to adult articular cartilage was validated at 3.75-fold by RT-qPCR (p < 0.01, Figure 2).

In neonatal articular chondrocytes, there is also an upregulation of collagen-modifying enzymes necessary for the assembly of ECM; this includes proline 4-hydroxylase (P4H) and lysyl oxidase. P4H is a catalyst necessary for the modification of proline for collagen triple helix formation 32. Increased transcript abundance for P4HA1 indicated by microarray in neonatal articular cartilage relative to adult articular cartilage was validated at 3.71-fold by RT-qPCR (p < 0.01, Figure 2). Lysyl oxidases catalyze the cross-linking and condensation of collagen fibrils by oxidizing lysine and hydroxylysine residues to become lysyl and hydroxylysyl aldehydes 33. The condensation of collagens helps increase tensile strength within cartilage. As such, the upregulation of LOXL2 is likely necessary for adaptation to shear and weight-bearing stresses. When immature bovine articular cartilage is treated with the lysyl oxidase inhibitor β-aminopropionitrile in an in vitro model, a reduction of tensile integrity was noted 34. These findings are consistent with neonatal expression of collagen modifying enzymes in parallel to higher levels of collagen synthesis during growth and biomechanical adaptation.

Increased steady state levels of tenascin-C (TNC) transcripts in neonatal cartilage are also consistent with a remodeling process. This protein is upregulated in tissues at developmental stages, during wound healing, and during tumorigenesis 353637. TNC has an anti-adhesive N-terminal domain and a pro-adhesive C-terminal domain; its multiple domains allow for interactions with many ECM components, including fibronectin 3839. Thus, the presence of TNC affects cells within the ECM, notably by inhibiting cell adhesion and focal adhesion kinase-signaling while stimulating growth 3537. The effects of TNC on matrix organization have been well characterized in the developing chick forebrain, in healing skin wounds, and in tumorigenesis 3740. Tenascin-C is expressed within the developing limb; however, it disappears throughout the limb and the growth plate during endochondral ossification, though expression can still be detected in postnatal articular cartilage 3841. As maturation occurs in normal articular cartilage and the provisional matrix is replaced by adapted anisogenous matrix, TNC expression decreases 3841. The current data are consistent with previous TNC findings for chicken, mouse, and rat. Theories for the postnatal presence and then reduction of TNC within the articular cartilage include maintenance of the chondrocyte phenotype, capability of the cells to attach and then detach in an expanding matrix, and the utilization of signal transduction for mediating chondrocyte developmental fate 38.

Another interesting finding was elevated steady state levels of transcripts encoding nidogen-2 (NID2) in neonatal chondrocytes (Table 3). NID2 is one of the two nidogen matrix-binding isoforms found to exist in mammals which are involved in basement membrane assembly 424344. Nidogens are typically localized to basement membranes, but have also been associated with development and tissue remodeling, including limb formation 4547. Thus far, it has been shown that nidogens interact with perlecan, collagen types I and IV, integrins, and laminins 444548. Matrix-binding proteins like nidogens also have the capacity to play a role in the adaptive maturation process of a growing neonate, which seems to be best characterized by the expansion of collagens within the provisional immature matrix and the modifications of the matrix postnatally to withstand stresses.

The spectrum of upregulated transcripts in mature articular cartilage is consistent with chondrocytes that have transitioned functionally from cartilage growth to cartilage maintenance. Besides elevated adult steady state transcript levels for matrix molecules like cartilage intermediate layer protein (CILP), cartilage oligomeric matrix protein (COMP), and fibronectin (FN), the data also reveal other clones that contribute to the roles of chondrocytes in mature cartilage (Tables 2 and 4). With regards to matrix proteins, differences in levels of CILP mRNA detected by microarray hybridization correspond to previous reports of protein accumulation in the extraterritorial matrix of adult cartilage with age 49. It is believed that this protein causes increased calcium pyrophosphate dihydrate deposition in cartilage, impairs chondrocyte responsive to IGF-1, and subsequently hinders matrix repair 50. Moreover, increased levels of mRNA encoding COMP were also found in adult cartilage; RT-qPCR quantified this difference as 3.5-fold (Figure 2). COMP is a homopentameric glycoprotein that binds to type II collagen interterritorially to stabilize the ECM 5152. COMP mRNA upregulation has been previously described during different stages of limb and joint development with species-dependence as well as with mechanical compression 5253. Increased fibronectin mRNA hybridization in adult articular cartilage parallels findings from Burton-Wurster et al. (Table 4) 54. Fibronectin is an important extracellular matrix adhesion glycoprotein for cell-matrix signaling. Besides genes which encode matrix proteins, categories of many other genes which typify the maintenance of a heterogeneous matrix are represented. For example, genes with functional roles in collagen-, glycosaminoglycan-, and proteoglycan-binding are upregulated in adult articular chondrocytes (Table 4). Once cartilage growth and adaptation are complete, type II collagen expression is sustained yet at lower levels as the chondrocyte's primary role transforms to tissue maintenance. Increased mRNA levels of matrix metalloproteinase-1 (MMP1), tissue inhibitor of metallopeptidases-2 (TIMP2), and chondroprotective nexin-1 (PN1) are also consistent with cartilage maintenance (Table 4). With maturation, gene expression in articular chondrocytes reflects tissue homeostasis and a perpetuation of the ability to withstand shear and weight-bearing stresses.

Histological examination of articular cartilage reveals increased cellularity per unit volume of cartilage in neonatal animals compared to adults. A stable chondrocyte number and expanding matrix may be a primary mechanism in achieving the lower cell density observed in adult articular cartilage. In comparing pro- vs. anti- cell proliferation, cell cycle, and apopoptic genes in the transcriptional profiles, questions with regards to cell proliferation and cell cycle regulation persist. From the transcriptional profiles, it is difficult to say definitively at either stage of maturation that the chondrocytes are in a state of proliferation or quiescence. Relatively few probes representing these functional categories were significantly up-regulated in neonates. Moreover, though many more (25) gene probes representing these ontology categories were upregulated in adults, distribution did not favor cell proliferation or growth factor activity. Growth factors such as TGFβ1 and IGF1 have been shown to induce chondrocyte proliferation and ECM production with limb development and in culture, and the importance of factors and receptors like BMPR1A, GDF5, and TGFβR2 in the maintenance of postnatal articular have also been demonstrated 4455565758. However, FGF10 and TGFβR3 were the only genes related to growth factor activity with > 2 fold difference between adult and neonatal articular cartilage (Table 5). Increased steady state levels of TGFβR3 in articular cartilage for adults were validated by qPCR at 2.70-fold (p < 0.01). While transcript levels for growth factors were relatively similar, it is possible that growth factor activity in postnatal articular cartilage maturation is regulated primarily at a post-transcriptional level. Growth factors sequestered within the extracellular matrix of articular cartilage have been shown to become functionally activated by enzymes like matrix metalloproteinases 5960.

A broad disparity existed in the number of differentially expressed genes between neonates and adults (Figure 1c). Of the 544 significant genes represented in the array data, a majority (499 genes, or 91.7%) represented increased steady state transcript levels in adult chondrocytes. This difference could be explained by one of two theories. It is possible that cellular activity is more complex in the adult chondrocyte which is thus represented by an increased abundance of transcripts. A second and perhaps more likely explanation is that the discrepancy is accounted for by an adult-biased representation of probe sets on the microarray. Articular cartilage from the 15-month old horse utilized in the generation of the cDNA library spotted on the array likely reflects an adult articular chondrocyte transcriptome with neonate-specific transcripts under-represented. Thus, it is possible that cell proliferation or cell cycle regulatory genes expressed in the neonate were absent from the array and thus were unable to be assessed. A further opportunity to extend the data from the current study would be to conduct time course experiments comparing transcriptional profiling data from immature and adolescent ages to neonatal and adult samples, while including joint-specific and location-specific comparisons in the articular cartilage maturation assessment 61.

Previous studies of postnatal articular cartilage maturation in mammals have focused on age-dependent changes in the structural and biochemical organization of the matrix. The current data extends this knowledge by using transcriptional profiling to compare gene expression between neonatal and adult chondrocytes in equine articular cartilage. Seven percent (642) of the 9,367 probe sets on an equine-specific microarray were differentially expressed (p < 0.01), organizing primarily into ontology categories related to extracellular matrix, skeletal development, and cell adhesion. Collagens, matrix-modifying enzymes, and provisional matrix proteins were expressed at higher levels in the articular cartilage of newborn foals. Transcripts encoding leucine-rich small proteoglycans, matrix assembly, and cartilage maintenance proteins were found at higher steady state levels in adult articular chondrocytes. Microarray data for COL2A1, COMP, P4HA1, TGFB1, TGFBR3, and TNC were validated by quantitative PCR. Overall, the findings support the hypothesis that gene expression changes in articular cartilage during postnatal maturation reflect a functional transition of chondrocytes from growth and matrix synthesis in the neonate to cartilage homeostasis in the adult.

The authors declare that they have no competing interests.

MJM and JNM carried out the sample collections and molecular biology studies. LH and AJS carried out the statistical analyses for microarray studies. MJM performed statistical analyses for validation studies. All authors contributed to the writing of this manuscript. All authors read and approved the final manuscript.