Traumatic cartilage lesions represent a common symptomatic and disabling problem1,2
that often requires surgical intervention to relieve pain and to prevent possible evolution towards secondary OA3,19,20
. The absence of relevant preclinical animal models of joint surface regeneration suitable for molecular studies and genetic manipulation has represented a bottleneck for the development of novel therapeutics to enhance/support joint surface repair. In this study, we have developed and characterized a mouse model of acute full thickness JSD with a strain and age dependent repair outcome. Adult DBA/1 mice reproducibly healed experimental joint surface lesions, whereas age-matched C57BL/6 healed poorly and instead developed features of secondary OA. Aged mice failed to repair, but, in contrast to age-matched C57BL/6 mice, DBA/1 developed no signs of secondary OA. The strain-dependent different outcome in young animals was associated with different regulation of apoptosis, proliferation and matrix remodeling.
Since the repair of JSD depends largely on their size and depth7,9,10
, the reproducibility of the injury in our model was an important concern. We therefore measured the size of the defects focusing the analysis on the interval between planes A and C (B) because, within this region, the articular cartilage is of uniform thickness and it is well exposed during surgery after patellar dislocation. Importantly, the intersection of the growth plate within these planes provided a useful anatomical landmark. We achieved a high reproducibility and consistency of the injury by combining a well controlled sampling technique within a selected anatomical area and the design of a simple and reliable device to induce the injury. Importantly, all the defects were full thickness in all the animals and slightly deeper than 100% of the cartilage thickness. Such consistency was largely ensured by the resistance offered by the subchondral bone, the resilience of the 26 G needle used to make the device, and the presence of the glass bead which by adapting to the concavity of the patellar groove, allowed the tip of the needle to run reproducibly in the centre of the patellar groove.
C57BL/6 mice develop spontaneous OA with ageing32
and they are more susceptible than DBA/1 to OA induced by destabilization of the medial meniscus (DMM)33
. However, spontaneous OA appears in C57BL/6 mice only after their first year of age, and no signs of OA were present in control or sham-operated joints in our study. It is therefore unlikely that pre-existing OA or joint instability inadvertently induced during surgery were the causes for repair failure. On the other hand, the generation of a JSD in C57BL/6 mice precipitated the appearance of secondary OA features as early as 1 week after injury. At 4 weeks, when the OA features in C57BL/6 mice had advanced, there was also a clear difference in repair outcome compared to DBA/1. It is possible that the rapid development of secondary OA may have conditioned repair failure. Our experimental setup does not discriminate whether the failure of repair and the development of OA are causally related, or if they represent distinct processes resulting from an inferior joint homeostatic mechanism in C57BL/6 than in DBA/1 mice. However, the observation that aged DBA/1 mice did not repair and did not develop OA either, under our experimental conditions and at the time points examined, demonstrates that repair failure alone may not be sufficient for the development of secondary OA and suggests that the two processes may be, at least in part, uncoupled.
On the other hand, DBA/1 mice are responsive to inflammatory arthritis models such as type II collagen-induced arthritis whereas C57BL/6 mice are resistant34
. A modest infiltrate was present in the synovial membrane of the two strains during the first week following surgery (data not shown); however, it is possible that a different inflammatory response may play a role in repair outcomes.
Non-differentiated spindle-like cells filled the lesions in DBA/1 mice at 1 week. In C57BL/6, morphologically similar cells appeared in the defects after 4 weeks when chondrogenesis had already occurred in DBA/1 mice. This delay in populating the wound could be due to differences reported in proliferation rate, cell surface epitopes and differentiation potential between bone marrow mesenchymal stem cells (MSCs) isolated from C57BL/6 and DBA/1 with higher chondrogenic potential of DBA/1 MSCs35
. Additional potential mechanism could be a delayed activation/migration of mesenchymal progenitors in response to cartilage signals released by injured cartilage27
. We recently showed that genes encoding morphogens, chemokines and other signaling molecules are activated in human adult articular cartilage after mechanical injury36
. Failure of either the cartilage to deploy such response to injury or the mesenchymal progenitor/stem cells to respond to such signaling molecules may contribute to the delay of C57BL/6 mesenchymal cells in filling the defect.
Chondrocyte apoptosis progressively resolved in DBA/1 mice and persisted in C57BL/6 for at least 8 weeks. Chondrocyte death following mechanical injury and its potential role in the pathogenesis of OA have been described in several studies28,29,37,38
. However, apoptotic cells may play a positive role in the repair responses by releasing chemo-attractants for stem/progenitor cells such as high mobility group box 1 (HMGB1)39
. Therefore, it is possible that controlled chondrocyte apoptosis, in the initial phases of repair, may play a similar function after cartilage injury; however, it could be detrimental if not tightly regulated and inappropriately prolonged. Our experimental setup does not allow discriminating whether the prolonged apoptosis in C57BL/6 is determining or a consequence of repair failure.
Proliferation in the cartilage surrounding the injury site has been reported following mechanical trauma28,40
. We did not detect chondrocyte proliferation in the articular cartilage surrounding the injury. However, proliferating cells were identified within the repair tissue in DBA/1 mice at 1 and 4 weeks post-surgery. This discrepancy with previous literature could be due to differences in the experimental conditions, the time points analyzed and the detection systems used. We examined the phosphorylation of histone H3 which is tightly correlated with the chromosome condensation phase during mitosis, thereby representing a snapshot of cells in M phase of cell cycle41
. Conversely, incorporation of labeled nucleosides marks all the cells undergoing DNA duplication during the labeling period. Furthermore, cell proliferation in adult tissues occurs at a very low rate, and may take place in a short time-span after repair, thus our time points might have missed proliferation within the cartilage surrounding the injury. Surrounding tissues such as bone marrow and synovium have been proposed to contribute cells to the reparative process in different in vivo
. However, the origin of the cells populating the defects and ultimately forming the repair cartilage in DBA/1 mice remains to be clarified.
Our study shows a prompt activation of matrix remodeling after injury within the repair tissue in DBA/1 mice and in the remaining cartilage in both strains. Matrix remodeling has been described in response to injury28,43
, during OA44,45
and in human repair cartilage tissue obtained from ACI46
. Interestingly, in this study, DBA/1 mice maintained a high level of collagen type II neo-synthesis and a low collagen catabolic activity for 8 weeks after injury. However, C57BL/6 mice exhibited a reverse pattern with progressive decrease in the number of CP II positive cells and intense Col2-3/4Cshort
staining. Aggrecan degradation was prevalently driven by aggrecanases in C5BL/6 mice and by MMPs in DBA/1 mice.
Recently, ADAMTS-5 has been identified as the main aggrecanase in mouse cartilage and its ablation protected the mice from OA induced by DMM47
. Similarly, mutants harboring a genetic allele of the aggrecan gene that blocks aggrecanases mediated cleavage were also protected when challenged in DMM model, but, interestingly, mice harboring an aggrecan mutant allele that is resistant to MMP-mediated cleavage developed more severe OA than their wild-type littermates48
. Moreover, MMP9-deficient mice were more susceptible to OA than their wild-type littermates in the same model33
. Taken together, these data and our results confirm the critical role of aggrecanases in cartilage destruction and suggest that a controlled level of MMP-mediated aggrecanolysis may be needed for cartilage homeostasis, and possibly repair, as MMPs proteolysis can facilitate cell migration, regulate tissue architecture, release and activate ECM bound growth factors and signaling molecules49
. A caveat, in the interpretation of our data, is that aggrecan cleaved by aggrecanases may be internalized50
and thereby no longer available for MMPs cleavage even in the presence of active MMPs. This may explain why, in C57BL/6, although several MMPs can cleave collagen and aggrecan, we only detected cleaved type II collagen. Another explanation for this discrepancy is that different MMPs may be responsible for aggrecan and collagen type II cleavage. Understanding these events would be crucial for the identification of the correct window of therapeutic intervention with inhibitors of aggrecanases and MMPs that are being developed and tested in preclinical and clinical studies51
The JSD repair encompasses highly coordinated biological events that require a fine tuning of diverse processes including cellular apoptosis, proliferation and tissue patterning regulated in a temporal–spatial manner. The molecular mechanisms regulating this process are poorly understood and their study has been hampered by the lack of suitable models. We anticipate that this new model of joint surface injury and repair, combined with mouse genetics, will be instrumental to elucidate the molecular control of these events, thereby enabling the development of new therapeutic agents to improve joint surface healing. It must stressed that this model is not a model of primary OA, but rather a model of joint surface injury and repair. However, the occurrence of post-traumatic OA features in C57BL/6 mice suggest it could be used to study the mechanism by which repair influences progression of post-traumatic secondary OA.
We and others27,36,52,53
have demonstrated that the injured articular cartilage deploys a robust response to injury with activation of genes encoding signaling molecules and morphogens. This could be the effect of a putative repair response, or an adaptive response to damage, or could even play a role in post-traumatic secondary OA development. This model will allow addressing the functionality and hierarchy of molecules in the response to injury and subsequently represents a preclinical in vivo
model to develop novel molecular therapeutics to support joint surface healing in cell-free in situ
tissue engineering approaches.