ADAMTS4 and Spontaneous Integration – Mechanisms of Articular Cartilage Repair

Student thesis: Doctoral ThesisDoctor of Philosophy


Articular cartilage (AC) is the smooth shock absorbing and load distributing connective tissue found at the ends of bones in diarthrodial joints. AC demonstrates extremely poor regenerative abilities in adults once injured or diseased, e.g., osteoarthritis (OA), where regenerative therapies to restore functional AC after injury or disease remains an ongoing challenge. AC tissue engineering (TE) has emerged as a promising avenue for developing future therapies. However, AC graft implantation demonstrates poor integration with the host tissue; where full structural integration is critical for tissue homeostasis and operational functionality. Elucidating the mechanism/s involved in successful integration of native post-injury AC can therefore be leveraged for developing future TE strategies. The primary objective of this thesis was therefore to establish a model of AC post-injury integration so as to identify the mechanism/s involved in successful integration and further investigate how these mechanisms might be regulated by their mechanical environment. Using AC from juvenile porcine, a successful integration model was developed where we demonstrate by scanning electron microscopy (SEM) the restoration of the collagenous network across the integration interface. Using qPCR we ruled out the production of new fibrillar collagens, e.g., collagen type I and type II, enzymatic cross-linkers, e.g., lysyl oxidase, and small leucine-rich proteoglycans (known to be involved in collagen assembly) as mechanisms involved in integration. By devitalising AC (one freeze/thaw cycle), we nevertheless observed biologically inactive AC to integrate post-injury. This integration was further observed to become enhanced by serial enzymatic removal of proteoglycans (PGs) at the injury interface: this was confirmed via histology, SEM, and tensile stress failure testing. This suggested a spontaneous mechanism of collagen cross-linking mediated by localised PGs depletion. We therefore returned to our living AC model to investigate if local PGs were depleted in response to injury. Here, we found significant loss of PGs, as well as, significant overlapping upregulation of aggrecanase ADAMTS4 at the integration interface. By significantly inhibiting ADAMTS4 with broad spectrum inhibitor TIMP3, we demonstrated a preservation of PGs at the integration interface and significantly weaker integration by tensile stress failure testing compared to non-treated controls. This correlation suggests that successful AC repair is, at least in part, mediated by ADAMTS4 through depleting PGs to remove barriers to promote collagen interaction for spontaneously cross-linking. Identifying ADAMTS4 to play a mechanistic role in successful AC integration, we finally aimed to assess if the cell and nuclear geometry of chondrocytes regulated ADAMTS4. By seeding primary porcine chondrocytes on fibronectin coated ‘soft’ and ‘stiff’ polyacrylamide hydrogels, we observed ADAMTS4 expression to correlate with nuclear spreading. We finally aimed to investigate the role of nuclear compression, by seeding chondrocytes on fibronectin coated flat and 10μm-by-10μm microgroove PDMS substrates; no significant ADAMTS4 expression differences were identified between the conditions.

ADAMTS4 is currently considered deleterious to AC and a pathophysiological marker of OA. However, the work presented in this thesis suggests that ADAMTS4 plays a critical role in the successful post-injury regenerative response, suggesting there is likely a transition from a repair mechanism to pathological destruction of AC when over expressed for extended periods of time. Collectively, this thesis offers insights into a process of spontaneous integration of the collagenous network facilitated by local ADAMTS4 mediated PGs depletion. This thesis additionally demonstrates that ADAMTS4 is in part regulated by nuclear deformation of chondrocytes. To this end, our findings have elucidated criteria for successful AC post-injury integration which may become relevant to developing future TE strategies for AC repair and regeneration.
Date of Award1 Jan 2022
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorEileen Gentleman (Supervisor) & Agamemnon Grigoriadis (Supervisor)

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