**5. Conclusions**

To conclude, we demonstrate that the HS proteoglycan, perlecan, is clearly present in repair tissue formed both via cell therapy repair of chondral defects and also naturally occurring repair tissue. The localisation of perlecan, as well as type III collagen, which is often found in developing or repairing tissue, is more diffuse for both molecules in the fibrocartilaginous tissue which forms initially, than in the more mature repair tissue. This more mature repair tissue has morphology resembling hyaline cartilage with has more of the typical cell-associated staining pattern seen in adult articular cartilage. The co-localisation of perlecan and collagen type VI and its biomechanical role in the PCM in repair cartilage remains unclear and further research could reveal a key mechanism that incorporates the different loading forces in the articular joint. The strong perlecan staining observed in chondrocyte clusters could be mediated via its domain IV-3 and the suppression of Erk1/2 signalling. We have also shown that heparanase treatment increases the proliferation of chondrocytes, without altering their phenotypical features, at least, as assessed in this study. Taken together, it is plausible to assume that perlecan has an important role in cartilage repair. Further work is required to fully comprehend how heparanase influences different types of repair, and whether this enzyme can be harnessed to enhance the quality of de novo cartilage repair in vivo.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2218-273 X/11/1/92/s1, Figure S1: Representative negative controls for immunohistochemistry studies.

**Author Contributions:** Conceptualization, J.G., J.M., S.R., and J.H.K.; Data Acquisition, Data Analysis; J.G., H.S.M., J.M., and J.H.K.; Manuscript Preparation, J.G., H.S.M., J.M., J.H.K., and S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Medical Research Council (MR/L010453/1 and MR/N02706X/1), Versus Arthritis (grants 18480, 19429, 21156) and NHMRC Project Grant 51267 The role of perlecan in tensional connective tissues.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the National Research Ethics Service—Coventry and Warwickshire (REC reference 11/WM/0175) in 2011.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data available on request due to restrictions eg privacy or ethical.

**Acknowledgments:** We acknowledge the intellectual and clinical contributions of the late James Richardson to this study. We are also grateful to the OsCell team at The John Charnley Laboratory, RJAH Orthopaedic Hospital, UK for the processing of harvested tissues for autologous cell therapy.

**Conflicts of Interest:** The authors declare no conflict of interest.
