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Orthopedic and Rehabilitation Engineering

Mechanisms, Diagnostics, and Therapies for Rehabilitation of Orthopaedic Tissues

Role of CCN2 in Achilles Tendon Healing

Friday, October 25, 2024

2:00 PM – 2:15 PM EST

Location: Room 339

Invited Speaker(s)

Natalie Leong, MD (she/her/hers)

Associate Professor
University of Maryland School of Medicine, Baltimore VA Medical Center
Ellicott City, Maryland, United States

Co-Author(s)

DS

Danielle Sim

Medical Student
University of Maryland School of Medicine, United States

Last Author(s)

JJ

Jie Jiang

Assistant Professor
University of Maryland School of Medicine, United States

Introduction: Tendon injuries are common orthopaedic conditions, with an estimated economic cost of over $40 billion annually in the United States. There is a considerable need for better clinical options to augment both surgical and non-surgical tendon injuries. Thus, a mechanistically-based biologic treatment to promote tendon healing would be beneficial. CCN2, also known as Connective Tissue Growth Factor, is a member of the CCN family of matricellular proteins. It has important roles in many biological processes, but most importantly, it is known to be associated with cutaneous wound healing and virtually all fibrotic pathology. It is believed that CCN2 can cooperate with TGF-β to induce sustained fibrosis and to increase extracellular matrix production in association with other fibrosis-inducing conditions.

Due to CCN2’s role in wound healing and fibrosis, several investigators have examined CCN2’s role in tendon healing. CCN2 expression is known to be upregulated during early flexor tendon injury and then to decrease over time. Exogenous CCN2 treatment promoted patellar tendon repair, while knocking down CCN2 limited flexor tendon scarring and adhesion. Additionally, many investigators have started using CCN2 in vitro to differentiate MSCs into tenocytes. While these studies have suggested important functions that CCN2 plays in tendon healing, much remains to be learned about the exact role of CCN2 during tendon repair. We hypothesized that knockout of CCN2 during tendon fibrous scar formation will result in less fibrous scar formation and better tendon healing.

Materials and

Methods: All animal studies were conducted after approval by the local IACUC. Inducible conditional CCN2 knockout mice were generated by crossing
CCN2 floxed mice with CAGACreERT2 mice (CCN2iKO). After complete deletion of CCN2 in adult mice with tamoxifen, the Achilles tendon was
completely transected while leaving the plantaris tendon intact. Following euthanasia after 28 days post-injury, the entire hindlimb was isolated and fixed in 4% paraformaldehyde overnight, then paraffin embedded and sectioned. Hematoxylin and eosin staining was performed using standard protocols. Immunohistochemistry was performed for αSma (myofibroblasts). Images of four representative areas were taken of each specimen at 4x and 20x magnification, with a n=4 animals per group, and then analyzed.

Results, Conclusions, and Discussions: After 28 days post-injury, dramatically improved Achilles tendon healing was observed CCN2iKO mice as compared to wild type littermates
(Figures 1, 2). Additionally, a drastic reduction of in alpha smooth muscle actin (αSma) positive myofibroblasts in CCN2iKO mice was observed as
compared to WT littermates (Figure 2). These results show that complete knockout of CCN2 enhances Achilles tendon healing and reduces αSma positive myofibroblast expression.

Previous studies have shown that exogenous CCN2 can enhance patella tendon healing. While these opposite results seem to be contradictory at first glance, the two different injury models have been shown to heal differently. The patella tendon injury model used by Lee et al. does not normally heal on its own. Other studies have shown that partial patella tendon injuries heal through a more regenerative intrinsic process in which resident progenitor cells proliferate and produce collagen to repair the injured tendon. This process is drastically different than the fibro-scar formation that occurs in the Achilles transection model, in which all fibroblasts in the healing tendon are believed to be from extrinsic sources. The Achilles tendon injury model used in this study is known to heal through fibrosis with functional scaring. The decreased αSma expression observed in CCN2iKO mice suggests that CCN2 is involved in fibrous scar formation. Howell et al compared Achilles injury models between neonatal and adult mice. Both sets of mice healed through extrinsic means, but that the neonates were able to regenerate the Achilles tendon while the adult mice formed a fibrous scar. In the neonate group, the fibrotic scar markers were transient, while in adult mice, these markers persisted throughout. This suggests that myofibroblasts may be involved in promoting fibrous scar formation, and that CCN2 improves tendon healing in part through downregulating their activity. While the data shows that knocking out CCN2 can reduce fibrotic markers, the exact mechanism behind this finding remains unclear. Further study of fibrotic markers of interest as well as structural and mechanical analyses at multiple time points are needed to improve our limited understanding of the role of CCN2 in fibrous scar tendon healing.

Acknowledgements (Optional): This work was supported in part by Career Development Award Number IK2 BX004879 from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service and the Orthopaedic Research and Education Foundation.