Richard H. Gelberman

Intrasynovial tendon injuries are among the most challenging in orthopedics. Despite significant improvements in operative and rehabilitation methods, functional outcomes continue to be limited by adhesions, gap formation, and rupture. Adhesions result from excessive inflammation, whereas tendon gapping and rupture result from inflammation-induced matrix degradation and insufficient regeneration. Therefore, this study used a combined treatment approach to modulate inflammation with adipose-derived mesenchymal stromal cells (ASCs) while stimulating tendon regeneration with connective tissue growth factor (CTGF). ASCs were applied to the repair surface via cell sheets and CTGF was delivered to the repair center via porous sutures. The effect of the combined treatment was assessed fourteen days after repair in a canine flexor tendon injury model. CTGF, either alone or with ASCs, reduced inflammatory (IL1B and IL6) and matrix degrading (MMP3 and MMP13) gene expression, while increasing anti-inflammatory gene (IL4) expression and collagen synthesis compared to control repairs. The combined treatment was more effective than CTGF treatment alone, reducing the inflammatory IFNG and scar-associated COL3A1 gene expression and increasing CD146⁺ tendon stem/progenitor cells at the tendon surface and interior along the core suture tracks. Therefore, the combined approach is promising in promoting early flexor tendon healing and worthy of further investigation.

Recent growth factor, cell, and scaffold‐based experimental interventions for intrasynovial flexor tendon repair have demonstrated therapeutic potential in rodent models. However, these approaches have not achieved consistent functional improvements in large animal trials due to deleterious inflammatory reactions to delivery materials and insufficient induction of targeted biological healing responses. In this study, we achieved porous suture‐based sustained delivery of connective tissue growth factor (CTGF) into flexor tendons in a clinically relevant canine model. Repairs with CTGF‐laden sutures were mechanically competent and did not show any evidence of adhesions or other negative inflammatory reactions based on histology, gene expression, or proteomics analyses at 14 days following repair. CTGF‐laden sutures induced local cellular infiltration and a significant biological response immediately adjacent to the suture, including histological signs of angiogenesis and collagen deposition. There were no evident widespread biological effects throughout the tendon substance. There were significant differences in gene expression of the macrophage marker CD163 and anti‐apoptotic factor BCL2L1; however, these differences were not corroborated by proteomics analysis. In summary, this study provided encouraging evidence of sustained delivery of biologically active CTGF from porous sutures without signs of a negative inflammatory reaction. With the development of a safe and effective method for generating a positive local biological response, future studies can explore additional methods for enhancing intrasynovial tendon repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2052–2063, 2018.

Background: The clinical outcomes following intrasynovial flexor tendon repair are highly variable. Excessive inflammation is a principal factor underlying the formation of adhesions at the repair surface and affecting matrix regeneration at the repair center that limit tendon excursion and impair tendon healing. A previous in-vitro study revealed that adipose-derived mesenchymal stromal cells (ASCs) modulate tendon fibroblast response to macrophage-induced inflammation. The goal of the current study was therefore to explore the effectiveness of autologous ASCs on the inflammatory stage of intrasynovial tendon healing in vivo using a clinically relevant animal model. Methods: Zone II flexor tendon transections and suture repairs were performed in a canine model. Autologous ASC sheets were delivered to the surface of repaired tendons. Seven days after repair, the effects of ASCs on tendon healing, with a focus on the inflammatory response, were evaluated using gene expression assays, immunostaining, and histological assessments. Results: ASCs delivered via the cell sheet infiltrated the host tendon, including the repair surface and the space between the tendon ends, as viewed histologically by tracking GFP-expressing ASCs. Gene expression results demonstrated that ASCs promoted a regenerative/anti-inflammatory M2 macrophage phenotype and regulated tendon matrix remodeling. Specifically, there were significant increases in M2-stimulator (IL-4), marker (CD163 and MRC1), and effector (VEGF) gene expression in ASC-sheet treated tendons compared with nontreated tendons. When examining changes in extracellular matrix expression, tendon injury caused a significant increase in scar-associated COL3A1 expression and reductions in COL2A1 and ACAN expression. The ASC treatment effectively counteracted these changes, returning the expression levels of these genes closer to normal. Immunostaining further confirmed that ASC treatment increased CD163⁺ M2 cells in the repaired tendons and suppressed cell apoptosis at the repair site. Conclusions: This study provides a novel approach for delivering ASCs with outcomes indicating potential for substantial modulation of the inflammatory environment and enhancement of tendon healing after flexor tendon repair.

Introduction: Macrophage-driven inflammation is a key feature of the early period following tendon repair, but excessive inflammation has been associated with poor clinical outcomes. Modulation of the inflammatory environment using molecular or cellular treatments may provide a means to enhance tendon healing. Methods: To examine the effect of pro-inflammatory cytokines secreted by macrophages on tendon fibroblasts (TF), we established in vitro models of cytokine and macrophage-induced inflammation. Gene expression, protein expression, and cell viability assays were used to examine TF responses. In an effort to reduce the negative effects of inflammatory cytokines on TFs, adipose-derived mesenchymal stromal cells (ASCs) were incorporated into the model and their ability to modulate inflammation was investigated. Results: The inflammatory cytokine interleukin 1 beta (IL-1β) and macrophages of varying phenotypes induced up-regulation of pro-inflammatory factors and matrix degradation factors and down-regulation of factors related to extracellular matrix formation by TFs in culture. ASCs did not suppress these presumably negative effects induced by IL-1β. However, ASC co-culture with M1 (pro-inflammatory) macrophages successfully suppressed the effects of M1 macrophages on TFs by inducing a phenotypic switch from a pro-inflammatory macrophage phenotype to an anti-inflammatory macrophage phenotype, thus resulting in exposure of TFs to lower levels of pro-inflammatory cytokines (e.g., IL-1β, tumor necrosis factor alpha (TNFα)). Conclusions: These findings suggest that IL-1β and M1 macrophages are detrimental to tendon healing and that ASC-mediated modulation of the post-operative inflammatory response may be beneficial for tendon healing.

To improve the functional outcomes of intrasynovial tendon suture, prior experiments evaluated individual technical modifications used in the repair process. Few studies, however, have assessed the combinatorial effects of those suture modifications in an integrated biomechanical manner, including a sample size sufficient to make definitive observations on repair technique. Two hundred fifty-six flexor tendon repairs were performed in human cadavera, and biomechanical properties were determined. The effects of five factors for flexor tendon repair were tested: core suture caliber (4-0 or 3-0), number of sutures crossing the repair site (four- or eight-strand), core suture purchase (0.75 or 1.2 cm), peripheral suture caliber (6-0 or 5-0), and peripheral suture purchase (superficial or 2 mm). Significant factors affecting the properties of the repair were the number of core suture strands and the peripheral suture purchase. The least significant factors were core suture purchase and peripheral suture caliber. The choice of core suture caliber affected the properties of repair marginally. Based on these results, we recommend that surgeons continue to focus on multi-strand repair methods, as the properties of eight-strand repairs were far better than those of four-strand repairs. To resist gap formation and enhance repair strength, a peripheral suture with 2 mm purchase is also recommended. Finally, since core suture caliber affected some biomechanical properties, including the failure mode, a 3-0 suture could be considered, provided that future in vivo studies can confirm that gliding properties are not adversely influenced. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1652–1659, 2012