Repaired intrasynovial flexor tendons are susceptible to gap formation and rupture during the first 3 weeks following suture, and to the formation of adhesions between the tendon and its surrounding synovial sheath [1–5]. Although low levels of inflammatory cytokines are likely necessary to attract FBs to the repair site [5, 6], a marked inflammatory response after tendon suture has been identified as a key factor leading to highly variable clinical outcomes [6, 8, 15]. Control of the inflammatory environment after operative repair is therefore a potential therapeutic target. In the current study, we demonstrated that the delivery of ASCs to the surface of a repaired tendon via a new ASC sheet approach was effective in modulating the inflammatory environment in the early period after repair. Because recent evidence suggests that fine modulation of inflammation in the earliest stages following operative repair can improve healing [12, 15, 16, 27], the approach presented here holds great promise for enhancing flexor tendon repair.
The findings in the current study are consistent with a prior in-vitro report demonstrating that ASCs suppress the negative effects of macrophages on TFs by inducing a phenotypic switch from the proinflammatory M1 macrophage phenotype to the anti-inflammatory M2 macrophage phenotype . Prior in vitro and in vivo studies in other tissues have also shown that MSCs have the capacity to alter the inflammatory environment and improve the healing response [16, 27–29]. However, it remains unclear how ASCs regulate inflammation during tendon healing. ASCs may secrete factors that modulate macrophage activity by promoting changes in macrophage differentiation or cytokine expression patterns, may secrete factors that modulate TF activity leading to reduced sensitivity to cytokines such as IL-1B, and/or may inactivate circulating proinflammatory factors (e.g., by releasing factors that degrade or sequester circulating proinflammatory cytokines). The results of the current study support the premise that the primary effect of ASC application is modulation of macrophage differentiation. The application of ASCs in vivo led to increased expression of the M2 stimulator genes IL-4 and IL-13 and the M2 marker genes CD163 and MRC1. This concept is supported further by the histological results, which demonstrated that implanted ASCs accumulated at the tendon surface and within the repair site, where infiltrating monocytes first arrived and CD163+ cells were initially detected. The coappearance of ASCs and macrophages indicates a likely interaction between the two cell types. Previous reports have indicated that the interaction may be mediated via a paracrine mechanism . Nevertheless, the underlining mechanisms are still unclear. Besides macrophages, ASCs may have also influenced the function of other neighboring cells by modulating the local molecular environment.
The functional significance of the M2 phenotype is its potential to improve tendon healing by promoting angiogenesis, cell proliferation, and matrix remodeling while concurrently suppressing cell death and tissue damage [11, 15, 28]. Consistently, the current study revealed that ASC treatment promoted VEGF expression, retained expression of tendon matrix genes COL2A1 and COL3A1 levels closer to the levels found in the healthy tissue, and suppressed tendon cell apoptosis. The ability of ASCs to promote the M2 phenotype and modulate the inflammatory environment may be influenced by factors such as the magnitude and chronicity of the injury, the age of patient, and the source of the cells. However, it has been shown that the potency of ASCs can also be modified by exogenous stimuli , thus broadening the potential applications of ASCs to multiple tendon pathologies and patient populations. Longer time points of healing are necessary, however, to determine whether these findings will result in functional improvements.
Despite the apparent induction of M2 macrophages by ASCs, there was no significant reduction in major proinflammatory cytokine genes (e.g., IL-1B and IFNG) in the current study. This discrepancy may have resulted from a variation in individual responses to injury and ASCs, as indicated by the relatively high standard deviations seen in the gene expression results. In addition, given the massive scale of initial inflammatory responses after tendon repair , modulation of the natural response may require a longer interval. In prior studies, a suppressive effect of ASCs on IL-1B expression was undetectable until 5 days after ASC–macrophage coculture . Because both implanted ASCs and macrophages need to migrate to the repair site, it may take even longer time to detect this effect in vivo.
A major challenge in biological enhancement of intrasynovial tendon healing is the necessity to stimulate extracellular matrix formation at the repair site (to improve tendon strength) while at the same time suppressing extracellular matrix formation at the tendon surface (to allow for gliding). Delivery of cells and/or growth factors to repaired tendons can stimulate adhesions if they are not localized to the repair site and effectively isolated from the intrasynovial space . The current study explores a new solution to this problem. ASCs were formed into sheets and wrapped around the tendon at the repair site. To prevent stimulation of adhesions and to anchor the ASC sheet to the tendon, a thin layer of HA hydrogel was applied superficially to the ASC sheet. The approach resulted in viable ASCs infiltrating the tendon repair site by 7 days post operation. Of importance, there were no adhesions noted in any of the treated repairs. This is in contrast to a recent study where ASCs and the growth factor BMP-12 were implanted in the interior of the tendon via surgically generated slits . In that study, a low-grade inflammatory reaction developed due to the polymer-based scaffold, negating any positive effects from the treatment. Future studies using the new cell sheet platform will incorporate growth factors along with stem cells and will test the potential to improve both functional and structural outcomes.
There were a number of limitations to the current study. First, due to the study’s focus on demonstrating the feasibility of a new approach and the emphasis on the early inflammatory processes, only one early time point was examined. Although the ASCs were shown to influence the inflammatory response, it is not known whether this modulation will result in longer-term functional benefits. However, now that the novel method has been established as safe and effective in the short term, the efficacy of the approach can be explored further in longer-term studies. Second, gene expression results do not always correlate with protein expression levels and activities. However, current immunohistochemical results strongly supported one of the primary gene expression outcomes of the study: the M2 phenotypic marker CD163 was significantly increased in the ASC-treated group compared with control. This result is strengthened by prior proteomics data from healing FDP tendons, which confirmed the substantial accumulation of CD163 proteins after ASC treatment . Third, the mechanisms by which ASCs promote the M2 phenotype were not explored in the current study. Furthermore, M1 and M2 phenotypes lie at the extremes of a continuum of macrophage expression profiles, and it remains unclear whether certain M1 characteristics remain after ASC treatment.