Adipose-derived mesenchymal stromal cells modulate tendon fibroblast responses to macrophage-induced inflammation in vitro.

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-1ß) 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-1ß. 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-1ß, tumor necrosis factor alpha (TNFα)). CONCLUSIONS: These findings suggest that IL-1ß 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.

The early inflammatory response after flexor tendon healing: a gene expression and histological analysis.

Despite advances in surgical techniques over the past three decades, tendon repairs remain prone to poor clinical outcomes. Previous attempts to improve tendon healing have focused on the later stages of healing (i.e., proliferation and matrix synthesis). The early inflammatory phase of tendon healing, however, is not fully understood and its modulation during healing has not yet been studied. Therefore, the purpose of this work was to characterize the early inflammatory phase of flexor tendon healing with the goal of identifying inflammation-related targets for future treatments. Canine flexor tendons were transected and repaired using techniques identical to those used clinically. The inflammatory response was monitored for 9 days. Temporal changes in immune cell populations and gene expression of inflammation-, matrix degradation-, and extracellular matrix-related factors were examined. Gene expression patterns paralleled changes in repair-site cell populations. Of the observed changes, the most dramatic effect was a greater than 4,000-fold up-regulation in the expression of the pro-inflammatory factor IL-1ß. While an inflammatory response is likely necessary for healing to occur, high levels of pro-inflammatory cytokines may result in collateral tissue damage and impaired tendon healing. These findings suggest that future tendon treatment approaches consider modulation of the inflammatory phase of healing.

Nanofiber scaffolds with gradients in mineral content for spatial control of osteogenesis.

Reattachment of tendon to bone has been a challenge in orthopedic surgery. The disparate mechanical properties of the two tissues make it difficult to achieve direct surgical repair of the tendon-to-bone insertion. Healing after surgical repair typically does not regenerate the natural attachment, a complex tissue that connects tendon and bone across a gradient in both mineral content and cell phenotypes. To facilitate the regeneration of the attachment, our groups have developed a nanofiber-based scaffold with a graded mineral coating to mimic the mineral composition of the native tendon-to-bone insertion. In the present work, we evaluated the ability of this scaffold to induce graded osteogenesis of adipose-derived mesenchymal stem cells (ASCs). Results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferating cell nuclear antigen staining indicated that cell proliferation was negatively correlated with the mineral content. In contrast, alkaline phosphatase staining, an indicator of osteogenesis, was positively correlated with the mineral content. Likewise, runt-related transcription factor 2 (an early marker of osteoblast differentiation) and osteocalcin (a late marker of osteoblast differentiation) immunostaining were both positively correlated with the mineral content. These results indicate that a gradient in mineral content on the surface of a nanofiber scaffold is capable of inducing graded differentiation of ASCs into osteoblasts for enthesis repair.

Effect of bone morphogenetic protein 2 on tendon-to-bone healing in a canine flexor tendon model.

Tendon-to-bone healing is typically poor, with a high rate of repair-site rupture. Bone loss after tendon-to-bone repair may contribute to poor outcomes. Therefore, we hypothesized that the local application of the osteogenic growth factor bone morphogenetic protein 2 (BMP-2) would promote bone formation, leading to improved repair-site mechanical properties. Intrasynovial canine flexor tendons were injured in Zone 1 and repaired into bone tunnels in the distal phalanx. BMP-2 was delivered to the repair site using either a calcium phosphate matrix (CPM) or a collagen sponge (COL) carrier. Each animal also received carrier alone in an adjacent repair to serve as an internal control. Repairs were evaluated at 21 days using biomechanical, radiographic, and histologic assays. Although an increase in osteoid formation was noted histologically, no significant increases in bone mineral density occurred. When excluding functional failures (i.e., ruptured and gapped repairs), mechanical properties were not different when comparing BMP-2/CPM groups with carrier controls. A significantly higher percentage of BMP-2 treated specimens had a maximum force <20 N compared to carrier controls. While tendon-to-bone healing can be enhanced by addressing the bone loss that typically occurs after surgical repair, the delivery of BMP-2 using the concentrations and methods of the current study did not improve mechanical properties over carrier alone. The anticipated anabolic effect of BMP-2 was insufficient in the short time frame of this study to counter the post-repair loss of bone.

Sustained delivery of transforming growth factor beta three enhances tendon-to-bone healing in a rat model.

Despite advances in surgical technique, rotator cuff repairs are plagued by a high rate of failure. This failure rate is in part due to poor tendon-to-bone healing; rather than regeneration of a fibrocartilaginous attachment, the repair is filled with disorganized fibrovascular (scar) tissue. Transforming growth factor beta 3 (TGF-ß3) has been implicated in fetal development and scarless fetal healing and, thus, exogenous addition of TGF-ß3 may enhance tendon-to-bone healing. We hypothesized that: TGF-ß3 could be released in a controlled manner using a heparin/fibrin-based delivery system (HBDS); and delivery of TGF-ß3 at the healing tendon-to-bone insertion would lead to improvements in biomechanical properties compared to untreated controls. After demonstrating that the release kinetics of TGF-ß3 could be controlled using a HBDS in vitro, matrices were incorporated at the repaired supraspinatus tendon-to-bone insertions of rats. Animals were sacrificed at 14-56 days. Repaired insertions were assessed using histology (for inflammation, vascularity, and cell proliferation) and biomechanics (for structural and mechanical properties). TGF-ß3 treatment in vivo accelerated the healing process, with increases in inflammation, cellularity, vascularity, and cell proliferation at the early timepoints. Moreover, sustained delivery of TGF-ß3 to the healing tendon-to-bone insertion led to significant improvements in structural properties at 28 days and in material properties at 56 days compared to controls. We concluded that TGF-ß3 delivered at a sustained rate using a HBDS enhanced tendon-to-bone healing in a rat model.

"Aligned-to-random" nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site.

We have demonstrated the fabrication of "aligned-to-random" electrospun nanofiber scaffolds that mimic the structural organization of collagen fibers at the tendon-to-bone insertion site. Tendon fibroblasts cultured on such a scaffold exhibited highly organized and haphazardly oriented morphologies, respectively, on the aligned and random portions.