Over the past few decades, flexor tendon repair research has focused on improving rehabilitation protocols and surgical techniques. This research has led to decreased adhesion formation and repair-site elongation. Despite these advances, clinical outcomes continue to be variable and result in 1.5 million days lost from work per year.[1-6] As research advances resulting from altering operative or rehabilitation techniques have plateaued, the field has shifted to biologic manipulation of tendon healing, with the goals of further improving tendon strength, decreasing gap formation, and preventing adhesion formation.
After surgical repair, tendon healing progresses through three overlapping phases: inflammation (days 1–7), proliferation (days 3–14), and remodeling (day 10 onward). Prior attempts to improve tendon repair have focused on the later stages of healing by enhancing tendon fibroblast proliferation or promoting extracellular matrix (ECM) synthesis.[7-9] Growth factors have successfully been used to stimulate biologic activity during flexor tendon healing.[7-9] Despite increased proliferation and matrix remodeling, however, healing tendons failed to accrue strength during the first 3 weeks following suture. Therefore, the repair site remains at risk for gap formation or rupture following current treatment approaches.
In contrast to vascular and highly cellular tissues such as skin, where healing has been described in detail, tendons are relatively avascular[10-12] and paucicellular. Given that a typical wound healing response is initiated by an infiltration of inflammatory cells from the nearby vasculature, tendons may have a diminished healing response compared to skin. Articular cartilage, for example, which is avascular, has a very limited wound healing response.[14, 15] Furthermore, due to the relative paucity of cells in the intrasynovial flexor tendon, early tendon healing relies on the migration of tendon surface cells and/or extrinsic cells from the synovial sheath to the repair site. This process may delay the overall healing process in comparison to highly cellular skin healing models.
Based on these results, the focus of the current study is to examine the early inflammatory stage of healing, including enhancement of cell survival, migration, and proliferation immediately following tendon suture, in order to provide a basis for future treatments. A clinically relevant large animal model of flexor tendon injury and repair was used to investigate temporal changes in immune cell populations, and gene expression of inflammation-, matrix remodeling-, ECM, and differentiation-related factors during the early post-repair period.
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- MATERIALS AND METHODS
The temporal changes in cell population and gene expression of inflammation-, matrix remodeling-, ECM-, and differentiation-related factors were examined during the first 9 post-operative days following intrasynovial flexor tendon injury and repair. During this period, the expression of pro-inflammatory and matrix remodeling genes was significantly up-regulated. TNFα, IL-1β, and COX2 levels rose dramatically as early as Day 1. Expression of these genes subsequently decreased over time, coincident with changes in cell populations from immune cells to tendon fibroblasts. In contrast to the marked elevation of pro-inflammatory genes, ECM- and differentiation-related genes were significantly down-regulated on Day 1, an interval during which immune cells were prominent at the repair site. Matrix and differentiation gene expression values increased toward baseline levels as the number of PMNs decreased and tendon fibroblasts began to populate the repair site. Simultaneous with these early changes in gene expression, cellular proliferation, and cellular migration, MMP levels increased beginning at the time of injury. MMP levels increased further as macrophages cleared the wound of debris and dead tissue and as fibroblasts proliferated within the repair, initiating the remodeling phase of healing. The temporal increase in MMP expression coincided well with morphological changes in collagen fiber alignment. VEGF gene expression, which is related to the onset of neovascularization, was up-regulated on Day 1. This is consistent with the appearance of newly formed blood vessels 9 days post-operatively. On the other hand, LOX and LUB remained down-regulated throughout the 9-day study. Given that LOX is important in cross-linking collagen and LUB is involved in tendon lubrication and gliding function, these results are not surprising; we anticipate that these genes to play a larger role in the remodeling stage of healing (i.e., 10 days after injury or later), which our study did not examine.
Prior studies of flexor tendon healing have focused for the most part on morphological changes at the repair site,[18, 19] and on the cellular source contributing to tendon healing.[19-21] While these studies have provided key data supporting advances in surgical techniques and rehabilitation protocols, the early hemorrhagic and inflammatory processes have not been explored thoroughly. While Gelberman et al. described fibrinous bridges overlying a mesh of erythrocytes, macrophages, and other inflammatory-type cells on Day 3, earlier timepoints were not investigated. The expression of inflammatory and catabolic factors was not described until the past decade. Using a rabbit model of extrasynovial flexor tendon injury and repair, Berglund et al. revealed a significant elevation in the expression of pro-inflammatory factors (IL-1β and COX2) 3 days post-operatively. IL-1β and COX2 levels were noted to remain elevated through Day 6 and returning to baseline by Day 12. Concurrently, the matrix degradation factor, MMP13, was significantly up-regulated on Day 3 and remained elevated through Day 24. These results are generally in agreement with those of the current study. However, the animal model used in the previous study was limited with regard to clinical relevance: (1) the small size of rabbit flexor tendons precluded the use of standard repair methods used on humans, (2) a clinically appropriate postoperative rehabilitation protocol was not implemented, and (3) an extrasynovial tendon was used rather than the more commonly injured intrasynovial flexor tendon. In the current study, using a clinically relevant canine model, histological and gene expression experiments were performed to examine the cellular populations and gene expression patterns at in the early phase of flexor tendon healing.
Further experiments are needed to elucidate the cellular sources contributing to the changes in gene expression. As the gene expression changes were due to a combination of inflammatory cells and fibroblasts, the specific expression patterns for individual cell types remain unknown. However, histological assessment of the various cell types partly addresses this uncertainty. The percentage of fibroblasts (compared to overall cellularity) was estimated based on a morphological assessment and quantification. Given the striking rise in cellularity at the repair site and the relatively low number of fibroblasts noted on Day 1, we postulate that the up-regulation of pro-inflammatory factors at this early timepoint was primarily due to the infiltration of immune cells (i.e., PMNs and monocytes/macrophages). Similarly, the initial down-regulation of tendon-specific and tendon ECM genes is likely due to a diluted fibroblast population. RNA extracted from normal/uninjured tendons was mainly from fibroblasts. These cells express SCX, TNMD, COL1, and COL3. In contrast, RNA extracted from the repaired tendons on Day 1 was mainly derived from cells that do not express those genes. As the cell population shifted from immune cell-dominant to fibroblast-dominant, the expression of tendon-specific and tendon ECM-related factors returned to baseline levels. While the histological assessment of fibroblast percentage helped to interpret the gene expression data, immunohistochemical analyses would further elucidate the cellular source of the differential gene expression seen over time. Immunohistochemistry also would assist in verifying the relationship of the gene expression levels and subsequent changes in protein expression. However, the canine antibodies required to carry out these studies are not yet commercially available.
There were a number of limitations to the current study. First, additional timepoints are needed to more precisely determine temporal changes, as there appeared to be dramatic changes in cellular and gene expression patterns between Days 3 and 9. Second, protein-level assessment is necessary to validate gene expression changes. However, protein expression assessment using techniques such as Western immunoblot analysis requires canine-specific antibodies, which have limited availability. Third, due to the use of digits from the contralateral (uncasted) paw for normal controls, we are unable to say with certainty that the effects seen in the injured and repaired digits were solely due to the injury and not due to post-operative immobilization. This is a minor concern, however, as the cast-immobilized limbs received twice-daily passive motion rehabilitation, as is done clinically. There is substantial evidence demonstrating that the optimal scenario for flexor tendon healing is low (but not zero) loading. Cast immobilization without passive motion rehabilitation or complete unloading of the repair site is detrimental to healing, but high loads can also lead to gapping or rupture and poor healing. Furthermore, the timepoints analyzed in the current study are relatively short, especially when considering the time frames within which negative immobilization effects are typically observed. The most remarkable effects in the current study were seen on Day 1 and lessened with time, further supporting the conclusion that they were related to the injury and not to the immobilization.
The results of our study have implications for developing strategies to improve tendon repair outcomes. Previous in vitro studies have shown that pro-inflammatory factors, such as IL-1β and TNFα, induce tendon fibroblasts to up-regulate their own expression of inflammatory and catabolic enzymes and to down-regulate their expression of type I collagen. At the tissue level, these changes may result in reductions in ultimate tensile strength and elastic modulus and increases in the maximum strain of repaired tendons. Gulotta et al. demonstrated that inhibition of TNFα during tendon healing improved the overall strength of tendon repair repair. Similarly, De la Durantaye et al. found that macrophage depletion during tendon healing leads to enhanced material properties of the healed tendons. Dagher et al. showed a correlation between a shift in the macrophage population (from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages) improved ligament healing. Classical dermal wound healing literature suggests that neutrophils are not necessary for proper wound healing and more recent evidence suggests that neutrophils may impede skin healing. Modulation of inflammation in a skin wound healing model resulted in an enhanced healing response characterized by accelerated wound healing and organized dermis and collagen bundles. Taken together, these studies suggest that high levels of pro-inflammatory cytokines may be detrimental to tendon healing and modulation of the early inflammatory phase of healing may be beneficial to tendon healing.
Despite evidence that suppressing inflammation can lead to improved wound healing, some pro-inflammatory cytokines are likely necessary for recruitment of immune cells to the site of injury and subsequent attraction of tendon fibroblasts.[33, 34] Synthesis of numerous potent growth factors, such as TGF-β and PDGF, by immune cells promotes cell proliferation and synthesis of ECM.[33, 34] Immune cells also play a pivotal role in angiogenesis through the secretion of VEGF.[33, 34] Several prior experiments have shown that complete depletion of certain immune cells and pro-inflammatory factors during wound healing leads to retarded wound repair.[35, 36] Specifically, macrophages appear to play an important role in debridement of the wound.[37, 38] Therefore, based on these studies, global IL-1β blockade or immune cell depletion is unlikely to be an effective strategy for improving tendon healing. Fine modulation of the inflammatory environment is likely necessary to enhance tendon healing. This could potentially be achieved using mesenchymal stem cells, which have recently been shown to modulate inflammation by controlling macrophages phenotype,[39, 40] or targeted therapies that protect tendon fibroblasts from the detrimental effects of cytokines such as IL-1β.