Connie S. Chamberlain

The spatio‐temporal dynamics of ligament healing

Ligament injury commonly occurs with no effective treatment to restore its original state. Numerous studies have examined wound healing after injury, reporting a provisional matrix and scar formation within the wound. Few studies however report the inflammatory, proliferative, and remodeling process during ligament healing in a spatio‐temporal manner. Our goal was then to more completely elucidate this process in a rat medial collateral ligament (MCL) healing model. In this study, medial collateral ligaments were surgically transected and allowed to heal. At 1, 3, 5, 7, 9, 11, 14, and 28 days postinjury ligaments were collected and examined with microangiography or immunohistochemistry. We demonstrate that neutrophils and mitotic cells peak between 1 and 5 days postinjury. The majority of factors crest between 5 and 9 days postinjury, including circulating macrophages, resident macrophages, T lymphocytes, hematopoietic cells, vascular endothelial growth factor, and blood vessels. The apoptotic cells predominate from day 9 to the end of the study (day 28). Initially, most assayed markers localize to the epiligament and to granulation tissue at the site of damage. Later, the healing region with its granulation tissue and cells continues to expand into the uninjured tissue. From these results, we have expanded current descriptions of ligament healing and offer a more complete representation of the healing process.

The Influence of Macrophage Depletion on Ligament Healing

Despite a complex cascade of cellular events to reconstruct damaged extracellular matrix (ECM), ligament healing results in a mechanically inferior, scar-like tissue. During normal healing, the number of macrophages significantly increases within the wound site. Then, granulation tissue expands into any residual, normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise, and an inefficient repair process. To study the effects of macrophages on the repair process, bilateral, surgical rupture of their medial collateral ligaments (MCLs) was done on rats. Treatment animals received liposome-encapsulated clodronate, 2 days before rupture to ablate phagocytosing macrophages. Ligaments were then collected at days 5, 11, and 28 for immunohistochemistry (IHC) and/or mechanical testing. Clodronate treatment reduced both the M1 and M2 macrophages at day 5 and altered early healing. However, the macrophages effectively returned to control levels after day 5 and reinitiated a wound-healing response. Our results suggest that an early macrophage response, which is necessary for debridement of damaged tissue in the wound, is also important for cytokine release to mediate normal repair processes. Additionally, nonspecific inhibition of macrophages (without regard to specific macrophage populations) can control excessive granulation tissue formation but is detrimental to early matrix formation and ligament strength.

Satellite cells express distinct patterns of myogenic proteins in immature skeletal muscle

Satellite cells are the myogenic cells lying between the myofiber sarcolemma and basal lamina. The objective of this study was to determine the expression patterns of MyoD, myogenin, and Pax7 within the satellite cell population in the growing rat soleus and extensor digitorum longus (EDL) muscles. Secondly, the expression of the myogenic markers was also studied within the interstitial cell compartment and myonuclei. It was discovered that the soleus contained a higher number of Pax7, MyoD, or myogenin‐positive nuclei compared with the EDL. Similarly, myogenin was expressed at a lower level in the myonuclei of the soleus compared with the EDL, and myogenin was expressed at a higher level in the interstitial compartment of the soleus compared with the EDL. When interstitial nuclei, myonuclei, and double‐labeled nuclei were used in the estimate of the satellite cell population, it was discovered that approximately of 13% of the myofibers in a transverse section of the soleus muscle and 4.1% of EDL myofibers exhibit a labeled satellite cell nucleus. Overall, results from this study suggest that expression patterns of these markers vary predictably among muscles with different growth dynamics and phenotypic characteristics. Developmental Dynamics 235:3230–3239, 2006.

Quantification of Collagen Organization and Extracellular Matrix Factors within the Healing Ligament

Ligament healing of a grade III injury (i.e., a complete tear) involves a multifaceted chain of events that forms a neoligament, which is more scar-like in character than the native tissue. The remodeling process may last months or even years with the injured ligament never fully recovering pre-injury mechanical properties. With tissue engineering and regenerative medicine, understanding the normal healing process in ligament and quantifying it provide a basis to create and assess innovative treatments. Ligament fibroblasts produce a number of extracellular matrix (ECM) components, including collagen types I and III, decorin and fibromodulin. Using a combination of advanced histology, molecular biology, and nonlinear optical imaging approaches, the early ECM events during ligament healing have been better characterized and defined. First, the dynamic changes in ECM factors after injury are shown. Second, the factors associated with creeping substitution are identified. Finally, a method to quantify collagen organization is developed and used. Each ECM factor described herein as well as the temporal quantification of fiber organization helps elucidate the complexity of ligament healing.

Quantification of collagen organization using fractal dimensions and Fourier transforms

Collagen fibers and fibrils that comprise tendons and ligaments are disrupted or damaged during injury. Fibrillogenesis during healing produces a matrix that is initially quite disorganized, but remodels over time to resemble, but not replicate, the original roughly parallel microstructure. Quantification of these changes is traditionally a laborious and subjective task. In this work we applied two automated techniques, fast Fourier transformation (FFT) and fractal dimension analysis (FA) to quantify the organization of collagen fibers or fibrils. Using multi-photon images of collagen fibers obtained from rat ligament we showed that for healing ligaments, FA differentiates more clearly between the different time-points during healing. Using scanning electron microscopy images of overstretched porcine flexor tendon, we showed that combining FFT and FA measures distinguishes the damaged and undamaged groups more clearly than either method separately.

Interleukin expression after injury and the effects of interleukin-1 receptor antagonist.

Ligament healing follows a series of complex coordinated events involving various cell types, cytokines, as well as other factors, producing a mechanically inferior tissue more scar-like than native tissue. Macrophages provide an ongoing source of cytokines to modulate inflammatory cell adhesion and migration as well as fibroblast proliferation. Studying interleukins inherent to ligament healing during peak macrophage activation and angiogenesis may elucidate inflammatory mediators involved in subsequent scar formation. Herein, we used a rat healing model assayed after surgical transection of their medial collateral ligaments (MCLs). On days 3 and 7 post-injury, ligaments were collected and used for microarray analysis. Of the 12 significantly modified interleukins, components of the interleukin-1 family were significantly up-regulated. We therefore examined the influence of interleukin-1 receptor antagonist (IL-1Ra) on MCL healing. Transected rat MCLs received PBS or IL-1Ra at the time of surgery. Inhibition of IL-1 activation decreased pro-inflammatory cytokines (IL-1α, IL-1β, IL-12, IL-2, and IFN-γ), myofibroblasts, and proliferating cells, as well as increased anti-inflammatory cytokines (IL-10), endothelial cells/blood vessel lumen, M2 macrophages, and granulation tissue size without compromising the mechanical properties. These results support the concept that IL-1Ra modulates MCL-localized granulation tissue components and cytokine production to create a transient environment that is less inflammatory. Overall, IL-1Ra may have therapeutic potential early in the healing cascade by stimulating the M2 macrophages and altering the granulation tissue components. However, the single dose of IL-1Ra used in this study was insufficient to maintain the more regenerative early response. Due to the transient influence on most of the healing components tested, IL-1Ra may have greater therapeutic potential with sustained delivery.

The Influence of Interleukin-4 on Ligament Healing

Despite a complex cascade of cellular events to reconstruct the damaged extracellular matrix, ligament healing results in a mechanically inferior scarred ligament. During normal healing, granulation tissue expands into any residual normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise and an inefficient repair process. To control creeping substitution and possibly enhance the repair process, the antiinflammatory cytokine, interleukin‐4 (IL‐4), was administered to rats before and after rupture of their medial collateral ligaments. In vitro experiments showed a time‐dependent effect on fibroblast proliferation after IL‐4 treatment. In vivo treatments with IL‐4 (100 ng/mL IV) for 5 days resulted in decreased wound size and type III collagen and increased type I procollagen, indicating a more regenerative early healing in response to the IL‐4 treatment. However, continued treatment of IL‐4 to day 11 antagonized this early benefit and slowed healing. Together, these results suggest that IL‐4 not only influences the macrophages and T lymphocytes but also stimulates fibroblasts associated with the proliferative phase of healing in a dose‐, cell‐, and time‐dependent manner. Although treatment significantly influenced healing in the first week after injury, IL‐4 alone was unable to maintain this early regenerative response.

Immune modulation with primed mesenchymal stem cells delivered via biodegradable scaffold to repair an Achilles tendon segmental defect

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1β may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF‐α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC‐seeded PLG scaffold, or TNF‐α‐primed MSC‐seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post‐injury. In vivo, MSCs, regardless of priming, increased IL‐10 production and reduced the inflammatory factor, IL‐1α. Primed MSCs reduced IL‐12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti‐inflammatory factor IL‐4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post‐injury. Taken together delivery of TNF‐α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC‐induced healing response.

Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile.

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1 × 106 or high dose 4 × 106 MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1β, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.

Interleukin-1 Receptor Antagonist Modulates Inflammation and Scarring after Ligament Injury

Abstract Ligaments have limited regenerative potential and as a consequence, repair is protracted and results in a mechanically inferior tissue more scar-like than native ligament. We previously reported that a single injection of interleukin-1 receptor antagonist (IL-1Ra) delivered at the time of injury, decreased the number of M2 macrophage-associated inflammatory cytokines. Based on these results, we hypothesized that IL-1Ra administered after injury and closer to peak inflammation (as would occur clinically), would more effectively decrease inflammation and thereby improve healing. Since IL-1Ra has a short half-life, we also investigated the effect of multiple injections. The objective of this study was to elucidate healing of a medial collateral ligament (MCL) with either a single IL-1Ra injection delivered one day after injury or with multiple injections of IL-1Ra on days 1, 2, 3, and 4. One day after MCL injury, rats received either single or multiple injections of IL-1Ra or PBS. Tissue was then collected at days 5 and 11. Both single and multiple IL-1Ra injections reduced inflammatory cytokines, but did not change mechanical behavior. A single injection of IL-1Ra also reduced the number of myofibroblasts and increased type I procollagen. Multiple IL-1Ra doses provided no additive response and, in fact, reduced the M2 macrophages. Based on these results, a single dose of IL-1Ra was better at reducing the MCL-derived inflammatory cytokines compared to multiple injections. The changes in type I procollagen and myofibroblasts further suggest a single injection of IL-1Ra enhanced repair of the ligament but not sufficiently to improve functional behavior.