Camila B. Carballo

Indian hedgehog signaling and the role of graft tension in tendon‐to‐bone healing: Evaluation in a rat ACL reconstruction model

The structure and composition of the native enthesis is not recapitulated following tendon‐to‐bone repair. Indian Hedgehog (IHH) signaling has recently been shown to be important in enthesis development in a mouse model but no studies have evaluated IHH signaling in a healing model. Fourteen adult male rats underwent ACL reconstruction using a flexor tendon graft. Rats were assigned to two groups based on whether or not they received 0N or 10N of pre‐tension of the graft. Specimens were evaluated at 3 and 6 weeks post‐operatively using immunohistochemistry for three different protein markers of IHH signaling. Quantitative analysis of staining area and intensity using custom software demonstrated that IHH signaling was active in interface tissue formed at the healing tendon‐bone interface. We also found increased staining area and intensity of IHH signaling proteins at 3 weeks in animals that received a pre‐tensioned tendon graft. No significant differences were seen between the 3‐week and 6‐week time points. Our data suggests that the IHH signaling pathway is active during the tendon‐bone healing process and appears to be mechanosensitive, as pre‐tensioning of the graft at the time of surgery resulted in increased IHH signaling at three weeks.

Dendritic cells maintain dermal adipose–derived stromal cells in skin fibrosis

Scleroderma is a group of skin-fibrosing diseases for which there are no effective treatments. A feature of the skin fibrosis typical of scleroderma is atrophy of the dermal white adipose tissue (DWAT). Adipose tissue contains adipose-derived mesenchymal stromal cells (ADSCs) that have regenerative and reparative functions; however, whether DWAT atrophy in fibrosis is accompanied by ADSC loss is poorly understood, as are the mechanisms that might maintain ADSC survival in fibrotic skin. Here, we have shown that DWAT ADSC numbers were reduced, likely because of cell death, in 2 murine models of scleroderma skin fibrosis. The remaining ADSCs showed a partial dependence on dendritic cells (DCs) for survival. Lymphotoxin β (LTβ) expression in DCs maintained ADSC survival in fibrotic skin by activating an LTβ receptor/β1 integrin (LTβR/β1 integrin) pathway on ADSCs. Stimulation of LTβR augmented the engraftment of therapeutically injected ADSCs, which was associated with reductions in skin fibrosis and improved skin function. These findings provide insight into the effects of skin fibrosis on DWAT ADSCs, identify a DC-ADSC survival axis in fibrotic skin, and suggest an approach for improving mesenchymal stromal cell therapy in scleroderma and other diseases.

Basic Science of Articular Cartilage

The most challenging aspects in treating articular cartilage injury include identifying the cellular and molecular mechanism(s) that lead to matrix changes and the differentiation and dedifferentiation behavior of chondrocytes, and understanding how they affect the structural integrity of the articular cartilage and tissue remodeling. Several treatment strategies have been proposed. A better understanding of the signaling pathways and growth and transcription factors for genes responsible for chondrogenesis is an important component in the development of new therapies to prevent cartilage degeneration or promote repair to replicate the physiologic and functional properties of the original cartilage.

Animal models for rotator cuff repair

Rotator cuff (RC) injuries represent a significant source of pain, functional impairment, and morbidity. The large disease burden of RC pathologies necessitates rapid development of research methodologies to treat these conditions. Given their ability to model anatomic, biomechanical, cellular, and molecular aspects of the human RC, animal models have played an indispensable role in reducing injury burden and advancing this field of research for many years. The development of animal models in the musculoskeletal (MSK) research arena is uniquely different from that in other fields in that the similarity of macrostructures and functions is as critical to replicate as cellular and molecular functions. Traditionally, larger animals have been used because of their anatomic similarity to humans and the ease of carrying out realistic surgical procedures. However, refinement of current molecular methods, introduction of novel research tools, and advancements in microsurgical techniques have increased the applicability of small animal models in MSK research. In this paper, we review RC animal models and emphasize a murine model that may serve as a valuable instrument for future RC tendon repair investigations.

Involvement of Indian hedgehog signaling in mesenchymal stem cell–augmented rotator cuff tendon repair in an athymic rat model

BACKGROUND Bone marrow aspirate has been used in recent years to augment tendon-to-bone healing, including in rotator cuff repair. However, the healing mechanism in cell-based therapy has not been elucidated in detail. METHODS Sixteen athymic nude rats were randomly allocated to 2 groups: experimental (human mesenchymal stem cells in fibrin glue carrier) and control (fibrin glue only). Animals were sacrificed at 2 and 4 weeks. Immunohistochemical staining was performed to evaluate Indian hedgehog (Ihh) signaling and SOX9 signaling in the healing enthesis. Macrophages were identified using CD68 and CD163 staining, and proliferating cells were identified using proliferating cell nuclear antigen staining. RESULTS More organized and stronger staining for collagen II and a higher abundance of SOX9+ cells were observed at the enthesis in the experimental group at 2 weeks. There was significantly higher Gli1 and Patched1 expression in the experimental group at the enthesis at 2 weeks and higher numbers of Ihh+ cells in the enthesis of the experimental group vs control at both 2 weeks and 4 weeks postoperatively. There were more CD68+ cells localized to the tendon midsubstance at 2 weeks compared with 4 weeks, and there was a higher level of CD163 staining in the tendon midsubstance in the experimental group than in the control group at 4 weeks. CONCLUSION Stem cell application had a positive effect on fibrocartilage formation at the healing rotator cuff repair site. Both SOX9 and Ihh signaling appear to play an important role in the healing process.

Timing of Postoperative Mechanical Loading Affects Healing Following Anterior Cruciate Ligament Reconstruction: Analysis in a Murine Model

Background: Following anterior cruciate ligament (ACL) reconstruction, the mechanical loading of the tissues has a significant impact on tendon-to-bone healing. The purpose of this study was to determine the effect of the timing of the initiation of mechanical loading on healing of a tendon graft in a bone tunnel. Methods: ACL reconstruction using a flexor tendon autograft was performed in 56 mice randomized to 4 groups with differing times to initiation of postoperative mechanical loading: (1) immediate, (2) 5 days, (3) 10 days, or (4) 21 days following surgery. An external fixator was placed across the knee at the time of surgery and removed when mechanical loading was scheduled to commence. Following removal of the external fixator, animals were permitted free, unrestricted cage activity. All mice were killed on postoperative day 28, and tendon-to-bone healing was assessed by biomechanical testing, microcomputed tomography (micro-CT), and histological analysis. Results: The mean failure force (and standard deviation) of the reconstructed ACL at the time of sacrifice was highest for Group 2 (3.29 ± 0.68 N) compared with Groups 1, 3, and 4 (p = 0.008). Micro-CT bone volume fraction was greatest for Group 2 in the femoral tunnel (p = 0.001), tibial tunnel (p = 0.063), and both bones (p < 0.001). Similarly, histological analysis demonstrated a narrower scar tissue interface and increased direct contact at the tendon-bone interface (p = 0.012) for Group 2. Conclusions: Following ACL reconstruction, a defined period of immobilization without weight-bearing appears to improve biomechanical strength of the healing tendon-bone interface, while prolonged periods without mechanical load and motion decrease the ultimate load to failure in this murine model. Clinical Relevance: The ideal period of restricted weight-bearing and motion following ACL reconstruction remains undefined. In a murine model, improved healing was noted for animals immobilized for a brief period of 5 days. This work may serve as an initial step in determining the ideal time period in a clinical population.

Evaluating the role of subacromial impingement in rotator cuff tendinopathy: Development and analysis of a novel murine model: EVALUATING THE ROLE OF SUBACROMIAL IMPINGEMENT

Subacromial impingement of the rotator cuff is understood as a contributing factor in the development of rotator cuff tendinopathy. However, changes that occur in the impinged tendon are poorly understood and warrant further study. To enable further study of rotator cuff tendinopathy, we performed a controlled laboratory study to determine feasibility and baseline characteristics of a new murine model for subacromial impingement. This model involves surgically inserting a microvascular clip into the subacromial space in adult C57Bl/6 mice. Along with a sham surgery arm, 90 study animals were distributed among time point groups for sacrifice up to 6 weeks. All animals underwent bilateral surgery (total N = 180). Biomechanical, histologic, and molecular analyses were performed to identify and quantify the progression of changes in the supraspinatus tendon. Decreases in failure force and stiffness were found in impinged tendon specimens compared to sham and no‐surgery controls at all study time points. Semi‐quantitative scoring of histologic specimens demonstrated significant, persistent tendinopathic changes over 6 weeks. Quantitative real‐time polymerase chain reaction analysis of impinged tendon specimens demonstrated persistently increased expression of genes related to matrix remodeling, inflammation, and tendon development. Overall, this novel murine subacromial impingement model creates changes consistent with acute tendonitis, which may mimic the early stages of rotator cuff tendinopathy. A robust, simple, and reproducible animal model of rotator cuff tendinopathy is a valuable research tool to allow further studies of cellular and molecular mechanisms and evaluation of therapeutic interventions in rotator cuff tendinopathy.

Osteoarthritic Synovial Fluid and TGF-β1 Induce Interleukin-18 in Articular Chondrocytes

Objective Synovial fluid (SF) plays an important role in the maintenance of articular cartilage. SF is a dynamic reservoir of proteins derived from cartilage and synovial tissue; thus, its composition may serve as a biomarker that reflects the health and pathophysiological condition of the joint. The purpose of the current study was to evaluate the osteoarthritic synovial fluid (OASF) and transforming growth factor-β1 (TGF-β1) activity in articular chondrocytes catabolic and inflammatory responses. Design Chondrocytes were seeded at passage 2 and cultured for 72 hours under different conditions. Human chondrocytes were subjected to OASF while rat chondrocytes were subjected to either healthy synovial fluid (rSF) or TGF-β1 and then assigned for cell viability analysis. In addition, the effects of OASF and TGF-β1 on chondrocytes metalloprotease (MMP)-3 and MMP-13 and interleukin-18 (IL-18) expression were evaluated by immunocytochemistry, ELISA, and reverse transcriptase-polymerase chain reaction. Results SF from osteoarthritic patients significantly induced MMP-3, MMP-13, and IL-18 receptor expression in chondrocytes. To put in evidence the inflammatory activity of OASF, healthy chondrocytes from rat were cultured with TGF-β1. In the presence of TGF-β1 these cells started to express MMP-3, MMP-13, and IL-18 genes and attached to each other forming a chondrocyte aggregated structure. Healthy SF was able to maintain a typical monolayer of rounded chondrocytes with no inflammatory response. Conclusion In summary, these observations demonstrated that TGF-β1, one of the components of OASF, has a dual effect, acting in chondrocyte maintenance and also inducing inflammatory and catabolic properties of these cells.

Blood-induced bone loss in murine hemophilic arthropathy is prevented by blocking the iRhom2/ADAM17/TNF-α pathway

Hemophilic arthropathy (HA) is a debilitating degenerative joint disease that is a major manifestation of the bleeding disorder hemophilia A. HA typically begins with hemophilic synovitis that resembles inflammatory arthritides, such as rheumatoid arthritis, and frequently results in bone loss in patients. A major cause of rheumatoid arthritis is inappropriate release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by the TNF-α convertase (TACE; also referred to as ADAM17) and its regulator, iRhom2. Therefore, we hypothesized that iRhom2/ADAM17-dependent shedding of TNF-α also has a pivotal role in mediating HA. Here, we show that addition of blood or its components to macrophages activates iRhom2/ADAM17-dependent TNF-α shedding, providing the premise to study the activation of this pathway by blood in the joint in vivo. For this, we turned to hemophilic FVIII-deficient mice (F8-/- mice), which develop a hemarthrosis following needle puncture injury with synovial inflammation and significant osteopenia adjacent to the affected joint. We found that needle puncture-induced bleeding leads to increased TNF-α levels in the affected joint of F8-/- mice. Moreover, inactivation of TNF-α or iRhom2 in F8-/- mice reduced the osteopenia and synovial inflammation that develops in this mouse model for HA. Taken together, our results suggest that blood entering the joint activates the iRhom2/ADAM17/TNF-α pathway, thereby contributing to osteopenia and synovitis in mice. Therefore, this proinflammatory signaling pathway could emerge as an attractive new target to prevent osteoporosis and joint damage in HA patients.

Biomechanics and Microstructural Analysis of the Mouse Knee and Ligaments

Abstract The purpose of this study is to determine the feasibility of using murine models for translational study of knee ligament injury, repair, and reconstruction. To achieve this aim, we provide objective, quantitative data detailing the gross anatomy, biomechanical characteristics, and microscopic structure of knee ligaments of 44 male mice (C57BL6, 12 weeks of age). Biomechanical testing determined the load‐to‐failure force, stiffness, and the site of ligament failure for the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), and the medial and lateral collateral ligaments (MCL and LCL). These data are complemented by histological characterization of each of the knee ligaments. In addition, the osseous morphology of the mouse knee was examined using high‐resolution nanofocus computed tomography (CT), while standard micro‐CT was employed to measure bone morphometrics of the distal femur and proximal tibia. Collectively, our findings suggest that the gross anatomy of the mouse knee is similar to the human knee despite some minor differences and features unique to the murine knee. The ACL had the highest load to failure (5.60 ± 0.75 N), the MCL (3.33 ± 1.45 N), and the PCL (3.45 ± 0.84 N) were similar, and the LCL (1.44 ± 0.37 N) had the lowest load to failure and stiffness. Murine models provide a unique opportunity to focus on biological processes that impact ligament pathology and healing due to the availability of transgenic strains. Our data support their use as a translational platform for the in vivo study of ligament injury, repair, and reconstruction.