Amir Lebaschi

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.

The Effect of Graft Pretensioning on Bone Tunnel Diameter and Bone Formation After Anterior Cruciate Ligament Reconstruction in a Rat Model: Evaluation With Micro–Computed Tomography

Background: Moderate graft pretensioning in anterior cruciate ligament (ACL) reconstruction is paramount to restore knee stability and normalize knee kinematics. However, little is known about the effect of graft pretensioning on graft-to-bone healing after ACL reconstruction. Hypothesis: Moderate graft pretensioning will improve bone formation within the bone tunnel after ACL reconstruction, resulting in superior load to failure. Study Design: Controlled laboratory study. Methods: 67 male Sprague-Dawley rats underwent unilateral ACL reconstruction with a flexor digitorum longus tendon autograft. The graft was subjected to pretensioning forces of 0 N, 5 N, or 10 N. Custom-made external fixators were used for knee immobilization postoperatively. Rats were euthanized for biomechanical load-to-failure testing (n = 45) and micro–computed tomography (μCT) examination (n = 22) at 3 and 6 weeks after surgery. Three regions of each femoral and tibial bone tunnel (aperture, middle, and tunnel exit) were chosen for measurement of tunnel diameter and new bone formation. Results: Biomechanical tests revealed significantly higher load-to-failure in the 5-N graft pretensioned group compared with the 0- and 10-N groups at 3 weeks (8.58 ± 2.67 N vs 3.96 ± 1.83 N and 4.46 ± 2.62 N, respectively) and 6 weeks (16.56 ± 3.50 N vs 10.82 ± 1.97 N and 7.35 ± 2.85 N, respectively) after surgery (P < .05). The mean bone tunnel diameters at each of the 3 regions were significantly smaller in the 5-N group, at both the femoral and tibial tunnel sites, than in the 0- and 10-N groups (P < .05). At 3 and 6 weeks postoperatively, the bone mineral density, bone volume fraction, and connectivity density around the aperture and middle regions of the tibial bone tunnels were all significantly higher in the 5-N group compared with the 0- and 10-N groups (P < .05). In the aperture and middle regions of the femoral bone tunnels, pretensioning at either 5 or 10 N resulted in increased bone formation compared with the nonpretensioned group at 3 weeks postoperatively. No differences were found in bone formation between any of the 3 femoral tunnel regions at 6 weeks. Conclusion: Graft pretensioning can stimulate new bone formation and improve tendon-to-bone tunnel healing after ACL reconstruction. Clinical Relevance: Optimal graft pretensioning force in ACL reconstruction can improve bone tunnel healing. Further study is necessary to understand the mechanisms underlying the effect of graft pretensioning on healing at the bone-tunnel interface.

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.

Tissue‐specific endothelial cells: a promising approach for augmentation of soft tissue repair in orthopedics

Biologics are playing an increasingly significant role in the practice of modern medicine and surgery in general and orthopedics in particular. Cell‐based approaches are among the most important and widely used modalities in orthopedic biologics, with mesenchymal stem cells and other multi/pluripotent cells undergoing evaluation in numerous preclinical and clinical studies. On the other hand, fully differentiated endothelial cells (ECs) have been found to perform critical roles in homeostasis of visceral tissues through production of an adaptive panel of so‐called “angiocrine factors.” This newly discovered function of ECs renders them excellent candidates for novel approaches in cell‐based biologics. Here, we present a review of the role of ECs and angiocrine factors in some visceral tissues, followed by an overview of current cell‐based approaches and a discussion of the potential applications of ECs in soft tissue repair.

Murine Supraspinatus Tendon Detachment and Repair Model Augmented With Tendon-Derived, Activated Endothelial Cells: A New Concept in Biologic Enhancement of Tendon-to-Bone Healing

Objectives: Biologic interventions are being increasingly used to improve outcome of rotator cuff repair. Individual growth factors and stem cell-based augmentations are among the most popular approaches. In this study we investigated a novel approach in which tendon-derived, activated endothelial cells (tAECs) are used as a source of tissue-specific growth factors to stimulate tendon intrinsic stem cell niche in our established murine model of microsurgical supraspinatus tendon (SST) detachment and repair. Methods: Study protocol was approved by the IACUC. In stage 1, 20 C57BL/6 mice underwent tendon harvest from fore- and hind limbs and endothelial cells isolation and transfection with adenoviral E4 ORF1 and red fluorescent protein labelling using a proprietary protocol. In stage 2, 158 C57BL/6 mice underwent the procedure with implantation of a 10μm fibrin glue bead (FGB) at the repair site and were randomly allocated to study group (SG) and control group (CG). Study mice received 100,000 tAECs suspended in the FGB. Control mice received the FGB only. 3 mice were sacrificed on post-operative day (POD) #3 and #7 for cell viability study. 96 mice were sacrificed at 1, 2, and 4 weeks, with 10 and 6 mice in each group and time point for biomechanical and histologic evaluations, respectively. 56 mice were sacrificed on POD 5, 10, 14, and 28 for gene expression analysis using qPCR, with 7 mice in each group and time point. Biomechanical evaluation consisted of determination of failure force and site. Histologic parameters were cell count (H&E), number of vessels (factor VIII-specific antibody), number of chondrocytes and proteoglycan content (Alcian Blue), and collagen organization (picrosirius red and polarized light microscopy). 12 evaluated genes were aggrecan (ACAN), collagen1α1 (COL1), collagen3α1 (COL3), MKX, MMP-3, 13, and 14, Runx2, scleraxis (Scx), SOX9, tenomodulin (TNM) and VEGFa. Results: Fluorescent microscopy revealed viable tAECs at the repair site on POD #3 with No fluorescent activity on POD #7. Failure force in the SG was 50% higher at 2 weeks (2.50 ± 0.55 N vs 1.86 ± 0.82 N, p = 0.012) with 65% of all failures occurring at SST mid-substance, whereas 90% of failures in the CG occurred at the repair site. There was no significant difference in failure force at 4 weeks. Cell count, number of vessels, and proteoglycan content (Figure 1) were significantly higher in the SG at all time points. The number of chondrocytes was higher in the SG at 1 week (Figure 1). Quantitative analysis showed significantly superior collagen organization in the SG (Figure 2). SCX, MKX, TNM and COL3 expression were up-regulated on POD #10 and decreased on day #28 in both groups. Expression of TNM and COL3 in the SG showed significance difference at 4 weeks. ACAN, SOX9, COL1, MMP14 expression exhibited an increase starting from POD 5, followed by a significant decline towards baseline by POD 28. In the SG, SOX9 peaked at day 14. Expression of VEGFa showed an uptrend at earlier time points (Figure 3). Conclusion: tAECs increased repair strength of SST. Histologic findings were suggestive of a more vigorous cellular and vascular response, higher proteoglycan content, and ultimately, superior collagen organization. A commensurate pattern was observed in expression of relevant genes. This approach opens a new avenue for cell-based biologic augmentation of SST repair with the goal of stimulating the intrinsic stem cell niche in tendon, as opposed to incorporating exogenous cell sources.

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.

Umbilicoplasty with Vertical Incision in Abdominoplasty

The umbilicus occupies the central locus of the abdomen and is one of the most influential determinants of aesthetic outcome of abdominoplasty. The surgeon should diligently and meticulously address the issue of umbilicus reconstruction as an integral part of abdominoplasty. The author discusses anatomy, characterization of a beautiful umbilicus, the operative technique, and postoperative care. Possible complications are described.

Umbilical Pilonidal Disease

The exact pathogenesis of pilonidal disease remains unknown. Umbilical pilonidal disease is most commonly encountered in adults who have a hair tuft around their umbilicus and is more common in males. The authors describe nonsurgical management as well as surgical management. The authors’ preferred technique is discussed as is subfascial extension.

Restriction of Postoperative Joint Loading in a Murine Model of Anterior Cruciate Ligament Reconstruction: Botulinum Toxin Paralysis and External Fixation

Abstract Control of knee motion in small animal models is necessary to study the effect of mechanical load on the healing process. This can be especially challenging in mice, which are being increasingly used for various orthopedic reconstruction models. We explored the feasibility of botulinum toxin (Botox; Allergan, Dublin, Ireland) paralysis and a newly designed external fixator to restrict motion of the knee in mice undergoing anterior cruciate ligament (ACL) reconstruction. Nineteen C57BL/6 mice were allocated to two groups: (1) Botox group (n = 9) and (2) external fixator group (n = 10). Mice in Botox group received two different doses of Botox: 0.25 unit (n = 3) and 0.5 unit (n = 6). Injection was performed 72 hours prior to ACL reconstruction into the quadriceps, hamstring, and calf muscles of the right hind leg. Mice in external fixator group received an external fixator following ACL reconstruction. Mice were monitored for survival, tolerance, and achievement of complete knee immobilization. All mice were meant for sacrifice on day 14 postoperatively. No perceptible change in gait was observed with 0.25 unit of Botox. All mice that received 0.5 unit of Botox had complete hind limb paralysis documented by footprint analysis 2 days after injection but failed to tolerate anesthesia and were euthanized 24 hours after operation due to their critical condition. In contrast, the external fixator was well tolerated and effectively immobilized the limb. There was a single occurrence of intraoperative technical error in the external fixator group that led to euthanasia. No mechanical failure or complication was observed. Botox paralysis was not a viable option for postoperative restriction of motion and joint loading in mice. However, external fixation was an effective method for complete knee immobilization and can be used in murine models requiring postoperative control of knee loading. This study introduces a robust research tool to allow control of postoperative joint loading in animal models such as ACL reconstruction, permitting study of the effects of mechanical load on the biologic aspects of tendon‐to‐bone healing.