John R. Ehteshami

Application of Bone Marrow-Derived Mesenchymal Stem Cells in a Rotator Cuff Repair Model

Background Rotator cuff tendons heal to bone with interposed scar tissue, which makes repairs prone to failure. The purpose of this study was to determine if the application of bone marrow-derived mesenchymal stem cells (MSCs) can improve rotator cuff healing after repair. Hypothesis Application of MSCs to the repair site will result in superior results compared with controls on histologic and biomechanical testing. Study Design Controlled laboratory study. Methods Ninety-eight Lewis rats underwent unilateral detachment and repair of the supraspinatus tendon; 10 rats were used for MSC harvest. Eight animals were used for cell tracking with Ad-LacZ. The remaining animals received either 10 6 MSCs in a fibrin carrier, the carrier alone, or nothing at the repair site. Animals were sacrificed at 2 and 4 weeks for histologic analysis to determine the amount of fibrocartilage formation and the collagen organization at the insertion. Biomechanical testing was also performed. Results Specimens treated with Ad-LacZ–transduced MSCs exhibited more β-galactosidase activity at the repair site compared with controls at both 2 and 4 weeks, although activity at 4 weeks was less than that at 2 weeks. There were no differences in the amount of new cartilage formation or collagen fiber organization between groups at either time point. There were also no differences in the biomechanical strength of the repairs, the cross-sectional area, peak stress to failure, or stiffness. Conclusion The addition of MSCs to the healing rotator cuff insertion site did not improve the structure, composition, or strength of the healing tendon attachment site despite evidence that they are present and metabolically active. Clinical Relevance A biologic solution to the problem of tendon-to-bone healing in the rotator cuff remains elusive. The repair site may lack the cellular and/or molecular signals necessary to induce appropriate differentiation of transplanted cells. Further studies are needed to determine if cell-based strategies need to be combined with growth and differentiation factors to be effective.

Indomethacin and Celecoxib Impair Rotator Cuff Tendon-to-Bone Healing

Background Nonsteroidal anti-inflammatory drugs are commonly prescribed after rotator cuff repair. These agents can impair bone formation, but no studies have evaluated their impact on tendon-to-bone healing. Hypothesis Traditional nonselective nonsteroidal anti-inflammatory drugs and cyclooxygenase-2–specific nonsteroidal antiinflammatory drugs interfere with tendon-to-bone healing. Study Design Controlled laboratory study. Methods One hundred eighty Sprague-Dawley rats underwent acute rotator cuff repairs. Postoperatively, 60 rats received 14 days of celecoxib, a cyclooxygenase-2–specific nonsteroidal anti-inflammatory drug; 60 received indomethacin, a traditional nonselective nonsteroidal anti-inflammatory drug; and 60 received standard rat chow. Animals were sacrificed at 2, 4, and 8 weeks and evaluated by gross inspection, biomechanical testing, histologic analysis, and polarized light microscopy to quantify collagen formation and maturation. Results Five tendons completely failed to heal (4 celecoxib, 1 indomethacin). There were significantly lower failure loads in the celecoxib and indomethacin groups compared with the control groups at 2, 4, and 8 weeks (P< .001), with no significant difference between nonsteroidal anti-inflammatory drug groups. There were significant differences in collagen organization and maturation between the controls and both nonsteroidal anti-inflammatory drug groups at 4 and 8 weeks (P< .001). Controls demonstrated progressively increasing collagen organization during the course of the study (P< .001), whereas the nonsteroidal anti-inflammatory drug groups did not. Conclusion Traditional and cyclooxygenase-2–specific nonsteroidal anti-inflammatory drugs significantly inhibited tendon-tobone healing. This inhibition appears linked to cyclooxygenase-2. Clinical Relevance If the results of this study are verified in a larger animal model, the common practice of administering nonsteroidal anti-inflammatory drugs after rotator cuff repair should be reconsidered.

Stem cells genetically modified with the developmental gene MT1-MMP improve regeneration of the supraspinatus tendon-to-bone insertion site.

Background: Rotator cuffs heal through a scar tissue interface after repair, which makes them prone to failure. Membrane type 1 matrix metalloproteinase (MT1-MMP) is upregulated during embryogenesis in areas that develop into tendon-bone insertion sites. Hypothesis: Bone marrow-derived stem cells in the presence of the developmental signal from MT1-MMP will drive the healing process toward regeneration and away from scar formation. Study Design: Controlled laboratory study. Methods: Sixty Lewis rats underwent unilateral detachment and repair of the supraspinatus tendon. Thirty animals received mesenchymal stem cells (MSCs) in a fibrin glue carrier, and 30 received adenoviral MT1-MMP (Ad-MT1-MMP)–transduced MSCs. Animals were sacrificed at 2 weeks and 4 weeks and evaluated for the presence of fibrocartilage and collagen fiber organization at the insertion. Biomechanical testing was performed to determine the structural and material properties of the repaired tissue. Statistical analysis was performed with a Wilcoxon rank-sum test with significance set at P = .05. Results: There were no differences between the Ad-MT1-MMP and MSC groups in any outcome variable at 2 weeks. At 4 weeks, the Ad-MT1-MMP group had more fibrocartilage (P = .05), higher ultimate load to failure (P = .01), higher ultimate stress to failure (P = .005), and higher stiffness values (P = .02) as compared with the MSC group. Conclusion: Mesenchymal stem cells genetically modified to overexpress the developmental gene MT1-MMP can augment rotator cuff healing at 4 weeks by the presence of more fibrocartilage at the insertion and improved biomechanical strength. Clinical Relevance: Biologic augmentation of repaired rotator cuffs with MT1-MMP–transduced MSCs may reduce the incidence of retears. However, further studies are needed to determine if this remains safe and effective in larger models.

The effect of matrix metalloproteinase inhibition on tendon-to-bone healing in a rotator cuff repair model

HYPOTHESIS Recent studies have demonstrated a potentially critical role of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the pathophysiology of rotator cuff tears. We hypothesize that local delivery of a MMP inhibitor after surgical repair of the rotator cuff will improve healing at the tendon-to-bone surface interface. MATERIALS AND METHODS Sixty-two male Sprague-Dawley rats underwent acute supraspinatus detachment and repair. In the control group (n=31), the supraspinatus was repaired to its anatomic footprint. In the experimental group (n=31), recombinant alpha-2-macroglobulin (A2M) protein, a universal MMP inhibitor, was applied at the tendon-bone interface with an identical surgical repair. Animals were sacrificed at 2 and 4 weeks for histomorphometry, immunohistochemistry, and biomechanical testing. Statistical comparisons were performed using unpaired t tests. Significance was set at P < .05. RESULTS Significantly greater fibrocartilage was seen at the healing enthesis in the A2M-treated specimens compared with controls at 2 weeks (P < .05). Significantly greater collagen organization was observed in the A2M-treated animals compared with controls at 4 weeks (P < .01). A significant reduction in collagen degradation was observed at both 2 and 4 weeks in the experimental group (P < .05). Biomechanical testing revealed no significant differences in stiffness or ultimate load-to-failure. CONCLUSION Local delivery of an MMP inhibitor is associated with distinct histologic differences at the tendon-to-bone interface after rotator cuff repair. Modulation of MMP activity after rotator cuff repair may offer a novel biologic pathway to augment tendon-to-bone healing after rotator cuff repair.

Adenoviral-mediated gene transfer of human bone morphogenetic protein-13 does not improve rotator cuff healing in a rat model.

Background: Rotator cuff tendon-to-bone healing occurs by formation of a scar tissue interface after repair, which makes it prone to failure. Bone morphogenetic protein–13 (BMP-13) has been implicated in tendon and cartilage repair, and thus may augment rotator cuff repairs. The purpose of this study was to determine if the application of mesenchymal stem cells (MSCs) transduced with BMP-13 could improve regeneration of the tendon-bone insertion site in a rat rotator cuff repair model. Hypothesis: Mesenchymal stem cells genetically modified to overexpress BMP-13 will improve rotator cuff healing based on histologic and biomechanical outcomes. Study Design: Controlled laboratory study. Methods: Sixty Lewis rats underwent unilateral detachment and repair of the supraspinatus tendon and 10 rats were used for MSC harvest. Animals were randomized into 2 groups (30 animals/group). The experimental group received 106 MSCs transduced with adenoviral-mediated gene transfer of human BMP-13 (Ad-BMP-13). The second group received untransduced MSCs. Fifteen animals in each group were sacrificed at 2 and 4 weeks. At each time point, 12 animals were allocated for biomechanical testing, and 3 for histomorphometric analysis. Results: There were no differences in the amount of new cartilage formation or collagen fiber organization between groups at either time point. There were also no differences in the biomechanical strength of the repairs, the cross-sectional area, peak stress at failure, or stiffness. Conclusion: Application of MSCs genetically modified to overexpress BMP-13 did not improve healing in a rat model of rotator cuff repair. Clinical Relevance: Further studies are needed to evaluate various growth factors and combinations of growth factors to determine the optimal factor for the biologic augmentation of rotator cuff repairs.

Augmentation of Tendon-to-Bone Healing with a Magnesium-Based Bone Adhesive

Background Healing of an anterior cruciate ligament graft in a bone tunnel occurs by formation of fibrous scar tissue, which is weaker than the normal fibrocartilaginous insertion. Hypothesis We hypothesized that a magnesium-based bone adhesive would improve tendon-to-bone healing in a rabbit anterior cruciate ligament reconstruction model. Study Design Controlled laboratory study. Methods Thirty-five New Zealand White rabbits underwent bilateral anterior cruciate ligament reconstructions with semitendinosus autografts. A total of 12.5 g of bone adhesive was placed in the intraosseous tunnel around the graft in one limb, while the tunnels in the contralateral limb received no implant. Sixteen animals each were sacrificed at 3 weeks and at 6 weeks (12 biomechanical testing/4 histology). Outcomes included semiquantitative histologic analyses for new cartilage formation and fibrous tissue formation in the tendon-bone interface, microcomputed tomography to quantify new bone formation along the bone tunnel, and biomechanical testing of load-to-failure and stiffness. Three animals were sacrificed at time 0 to confirm adequate tunnel fill with the bone adhesive on microcomputed tomography. Results All specimens had adequate tunnel fill with the bone adhesive at time 0. Application of the bone adhesive resulted in more cartilage formation and less fibrous tissue formation at the tendon-bone interface at 6 weeks compared with controls (P < .05). There was significantly more bone formation in the tibia of the treated limbs at 6 weeks (P = .01). The load-to-failure was significantly higher in the treated group at 6 weeks (71.8 ± 31.8 N vs 43.4 ± 14.8 N; P = .04). There were no differences in stiffness at either time point, and there were no differences at 3 weeks in any outcome variable. Conclusion The magnesium-based bone adhesive improves tendon-to-bone healing based on histologic and biomechanical testing at 6 weeks in a rabbit model of anterior cruciate ligament reconstruction. Clinical Relevance Further studies are needed to investigate the clinical potential of this bone adhesive to enhance healing and decrease recovery time in soft-tissue ligament reconstruction.

The effect of muscle paralysis using Botox on the healing of tendon to bone in a rat model.

HYPOTHESIS Despite good clinical results after rotator cuff repair, follow-up studies show significant rates of failed healing. This may be because of excessive tension on the repaired tendon due to shoulder motion. We hypothesized that botulinum toxin A injections would result in improved attachment strength and collagen organization at the tendon-bone interface at early time points but may result in decreased mechanical properties at later time points because of the negative effects of stress deprivation. MATERIALS AND METHODS We performed division and repair of the supraspinatus tendon in 132 rats: 66 underwent repair alone and 66 received injections of botulinum toxin into the muscle before repair. Rats were killed at 4, 8, and 24 weeks and were evaluated by use of histologic, biomechanical, and micro-computed tomography analyses. RESULTS At 4 and 24 weeks, there was no significant difference in load to failure between groups. At 8 weeks, the botulinum group had a significantly lower load to failure compared with controls (27.7 N vs 46.7 N, P < .01). The weight of the supraspinatus muscle was significantly decreased at 4 and 8 weeks in the botulinum group, but it recovered by 24 weeks. Micro-computed tomography analysis showed the botulinum group to have significantly less bone volume, total mineral content, and total mineral density at 8 weeks. Histologic analysis showed formation of a more normal tidemark and increased collagen fiber organization in the botulinum specimens at 4 weeks. DISCUSSION Botulinum toxin A-treated specimens had increased collagen fiber organization at 4 weeks and decreased mechanical properties at later time points. The rapid healing of the rat rotator cuff likely makes it difficult to realize benefits from reduction in strain.

Comparison of Neurotization Versus Nerve Repair in an Animal Model of Chronically Denervated Muscle

Purpose Reinnervation of chronically denervated muscle is clinically unpredictable and poorly understood. Current operative strategies include either direct nerve repair, nerve grafting, nerve transfer, or neurotization. The goal of this study is to compare muscle recovery using microneural repair versus neurotization in a rat model of chronic denervation. Methods Fifty-eight Sprague-Dawley rats had surgical denervation of the tibialis anterior muscle by transecting the common peroneal nerve. After 0, 8, 12, or 22 weeks of denervation, animals were assigned to either a direct repair or a neurotization cohort. An additional 7 animals were used for a sham cohort, and 7 of the 58 were used as controls. After a 12-week recovery period, animals had contractile strength and EMG testing of the tibialis anterior muscle. Peak force and characteristics were compared to the unoperated, contralateral limb. Tibialis anterior muscles were then harvested for mass and histologic evaluation. Results Sixty-two animals completed testing. Denervated controls demonstrated a significant decrease in muscle mass, contractile strength, and peak motor nerve conduction amplitude compared to sham animals. In all groups, chronicity of denervation adversely affected functional recovery. On average, repair animals performed better than neurotization animals with respect to muscle mass, contractile strength, and peak motor amplitude. Differences in contractile force, however, were significant only at the 0 week denervation group (94% ± 30 vs 50% ± 20, repair vs neurotization). Neurotized muscles processed for histologic analysis demonstrated acetylcholinesterase activity at the nerve-muscle interface, confirming the formation of motor end plates de novo. Conclusions We demonstrated that neurotization is capable of reinnervating de novo end plates in chronically denervated muscle. Our data do not support the hypothesis that direct muscle neurotization is superior to nerve repair for functional restoration of chronically denervated muscle. However, as the duration of denervation increases, the difference between outcomes of the neurotization and repair group narrows, suggesting that neurotization may offer a viable surgical alternative in the setting of prolonged denervation.