Lawrence V. Gulotta

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.

Bone Marrow–Derived Mesenchymal Stem Cells Transduced With Scleraxis Improve Rotator Cuff Healing in a Rat Model

Background: Rotator cuffs heal through a scar tissue interface after repair that makes them prone to failure. Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is thought to direct tendon development during embryogenesis. The purpose of this study was to determine if the application of mesenchymal stem cells (MSCs) transduced with adenoviral-mediated scleraxis (Ad-Scx) could improve regeneration of the tendon-bone insertion site in a rat rotator cuff repair model. Hypothesis: Bone marrow–derived cells transduced with Scx would improve the structure of the healing tendon-bone interface and result in increased tendon attachment strength. Study Design: Controlled laboratory study. Methods: Sixty Lewis rats underwent unilateral detachment and repair of the supraspinatus tendon. Thirty animals received MSCs in a fibrin glue carrier, and 30 received Ad-Scx-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 Scx and MSC groups in terms of histologic appearance at 2 weeks. However, the Scx group had higher ultimate stress-to-failure (2.6 ± 0.9 vs 1.7 ± 0.3 MPa; P = .03) and stiffness (8.4 ± 2.9 vs 5.0 ± 1.9 N/mm; P = .01) compared with the MSC group. At 4 weeks, the Scx group had more fibrocartilage (728.7 ± 50.4 vs 342.6 ± 217.0 mm2; P = .04), higher ultimate load to failure (26.7 ± 4.6 vs 20.8 ± 4.4 N; P = .01), higher ultimate stress to failure (4.7 ± 1.3 vs 3.5 ± 1.0 MPa; P < .04), and higher stiffness values (15.3 ± 3.4 vs 9.3 ± 2.2 N/mm; P < .001) as compared with the MSC group. Conclusion: Mesenchymal stem cells genetically modified with Scx can augment rotator cuff healing at early time points. Clinical Relevance: Biologic augmentation of acutely injured rotator cuffs with Scx-transduced MSCs may improve rotator cuff tendon healing and reduce the incidence of re-tears. However, further studies are needed to determine if this remains safe and effective in larger models.

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.

Growth factors for rotator cuff repair.

The 4 fibrocartilagenous transition zones of the rotator cuff insertion site are not recreated following surgical repair. Instead, a layer of scar tissue is formed between the tendon and the bone, which makes repairs prone to failure. Growth factors are a group of cytokines that induce mitosis, extracellular matrix production, neovascularization, cell maturation, and differentiation. Research has focused on their ability to augment rotator cuff repairs. Studies have shown that several factors are capable of increasing the strength of repairs in animal models. However, this appears to be accomplished through the production of more scar tissue, as opposed to regeneration of native tissue. It is becoming clear that multiple factors may be needed to regenerate the native tendon-bone insertion site. The optimal timing and vehicle for growth factor delivery have remained elusive. Gene therapy and tissue scaffolds provide promising options for the future, but the engineering still needs to be optimized for clinical use. Growth factor therapy for rotator cuff repairs remains a promising therapeutic for the future; however, much work needs to be done to optimize its effectiveness.

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.

Humeral component retroversion in reverse total shoulder arthroplasty: a biomechanical study

BACKGROUND Reverse total shoulder arthroplasty offers pain relief and functional improvement for patients with rotator cuff-deficient shoulders. The purpose of this study was to determine the optimal amount of humeral retroversion for this prosthesis. MATERIALS AND METHODS Six cadaveric shoulders underwent computed tomography (CT) imaging and were then dissected of soft tissues, except for their tendinous attachments. A reverse total shoulder arthroplasty was implanted in 0°, 20°, 30°, and 40° of retroversion, and the shoulders were mounted on a simulator to determine the muscle forces required to achieve 30° and 60° of scaption. CT images were converted into 3-dimensional models, and the amount of internal and external rotation was determined with computer modeling at various scaption angles. RESULTS No differences were found in the forces required for 30° or 60° of scaption for any muscle, at any retroversion. With increasing retroversion, more impingement-free external rotation was obtained, with a concomitant decrease in the amount of internal rotation. Above 60°, the humerus was allowed to rotate around the glenosphere unencumbered. CONCLUSIONS Increasing retroversion did not affect the muscle force requirements for scaption across the shoulder. Placing the humeral component in 0° to 20° of retroversion allows maximum internal rotation with the arm at the side, a movement that is required for daily activities. This limits external rotation with the arm at the side, but has no effect on external rotation with the arm elevated.

Regulation of gene expression in human tendinopathy

BackgroundChronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy.MethodsWe have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy.ResultsDiseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response.ConclusionsIdentification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics.

Prospective evaluation of arthroscopic rotator cuff repairs at 5 years: part I - Functional outcomes and radiographic healing rates

BACKGROUND A rotator cuff registry was established to prospectively evaluate the effectiveness of all-arthroscopic repairs. This study reports those results at 5 years. MATERIALS AND METHODS The study enrolled 193 patients who underwent all-arthroscopic rotator cuff repairs. Patients were evaluated preoperatively and at 1, 2, and 5 years postoperatively. Outcome measurements included the American Shoulder and Elbow Surgeons (ASES) score, range of motion, manual muscle testing, and ultrasonography. RESULTS At 5-years, 106 patients completed follow-up, representing 55% of the patients originally enrolled but 77% of those who returned for evaluation at 1 year. ASES scores improved from 52.6 ± 23.2 preoperatively to 92.6 ± 14.8 at 5 years (P < .001). Paired analyses showed no differences between the ASES scores at 2 and 5 years, but the scores improved from 1 to 5 years (P = .002). Between years 2 and 5, passive forward elevation decreased from 173° ± 10.3° to 168.6° ± 16.8° (P = .02) and external rotation decreased from 73.6° ± 21.3° to 67.8° ± 19.6° (P = .04). Patients improved a full motor grade in forward elevation and external rotation and this remained stable over time. The healing rates for all patients were 64.3% at 1 year, 75.4% at 2 years, and 81.2% at 5 years. Paired analyses showed increased healing rates from 1 to 5 years (P = .001) and from 2 to 5 years (P = .05). DISCUSSION The midrange results of all-arthroscopic rotator cuff repairs are good, and functional results remain constant over 5 years. The ultrasound healing rates continued to increase with time; however, the mechanism and the clinical significance of this are uncertain at this time.