David Kovacevic

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.

Biological Augmentation of Rotator Cuff Tendon Repair

A histologically normal insertion site does not regenerate following rotator cuff tendon-to-bone repair, which is likely due to abnormal or insufficient gene expression and/or cell differentiation at the repair site. Techniques to manipulate the biologic events following tendon repair may improve healing. We used a sheep infraspinatus repair model to evaluate the effect of osteoinductive growth factors and BMP-12 on tendon-to-bone healing. Magnetic resonance imaging and histology showed increased formation of new bone and fibrocartilage at the healing tendon attachment site in the treated animals, and biomechanical testing showed improved load-to-failure. Other techniques with potential to augment repair site biology include use of platelets isolated from autologous blood to deliver growth factors to a tendon repair site. Modalities that improve local vascularity, such as pulsed ultrasound, have the potential to augment rotator cuff healing. Important information about the biology of tendon healing can also be gained from studies of substances that inhibit healing, such as nicotine and antiinflammatory medications. Future approaches may include the use of stem cells and transcription factors to induce formation of the native tendon-bone insertion site after rotator cuff repair surgery.

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.

Doxycycline-Mediated Inhibition of Matrix Metalloproteinases Improves Healing After Rotator Cuff Repair

Background Recent studies demonstrate a potentially critical role of matrix metalloproteinases (MMPs) and their inhibitors in the pathophysiology of rotator cuff tears. Hypothesis Doxycycline-mediated MMP inhibition after rotator cuff repair will improve tendon-to-bone healing. Study Design Controlled laboratory study. Methods Rats (n = 183) underwent acute detachment and repair of the supraspinatus tendon and the animals were divided into 4 groups: In controls (n = 66), the supraspinatus was repaired to its anatomical footprint. In experimental groups, an identical surgery was performed with doxycycline (130 mg/kg/d) administered orally at (1) preoperative day 1 (n = 66), (2) postoperative day (POD) 5 (n = 28), or (3) POD 14 (n = 23). Animals were sacrificed at 5 days, 8 days, 2 weeks, and 4 weeks. Tendon-bone interface was evaluated with histomorphometry. Enzyme-linked immunosorbent assay for local MMP-13 activity was performed at 8 days and 4 weeks. Biomechanical testing of the healing enthesis was performed at 8 days, 2 weeks, and 4 weeks. Serum doxycycline levels were measured at sacrifice. Statistical analysis was performed using unpaired t tests and 2-way analysis of variance (P < .05). Results Serum doxycycline levels were significantly higher in all treated groups compared with controls (1830 ± 835 vs 3 ± 3 ng/mL, respectively; P < .001). Doxycycline-treated animals demonstrated greater metachromasia and improved collagen organization at the healing enthesis at POD 5 (P < .06), POD 8 (P < .03), and 2 weeks (P < .04). The MMP-13 activity was significantly reduced in doxycycline-treated compared with control animals at POD 8 (6740 ± 2770 vs 10400 ± 2930 relative fluorescent units [RFU], respectively; P < .02) but not at 4 weeks (3600 ± 3280 vs 4530 ± 2720 RFU, respectively). The healing enthesis of animals started on doxycycline preoperatively or at POD 5 had an increased load to failure compared to controls at 2 weeks (13.6 ± 1.8 and 13.2 ± 1.94 N vs 9.1 ± 2.5 N, respectively; P < .01). Conclusion/Clinical Relevance Modulation of MMP-13 activity after rotator cuff repair may offer a novel biological pathway to augment tendon-to-bone healing.

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.

Calcium-Phosphate Matrix With or Without TGF-β3 Improves Tendon-Bone Healing After Rotator Cuff Repair

Background: Rotator cuff tendon heals by formation of an interposed zone of fibrovascular scar tissue. Recent studies demonstrate that transforming growth factor–beta 3 (TGF-β3) is associated with tissue regeneration and “scarless” healing, in contrast to scar-mediated healing that occurs with TGF-β1. Hypothesis: Delivery of TGF-β3 in an injectable calcium-phosphate matrix to the healing tendon-bone interface after rotator cuff repair will result in increased attachment strength secondary to improved bone formation and collagen organization and reduced scar formation of the healing enthesis. Study Design: Controlled laboratory study. Methods: Ninety-six male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon followed by acute repair using transosseous suture fixation. Animals were allocated into 1 of 3 groups: (1) repair alone (controls, n = 32), (2) repair augmented by application of an osteoconductive calcium-phosphate (Ca-P) matrix only (n = 32), or (3) repair augmented with Ca-P matrix + TGF-β3 (2.75 µg) at the tendon-bone interface (n = 32). Animals were euthanized at either 2 weeks or 4 weeks postoperatively. Biomechanical testing of the supraspinatus tendon-bone complex was performed at 2 and 4 weeks (n = 8 per group). Microcomputed tomography was utilized to quantitate bone microstructure at the repair site. The healing tendon-bone interface was evaluated with histomorphometry and immunohistochemical localization of collagen types I (COLI) and III (COLIII). Statistical analysis was performed using 2-way analysis of variance with significance set at P < .05. Results: There was significantly greater load to failure of the Ca-P matrix + TGF-β3 group compared with matrix alone or untreated controls at 4 weeks postoperatively (P = .04). At 2 weeks, microcomputed tomography revealed a larger volume of newly formed bone present at the healing enthesis in both experimental groups compared with the control group. By 4 weeks, this newly formed, woven bone had matured into calcified, lamellar bone. Histomorphometric analysis demonstrated significantly greater fibrocartilage and increased collagen organization at the healing tendon-bone insertion site in both experimental groups compared with the control group at 2 weeks (P = .04). Over time, TGF-β3 delivery led to greater COLI expression compared with COLIII at the healing enthesis, indicating a more favorable COLI to COLIII ratio with administration of TGF-β3. Conclusion: Augmentation with an osteoconductive Ca-P matrix at the tendon-bone repair site is associated with new bone formation, increased fibrocartilage, and improved collagen organization at the healing tendon-bone interface in the early postoperative period after rotator cuff repair. The addition of TGF-β3 significantly improved strength of the repair at 4 weeks postoperatively and resulted in a more favorable COLI/COLIII ratio. Clinical Relevance: The delivery of TGF-β3 with an injectable Ca-P matrix at the supraspinatus tendon footprint has promise to improve healing after soft tissue 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.

Thirty-Day Readmission Following Total Hip and Knee Arthroplasty – A Preliminary Single Institution Predictive Model

We sought to identify demographic or care process variables associated with increased 30-day readmission within the total hip and knee arthroplasty patient population. Using this information, we generated a model to predict 30-day readmission risk following total hip and knee arthroplasty procedures. Longer index length of stay, discharge disposition to a nursing facility, blood transfusion, general anesthesia, anemia, anticoagulation status prior to index admission, and Charlson Comorbidity Index greater than 2 were identified as independent risk factors for readmission. Care process factors during the hospital stay appear to have a large predictive value for 30-day readmission. Specific comorbidities and patient demographic factors showed less significance. The predictive nomogram constructed for primary total joint readmission had a bootstrap-corrected concordance statistic of 0.76.