Targeting the hedgehog signaling pathway to improve tendon-to-bone integration.

OBJECTIVE: While the role of hedgehog (Hh) signaling in promoting zonal fibrocartilage production during development is well-established, whether this pathway can be leveraged to improve tendon-to-bone repair in adults is unknown. Our objective was to genetically and pharmacologically stimulate the Hh pathway in cells that give rise to zonal fibrocartilaginous attachments to promote tendon-to-bone integration. DESIGN: Hh signaling was stimulated genetically via constitutive Smo (SmoM2 construct) activation of bone marrow stromal cells or pharmacologically via systemic agonist delivery to mice following anterior cruciate ligament reconstruction (ACLR). To assess tunnel integration, we measured mineralized fibrocartilage (MFC) formation in these mice 28 days post-surgery and performed tunnel pullout testing. RESULTS: Hh pathway-related genes increased in cells forming the zonal attachments in wild-type mice. Both genetic and pharmacologic stimulation of the Hh pathway increased MFC formation and integration strength 28 days post-surgery. We next conducted studies to define the role of Hh in specific stages of the tunnel integration process. We found Hh agonist treatment increased the proliferation of the progenitor pool in the first week post-surgery. Additionally, genetic stimulation led to continued MFC production in the later stages of the integration process. These results indicate that Hh signaling plays an important biphasic role in cell proliferation and differentiation towards fibrochondrocytes following ACLR. CONCLUSION: This study reveals a biphasic role for Hh signaling during the tendon-to-bone integration process after ACLR. In addition, the Hh pathway is a promising therapeutic target to improve tendon-to-bone repair outcomes.

Risk Factors for Cerebral Desaturation Events During Shoulder Surgery in the Beach Chair Position.

PURPOSE: The goals of this study were 2-fold: (1) to determine the risk factors for cerebral desaturation events (CDEs) after implementation of a comprehensive surgical and anesthetic protocol consisting of patient risk stratification, maintenance of normotensive anesthesia, and patient positioning in a staged fashion, and (2) to assess for subclinical neurologic decline associated with intraoperative ischemic events through cognitive testing. METHODS: One hundred patients undergoing shoulder surgery in the beach chair position were stratified for risk of CDE based on Framingham stroke criteria, body mass index (BMI), and history of cerebrovascular accidents. Cerebral oxygen saturation was monitored with near-infrared spectroscopy. As per a standardized protocol, mean arterial pressure was maintained between 70 and 90 mm Hg. The head was raised in 2 stages separated by 3 minutes. CDE were defined as >20% drop from baseline or <55% O2 absolute threshold. Patients completed a Mini-Mental State Examination during preoperative examination and at the first postoperative visit. RESULTS: The CDE rate was 4% overall and 4.3% in patients undergoing general anesthesia. Forty-five patients were in the higher risk category, and all CDEs occurred in that group. Patients with a Framingham score ≥ 10 or BMI ≥ 35 who underwent general anesthesia had an increased risk of CDE (P = .04). No significant change was noted in Mini-Mental State Examination scores between pre- and postoperative visits. No correlation was shown between CDE and history of diabetes, smoking, cardiovascular disease, or left ventricular hypertrophy. CONCLUSIONS: Our observed CDE rate was lower than previously reported rates, likely because of risk stratification, staged positioning, and normotensive anesthesia. Framingham score ≥ 10 and BMI ≥ 35 are risk factors for CDE in the beach chair position. LEVEL OF EVIDENCE: Level II, prospective observational study with >80% follow-up.

Effects of pulsed electromagnetic field therapy at different frequencies and durations on rotator cuff tendon-to-bone healing in a rat model.

BACKGROUND: Rotator cuff tears affect millions of individuals each year, often requiring surgical intervention. However, repair failure remains common. We have previously shown that pulsed electromagnetic field (PEMF) therapy improved tendon-to-bone healing in a rat rotator cuff model. The purpose of this study was to determine the influence of both PEMF frequency and exposure time on rotator cuff healing. METHODS: Two hundred ten Sprague-Dawley rats underwent acute bilateral supraspinatus injury and repair followed by either Physio-Stim PEMF or high-frequency PEMF therapy for 1, 3, or 6 hours daily. Control animals did not receive PEMF therapy. Mechanical and histologic properties were assessed at 4, 8, and 16 weeks. RESULTS: Improvements in different mechanical properties at various endpoints were identified for all treatment modalities when compared with untreated animals, regardless of PEMF frequency or duration. Of note, 1 hour of Physio-Stim treatment showed significant improvements in tendon mechanical properties across all time points, including increases in both modulus and stiffness as early as 4 weeks. Collagen organization improved for several of the treatment groups compared with controls. In addition, improvements in type I collagen and fibronectin expression were identified with PEMF treatment. An important finding was that no adverse effects were identified in any mechanical or histologic property. CONCLUSIONS: Overall, our results suggest that PEMF therapy has a positive effect on rat rotator cuff healing for each electromagnetic fundamental pulse frequency and treatment duration tested in this study.

Incidence of peripheral nerve injury during shoulder arthroplasty when motor evoked potentials are monitored.

To report the incidence of clinically detectable nerve injuries when utilizing transcranial electrical motor evoked potentials (MEPs) during shoulder arthroplasty. A retrospective review of patients undergoing shoulder arthroplasty with continuous IONM was performed. The criteria for nerve alerts was an 80% amplitude reduction in MEPs. The primary outcome measure was post-operative clinically detectable nerve deficit. An additional retrospective analysis on a subset of cases using an all-or-none (100% amplitude reduction) criterion applied to the deltoid was performed. Two hundred eighty four arthroplasty cases were included. There were no permanent post-operative nerve injuries and two transient nerve injuries (0.7%). MEP alerts occurred in 102 cases (36.2%). Nineteen (6.7%) cases did not have signals return above alert threshold at closure. These cases were significantly associated with post-operative nerve injury (p = 0.03). There were no false negatives, making sensitivity 100% and specificity was 93.9%. In the subset of cases in which an all-or-none criterion was retrospectively applied to just the deltoid, MEP alerts occurred in just 17.9% of cases; specificity improved to 98.0%. We conclude that utilization of the real-time diagnostic MEP data during shoulder arthroplasty aids surgeons in decision making regarding impending peripheral nerve injuries.

The timing of retears after arthroscopic rotator cuff repair.

BACKGROUND: Little is known about the time dependence of the failure rate of surgically repaired rotator cuffs. Retears are significant, as they are common and may lead to less satisfactory outcomes and additional operations. Their timing is critical foundational information for understanding failure mechanisms. However, this remains unclear. Currently, there exist a number of studies that have reported retear rates at specific time points. Combining data from these publications can reveal when cuffs retear, which will help inform expectations and guidelines for progression of activity after surgery. METHODS: PubMed, Medline, and Embase were searched for studies relating to rotator cuff repair. Abstracts and articles were evaluated on the basis of predefined inclusion and exclusion criteria. Data were extracted from those publications that satisfied all requirements, and regression analysis was performed. RESULTS: Thirteen articles were included in the final meta-analysis. Retear rates for medium tears increased for approximately 15 months and leveled off at approximately 20%. Retear rates for large tears progressed steadily for about 12 months and approached an upper limit of approximately 40%. Retear rates for massive tears ranged from 20% to 60%, but the distribution of retear rate over time for these cuff tears is not clear from these data. CONCLUSION: Retear rates for medium and large tears generally increase until at least 10-15 months after surgery, after which they are likely to level off. Retear rates for massive tears are variable and may follow a time course different from that of other tear sizes. Retear rates depend on size of the original tear.

Effect of scapular dyskinesis on supraspinatus repair healing in a rat model.

BACKGROUND: Rotator cuff tears are common conditions that often require surgical repair to improve function and to relieve pain. Unfortunately, repair failure remains a common problem after rotator cuff repair surgery. Several factors may contribute to repair failure, including age, tear size, and time from injury. However, the mechanical mechanisms resulting in repair failure are not well understood, making clinical management difficult. Specifically, altered scapular motion (termed scapular dyskinesis) may be one important and modifiable factor contributing to the risk of repair failure. Therefore, the objective of this study was to determine the effect of scapular dyskinesis on supraspinatus tendon healing after repair. METHODS: A rat model of scapular dyskinesis was used. Seventy adult male Sprague-Dawley rats (400-450 g) were randomized into 2 groups: nerve transection of the accessory and long thoracic nerves (SD) or sham nerve transection (Sham control). After this procedure, all rats underwent unilateral detachment and repair of the supraspinatus tendon. All rats were sacrificed at 2, 4, and 8 weeks after surgery. Shoulder function, passive joint mechanics, and tendon properties (mechanical, histologic, organizational, and compositional) were evaluated. RESULTS: Scapular dyskinesis alters joint function and may lead to compromised supraspinatus tendon properties. Specifically, diminished mechanical properties, altered histology, and decreased tendon organization were observed for some parameters. CONCLUSION: This study identifies scapular dyskinesis as one underlying mechanism leading to compromise of supraspinatus healing after repair. Identifying modifiable factors that lead to compromised tendon healing will help improve clinical outcomes after repair.

Arthroscopic subscapularis repair.

As the largest and most powerful rotator cuff muscle, the subscapularis plays a critical role in proper shoulder function. The diagnosis of subscapularis tears is made based on history and physical examination and confirmed with imaging studies. Historically, subscapularis tears have been addressed in an open fashion. Recent literature has demonstrated successful all-arthroscopic repair of isolated subscapularis tears and anterosuperior rotator cuff tears. Successful arthroscopic treatment follows several critical steps: patient positioning, portal placement, obtaining adequate visualization and working space, identifying and mobilizing the subscapularis tendon, preparation of the tendon edge and lesser tuberosity, appropriate anchor placement, suture passage, and finally, secure knot-tying and fixation. Excellent functional and clinical outcomes following these steps have been demonstrated by various investigators. Additional research is needed, however, because direct comparison between all-arthroscopic and open subscapularis repair is limited, and neither technique has proved to be superior.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

Pulsed electromagnetic field therapy alters early healing in a rat model of rotator cuff injury and repair: Potential mechanisms.

Rotator cuff repair failure remains common due to poor tendon healing, particularly at the enthesis. We previously showed that pulsed electromagnetic field (PEMF) therapy improved the mechanical properties of the rat supraspinatus tendon postoperatively. However, little is known about the mechanisms behind PEMF-dependent contributions to improved healing in this injury model. The objective of this study was to determine the influence of PEMF treatment on tendon gene expression and cell composition, as well as bone microarchitecture and dynamic bone metabolism during early stages of healing. We hypothesized that PEMF treatment would amplify tendon-healing related signaling pathways while mitigating inflammation and improve bone metabolism at the repair site. Rats underwent rotator cuff injury and repair followed by assignment to either control (non-PEMF) or PEMF treatment groups. Gene and protein expression as well as tendon and bone histological assessments were performed 3, 7, 14, 21, and 28 days after injury. Gene expression data demonstrated an upregulation in the bone morphogenetic protein 2 signaling pathway and increases in pro-osteogenic genes at the insertion, supporting important processes to re-establish the tendon-bone interface. PEMF also downregulated genes related to a fibrotic healing response. Anti-inflammatory effects were demonstrated by both gene expression and macrophage phenotype. PEMF significantly increased the rate of kinetic bone formation directly adjacent to the tendon enthesis as well as the number of cuboidal surface osteoblasts (active osteoblasts) in the humeral head. This study has provided insight into how PEMF affects cellular and molecular processes in the supraspinatus tendon and adjacent bone after injury and repair.

Effect of supraspinatus tendon repair technique on the infraspinatus tendon.

Supraspinatus tendon tears are common and often propagate into larger tears that include the infraspinatus tendon, resulting in loss of function and increased pain. Previously, we showed that the supraspinatus and infraspinatus tendons mechanically interact through a range of rotation angles, potentially shielding the torn supraspinatus tendon from further injury while subjecting the infraspinatus tendon to increased risk of injury. Surgical repair of torn supraspinatus tendons is common, yet the effect of the repair on the infraspinatus tendon is unknown. Since we have established a relationship between strain in the supraspinatus and infraspinatus tendons the success of a supraspinatus tendon repair depends on its effect on the loading environment in the infraspinatus tendon. More specifically, the effect of transosseous supraspinatus tendon repair in comparison to one that utilizes suture anchors, as is commonly done with arthroscopic repairs, on this interaction through these joint positions will be evaluated. We hypothesize that at all joint positions evaluated, both repairs will restore the interaction between the two tendons. For both repairs, (1) increasing supraspinatus tendon load will increase infraspinatus tendon strain and (2) altering the rotation angle from internal to external will increase strain in the infraspinatus tendon. Strains were measured in the infraspinatus tendon insertion through a range of joint rotation angles and supraspinatus tendon loads, for the intact, transosseous, and suture anchor repaired supraspinatus tendons. Images corresponding to specific supraspinatus tendon loads were isolated for the infraspinatus tendon insertion for analysis. The effect of supraspinatus tendon repair on infraspinatus tendon strain differed with joint position. Altering the joint rotation did not change strain in the infraspinatus tendon for any supraspinatus tendon condition. Finally, increasing supraspinatus tendon load resulted in an increase in average maximum and decrease in average minimum principal strain in the infraspinatus tendon. There is a significant difference in infraspinatus tendon strain between the intact and arthroscopically (but not transosseous) repaired supraspinatus tendons that increases with greater loads. Results suggest that at low loads neither supraspinatus tendon repair technique subjects the infraspinatus tendon to potentially detrimental loads; however, at high loads, transosseous repairs may be more advantageous over arthroscopic repairs for the health of the infraspinatus tendon. Results emphasize the importance of limiting loading of the repaired supraspinatus tendon and that at low loads, both repair techniques restore the interaction to the intact supraspinatus tendon case.

Total shoulder arthroplasty in older patients: increased perioperative morbidity?

BACKGROUND: More elderly patients are becoming candidates for total shoulder arthroplasty with an increase in frequency of the procedure paralleling the rise in other total joint arthroplasties. Controversy still exists, however, regarding the perioperative morbidity of total joint arthroplasty in elderly patients, particularly those 80 years of age and older. QUESTIONS/PURPOSES: We asked whether perioperative complications and mortality, transfusion requirements, inpatient length of stay, and discharge disposition after total shoulder arthroplasty were similar in patients 80 years and older compared with those in younger patients. METHODS: We retrospectively compared the 90-day complications, mortality, and other perioperative variables after total shoulder arthroplasty in 40 patients (43 shoulders) aged 80 years and older (Group A; mean age, 82 years) with 46 patients (47 shoulders) younger than 70 years (Group B; mean age, 61 years). RESULTS: We found no differences in complication rates between Group A and B, including systemic (26% versus 11%) and local (5% versus 9%) complications or major (7% versus 2%) and minor (23% versus 17%) complications. There were no deaths in either group. Group A had an increased transfusion requirement (16% versus 2%) and a decreased number of direct to home discharges (67% versus 98%). Presence of systemic complications predicted increased length of stay in patients overall and in Group A patients. CONCLUSIONS: Total shoulder arthroplasty can be performed in patients 80 years and older with rates of perioperative complications and mortalities comparable to those of younger patients, although these older patients may require a longer period of institutional care before return to home and may be more likely to require a blood transfusion. LEVEL OF EVIDENCE: Level III, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Mechanical properties of the different rotator cuff tendons in the rat are similarly and adversely affected by age.

Rotator cuff tendon tears and tendinopathies are common injuries affecting a large portion of the population and can result in pain and joint dysfunction. Incidence of rotator cuff tears significantly increases with advancing age, and up to 90% of these tears involve the supraspinatus. Previous literature has shown that aging can lead to inferior mechanics, altered composition, and changes in structural properties of the supraspinatus. However, there is little known about changes in supraspinatus mechanical properties in context of other rotator cuff tendons. Alterations in tendon mechanical properties may indicate damage and an increased risk of rupture, and thus, the purpose of this study was to use a rat model to define age-related alterations in rotator cuff tendon mechanics to determine why the supraspinatus is more susceptible to tears due to aging than the infraspinatus, subscapularis, and teres minor. Fatigue, viscoelastic, and quasi-static properties were evaluated in juvenile, adult, aged, and geriatric rats. Aging ubiquitously and adversely affected all rotator cuff tendons tested, particularly leading to increased stiffness, decreased stress relaxation, and decreased fatigue secant and tangent moduli in geriatric animals, suggesting a common intrinsic mechanism due to aging in all rotator cuff tendons. This study demonstrates that aging has a significant effect on rotator cuff tendon mechanical properties, though the supraspinatus was not preferentially affected. Thus, we are unable to attribute the aging-associated increase in supraspinatus tears to its mechanical response alone.

Risk factors associated with atraumatic posterolateral rotatory instability.

BACKGROUND: Traumatic posterolateral rotatory instability after elbow dislocation or fracture dislocation has been well described. However, few reports cover atraumatic posterolateral rotatory instability as a cause of lateral-sided elbow pain. We assessed the risk factors and epidemiology of atraumatic posterolateral rotatory instability in a case-control study. METHODS: A retrospective review of all patients treated operatively for atraumatic posterolateral rotatory instability during a 6-year period was compared with a group of patients with extensor carpi radialis brevis tendinopathy without instability treated during the same time period. Bivariate and multiple logistic regression statistical analyses were used to investigate the following risk factors: gender, age, hand dominance, diabetes, smoking, body mass index, corticosteroid injection history, and duration of symptoms. Disabilities of the Arm, Shoulder, and Hand and pain scores were obtained preoperatively and postoperatively. RESULTS: Thirteen patients with atraumatic posterolateral rotatory instability were compared with 12 patients with extensor carpi radialis brevis tendinopathy. Multivariate analysis revealed patients with atraumatic posterolateral rotatory instability were more likely to have multiple corticosteroid injections (P = .05) and present with a longer duration of symptoms (P = .03). Postoperative pain scores improved in both groups. CONCLUSIONS: Atraumatic posterolateral rotatory instability should be considered in the differential diagnosis of lateral elbow when patients present with a protracted clinical course. Statistically, posterolateral rotatory instability patients more often present with a history of multiple corticosteroid injections.

Chronic Nicotine Exposure Minimally Affects Rat Supraspinatus Tendon Properties and Bone Microstructure.

Cigarette smoking is the largest cause of preventable deaths, and a known risk factor for musculoskeletal issues including rotator cuff tendon tears. Tendon degeneration is believed to be due in part to changes in tendon cell health and collagen structure. Several studies have demonstrated that exposure to nicotine negatively impacts tendon healing, but surprisingly, nicotine exposure was shown to increase rat supraspinatus tendon stiffness. In order to address this seeming contradiction, the objective of this study was to comprehensively investigate the effects of long-term (18 weeks) exposure of nicotine on tendon-to-bone microstructural properties in a rat model. We hypothesized that long term subcutaneous nicotine delivery would lead to diminished tendon mechanical properties, decreased bone microstructure in the humeral head, and altered tendon cell morphology compared to age-matched control rats receiving saline. Results demonstrated a small decrease in tendon size and stiffness, with decreased cell density in the tendon midsubstance. However, no differences were found in the enthesis fibrocartilage or in the underlying subchondral or trabecular bone. In conclusion, our study revealed limited effects of nicotine on the homeostatic condition of the supraspinatus tendon, enthesis, and underlying bone. Future studies are needed to ascertain effects of other components of tobacco products.

Preoperative tendon retraction, not smoking, is a risk factor for failure with continuity after rotator cuff repair.

BACKGROUND: Smoking is a poor prognostic factor for healing after rotator cuff repair and is associated with inferior results. We hypothesized that smokers would have higher recurrent tear rates and more postoperative myotendinous junction (MTJ) retraction in healed repairs than nonsmokers three months postoperatively. METHODS: Rotator cuff repairs (RCRs) were retrospectively reviewed over a 2-year period. Patients underwent magnetic resonance imaging (MRI) within 6 months prior to surgery and again at 3 months postoperatively. Seventy-nine patients were included and stratified by smokers versus nonsmokers. Baseline patient demographics, tear characteristics, and surgical factors were collected. Preoperative and postoperative MRIs were assessed to quantify the MTJ position and to establish the recurrent tear rate. RESULTS: For the total cohort (nonsmokers, n = 56; smokers, n = 23), significant differences in age, race, and traumatic onset of injury existed between groups. There were no significant differences in recurrent tear between smokers (26%) and nonsmokers (27%), but nonsmokers were more satisfied. For patients with healed RCRs (nonsmokers, n = 41; smokers, n = 17), there were significant differences in race. On univariate analysis, nonsmokers had a significantly more lateral MTJ postoperatively (P = 0.05). On multivariable regression analysis, medialized postoperative MTJ position in healed cuffs was driven only by greater preoperative rotator cuff retraction preoperatively. There were no significant differences in MTJ position based on smoking status for patients with healed RCRs. CONCLUSION: Smoking does not appear to be an independent risk factor for postoperative MTJ retraction in healed RCRs, also known as failure in continuity. Preoperative tear size and retraction play the biggest roles in predicting postoperative MTJ position, regardless of smoking status. There are no significant differences in patient-reported outcomes for patients with healed RCRs, but nonsmokers had more satisfaction following RCR in the total cohort. LEVEL OF EVIDENCE: Level III; Retrospective cohort study; Diagnostic study.

Effect of anterior supraspinatus tendon partial-thickness tears on infraspinatus tendon strain through a range of joint rotation angles.

BACKGROUND: Rotator cuff tears are common shoulder problems whose propagation is difficult to predict because of the structural and mechanical inhomogeneity of the supraspinatus tendon. We have previously shown that the supraspinatus and infraspinatus tendons interact mechanically when the supraspinatus tendon is intact or exhibits a full-thickness tear, so that an increase in supraspinatus tendon strain is paralleled by an increase in infraspinatus tendon strain. Such interaction is critical and suggests that an increase in infraspinatus tendon strain that accompanies an increase in supraspinatus tendon strain may shield the supraspinatus tendon from further injury, but increase the risk of injury to the infraspinatus tendon. In this study, the mechanical interactions between the supraspinatus and infraspinatus tendons were evaluated for the commonly occurring supraspinatus tendon partial-thickness tears through a range of rotation angles. METHODS: For each joint rotation and supraspinatus tendon tear size evaluated, the supraspinatus tendon was loaded, and images corresponding to 5 N, 30 N, 60 N, and 90 N of supraspinatus tendon load were isolated for the speckle painted supraspinatus and infraspinatus tendons. A region of interest outlining the insertion site was isolated and displacements between the 5 N loaded image and each of the others were measured, from which normalized average principal strains were quantified in both tendons. RESULTS: The observed effect on infraspinatus tendon strain paralleled that observed on strain in the supraspinatus tendon. Introducing a supraspinatus tendon partial-thickness tear and increasing load caused an increase in normalized average maximum and a decrease in normalized average minimum principal strain in the infraspinatus tendon. Increasing rotation angle from internal to external rotation caused a general decrease in normalized average maximum and increase in normalized average minimum principal strain in both tendons. CONCLUSION: The supraspinatus and infraspinatus tendons mechanically interact for the intact and partially torn supraspinatus tendons for neutral and rotated glenohumeral joint.

Cells from a GDF5 origin produce zonal tendon-to-bone attachments following anterior cruciate ligament reconstruction.

Following anterior cruciate ligament (ACL) reconstruction surgery, a staged repair response occurs where cells from outside the tendon graft participate in tunnel integration. The mechanisms that regulate this process, including the specific cellular origin, are poorly understood. Embryonic cells expressing growth and differentiation factor 5 (GDF5) give rise to several mesenchymal tissues in the joint and epiphyses. We hypothesized that cells from a GDF5 origin, even in the adult tissue, would give rise to cells that contribute to the stages of repair. ACLs were reconstructed in Gdf5-Cre;R26R-tdTomato lineage tracing mice to monitor the contribution of Gdf5-Cre;tdTom+ cells to the tunnel integration process. Anterior-posterior drawer tests demonstrated 58% restoration in anterior-posterior stability. Gdf5-Cre;tdTom+ cells within the epiphyseal bone marrow adjacent to tunnels expanded in response to the injury by 135-fold compared with intact controls to initiate tendon-to-bone attachments. They continued to mature the attachments yielding zonal insertion sites at 4 weeks with collagen fibers spanning across unmineralized and mineralized fibrocartilage and anchored to the adjacent bone. The zonal attachments possessed tidemarks with concentrated alkaline phosphatase activity similar to native entheses. This study established that mesenchymal cells from a GDF5 origin can contribute to zonal tendon-to-bone attachments within bone tunnels following ACL reconstruction.

Biocompatibility and bioactivity of an FGF-loaded microsphere-based bilayer delivery system.

Many drug delivery systems rely on degradation or dissolution of the carrier material to regulate release. In cases where mechanical support is required during regeneration, this necessitates composite systems in which the mechanics of the implant are decoupled from the drug release profile. To address this need, we developed a system in which microspheres (MS) were sequestered in a defined location between two nanofibrous layers. This bilayer delivery system (BiLDS) enables simultaneous structural support and decoupled release profiles. To test this new system, PLGA (poly-lactide-co-glycolic acid) microspheres were prepared using a water-in-oil-in-water (w/o/w) emulsion technique and incorporated Alexa Fluor-tagged bovine serum albumin (BSA) and basic fibroblast growth factor (bFGF). These MS were secured in a defined pocket between two polycaprolactone (PCL) nanofibrous scaffolds, where the layered scaffolds provide a template for new tissue formation while enabling independent and local release from the co-delivered MS. Scanning electron microscopy (SEM) images showed that the assembled BiLDS could localize and retain MS in the central pocket that was surrounded by a continuous seal formed along the margin. Cell viability and proliferation assays showed enhanced cell activity when exposed to BiLDS containing Alexa Fluor-BSA/bFGF-loaded MS, both in vitro and in vivo. MS delivered via the BiLDS system persisted in a localized area after subcutaneous implantation for at least 4 weeks, and bFGF release increased colonization of the implant. These data establish the BiLDS technology as a sustained in vivo drug delivery platform that can localize protein and other growth factor release to a surgical site while providing a structural template for new tissue formation. STATEMENT OF SIGNIFICANCE: Localized and controlled delivery systems for the sustained release of drugs are essential. Many strategies have been developed for this purpose, but most rely on degradation (and loss of material properties) for delivery. Here, we developed a bilayer delivery system (BiLDS) that decouples the physical properties of a scaffold from its delivery kinetics. For this, biodegradable PLGA microspheres were sequestered within a central pocket of a slowly degrading nanofibrous bilayer. Using this device, we show enhanced cell activity with FGF delivery from the BiLDS both in vitro and in vivo. These data support that BiLDS can localize sustained protein and biofactor delivery to a surgical site while also serving as a mechanical scaffold for tissue repair and regeneration.

Localized delivery of ibuprofen via a bilayer delivery system (BiLDS) for supraspinatus tendon healing in a rat model.

The high prevalence of tendon retear following rotator cuff repair motivates the development of new therapeutics to promote improved tendon healing. Controlled delivery of non-steroidal anti-inflammatory drugs to the repair site via an implanted scaffold is a promising option for modulating inflammation in the healing environment. Furthermore, biodegradable nanofibrous delivery systems offer an optimized architecture and surface area for cellular attachment, proliferation, and infiltration while releasing soluble factors to promote tendon regeneration. To this end, we developed a bilayer delivery system (BiLDS) for localized and controlled release of ibuprofen (IBP) to temporally mitigate inflammation and enhance tendon remodeling following surgical repair by promoting organized tissue formation. In vitro evaluation confirmed the delayed and sustained release of IBP from Labrafil-modified poly(lactic-co-glycolic) acid microspheres within sintered poly(ε-caprolactone) electrospun scaffolds. Biocompatibility of the BiLDS was demonstrated with primary Achilles tendon cells in vitro. Implantation of the IBP-releasing BiLDS at the repair site in a rat rotator cuff injury and repair model led to decreased expression of proinflammatory cytokine, tumor necrotic factor-α, and increased anti-inflammatory cytokine, transforming growth factor-ß1. The BiLDS remained intact for mechanical reinforcement and recovered the tendon structural properties by 8 weeks. These results suggest the therapeutic potential of a novel biocompatible nanofibrous BiLDS for localized and tailored delivery of IBP to mitigate tendon inflammation and improve repair outcomes. Future studies are required to define the mechanical implications of an optimized BiLDS in a rat model beyond 8 weeks or in a larger animal model.

Amplifying Bone Marrow Progenitors Expressing α-Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon-to-Bone Repair.

Traditional tendon-to-bone repair where the tendon is reattached to bone via suture anchors often results in disorganized scar production rather than the formation of a zonal insertion. In contrast, ligament reconstructions where tendon grafts are passed through bone tunnels can yield zonal tendon-to-bone attachments between the graft and adjacent bone. Therefore, ligament reconstructions can be used to study mechanisms that regulate zonal tendon-to-bone repair in the adult. Anterior cruciate ligament (ACL) reconstructions are one of the most common reconstruction procedures and while we know that cells from outside the graft produce the attachments, we have not yet established specific cell populations that give rise to this tissue. To address this knowledge gap, we performed ACL reconstructions in lineage tracing mice where α-smooth muscle actin (αSMACreERT2) was used to label αSMA-expressing progenitors within the bone marrow that produced zonal attachments. Expression of αSMA was increased during early stages of the repair process such that the contribution of SMA-labeled cells to the tunnel integration was highest when tamoxifen was delivered in the first week post-surgery. The zonal attachments shared features with normal entheses, including tidemarks oriented perpendicularly to collagen fibers, Col1a1-expressing cells, alkaline phosphatase activity, and proteoglycan-rich staining. Finally, the integration strength increased with time, requiring 112% greater force to remove the graft from the tunnel at 28 days compared with 14 days post-surgery. Future studies will target these progenitor cells to define the pathways that regulate zonal tendon-to-bone repair in the adult. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:105-116, 2020.