The pathology of oxidative stress-induced autophagy in a chronic rotator cuff enthesis tear.

Partial-thickness rotator cuff tears (PTRCTs) are often found in daily orthopedic practice, with most of the tears occurring in middle-aged patients. An anaerobic process and imbalanced oxygenation have been observed in PTRCTs, resulting in oxidative stress. Studies have shown the roles of oxidative stress in autophagy and the potential of unregulated mechanisms causing disturbance in soft tissue healing. This article aims to review literature works and summarize the potential pathology of oxidative stress and unregulated autophagy in the rotator cuff enthesis correlated with chronicity. We collected and reviewed the literature using appropriate keywords, in addition to the manually retrieved literature. Autophagy is a normal mechanism of tissue repair or conversion to energy needed for the repair of rotator cuff tears. However, excessive mechanisms will degenerate the tendon, resulting in an abnormal state. Chronic overloading of the enthesis in PTRCTs and the hypovascular nature of the proximal tendon insertion will lead to hypoxia. The hypoxia state results in oxidative stress. An autophagy mechanism is induced in hypoxia via hypoxia-inducible factors (HIFs) 1/Bcl-2 adenovirus E1B 19-kDa interacting protein (BNIP) 3, releasing beclin-1, which results in autophagy induction. Reactive oxygen species (ROS) accumulation would induce autophagy as the regulator of cell oxidation. Oxidative stress will also remove the mammalian target of rapamycin (mTOR) from the induction complex, causing phosphorylation and initiating autophagy. Hypoxia and endoplasmic reticulum (ER) stress would initiate unfolded protein response (UPR) through protein kinase RNA-like ER kinase (PERK) and activate transcription factor 4, which induces autophagy. Oxidative stress occurring in the hypovascularized chronic rotator cuff tear due to hypoxia and ROS accumulation would result in unregulated autophagy directly or autophagy mediated by HIF-1, mTOR, and UPR. These mechanisms would disrupt enthesis healing.

Combined Growth Factor Hydrogel Enhances Rotator Cuff Enthesis Healing in Rat But Not Sheep Model.

We hypothesized that a combined growth factor hydrogel would improve chronic rotator cuff tear healing in a rat and sheep model. Insulin-like growth factor 1, transforming growth factor ß1, and parathyroid hormone were combined into a tyraminated poly-vinyl-alcohol (PVA-Tyr) hydrogel and applied directly at the enthesis. In total, 30 Sprague-Dawley rats and 16 Romney ewes underwent unilateral rotator cuff tenotomy and then delayed repairs were performed after 3-4 weeks. The animals were divided into a control group (repair alone) and treatment group. The rotator cuffs were harvested at 12 weeks after surgery for biomechanical and histological analyses of the repair site. In the rat model, the stress at failure and Young's modulus were higher in the treatment group in comparison with the control group (73% improvement, p = 0.010 and 56% improvement, p = 0.028, respectively). Histologically, the repaired entheses in the treatment group demonstrated improved healing with higher semi-quantitative scores (10.1 vs. 6.55 of 15, p = 0.032). In the large animal model, there was no observable treatment effect. This PVA-Tyr bound growth factor system holds promise for improving rotator cuff healing. However, our approach was not scalable from a small to a large animal model. Further tailoring of this growth factor delivery system is still required. Level of Evidence: Basic Science Study; Biomechanics and Histology; Animal Model Impact Statement Previous studies using single-growth factor treatment to improve enthesis healing after rotator cuff repair have reported promising, but inconsistent results. A novel approach is to combine multiple growth factors using controlled-release hydrogels that mimic the normal healing process. In this study, we report that a combined growth factor hydrogel can improve the histological quality and strength of rotator cuff repair in a rat chronic tear model. This novel hydrogel growth factor treatment has the potential to be used in human clinical applications to improve healing after rotator cuff repair.

Effect of PARP-1 Inhibition on Rotator Cuff Healing: A Feasibility Study Using Veliparib in a Rat Model of Acute Rotator Cuff Repair.

BACKGROUND: PARP-1 (poly[ADP-ribose]) was shown to influence the inflammatory response after rotator cuff tear, leading to fibrosis, muscular atrophy, and fatty infiltration in mouse rotator cuff degeneration. So far, it is not known how PARP-1 influences enthesis healing after rotator cuff tear repair. HYPOTHESIS/PURPOSE: This study aimed to examine the feasibility of oral PARP-1 inhibition and investigate its influence on rat supraspinatus enthesis and muscle healing after rotator cuff repair. The hypothesis was that oral PARP-1 inhibition would improve enthesis healing after acute rotator cuff repair in a rat model. STUDY DESIGN: Controlled laboratory study. METHODS: In 24 Sprague-Dawley rats, the supraspinatus tendon was sharply detached and immediately repaired with a single transosseous suture. The rats were randomly allocated into 2 groups, with the rats in the inhibitor group receiving veliparib with a target dose of 12.5 mg/kg/d via drinking water during the postoperative recovery period. The animals were sacrificed 8 weeks after surgery. For the analysis, macroscopic, biomechanical, and histologic methods were used. RESULTS: Oral veliparib was safe for the rats, with no adverse effects observed. In total, the inhibitor group had a significantly better histologic grading of the enthesis with less scar tissue formation. The macroscopic cross-sectional area of the supraspinatus muscles was 10.5% higher (P = .034) in the inhibitor group, which was in agreement with an 8.7% higher microscopic muscle fiber diameter on histologic sections (P < .0001). There were no statistically significant differences in the biomechanical properties between the groups. CONCLUSION: This study is the first to investigate the influence of PARP-1 inhibition on healing enthesis. On the basis of these findings, we conclude that oral veliparib, which was previously shown to inhibit PARP-1 effectively, is safe to apply and has beneficial effects on morphologic enthesis healing and muscle fiber size. CLINICAL RELEVANCE: Modulating the inflammatory response through PARP-1 inhibition during the postoperative healing period is a promising approach to improve enthesis healing and reduce rotator cuff retearing. With substances already approved by the Food and Drug Administration, PARP-1 inhibition bears high potential for future translation into clinical application.

The recombinant human fibroblast growth factor-18 (sprifermin) improves tendon-to-bone healing by promoting chondrogenesis in a rat rotator cuff repair model.

BACKGROUND: Rotator cuff healing is improved by reconstructing the fibrocartilaginous structure of the tendon-to-bone enthesis. Fibroblast growth factor (FGF)-18 (sprifermin) is a well-known growth factor that improves articular cartilage repair via its anabolic effect. This study aimed to investigate the effect of recombinant human FGF-18 (rhFGF-18) on the chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro and tendon-to-bone healing in a rat model of rotator cuff repair. METHODS: Histological and reverse transcription-quantitative real-time polymerase chain reaction analyses of chondral pellets cultured with different concentrations of rhFGF-18 were performed. Bilateral detachment and repair of the supraspinatus tendon were performed on rats. The rats were administered 0.2 mL of sodium alginate (SA) hydrogel with (rhFGF-18/SA group, n = 12) or without (SA group, n = 12) 20 µg of rhFGF-18 into the repaired side. The simple repair group (n = 12) served as a control. At 4 and 8 weeks after surgery, histological analysis and biomechanical tests were performed. RESULTS: After chondrogenesis induction, compared with the control group, 10 ng/mL of rhFGF-18 increased pellet volume significantly (P = .002), with improved histological staining. It was noted that 10 ng/mL of rhFGF-18 upregulated the mRNA expression (relative ratio to control) of aggrecan (2.59 ± 0.29, P < .001), SRY-box transcription factor 9 (1.88 ± 0.05, P < .001), and type II collagen (1.46 ± 0.18, P = .009). At 4 and 8 weeks after surgery, more fibrocartilage and cartilaginous extracellular matrix was observed in rhFGF-18/SA-treated rats. The semiquantitative data from picrosirius red staining test were 31.1 ± 4.5 vs. 61.2 ± 4.1 at 4 weeks (P < .001) and 61.5 ± 2.8 vs. 80.5 ± 10.5 at 8 weeks (P = .002) (control vs. rhFGF-18/SA). Ultimate failure load (25.42 ± 3.61 N vs. 18.87 ± 2.71 N at 4 weeks and 28.63 ± 5.22 N vs. 22.15 ± 3.11 N at 8 weeks; P = .006 and P = .03, respectively) and stiffness (18.49 ± 1.38 N/mm vs. 14.48 ± 2.01 N/mm at 8 weeks, P = .01) were higher in the rhFGF-18/SA group than in the control group. CONCLUSION: rhFGF-18 promoted chondrogenesis in the hBMSCs in vitro. rhFGF-18/SA improved tendon-to-bone healing in the rats by promoting regeneration of the fibrocartilage enthesis. rhFGF-18 (sprifermin) may be beneficial in improving tendon-to-bone healing after rotator cuff repair.

Obesity Impairs Enthesis Healing After Rotator Cuff Repair in a Rat Model.

BACKGROUND: Being overweight or obese is associated with poor outcomes and an increased risk of failure after rotator cuff (RC) surgery. However, the effect of obesity on enthesis healing has not been well characterized. HYPOTHESES: Diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of RC repair, and a dietary intervention in the perioperative period would improve enthesis healing. STUDY DESIGN: Controlled laboratory study. METHODS: Male Sprague-Dawley rats were divided into 3 weight-matched groups (n = 26 per group): control diet (CD), high-fat diet (HFD), or HFD until surgery and then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate repair. Animals were sacrificed, and RCs were harvested at 2 and 12 weeks after surgery for biomechanical and histological evaluations. Metabolic end points were assessed using dual-energy X-ray absorptiometry and plasma analyses. RESULTS: DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. At 12 weeks after surgery, the body fat percentage (P = .0021) and plasma leptin concentration (P = .0025) were higher in the HFD group compared with the CD group. Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD groups compared with the CD group at 12 weeks after surgery, with semiquantitative scores of 6.20 (P = .0078), 4.98 (P = .0003), and 8.68 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure (P = .0278) at 12 weeks after surgery compared with the CD group, while the load to failure in the HFD group was low but not significantly different (P = .0960). There were no differences in the biomechanical and histological results between the groups at 2 weeks after surgery. Body mass at the time of surgery, plasma leptin concentration, and body fat percentage were negatively correlated with histology scores and plasma leptin concentration was correlated with load to failure at 12 weeks after surgery. CONCLUSION: DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring a normal weight with dietary changes after surgery did not improve healing outcomes. CLINICAL RELEVANCE: Obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after surgery. Exploring interventions that improve the metabolic state of obese patients and counseling patients appropriately about their modest expectations after repair should be considered.

Cellular Complexity at the Interface: Challenges in Enthesis Tissue Engineering.

The complex heterogeneous cellular environment found in tendon-to-bone interface makes this structure a challenge for interface tissue engineering. Orthopedic surgeons still face some problems associated with the formation of fibrotic tissue or re-tear occurring after surgical re-attachment of tendons to the bony insertion or the application of grafts. Unfortunately, an understanding of the cellular component of enthesis lags far behind of other well-known musculoskeletal interfaces, which blocks the development of new treatment options for the healing and regeneration of this multifaceted junction. In this chapter, the main characteristics of tendon and bone cell populations are introduced, followed by a brief description of the interfacial cellular niche, highlighting molecular mechanisms governing tendon-to-bone attachment and mineralization. Finally, we describe and critically assess some challenges faced concerning the use of cell-based strategies in tendon-to-bone healing and regeneration.

Von Hippel-Lindau (VHL) protein antagonist, VH298, promotes functional activities of tendon-derived stem cells and accelerates healing of entheses in rats by inhibiting ubiquitination of hydroxy-HIF-1α.

Enthesis is the region where a tendon attaches to a bone. It is a relatively vulnerable position, and in most cases surgical treatment is required upon rupture. The reconstructed enthesis is usually weaker compared to the original, and is prone to rupture again. Hypoxia-inducible factor-1 α (HIF-1α) is known to be involved in extensive activities in cells. It is inhibited under normoxic conditions, and undergoes two essential processes, hydroxylation and ubiquitination, the latter of which has been largely unexplored. Herein, we measured the levels of HIF-1α and hydroxy-HIF-1α in VH298-treated rat tendon-derived stem cells (TDSCs) by immunoblotting. We also detected the proliferation of TDSCs using CCK-8 assay and the mRNA levels of related genes by quantitative RT-PCR. The TDSCs were observed to be induced and the chondrogenic differentiation related genes were found to be enhanced. We also simulated in-vitro wounding in a scratch test and reconstructed the enthesis in a rat model of Achilles tendon by classical surgery followed by administration of phosphate buffer saline (PBS) injection or VH298 injection. We observed that HIF-1α and hydroxy-HIF-1α levels were increased in VH298-treated TDSCs in a dose- and time-dependent manner. Thirty micromolar VH298 could significantly increase cell proliferation, migration, and expression of collagen-1α, collagen-3α, decorin, tenomodulin, tenascin C genes, and chondrogenic differentiation-related genes, collagen-2α, SRY-box9, aggrecan. VH298-treated enthesis could tolerate more load-to-failure, had a better healing pattern, and activation of HIF signaling pathway. VH298 can thus enhance the functional activities of TDSCs, enhance their chondrogenic differentiation potential, and accelerate enthesis healing by inhibiting the ubiquitination of hydroxy-HIF-1α.

The effectiveness of demineralized cortical bone matrix in a chronic rotator cuff tear model.

BACKGROUND: The purpose of this study was to assess the effect of demineralized bone matrix (DBM) on rotator cuff tendon-bone healing. The hypothesis was that compared with a commercially available dermal matrix scaffold, DBM would result in a higher bone mineral density and regenerate a morphologically superior enthesis in a rat model of chronic rotator cuff degeneration. METHODS: Eighteen female Wistar rats underwent unilateral detachment of the supraspinatus tendon. Three weeks later, tendon repair was carried out in animals randomized into 3 groups: group 1 animals were repaired with DBM (n = 6); group 2 received augmentation with the dermal scaffold (n = 6); and group 3 (controls) underwent nonaugmented tendon-bone repair (n = 6). Specimens were retrieved at 6 weeks postoperatively for histologic analysis and evaluation of bone mineral density. RESULTS: No failures of tendon-bone healing were noted throughout the study. All groups demonstrated closure of the tendon-bone gap with a fibrocartilaginous interface. Dermal collagen specimens exhibited a disorganized structure with significantly more abnormal collagen fiber arrangement and cellularity than in the DBM-based repairs. Nonaugmented repairs exhibited a significantly higher bone mineral density than in DBM and the dermal collagen specimens and were not significantly different from control limbs that were not operated on. CONCLUSION: The application of DBM to a rat model of chronic rotator cuff degeneration did not improve the composition of the healing enthesis compared with nonaugmented controls and a commercially available scaffold. However, perhaps the most important finding of this study was that the control group demonstrated a similar outcome to augmented repairs.