Charge-Reversed Exosomes for Targeted Gene Delivery to Cartilage for Osteoarthritis Treatment.

Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan (GAG) rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address these issues, this study develops surface-modified cartilage-targeting exosomes as non-viral carriers for gene therapy. Charge-reversed cationic exosomes are engineered for mRNA delivery by anchoring cartilage targeting optimally charged arginine-rich cationic motifs into the anionic exosome bilayer by using buffer pH as a charge-reversal switch. Cationic exosomes penetrated through the full-thickness of early-stage arthritic human cartilage owing to weak-reversible ionic binding with GAGs and efficiently delivered the encapsulated eGFP mRNA to chondrocytes residing in tissue deep layers, while unmodified anionic exosomes do not. When intra-articularly injected into destabilized medial meniscus mice knees with early-stage OA, mRNA loaded charge-reversed exosomes overcame joint clearance and rapidly penetrated into cartilage, creating an intra-tissue depot and efficiently expressing eGFP; native exosomes remained unsuccessful. Cationic exosomes thus hold strong translational potential as a platform technology for cartilage-targeted non-viral delivery of any relevant mRNA targets for OA treatment.

Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes.

The nuclear localized protein deacetylase, SIRT6, has been identified as a crucial regulator of biological processes that drive aging. Among these processes, SIRT6 can promote resistance to oxidative stress conditions, but the precise mechanisms remain unclear. The objectives of this study were to examine the regulation of SIRT6 activity by age and oxidative stress and define the role of SIRT6 in maintaining redox homeostasis in articular chondrocytes. Although SIRT6 levels did not change with age, SIRT6 activity was significantly reduced in chondrocytes isolated from older adults. Using dimedone-based chemical probes that detect oxidized cysteines, we identified that SIRT6 is oxidized in response to oxidative stress conditions, an effect that was associated with reduced SIRT6 activity. Enhancement of SIRT6 activity through adenoviral SIRT6 overexpression specifically increased the basal levels of two antioxidant proteins, peroxiredoxin 1 (Prx1) and sulfiredoxin (Srx) and decreased the levels of an inhibitor of antioxidant activity, thioredoxin interacting protein (TXNIP). Conversely, in chondrocytes derived from mice with cartilage specific Sirt6 knockout, Sirt6 loss decreased Prx1 levels and increased TXNIP levels. SIRT6 overexpression decreased nuclear-generated H2O2 levels and oxidative stress-induced accumulation of nuclear phosphorylated p65. Our data demonstrate that SIRT6 activity is altered with age and oxidative stress conditions associated with aging. SIRT6 contributes to chondrocyte redox homeostasis by regulating specific members of the Prx catalytic cycle. Targeted therapies aimed at preventing the age-related decline in SIRT6 activity may represent a novel strategy to maintain redox balance in joint tissues and decrease catabolic signaling events implicated in osteoarthritis (OA).

Role of the Hypoxia-Inducible Factor Pathway in Normal and Osteoarthritic Meniscus and in Mice after Destabilization of the Medial Meniscus.

OBJECTIVE: Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development. METHODS: Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway. Meniscus cells were treated with pro-inflammatory stimuli, including interleukins (IL)-1ß, IL-6, transforming growth factor (TGF)-α, and fibronectin fragments (FnF). Target genes were also evaluated with HIF-1α and HIF-2α (Epas1) overexpression and knockdown. Wild-type (n = 36) and Epas1+/- (n = 30) heterozygous mice underwent destabilization of the medial meniscus (DMM) surgery and were evaluated at 2 and 4 weeks postoperatively for osteoarthritis development using histology. RESULTS: HIF-1α and HIF-2α immunostaining and gene expression did not differ between normal and osteoarthritic meniscus. While pro-inflammatory stimulation significantly increased both catabolic and anabolic gene expression in the meniscus, HIF-1α and Epas1 expression levels were not significantly altered. Epas1 overexpression significantly increased Col2a1 expression. Both wild-type and Epas1+/- mice developed osteoarthritis following DMM surgery. There were no significant differences between genotypes at either time point. CONCLUSION: The HIF pathway is likely not responsible for osteoarthritic changes in the human meniscus. Additionally, Epas1 insufficiency does not protect against osteoarthritis development in the mouse at early time points after DMM surgery. The HIF pathway may be more important for protection against catabolic stress.

Bony Ingrowth of Coil-Type Open-Architecture Anchors Compared With Screw-Type PEEK Anchors for the Medial Row in Rotator Cuff Repair: A Randomized Controlled Trial.

PURPOSE: To evaluate outcomes of screw-type and coil-type open-architecture suture anchors with respect to bony ingrowth, release of biological markers, and patient-reported outcome measures when used in rotator cuff repair (RCR). METHODS: Forty patients undergoing arthroscopic RCR for full-thickness rotator cuff tears were enrolled and prospectively randomized to receive a screw-type (19 patients) or coil-type (21 patients) suture anchor for the medial row during repair. All repairs used a transosseous-equivalent configuration with footprint anchors laterally. Marrow elements released during surgery were evaluated for 9 cytokine markers (insulin-like growth factor 1, fibroblast growth factor 2, bone morphogenetic proteins 7 and 2, platelet-derived growth factors AA and BB, epidermal growth factor, transforming growth factor beta1, and vascular endothelial growth factor). Postoperative computed tomography scans were performed at 6 months. Range of motion, strength, and validated patient-reported outcome measures (Simple Shoulder Test, Single Assessment Numeric Evaluation, visual analog scale, and American Shoulder and Elbow Surgeons scores) were gathered before the operation and at 6 months and 1 year postoperatively. RESULTS: Bone mineral density surrounding the coil-type anchor was significantly greater than that surrounding the screw-type anchor (P = .005). Bone mineral density values within the coil-type and screw-type anchors were comparable (P = .527); however, a larger amount of total bone mineral mass (in milligrams) was shown within the coil-type anchor owing to its larger volume (P < .01). Marrow elements released at the repair site were similar between groups (P > .05). Postoperatively, no statistically significant difference was found between groups for clinical outcome measures at 6 months or 1 year. Retear and complication rates were similar between groups (P > .05). CONCLUSIONS: Both the coil-type and screw-type anchors can be reliably used for RCR and produce similar clinical outcomes. The coil-type anchor resulted in superior bony growth surrounding the anchor and a larger total bone mineral mass within the anchor owing to its larger volume. LEVEL OF EVIDENCE: Level II, randomized prospective comparative study.

Differential peroxiredoxin hyperoxidation regulates MAP kinase signaling in human articular chondrocytes.

The peroxiredoxin (Prx) family of Cys-dependent peroxidases control intracellular levels of H2O2 and can regulate signal transduction. Inhibition of the Prxs, through hyperoxidation amongst other mechanisms, leads to oxidative stress conditions that can alter homeostatic signaling. To determine the effects oxidation of Prx1-Prx3 has on MAP kinase and IGF-1 signaling events in human chondrocytes, this study used 2-methyl-1,4-naphthoquinone (menadione) and 2,3-dimethyl-1,4-naphthoquinone (DMNQ) as H2O2-generating tools due to their differential mechanisms of action. Menadione and DMNQ generated similar levels of intracellular H2O2 as determined using the biosensor Orp1-roGFP and by measuring Prx redox status. However, menadione generated higher levels of mitochondrial H2O2 associated with Prx3 hyperoxidation and phosphorylation of Prx1 while DMNQ treatment was associated with hyperoxidation of cytosolic Prx1 and Prx2 but not mitochondrial Prx3. Both menadione and DMNQ induced sustained phosphorylation of p38 but only DMNQ activated JNK. Menadione but not DMNQ inhibited IGF-1-induced Akt phosphorylation. Chondrocytes transduced with an adenoviral vector to overexpress Prx3 displayed decreased PrxSO2/3 formation in response to menadione which was associated with restoration of IGF-1-mediated Akt signaling and inhibition of p38 phosphorylation. Prx1 and Prx2 overexpression had no effects on Prx redox status but Prx1 overexpression enhanced basal Akt phosphorylation. These results suggest that hyperoxidation of specific Prx isoforms is associated with distinct cell signaling events and identify Prx3 redox status as an important regulator of anabolic and catabolic signal transduction. Targeted strategies to prevent mitochondrial Prx3 hyperoxidation could be useful in maintaining cellular redox balance and homeostatic signaling.

Agili-C implant promotes the regenerative capacity of articular cartilage defects in an ex vivo model.

PURPOSE: Osteochondral implants are currently adopted for the treatment of symptomatic full-thickness chondral and osteochondral defects. Agili-C™ is a cell-free aragonite-based scaffold which aims to reproduce the original structure and function of the articular joint while directing the growth and regeneration of both cartilage and its underlying subchondral bone. The goal of the present study was to investigate the ex vivo mechanisms of action (MOA) of the Agili-C™ implant in the repair of full-thickness cartilage defects. In particular, we tested whether Agili-C™ implant has the potential to stimulate cartilage ingrowth through chondrocytes migration into the 3D interconnected porous structure of the scaffold, along with maintaining their viability and phenotype and the deposition of hyaline cartilage matrix. METHODS: Articular cartilage samples were collected through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL) within 24 h from death. For this study, cartilage from a total of 14 donors was used. To model a chondral defect, donut-shaped cartilage explants were prepared from each tissue specimen. The chondral phase of the Agili-C™ implant was placed inside the tissue in full contact and press fit manner. Cartilage explants with the Agili-C™ implant inside were cultured for 60 days. As a control, the same donut-shaped cartilage explants were cultured without Agili-C™, under the same culture conditions. RESULTS: Using fresh human cadaveric articular cartilage tissue in a 60-day culture, it was demonstrated that chondrocytes were able to migrate into the Agili-C™ scaffold and contribute to the deposition of the extracellular matrix (ECM) rich in collagen type II and aggrecan, and lacking collagen type I. Additionally, we were able to show the formation of a layer populated by progenitor-like cells on the articular surface of the implant. CONCLUSIONS: The analysis of samples taken from knee and ankle joints of human donors with a wide age range and both genders supports the potential of Agili-C™ scaffold to stimulate cartilage regeneration and repair. Based on these results, the present scaffold can be used in the clinical practice as a one-step procedure to treat full-thickness chondral defects.

Bone Marrow Aspirate Concentrate for Cartilage Defects of the Knee: From Bench to Bedside Evidence.

Objective To critically evaluate the current basic science, translational, and clinical data regarding bone marrow aspirate concentrate (BMAC) in the setting of focal cartilage defects of the knee and describe clinical indications and future research questions surrounding the clinical utility of BMAC for treatment of these lesions. Design A literature search was performed using the PubMed and Ovid MEDLINE databases for studies in English (1980-2017) using keywords, including ["bone marrow aspirate" and "cartilage"], ["mesenchymal stem cells" and "cartilage"], and ["bone marrow aspirate" and "mesenchymal stem cells" and "orthopedics"]. A total of 1832 articles were reviewed by 2 independent authors and additional literature found through scanning references of cited articles. Results BMAC has demonstrated promising results in the clinical application for repair of chondral defects as an adjuvant procedure or as an independent management technique. A subcomponent of BMAC, bone marrow derived-mesenchymal stem cells (MSCs) possess the ability to differentiate into cells important for osteogenesis and chondrogenesis. Modulation of paracrine signaling is perhaps the most important function of BM-MSCs in this setting. In an effort to increase the cellular yield, authors have shown the ability to expand BM-MSCs in culture while maintaining phenotype. Conclusions Translational studies have demonstrated good clinical efficacy of BMAC both concomitant with cartilage restoration procedures, at defined time points after surgery, and as isolated injections. Early clinical data suggests BMAC may help stimulate a more robust hyaline cartilage repair tissue response. Numerous questions remain regarding BMAC usage, including cell source, cell expansion, optimal pathology, and injection timing and quantity.

Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes.

Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1-3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observedin situin human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism.

Assessment of glenoid chondral healing: comparison of microfracture to autologous matrix-induced chondrogenesis in a novel rabbit shoulder model.

BACKGROUND: Management of glenohumeral arthrosis in young patients is a considerable challenge, with a growing need for non-arthroplasty alternatives. The objectives of this study were to develop an animal model to study glenoid cartilage repair and to compare surgical repair strategies to promote glenoid chondral healing. METHODS: Forty-five rabbits underwent unilateral removal of the entire glenoid articular surface and were divided into 3 groups--untreated defect (UD), microfracture (MFx), and MFx plus type I/III collagen scaffold (autologous matrix-induced chondrogenesis [AMIC])--for the evaluation of healing at 8 weeks (12 rabbits) and 32 weeks (33 rabbits) after injury. Contralateral shoulders served as unoperated controls. Tissue assessments included 11.7-T magnetic resonance imaging (long-term healing group only), equilibrium partitioning of an ionic contrast agent via micro-computed tomography (EPIC-µCT), and histologic investigation (grades on International Cartilage Repair Society II scoring system). RESULTS: At 8 weeks, x-ray attenuation, thickness, and volume did not differ by treatment group. At 32 weeks, the T2 index (ratio of T2 values of healing to intact glenoids) was significantly lower for the MFx group relative to the AMIC group (P = .01) whereas the T1ρ index was significantly lower for AMIC relative to MFx (P = .01). The micro-computed tomography-derived repair tissue volume was significantly higher for MFx than for UD. Histologic investigation generally suggested inferior healing in the AMIC and UD groups relative to the MFx group, which exhibited improvements in both integration of repair tissue with subchondral bone and tidemark formation over time. DISCUSSION: Improvements conferred by AMIC were limited to magnetic resonance imaging outcomes, whereas MFx appeared to promote increased fibrous tissue deposition via micro-computed tomography and more hyaline-like repair histologically. The findings from this novel model suggest that MFx promotes biologic resurfacing of full-thickness glenoid articular injury.

Effect of highly purified capsaicin on articular cartilage and rotator cuff tendon healing: An in vivo rabbit study.

Highly purified capsaicin has emerged as a promising injectable compound capable of providing sustained pain relief following a single localized treatment during orthopedic surgical procedures. To further assess its reliability for clinical use, the potential effect of highly purified capsaicin on articular cartilage metabolism as well as tendon structure and function warrants clarification. In the current study, rabbits received unilateral supraspinatus transection and repair with a single 1 ml injection of capsaicin (R+C), PEG-only placebo (R+P), or saline (R+S) into the glenohumeral joint (GHJ). An additional group received 1 ml capsaicin onto an intact rotator cuff (I+C). At 18 weeks post-op, cartilage proteoglycan (PG) synthesis and content as well as cell viability were similar (p>0.05) across treatment groups. Biomechanical testing revealed no differences (p>0.05) among tendon repair treatment groups. Similarly, histologic features of both cartilage and repaired tendons showed minimal differences across groups. Hence, in this rabbit model, a single injection of highly purified capsaicin into the GHJ does not induce a deleterious response with regard to cartilage matrix metabolism and cell viability, or rotator cuff healing. These data provide further evidence supporting the use of injectable, highly purified capsaicin as a safe alternative for management of postoperative pain following GHJ surgery.

Restoration of articular cartilage.

➤ Novel (i.e., quantitative and semiquantitative) cartilage imaging techniques can evaluate cartilage composition to augment information obtained from traditional magnetic resonance imaging sequences that detail morphology.➤ A well-defined role for drugs leading to chondroprotection has not yet been determined.➤ Shortcomings of bone marrow stimulation include limited production of hyaline repair tissue, unpredictable repair cartilage volume, and a negative impact on later cellular transplantation if required.➤ The role of biological augments, such as cellular concentrates or platelet-rich plasma, remains undefined. When their use is reported in the literature, it is important that their process of production and characterization be detailed.➤ Rehabilitation programs, incorporating controlled exercise and progressive partial weight-bearing, are an important part of cartilage repair surgery and should be detailed in reports on operative techniques applied.➤ Malalignment, meniscal injury, and ligament deficiency should be corrected in a staged or concomitant fashion to reduce the overall likelihood of mechanical failure in cartilage repair surgery.

Rotator cuff healing after continuous subacromial bupivacaine infusion: an in vivo rabbit study.

BACKGROUND: The objective of this study was to evaluate the effects of continuous subacromial bupivacaine infusion on supraspinatus muscle and rotator cuff tendon healing using gross, biomechanical, and histologic analyses. MATERIALS AND METHODS: Thirty-three New Zealand White rabbits underwent unilateral supraspinatus transection and rotator cuff repair (RCR). Rabbits were assigned to 1 of 3 groups: (1) RCR only, (2) RCR with continuous saline infusion for 48 hours, or (3) RCR with continuous 0.25% bupivacaine with epinephrine (1:200,000) infusion for 48 hours. Rabbits were euthanized postoperatively at 2 weeks (for histologic assessment) or 8 weeks (for biomechanical and histologic assessment). RESULTS: Tensile testing showed a significantly higher load to failure in intact tendons compared with repaired tendons (P < .01); however, no statistical differences were detected among RCR only, RCR saline, and RCR bupivacaine groups. Histologically, the enthesis of repaired tendons showed increased cellularity and disorganized collagen fibers compared with intact tendons, with no differences between treatment groups. Muscle histology demonstrated scattered degenerative muscle fibers at 2 weeks in RCR saline and RCR bupivacaine groups, but no degeneration was noted at 8 weeks. CONCLUSIONS: The healing supraspinatus tendons exposed to bupivacaine infusion showed similar histologic and biomechanical characteristics compared with untreated and saline-infused RCR groups. Muscle histology showed fiber damage at 2 weeks for the saline and bupivacaine-treated groups, with no apparent disruption at 8 weeks, suggesting a recovery process. Therefore, subacromial bupivacaine infusion in this rabbit rotator cuff model does not appear to impair muscle or tendon after acute injury and repair.

Synovial fluid from patients with early osteoarthritis modulates fibroblast-like synoviocyte responses to toll-like receptor 4 and toll-like receptor 2 ligands via soluble CD14.

OBJECTIVE: Synovial inflammation, a feature of both osteoarthritis (OA) and meniscal injury, is hypothesized to be triggered in part via stimulation of Toll-like receptors (TLRs). We undertook this study to test whether a TLR-2- or TLR-4-stimulating factor in synovial fluid (SF) from patients with early knee OA with meniscal injury could lead to inflammatory activation of synoviocytes. METHODS: SF was obtained from patients with early OA cartilage damage undergoing arthroscopic meniscal procedures. SF was used to stimulate primary cultures of fibroblast-like synoviocytes (FLS) and cell lines transfected with TLR-2 or TLR-4. SF was used either alone or in combination with a TLR-2 stimulus (palmitoyl-3-cysteine-serine-lysine-4 [Pam3CSK4]) or a TLR-4 stimulus (lipopolysaccharide [LPS]). In blocking experiments, SF was preincubated with anti-CD14 antibody. RESULTS: SF from these patients did not stimulate interleukin-8 (IL-8) release from TLR transfectants. Compared with SF on its own, SF (at concentrations of 0.09-25%) in combination with TLR-2 or TLR-4 ligands resulted in significant augmentation of IL-8 release from both transfectants and primary FLS. Soluble CD14 (sCD14), a coreceptor for TLRs, was measured in SF from patients with early OA at levels comparable to those in patients with advanced OA and patients with rheumatoid arthritis. Blockade with anti-CD14 antibody abolished the ability of SF to augment IL-8 production in response to LPS, and diminished Pam3CSK4 responses. CONCLUSION: SF augments FLS responses to TLR-2 and TLR-4 ligands. This effect was largely due to sCD14. Our results demonstrate that sCD14 in the setting of OA and meniscal injury sensitizes FLS to respond to inflammatory stimuli such as TLR ligands.

The role of synovial fluid markers of catabolism and anabolism in osteoarthritis, rheumatoid arthritis and asymptomatic organ donors.

INTRODUCTION: The purpose of this study was to correlate the level of anabolic and catabolic biomarkers in synovial fluid (SF) from patients with rheumatoid arthritis (RA), patients with osteoarthritis (OA) and asymptomatic organ donors. METHODS: SF was collected from the knees of 45 OA, 22 RA patients and 20 asymptomatic organ donors. Eight biomarkers were selected and analyzed by using an enzyme-linked immunosorbent assay: interleukin (IL)-1, IL-6, IL-8 and IL-11; leukemia-inhibitory factor (LIF); cartilage oligomeric protein (COMP); osteocalcin; and osteogenic protein 1 (OP-1). Data are expressed as medians (interquartile ranges). The effects of sex and disease activity were assessed on the basis of the Western Ontario and McMaster Universities index score for patients with OA and on the basis of white blood cell count, erythrocyte sedimentation rate and C-reactive protein level for patients with RA. RESULTS: The mean ages (± SD) of the patients were as follows: 53 ± 9 years for patients with OA, 54 ± 11 years for patients with RA and 52 ± 7 years for asymptomatic organ donors. No effect of participants' sex was identified. In the SF of patients with RA, four of five cytokines were higher than those in the SF of patients with OA and those of asymptomatic organ donors. The most significant differences were found for IL-6 and IL-8, where IL-6 concentration in SF of patients with RA was almost threefold higher than that in patients with OA and fourfold higher than that in asymptomatic donor controls: 354.7 pg/ml (1,851.6) vs. 119.4 pg/ml (193.2) vs. 86.97 pg/ml (82.0) (P < 0.05 and P < 0.05, respectively). IL-8 concentrations were higher in SF of patients with RA than that in patients with OA as well as that in asymptomatic donor controls: 583.6 pg/ml (1,086.4) vs. 429 pg/ml (87.3) vs. 451 pg/ml (170.1) (P < 0.05 and P < 0.05, respectively). No differences were found for IL-11 in the SF of patients with RA and that of patients with OA, while a 1.4-fold difference was detected in the SF of patients with OA and that of asymptomatic donor controls: 296.2 pg/ml (257.2) vs. 211.6 pg/ml (40.8) (P < 0.05). IL-1 concentrations were the highest in the SF of RA patients (9.26 pg/ml (11.1)); in the SF of asymptomatic donors, it was significantly higher than that in patients with OA (9.083 pg/ml (1.6) vs. 7.76 pg/ml (2.6); P < 0.05). Conversely, asymptomatic donor control samples had the highest LIF concentrations: 228.5 pg/ml (131.6) vs. 128.4 pg/ml (222.7) in the SF of patients with RA vs. 107.5 pg/ml (136.9) in the SF of patients with OA (P < 0.05). OP-1 concentrations were twofold higher in the SF of patients with RA than those in patients with OA and threefold higher than those in asymptomatic donor control samples (167.1 ng/ml (194.8) vs. 81.79 ng/ml (116.0) vs. 54.49 ng/ml (29.3), respectively; P < 0.05). The differences in COMP and osteocalcin were indistinguishable between the groups, as were the differences between active and inactive OA and RA. CONCLUSIONS: Activation of selected biomarkers corresponds to the mechanisms that drive each disease. IL-11, LIF and OP-1 may be viewed as a cluster of biomarkers significant for OA; while profiling of IL-1, IL-6, IL-8, LIF and OP-1 may be more significant in RA. Larger, better-defined patient cohorts are necessary to develop a biomarker algorithm for prognostic use.

Medial versus lateral supraspinatus tendon properties: implications for double-row rotator cuff repair.

BACKGROUND: Rotator cuff repair retear rates range from 25% to 90%, necessitating methods to improve repair strength. Although numerous laboratory studies have compared single-row with double-row fixation properties, little is known regarding regional (ie, medial vs lateral) suture retention properties in intact and torn tendons. HYPOTHESIS: A torn supraspinatus tendon will have reduced suture retention properties on the lateral aspect of the tendon compared with the more medial musculotendinous junction. STUDY DESIGN: Controlled laboratory study. METHODS: Human supraspinatus tendons (torn and intact) were randomly assigned for suture retention mechanical testing, ultrastructural collagen fibril analysis, or histologic testing after suture pullout testing. For biomechanical evaluation, sutures were placed either at the musculotendinous junction (medial) or 10 mm from the free margin (lateral), and tendons were elongated to failure. Collagen fibril assessments were performed using transmission electron microscopy. RESULTS: Intact tendons showed no regional differences with respect to suture retention properties. In contrast, among torn tendons, the medial region exhibited significantly higher stiffness and work values relative to the lateral region. For the lateral region, work to 10-mm displacement (1592 ± 261 N-mm) and maximum load (265 ± 44 N) for intact tendons were significantly higher (P < .05) than that of torn tendons (1086 ± 388 N-mm and 177 ± 71 N, respectively). For medial suture placement, maximum load, stiffness, and work of intact and torn tendons were similar (P > .05). Regression analyses for the intact and torn groups revealed generally low correlations between donor age and the 3 biomechanical indices. For both intact and torn tendons, the mean fibril diameter and area density were greater in the medial region relative to the lateral (P ≤ .05). In the lateral tendon, but not the medial region, torn specimens showed a significantly lower fibril area fraction (48.3% ± 3.8%) than intact specimens (56.7% ± 3.6%, P < .05). CONCLUSION: Superior pullout resistance of medially placed sutures may provide a strain shielding effect for the lateral row after double-row repair. Larger diameter collagen fibrils as well as greater fibril area fraction in the medial supraspinatus tendon may provide greater resistance to suture migration. CLINICAL RELEVANCE: While clinical factors such as musculotendinous integrity warrant strong consideration for surgical decision making, the present ultrastructural and biomechanical results appear to provide a scientific rationale for double-row rotator cuff repair where sutures are placed more medially at the muscle-tendon junction.

Protective effect of P188 in the model of acute trauma to human ankle cartilage: the mechanism of action.

OBJECTIVE: Because P188 poloxamer is effective in promoting cell survival in models of acute trauma, the objectives were to understand the mechanism of its action focusing on glycogen synthase kinase-3 (GSK3) activation, interleukin-6 (IL-6), and p38 signaling. DESIGN: Sixteen normal human tali were impacted using a 4-mm diameter indenter with an impulse of 1 Ns. Eight-millimeter cartilage plugs containing the 4-mm impacted core and 4-mm adjacent nonimpacted ring were removed and cultured with or without P188. Cell lysates were analyzed using Western blots with antibodies against total and phosphorylated extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, ATF-2, GSK3, Stat1, and Stat3. Additional tests were performed with the p38 inhibitor (p38i) SB203580. RESULTS: Studied pathways were activated after impaction with the peak of activity at 1 hour. P188 completely attenuated phosphorylation of Stat1 and ATF-2 and inhibited p38, Stat3, JNK, ERK, and GSK3. The p38i partially offset phosphorylation of Stat3, GSK3, and ERK suggesting a role of p38 in these three pathways. Additionally, the p38i improved cell survival (P = 0.053) and reduced apoptosis (by approximately 20%, P = 0.046, versus almost 40% by P188), thus confirming that P188 acts (at least in part) through the p38 pathway. CONCLUSION: Our results report a novel mechanism through which P188 exerts its protective effects on cartilage in the model of acute injury. In addition to its effect on cellular membrane, P188 affects stress-related p38 signaling, apoptosis-related GSK3, and inflammation-related IL-6 signaling. Taken together, these findings suggest that P188 alone or in combination with proanabolic agents may have a therapeutic potential in preventing progressive cartilage degeneration and the development of posttraumatic osteoarthritis.

Biceps tendinitis in chronic rotator cuff tears: a histologic perspective.

Patients with chronic rotator cuff tears frequently have anterior shoulder pain attributed to the long head of the biceps brachii (LHBB) tendon. In this study, tenodesis or tenotomy samples and cadaveric controls were assessed by use of immunohistochemical and histologic methods to quantify inflammation, vascularity, and neuronal plasticity. Patients had moderate pain and positive results on at least 1 clinical test of shoulder function. The number of axons in the distal LHBB was significantly less in patients with biceps tendinitis. Calcitonin gene-related peptide and substance P immunostaining was predominantly within nerve roots and blood vessels. A moderate correlation (R = 0.5) was identified between LHBB vascularity and pain scores. On the basis of these results, we conclude that, in the context of rotator cuff disease, the etiology of anterior shoulder pain with macroscopic changes in the biceps tendon is related to the complex interaction of the tendon and surrounding soft tissues, rather than a single entity.

Response of human chondrocytes prepared for autologous implantation to growth factors.

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.

Safety of epidural administration of Osteogenic Protein-1 (OP-1/BMP-7): behavioral and macroscopic observation.

STUDY DESIGN: To assess the safety of epidural administration of Osteogenic Protein-1 (OP-1). OBJECTIVES: To examine if epidural administration of OP-1 or administration into the nucleus pulposus (NP) resulted in ectopic bone formation and/or pain-related behavior. SUMMARY OF BACKGROUND DATA: OP-1 has the potential for treatment of degenerative disc disease. However, OP-1's safety, when it is applied into the epidural space or herniated nucleus pulposus, is not well established. METHODS: Forty rats were divided into 5 groups. Sham group: Left L4 and L5 nerve roots were exposed. NP group: The NP obtained from the tail was relocated onto the exposed nerve roots. NP+OP group: The NP obtained from the tail and injected with 0.2 microg of OP-1 in 1 microL of 5% lactose-buffered solution was placed on the nerve roots. GS group: A gelatin sponge was applied on the nerve roots. GS+OP group: A gelatin sponge soaked with 0.2 microg of OP-1 in 1 microL of 5% lactose-buffered solution was placed onto the nerve roots. Motor function and reflex responses to mechanical noxious stimuli were measured in all rats up to 3 weeks after surgery. Three weeks after surgery, all rats were killed for analysis of ectopic bone formation and magnitude of neural compression. RESULTS: Motor paresis was not observed in any groups. Only rats in the NP group showed evidence of irreversible mechanical hyperalgesia after surgery. There were no differences in the mechanical stimuli response among all groups except the NP group. Macroscopic examination revealed no ectopic bone formation or differences in neural compression among the groups. CONCLUSION: OP-1 application in the epidural space is safe based on behavioral measures and macroscopic observation on ectopic bone formation at 21 days after surgery.

Anti-catabolic effect of OP-1 in chronically compressed intervertebral discs.

Experimental animal models of disc degeneration have been used to assess the biomechanical behavior, biochemical composition, and biological changes in the intervertebral discs. The objective of our study was to evaluate the anabolic and anti-catabolic effects of intradiscal injection of Osteogenic Protein-1 (OP-1) by histology and immunohistochemistry in disc degeneration model. Thirty-four rats were divided into five groups: intact control; sham control; compressed nucleus pulposus (NP) injected with saline; and two OP-1 groups: COP-1 group (compression was continued after intradiscal OP-1 injection) and ROP-1 group (compression was released at the time of OP-1 injection). Anabolic and anti-catabolic effects of OP-1 were evaluated by histology and immunohistochemistry with the following antibodies: anti-pro- and anti-mature OP-1, anti-MMP-13, anti-aggrecanase, anti-substance P, anti-tumor necrosis factor-alpha (TNF-alpha), and anti-interleukin-1beta (IL-1beta). The OP-1 injection to the degenerative disc stimulated an anabolic response characterized by the restoration of the normal morphology of the disc, increased Safranin O staining in the NP, extention of the extracellular matrix, and stimulation of endogenous OP-1 synthesis in the NP, annulus fibrosis (AF), and end-plate. The anti-catabolic effect of OP-1 was documented by reduced immunostaining for aggrecanase, MMP-13, substance P, TNF-alpha, and IL-1beta. This study confirmed the anti-catabolic activity of OP-1 as demonstrated previously in human articular cartilage and provided critical evidence for the potential of OP-1 therapy in the treatment of disc degeneration. Because substance P is a neuropeptide linked with inflammation and pain, a reduction in the level of this protein may support our previously reported results on the effect of OP-1 on pain-related behavior.