Toward designing human intervention studies to prevent osteoarthritis after knee injury: A report from an interdisciplinary OARSI 2023 workshop.

Objective: The global impact of osteoarthritis is growing. Currently no disease modifying osteoarthritis drugs/therapies exist, increasing the need for preventative strategies. Knee injuries have a high prevalence, distinct onset, and strong independent association with post-traumatic osteoarthritis (PTOA). Numerous groups are embarking upon research that will culminate in clinical trials to assess the effect of interventions to prevent knee PTOA despite challenges and lack of consensus about trial design in this population. Our objectives were to improve awareness of knee PTOA prevention trial design and discuss state-of-the art methods to address the unique opportunities and challenges of these studies. Design: An international interdisciplinary group developed a workshop, hosted at the 2023 Osteoarthritis Research Society International Congress. Here we summarize the workshop content and outputs, with the goal of moving the field of PTOA prevention trial design forward. Results: Workshop highlights included discussions about target population (considering risk, homogeneity, and possibility of modifying osteoarthritis outcome); target treatment (considering delivery, timing, feasibility and effectiveness); comparators (usual care, placebo), and primary symptomatic outcomes considering surrogates and the importance of knee function and symptoms other than pain to this population. Conclusions: Opportunities to test multimodal PTOA prevention interventions across preclinical models and clinical trials exist. As improving symptomatic outcomes aligns with patient and regulator priorities, co-primary symptomatic (single or aggregate/multidimensional outcome considering function and symptoms beyond pain) and structural/physiological outcomes may be appropriate for these trials. To ensure PTOA prevention trials are relevant and acceptable to all stakeholders, future research should address critical knowledge gaps and challenges.

Evaluation of tendon and ligament microstructure and mechanical properties in a canine model of mucopolysaccharidosis I.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disorder characterized by deficient alpha-l-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans (GAGs) in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a strong clinical need for improved treatment approaches that specifically target joint tissues; however, their development is hampered by poor understanding of underlying disease pathophysiology, including how pathological changes to component tissues contribute to overall joint dysfunction. Ligaments and tendons, in particular, have received very little attention, despite the critical roles of these tissues in joint stability and biomechanical function. The goal of this study was to leverage the naturally canine model to undertake functional and structural assessments of the anterior (cranial) cruciate ligament (CCL) and Achilles tendon in MPS I. Tissues were obtained postmortem from 12-month-old MPS I and control dogs and tested to failure in uniaxial tension. Both CCLs and Achilles tendons from MPS I animals exhibited significantly lower stiffness and failure properties compared to those from healthy controls. Histological examination revealed multiple pathological abnormalities, including collagen fiber disorganization, increased cellularity and vascularity, and elevated GAG content in both tissues. Clinically, animals exhibited mobility deficits, including abnormal gait, which was associated with hyperextensibility of the stifle and hock joints. These findings demonstrate that pathological changes to both ligaments and tendons contribute to abnormal joint function in MPS I, and suggest that effective clinical management of joint disease in patients should incorporate treatments targeting these tissues.

Tension-activated nanofiber patches delivering an anti-inflammatory drug improve repair in a goat intervertebral disc herniation model.

Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.

Arthroscopic Excision of Pigmented Villonodular Synovitis of the Trochanteric Bursa.

Trochanteric bursitis is a common disorder affecting middle-aged adults and usually presents with lateral-based hip pain and swelling. It usually responds to conservative measures, including adductor stretching, abductor strengthening, and select injections of corticosteroid or platelet-rich plasma. For refractory cases, excision, open or arthroscopic, is usually recommended. We observed a 55-year-old woman who had lateral hip pain and longstanding swelling consistent with refractory trochanteric bursitis. Her persistent symptoms, coupled with atypical findings on imaging, prompted an arthroscopic evaluation. Arthroscopic examination of the peritrochanteric space revealed a fulminant bursal inflammation that pierced through the iliotibial band. The bursal inflammation was excised arthroscopically and biopsy of the tissue revealed a diagnosis of pigmented villonodular synovitis (PVNS). The patient had an uneventful recovery and had a full resolution of symptoms with no recurrence noted at 3-year follow-up. This is the first reported case of arthroscopic excision of PVNS of the trochanteric bursa. Given that it may mimic trochanteric bursitis, it is important for clinicians to be aware of the possibility of this progressive condition for appropriate clinical intervention. [Orthopedics. 2023;46(6):e381-e383.].

Proteomics identifies novel biomarkers of synovial joint disease in a canine model of mucopolysaccharidosis I.

Mucopolysaccharidosis I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a critical need for improved understanding of joint disease pathophysiology in MPS I, including specific biomarkers to predict and monitor joint disease progression, and response to treatment. The objective of this study was to leverage the naturally-occurring MPS I canine model and undertake an unbiased proteomic screen to identify systemic biomarkers predictive of local joint disease in MPS I. Synovial fluid and serum samples were collected from MPS I and healthy dogs at 12 months-of-age, and protein abundance characterized using liquid chromatography tandem mass spectrometry. Stifle joints were evaluated postmortem using magnetic resonance imaging (MRI) and histology. Proteomics identified 40 proteins for which abundance was significantly correlated between serum and synovial fluid, including markers of inflammatory joint disease and lysosomal dysfunction. Elevated expression of three biomarker candidates, matrix metalloproteinase 19, inter-alpha-trypsin inhibitor heavy-chain 3 and alpha-1-microglobulin, was confirmed in MPS I cartilage, and serum abundance of these molecules was found to correlate with MRI and histological degenerative grades. The candidate biomarkers identified have the potential to improve patient care by facilitating minimally-invasive, specific assessment of joint disease progression and response to therapeutic intervention.

Preclinical Use of FGF-18 Augmentation for Improving Cartilage Healing Following Surgical Repair: A Systematic Review.

OBJECTIVE: To evaluate the efficacy of fibroblast growth factor-18 (FGF-18) augmentation for improving articular cartilage healing following surgical repair in preclinical (in vivo) animal models. DESIGN: A systematic review was performed evaluating the efficacy of FGF-18 augmentation with cartilage surgery compared with cartilage surgery without FGF-18 augmentation in living animal models. Eligible intervention groups were FGF-18 treatment in conjunction with orthopedic procedures, including microfracture, osteochondral auto/allograft transplantation, and cellular-based repair. Outcome variables were: International Cartilage Repair Society (ICRS) score, modified O'Driscoll histology score, tissue infill score, qualitative histology, and adverse events. Descriptive statistics were recorded and summarized for each included study. RESULTS: In total, 493 studies were identified and 4 studies were included in the final analysis. All studies were randomized controlled trials evaluating in vivo use of recombinant human FGF-18 (rhFGF-18). Animal models included ovine (n = 3) and equine (n = 1), with rhFGF-18 use following microfracture (n = 3) or osteochondral defect repair (n = 1). The rhFGF-18 was delivered via intra-articular injection (n = 2), collagen membrane scaffold (n = 1), or both (n = 1). All studies reported significant, positive improvements in cartilage defect repair with rhFGF-18 compared with controls based on ICRS score (n = 4), modified O'Driscoll score (n = 4), tissue infill (n = 3), and expression of collagen type II (n = 4) (P < 0.05). No adverse events were reported with the intra-articular administration of this growth factor, indicating short-term safety and efficacy of rhFGF-18 in vivo. CONCLUSION: This systematic review provides evidence that rhFGF-18 significantly improves cartilage healing at 6 months postoperatively following microfracture or osteochondral defect repair in preclinical randomized controlled trials.

Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme ß-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human ß-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6 months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

Self-Assembly Culture Model for Engineering Musculoskeletal Tissues.

The goal of a self-assembly tissue engineering is to create functional tissue following a natural cell-driven process that mirrors natural development. This approach to tissue engineering has tremendous potential for the development of reparative strategies to treat musculoskeletal injuries and diseases, especially for articular cartilage which has poor regenerative capacity. Additionally, many bioengineering and culture methods fail to maintain the chondrocyte phenotype and contain the correct matrix composition in the long term. Existing cartilage-engineering approaches have been developed, but many approaches involve complicated culture techniques and require foreign substances and biomaterials as scaffolds. While these scaffold-based approaches have numerous advantages, such as an instant or rapid creation of biomechanical properties, they frequently result in dedifferentiation of cells in part, due to the adherence to foreign scaffold materials. In this chapter, we describe a novel approach of developing a scaffold-less cartilage-like biomaterial, using the simple principle that cells at high density bear a capacity to coalesce when they cannot attach to any culture substrate. We refer to the biomaterial formed as a cartilage tissue equivalent or CTA and have published to describe their characteristics and utility in high-throughput drug screening. The method is described to generate reproducible cartilage analogs using a specialized high-density suspension culture technique using a hydrogel poly-2-hydroxyethyl methacrylate (polyHEMA) coating of a culture dish. We have demonstrated that this approach can rapidly form biomass of chondrocytes that over time becomes very synthetically active producing a cartilage-like extracellular matrix that closely mimics the biochemical and biomechanical characteristics of native articular cartilage. The culture approach can also be used to form CTA from other than articular cartilage-derived chondrocytes as well as mesenchymal stem cells (MSCs) (while differentiating MSCs into chondrocytes). Some of the advantages are phenotype stability, reproducible CTA size, and biomechanical and biochemical characteristics similar to natural cartilage.

Identifying small molecules for protecting chondrocyte function and matrix integrity after controlled compressive injury.

Objective: Articular cartilage injury is central for the development of post-traumatic osteoarthritis (PTOA). With few disease-modifying therapies successful at offsetting progressive osteoarthritis (OA), our goal is to use a high throughput screening platform of cartilage injury to identify novel chondroprotective compounds. Targeting articular cartilage damage immediately after injury remains a promising therapeutic strategy to overcome irreversible tissue damage. Method: We constructed a single impact-cartilage screening method using a multi-platen system that simultaneously impacts 48 samples and makes use of engineered cartilage tissue analogs (known as CTAs). Drug libraries were screened and assessed for their ability to alter two crucial biological responses to impact injuries, namely matrix degradation and cell stress. Results: Over 500 small molecules were screened for their ability to alter proteoglycan loss, matrix metalloproteinase activity, and cell stress or death. Fifty-five compounds passed through secondary screening and were from commercial libraries of natural and redox, stem cell related compounds, as well as protease, kinase and phosphatase inhibitors. Through secondary screening, 16 promising candidates exhibited activity on one or more critical function of chondrocytes. While many are mechanistically known compounds, their function in joint diseases is not known. Conclusion: This platform was validated for screening drug activity against a tissue engineered model of PTOA. Multiple compounds identified in this manner have potential application as early protective therapy for treating PTOA, and require further study. We propose this screening platform can identify novel molecules that act on early chondrocyte responses to injury and provide an invaluable tool for therapeutic development.

Gravity-based patterning of osteogenic factors to preserve bone structure after osteochondral injury in a large animal model.

Chondral and osteochondral repair strategies are limited by adverse bony changes that occur after injury. Bone resorption can cause entire scaffolds, engineered tissues, or even endogenous repair tissues to subside below the cartilage surface. To address this translational issue, we fabricated thick-shelled poly(D,L-lactide-co-glycolide) microcapsules containing the pro-osteogenic agents triiodothyronine andß-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that the developed microcapsules rupturedin vitrounder increasing mechanical loads, and readily sink within a liquid solution, enabling gravity-based patterning along the osteochondral surface. In a large animal, these mechanically-activated microcapsules (MAMCs) were assessed through two different delivery strategies. Intra-articular injection of control MAMCs enabled fluorescent quantification of MAMC rupture and cargo release in a synovial joint setting over timein vivo. This joint-wide injection also confirmed that the MAMCs do not elicit an inflammatory response. In the contralateral hindlimbs, chondral defects were created, MAMCs were patternedin situ, and nanofracture (Nfx), a clinically utilized method to promote cartilage repair, was performed. The Nfx holes enabled marrow-derived stromal cells to enter the defect area and served as repeatable bone injury sites to monitor over time. Animals were evaluated one and two weeks after injection and surgery. Analysis of injected MAMCs showed that bioactive cargo was released in a controlled fashion over two weeks. A bone fluorochrome label injected at the time of surgery displayed maintenance of mineral labeling in the therapeutic group, but resorption in both control groups. Alkaline phosphatase (AP) staining at the osteochondral interface revealed higher AP activity in defects treated with therapeutic MAMCs. Overall, this study develops a gravity-based approach to pattern bioactive factors along the osteochondral interface, and applies this novel biofabrication strategy to preserve bone structure after osteochondral injury.

Recombinant fibroblast growth factor-18 (sprifermin) enhances microfracture-induced cartilage healing.

Posttraumatic osteoarthritis is a disabling condition impacting the mostly young and active population. In the present study, we investigated the impact of intra-articular sprifermin, a recombinant truncated fibroblast growth factor 18, on the outcome of microfracture treatment, a widely used surgical technique to enhance cartilage healing at the site of injury. For this study, we created a cartilage defect and performed microfracture treatment in fetlock joints of 18 horses, treated joints with one of three doses of sprifermin (10, 30, or 100 µg) or with saline, hyaluronan, and evaluated animals functional and structural outcomes over 24 weeks. For primary outcome measures, we performed histological evaluations and gene expression analysis of aggrecan, collagen types I and II, and cartilage oligomeric matrix protein in three regions of interest. As secondary outcome measures, we examined animals' lameness, performed arthroscopic, radiographic, and computed tomography (CT) scan imaging and gross morphology assessment. We detected the highest treatment benefit following 100 µg sprifermin treatment. The overall histological assessment showed an improvement in the kissing region, and the expression of constitutive genes showed a concentration-dependent enhancement, especially in the peri-lesion area. We detected a significant improvement in lameness scores, arthroscopic evaluations, radiography, and CT scans following sprifermin treatment when results from three dose-treatment groups were combined. Our results demonstrated, for the first time, an enhancement on microfracture outcomes following sprifermin treatment suggesting a cartilage regenerative role and a potential benefit of sprifermin treatment in early cartilage injuries.

Evaluating whole-genome expression differences in idiopathic and diabetic adhesive capsulitis.

BACKGROUND: Diabetic patients have a greater incidence of adhesive capsulitis (AC) and a more protracted disease course than patients with idiopathic AC. The purpose of this study was to compare gene expression differences between AC with diabetes mellitus and AC without diabetes mellitus. METHODS: Shoulder capsule samples were prospectively obtained from diabetic or nondiabetic patients who presented with shoulder dysfunction and underwent arthroscopy (N = 16). Shoulder samples of AC with and without diabetes (n = 8) were compared with normal shoulder samples with and without diabetes as the control group (n = 8). Shoulder capsule samples were subjected to whole-transcriptome RNA sequencing, and differential expression was analyzed with EdgeR. Only genes with a false discovery rate < 5% were included for further functional enrichment analysis. RESULTS: The sample population had a mean age of 47 years (range, 24-62 years), and the mean hemoglobin A1c level for nondiabetic and diabetic patients was 5.18% and 8.71%, respectively. RNA-sequencing analysis revealed that 66 genes were differentially expressed between diabetic patients and nondiabetic patients with AC whereas only 3 genes were differentially expressed when control patients with and without diabetes were compared. Furthermore, 286 genes were differentially expressed in idiopathic AC patients, and 61 genes were differentially expressed in diabetic AC patients. On gene clustering analysis, idiopathic AC was enriched with multiple structural and muscle-related pathways, such as muscle filament sliding, whereas diabetic AC included a greater number of hormonal and inflammatory signaling pathways, such as cellular response to corticotropin-releasing factor. CONCLUSIONS: Whole-transcriptome expression profiles demonstrate a fundamentally different underlying pathophysiology when comparing diabetic AC with idiopathic AC, suggesting that these conditions are distinct clinical entities. The new genes expressed explain the differences in the disease course and suggest new therapeutic targets that may lead to different treatment paradigms in these 2 subsets.

Stretch-responsive adhesive microcapsules for strain-regulated antibiotic release from fabric wound dressings.

Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement. Conventional non-woven fabric dressings, including gauzes, bandages and cotton wools, often fail in treating wound infections in a timely manner due to their passive release mechanism of antibiotics. Here, we propose adhesive mechanically-activated microcapsules (MAMCs) capable of strongly adhering to a fibrous matrix to achieve a self-regulated release of antibiotics upon uniaxial stretching of non-woven fabric dressings. To achieve this, a uniform population of polydopamine (PDA)-coated MAMCs (PDA-MAMCs) are prepared using a microfluidics technique and subsequent oxidative dopamine polymerization. The PDA-MAMC allows for robust mechano-activation within the fibrous network through high retention and effective transmission of mechanical force under stretching. By validating the potential of a PDA-MAMCs-laden gauze to release antibiotics in a tensile strain-dependent manner, we demonstrate that PDA-MAMCs can be successfully incorporated into a woven material and create a smart wound dressing for control of bacterial infections. This new mechano-activatable delivery approach will open up a new avenue for a stretch-triggered, on-demand release of therapeutic cargos in skin-mountable or wearable biomedical devices.

Combined Hydrogel and Mesenchymal Stem Cell Therapy for Moderate-Severity Disc Degeneration in Goats.

Intervertebral disc degeneration is a cascade of cellular, structural, and biomechanical changes that is strongly implicated as a cause of low-back pain. Current treatment strategies have poor long-term efficacy as they seek only to alleviate symptoms without preserving or restoring native tissue structure and function. The objective of this study was to evaluate the efficacy of a combined triple interpenetrating network hydrogel (comprising dextran, chitosan, and teleostean) and mesenchymal stem cell (MSC) therapy targeting moderate-severity disc degeneration in a clinically relevant goat model. Degeneration was induced in lumbar discs of 10 large frame goats by injection of chondroitinase ABC. After 12 weeks, degenerate discs were treated by injection of either hydrogel alone or hydrogel seeded with allogeneic, bone marrow-derived MSCs. Untreated healthy and degenerate discs served as controls, and animals were euthanized 2 weeks after treatment. Discs exhibited a significant loss of disc height 12 weeks after degeneration was induced. Two weeks after treatment, discs that received the combined hydrogel and MSC injection exhibited a significant, 10% improvement in disc height index, as well as improvements in histological condition. Discs that were treated with hydrogel alone exhibited reduced tumor necrosis factor-α expression in the nucleus pulposus (NP). Microcomputed tomography imaging revealed that the hydrogel remained localized to the central NP region of all treated discs after 2 weeks of unrestricted activity. These encouraging findings motivate further, longer term studies of therapeutic efficacy of hydrogel and MSC injections in this large animal model. Impact statement Low-back pain is the leading cause of disability worldwide, and degeneration of the intervertebral discs is considered to be one of the most common reasons for low-back pain. Current treatment strategies focus solely on alleviation of symptoms, and there is a critical need for new treatments that also restore disc structure and function. In this study, using a clinically relevant goat model of moderate-severity disc degeneration, we demonstrate that a combined interpenetrating network hydrogel and mesenchymal stem cell therapy provides acute improvements in disc height, histological condition, and local inflammation.

Resorbable Pins to Enhance Scaffold Retention in a Porcine Chondral Defect Model.

OBJECTIVE: Cartilage repair strategies have seen improvement in recent years, especially with the use of scaffolds that serve as a template for cartilage formation. However, current fixation strategies are inconsistent with regards to retention, may be technically challenging, or may damage adjacent tissues or the implant itself. Therefore, the goal of this study was to evaluate the retention and repair potential of cartilage scaffolds fixed with an easy-to-implement bioresorbable pin. DESIGN: Electrospun hyaluronic acid scaffolds were implanted into trochlear groove defects in 3 juvenile and 3 adult pigs to evaluate short-term retention (2 weeks; pin fixation vs. press-fit and fibrin fixation) and long-term repair (8 months; scaffold vs. microfracture), respectively. RESULTS: For the retention study, press-fit and fibrin fixation resulted in short-term scaffold dislodgment (n = 2 each), whereas pin fixation retained all scaffolds that were implanted (n = 6). Pin fixation did not cause any damage to the opposing patellar surface, and only minor changes in the subchondral bone were observed. For long-term repair, no differences were observed between microfracture and scaffold groups, in terms of second-look arthroscopy and indentation testing. On closer visualization with micro computed tomography and histology, a high degree of variability was observed between animals with regard to subchondral bone changes and cartilage repair quality, yet each Scaffold repair displayed similar properties to its matched microfracture control. CONCLUSIONS: In this study, pin fixation did not cause adverse events in either the short- or the long-term relative to controls, indicating that pin fixation successfully retained scaffolds within defects without inhibiting repair.

Hypoxic Preconditioning Enhances Bone Marrow-Derived Mesenchymal Stem Cell Survival in a Low Oxygen and Nutrient-Limited 3D Microenvironment.

OBJECTIVE: Skeletal tissues such as intervertebral disc and articular cartilage possess limited innate potential to regenerate, in part due to their avascularity and low cell density. Despite recent advances in mesenchymal stem cell (MSC)-based disc and cartilage regeneration, key challenges remain, including the sensitivity of these cells to in vivo microenvironmental stress such as low oxygen and limited nutrition. The objective of this study was to investigate whether preconditioning with hypoxia and/or transforming growth factor-ß 3 (TGF-ß3) can enhance MSC survival and extracellular matrix production in a low oxygen and nutrient-limited microenvironment. DESIGN: MSCs from multiple bovine donors were preconditioned in monolayer in normoxia or hypoxia, with or without TGF-ß3, and the global effects on gene expression were examined using microarrays. Subsequently, the effects of preconditioning on MSC survival and extracellular matrix production were examined using low oxygen and nutrient-limited pellet culture experiments. RESULTS: Hypoxic preconditioning resulted in upregulation of genes associated with growth, cell-cell signaling, metabolism, and cell stress response pathways, and significantly enhanced MSC survival for all donors in low oxygen and nutrient-limited pellet culture. In contrast, TGF-ß3 preconditioning diminished survival. The nature and magnitude of the effects of preconditioning with either hypoxia or TGF-ß3 on glycosaminoglycan production were donor dependent. CONCLUSIONS: These results strongly support the use of hypoxic preconditioning to improve postimplantation MSC survival in avascular tissues such as disc and cartilage.

Mechano-activated biomolecule release in regenerating load-bearing tissue microenvironments.

Although mechanical loads are integral for musculoskeletal tissue homeostasis, overloading and traumatic events can result in tissue injury. Conventional drug delivery approaches for musculoskeletal tissue repair employ localized drug injections. However, rapid drug clearance and inadequate synchronization of molecule provision with healing progression render these methods ineffective. To overcome this, a programmable mechanoresponsive drug delivery system was developed that utilizes the mechanical environment of the tissue during rehabilitation (for example, during cartilage repair) to trigger biomolecule provision. For this, a suite of mechanically-activated microcapsules (MAMCs) with different rupture profiles was generated in a single fabrication batch via osmotic annealing of double emulsions. MAMC physical dimensions were found to dictate mechano-activation in 2D and 3D environments and their stability in vitro and in vivo, demonstrating the tunability of this system. In models of cartilage regeneration, MAMCs did not interfere with tissue growth and activated depending on the mechanical properties of the regenerating tissue. Finally, biologically active anti-inflammatory agents were encapsulated and released from MAMCs, which counteracted degradative cues and prevented the loss of matrix in living tissue environments. This unique technology has tremendous potential for implementation across a wide array of musculoskeletal conditions for enhanced repair of load-bearing tissues.

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-ß, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

Characterization of Injury Induced by Routine Surgical Manipulations of Nasal Septal Cartilage.

IMPORTANCE: This study characterizes and compares common surgical manipulations' effects on septal cartilage to understand their implications for rhinoplasty outcomes based on cell viability and cartilage health. OBJECTIVE: To illustrate distinct differences in the impact of various surgical manipulations on septal cartilage in an in vitro septal cartilage model. A secondary objective is to better understand the chondrocyte's response to injury as well as how alterations in the extracellular matrix correspond to chondrocyte viability. DESIGN, SETTING, AND PARTICIPANTS: In this bench-top in vitro porcine model using juvenile bovine septal cartilage from bovine snouts, easily obtainable septal cartilage was used to generate large numbers of homogenous cartilage specimens. Quantitative outcomes at early and late time points were cell viability, cell stress, matrix loss, and qualitative assessment through histologic examination. The study was performed at a single academic tertiary care research hospital. INTERVENTIONS: Four common surgical manipulations were contrasted with a control group: crushed cartilage, scored cartilage, diced cartilage, and shaved cartilage. MAIN OUTCOMES AND MEASURES: Following the manipulation of the cartilage, the quantitative outcomes were glycosaminoglycan release to the media, lactate dehydrogenase release to the media, and cell death analysis through apoptosis staining. The qualitative outcomes were histologic staining of the manipulated cartilage with safranin-O/fast green stain to identify proteoglycan loss. RESULTS: The crushing followed by shaving manipulations were the most damaging as indicated by increased levels of lactate dehydrogenase release, glycosaminoglycans loss, and cell death. Matrix loss did not increase until after 48 hours postinjury. Furthermore, chondrocyte death was seen early after injury and accelerated to the late time point, day 9, in all manipulations. Conversely, cell stress was found to be greater at 48 hours postinjury, which then declined to the late time point, day 9. CONCLUSIONS AND RELEVANCE: The crushing manipulation followed by shaving and then dicing were the most destructive methods of cartilage manipulation relative to control specimens. Collectively, these outcomes demonstrate the range of injury which occurs with all septal cartilage manipulations and can inform rhinoplasty practice to use the least damaging effective surgical manipulation to obtain the desired outcome. LEVEL OF EVIDENCE: NA.

Mechanically-Activated Microcapsules for 'On-Demand' Drug Delivery in Dynamically Loaded Musculoskeletal Tissues.

Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimuli-responsive delivery systems can facilitate 'on-demand' release of therapeutics in response to a variety of physiologic triggering mechanisms (e.g. pH, temperature). However, few systems to date have taken advantage of mechanical inputs from the microenvironment to initiate drug release. Here, we developed mechanically-activated microcapsules (MAMCs) that are designed to deliver therapeutics in an on-demand fashion in response to the mechanically loaded environment of regenerating musculoskeletal tissues, with the ultimate goal of furthering tissue repair. To establish a suite of microcapsules with different thresholds for mechano-activation, we first manipulated MAMC physical dimensions and composition, and evaluated their mechano-response under both direct 2D compression and in 3D matrices mimicking the extracellular matrix properties and dynamic loading environment of regenerating tissue. To demonstrate the feasibility of this delivery system, we used an engineered cartilage model to test the efficacy of mechanically-instigated release of TGF-ß3 on the chondrogenesis of mesenchymal stem cells. These data establish a novel platform by which to tune the release of therapeutics and/or regenerative factors based on the physiologic dynamic mechanical loading environment, and will find widespread application in the repair and regeneration of numerous musculoskeletal tissues.