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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 . 202x;4x(x):xx–xx.]

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 A(1c) 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. LEVEL OF EVIDENCE: Basic Science Study; Molecular Biology

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.

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.

Conventional treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus (AF), resulting in a high incidence of recurrent herniation and persistent disfunction. The lack of repair and the acute inflammation that arise after injury further compromises the disc and can result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annular repair and the local delivery of bioactive anti-inflammatory factors. TARPs transmit physiologic strains to mechanically-activated microcapsules (MAMCs) embedded within, which activate and release encapsulated biomolecules in response to physiologic loading. Here, we demonstrate that the TARP design modulates implant biomechanical properties and regulates MAMC mechano-activation. Next, the FDA-approved anti-inflammatory molecule, interleukin 1 receptor antagonist, Anakinra, was loaded in TARPs and the effects of TARP-mediated annular repair and Anakinra delivery was evaluated in a model of annular injury in the goat cervical spine. TARPs showed robust integration with the native tissue and provided structural reinforcement at the injury site that prevented disc-wide aberrant remodeling resulting from AF detensioning. The delivery of Anakinra via TARP implantation improved the retention of disc biochemical composition through increased matrix deposition and retention at the site of annular injury. Anakinra delivery additionally attenuated the inflammatory response associated by scaffold implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the AF. These results demonstrate the translational and therapeutic potential of this novel TARP system for the treatment of intervertebral disc herniations.One Sentence SummaryTension-activated repair patches delivering bioactive anti-inflammatory factors improve healing in an in vivo goat cervical disc injury model.

Post-traumatic 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 (HA), 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 (COMP) in three regions of interest. As secondary outcome measures, we examined animals' lameness, performed arthroscopic, radiographic, and 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. This article is protected by copyright. All rights reserved.

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.

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 poly(D,L-lactide-co-glycolide) (PLGA) microcapsules containing the pro-osteogenic agents triiodothyronine and ß-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that developed microcapsules rupturedin vitrounder increasing mechanical loads, and readily sink within a liquid solution, allowing for gravity-based positioning onto the osteochondral surface. In a large animal, these mechano-active 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 time in 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 locally administered, 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 1 and 2 weeks after injection and surgery. Analysis of injected MAMCs showed that bioactive cargo was released in a controlled fashion over 2 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 establishes a new micro-fluidically generated delivery platform that releases therapeutic factors in an articulating joint, and reduces this to practice in the delivery of therapeutics that preserve bone structure after osteochondral injury.

Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement and other complications. 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.

Cartilage is a specialized tissue that has a relatively homogenous endogenous cell population but a diverse extracellular matrix (ECM) with depth-dependent mechanical properties. Cartilage repair remains an elusive clinical goal, with biological interventions preferred to arthroplasty in younger patients. Osteochondral transplantation (OCT) has emerged for the treatment of cartilage defects and osteoarthritis. Fresh allografts stored at 4 °C for periods of time have been utilized, though matrix and cell viability loss remained an issue. To address this, several studies have developed media formulations to maintain cartilage explants in vitro. One promising factor for these applications is sprifermin, a human-recombinant fibroblast growth factor-18 which stimulates chondrocyte proliferation and matrix synthesis and is in clinical trials for osteoarthritis treatment. We hypothesized that addition of sprifermin during storage would maintain the unique depth-dependent mechanical profile of cartilage explants, a feature of cartilage not often evaluated. In this study, cartilage explants were maintained for up to 6 weeks with or without a weekly 24-hour exposure to sprifermin (100 ng/ml) and the compressive modulus was assessed. Results showed that sprifermin treated samples maintained their depth-dependent mechanical profile through 3 weeks, whereas untreated samples lost their mechanical integrity over 1 week of culture. Sprifermin also affected ECM balance by maintaining levels of extracellular collagen and suppression of matrix metalloproteinase production. These findings support the use of sprifermin as a medium additive for OCT allografts during in vitro storage, and present a potential mechanism where sprifermin may impact a functional characteristic of cartilage in repair strategies.

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 and β-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that the developed microcapsules ruptured in vitro under 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 time in 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 patterned in 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.

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.

Background: Post traumatic osteoarthritis is a disabling condition impacting 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. Methods: For the purpose if 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 mg) or with saline, hyaluronan (HA), and evaluated animals functional and structural outcomes over 24 weeks. For primary outcome measures we performed histological evaluations and performed gene expression analysis of aggrecan, collagen types I and II, and cartilage oligomeric matrix protein (COMP) in three regions of interest. As secondary outcome measures we examined animal’s lameness, performed arthroscopic, radiographic, and CT scan imaging, and gross morphology assessment. Results: We detected the highest treatment benefit following 100 mg sprifermin treatment. Overall histological assessment showed an improvement in the kissing region and 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. Conclusion: Our results demonstrated for the first time, an enhancement in outcomes of the microfracture treatment following sprifermin treatment, suggesting a cartilage regenerative role and a potential benefit of sprifermin treatment in early cartilage injuries.

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.

Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cartilage exhibits limited healing capacity that declines with tissue maturation. Many approaches have been investigated for their ability to stimulate healing of mature cartilage or integration of repair tissue or tissue-engineered constructs with native cartilage. Growth factors present in immature tissue may enhance chondrogenesis and promote integrative repair of cartilage defects. In this study, we assessed the role of one such factor, fibroblast growth factor 18 (FGF18). Studies using FGF18 have shown a variety of positive effects on cartilage, including stimulation of chondrocyte proliferation, matrix biosynthesis, and suppression of proteinase activity. To explore the role of FGF18 on cartilage defect repair, we hypothesized that treatment with recombinant human FGF18 (sprifermin) would increase matrix synthesis in a defect model, thus improving integration strength. To test this hypothesis, 6 mm cartilage cylinders were harvested from juvenile bovine knees. A central 3 mm defect was created in each explant, and this core was removed and replaced. Resulting constructs were cultured in control or sprifermin-containing medium (weekly 24-h exposure of 100 ng/ml sprifermin) for 4 weeks. Mechanical testing, biochemical analysis, micro-CT, scanning electron microscopy, and histology were used to assess matrix production, adhesive strength, and structural properties of the cartilage-cartilage interface. Results showed greater adhesive strength, increased collagen content, and larger contact areas between core and annular cartilage in the sprifermin-treated group. These findings present a novel treatment for cartilage injuries that have potential to enhance defect healing and lateral cartilage-cartilage integration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2648-2656, 2018.

Tissue engineering is a promising approach for the repair of articular cartilage defects, with engineered constructs emerging that match native tissue properties. However, the inflammatory environment of the damaged joint might compromise outcomes, and this may be impacted by the choice of cell source in terms of their ability to operate anabolically in an inflamed environment. Here, we compared the response of engineered cartilage derived from native chondrocytes and mesenchymal stem cells (MSCs) to challenge by TNFα and IL-1β in order to determine if either cell type possessed an inherent advantage. Compositional (extracellular matrix) and functional (mechanical) characteristics, as well as the release of catabolic mediators (matrix metalloproteinases [MMPs], nitric oxide [NO]) were assessed to determine cell- and tissue-level changes following exposure to IL-1β or TNF-α. Results demonstrated that MSC-derived constructs were more sensitive to inflammatory mediators than chondrocyte-derived constructs, exhibiting a greater loss of proteoglycans and functional properties at lower cytokine concentrations. While MSCs and chondrocytes both have the capacity to form functional engineered cartilage in vitro, this study suggests that the presence of an inflammatory environment is more likely to impair the in vivo success of MSC-derived cartilage repair. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2901-2910, 2018.

The purpose of study was to investigate the maturation of mesenchymal stem cells (MSC) laden in HA constructs with various combinations of chemically defined medium (CM) components and determine the impact of dexamethasone and serum on construct properties. Constructs were cultured in CM with the addition or withdrawal of media components or were transferred to serum containing media that partially represents an in vivo-like condition where pro-inflammatory signals are present. Constructs cultured in CM+ (CM with TGF-β3) and DEX- (CM+ without dexamethasone) conditions produced robust matrix, while those in ITS/BSA/LA- (CM+ without ITS/BSA/LA) and Serum+ (10% FBS with TGF-β3) produced little matrix. While construct properties in DEX- were greater than those in CM+ at 4 weeks, properties in CM+ and DEX- reversed by 8 weeks. While construct properties in DEX- were greater than those in CM+ at 4 weeks, the continued absence or removal of dexamethasone resulted in marked GAG loss by 8 weeks. Conversely, the continued presence or new addition of dexamethasone at 4 weeks further improved or maintained construct properties through 8 weeks. Finally, when constructs were converted to Serum (in the continued presence of TGF-β3 with or without dexamethasone) after pre-culture in CM+ for 4 weeks, GAG loss was attenuated with addition of dexamethasone. Interestingly, however, collagen content and type was not impacted. In conclusion, dexamethasone influences the functional maturation of MSC-laden HA constructs, and may help to maintain properties during long-term culture or with in vivo translation by repressing pro-inflammatory signals. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1717-1727, 2018.

After cartilage injury and repair, the subchondral bone plate remodels. Skeletal maturity likely impacts both bone remodeling and inherent cartilage repair capacity. The objective of this study was to evaluate subchondral bone remodeling as a function of injury type, repair scenario, and skeletal maturity in a Yucatan minipig model. Cartilage defects (4 mm) were created bilaterally in the trochlear groove. Treatment conditions included a full thickness chondral defect (full chondral defect, n = 3 adult/3 juvenile), a partial thickness (∼50%) chondral defect (PCD, n = 3/3), and FCD treated with microfracture (MFX, n = 3/3). At 6 weeks postoperatively, osteochondral samples containing the lesion site were imaged by micro-computed tomography (CT) and analyzed by histology and immunohistochemistry. Via micro-CT, FCD and MFX groups showed increased bone loss in juveniles compared with adults. Quantification of histology using the ICRS II scoring system showed equal overall assessment for the FCD groups and better overall assessment in juvenile animals treated with MFX compared with adults. All FCD and MFX groups were inferior to control samples. For the PCD injury, both age groups had values close to the control values. For the FCD groups, there were greater alterations in the subchondral bone in juveniles compared with adults. Staining for collagen II showed more intense signals in juvenile FCD and MFX groups compared with adults. This large animal study of cartilage repair shows the significant impact of skeletal maturity on the propensity of subchondral bone to remodel as a result of chondral injury. This will improve selection criteria for animal models for studying cartilage injury, repair, and treatment.

Degeneration of the intervertebral discs is a progressive cascade of cellular, compositional and structural changes that is frequently associated with low back pain. As the first signs of disc degeneration typically arise in the disc’s central nucleus pulposus (NP), augmentation of the NP via hydrogel injection represents a promising strategy to treat early to mid-stage degeneration. The purpose of this study was to establish the translational feasibility of a triple interpenetrating network hydrogel composed of dextran, chitosan, and teleostean (DCT) for augmentation of the degenerative NP in a preclinical goat model. Ex vivo injection of the DCT hydrogel into degenerated goat lumbar motion segments restored range of motion and neutral zone modulus towards physiologic values. To facilitate non-invasive assessment of hydrogel delivery and distribution, zirconia nanoparticles were added to make the hydrogel radiopaque. Importantly, the addition of zirconia did not negatively impact viability or matrix producing capacity of goat mesenchymal stem cells or NP cells seeded within the hydrogel in vitro. In vivo studies demonstrated that the radiopaque DCT hydrogel was successfully delivered to degenerated goat lumbar intervertebral discs, where it was distributed throughout both the NP and annulus fibrosus, and that the hydrogel remained contained within the disc space for two weeks without evidence of extrusion. These results demonstrate the translational potential of this hydrogel for functional regeneration of degenerate intervertebral discs. Statement of Significance The results of this work demonstrate that a radiopaque hydrogel is capable of normalizing the mechanical function of the degenerative disc, is supportive of disc cell and mesenchymal stem cell viability and matrix production, and can be maintained in the disc space without extrusion following intradiscal delivery in a preclinical large animal model. These results support evaluation of this hydrogel as a minimally invasive disc therapeutic in long-term preclinical studies as a precursor to future clinical application in patients with disc degeneration and low back pain.

Collagen fibrils in tendon are believed to be discontinuous and transfer tensile loads through shear forces generated during interfibrillar sliding. However, the structures that transmit these interfibrillar forces are unknown. Various extrafibrillar tissue components (e.g., glycosaminoglycans, collagens XII and XIV) have been suggested to transmit interfibrillar loads by bridging collagen fibrils. Alternatively, collagen fibrils may interact directly through physical fusions and interfibrillar branching. The objective of this study was to test whether extrafibrillar proteins are necessary to transmit load between collagen fibrils or if interfibrillar load transfer is accomplished directly by the fibrils themselves. Trypsin digestions were used to remove a broad spectrum of extrafibrillar proteins and measure their contribution to the multiscale mechanics of rat tail tendon fascicles. Additionally, images obtained from serial block-face scanning electron microscopy were used to determine the three-dimensional fibrillar organization in tendon fascicles and identify any potential interfibrillar interactions. While trypsin successfully removed several extrafibrillar tissue components, there was no change in the macroscale fascicle mechanics or fibril:tissue strain ratio. Furthermore, the imaging data suggested that a network of smaller diameter fibrils (

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-1β [IL-1β], 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-β, 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.

ObjectiveSkeletal 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–β 3 (TGF-β3) can enhance MSC survival and extracellular matrix production in a low oxygen and nutrient-limited microenvironment.DesignMSCs from multiple bovine donors were preconditioned in monolayer in normoxia or hypoxia, with or without TGF-β3, 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.ResultsHypoxic 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-β3 preconditioning diminished survival. The nature and magnitude of the effects of preconditioning with either hypoxia or TGF-β3 on glycosaminoglycan production were donor dependent.ConclusionsThese results strongly support the use of hypoxic preconditioning to improve postimplantation MSC survival in avascular tissues such as disc and cartilage.

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-β3 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.

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.

Perlecan/heparan sulfate proteoglycan 2 (HSPG2), a large HSPG, is indispensable for the development of musculoskeletal tissues, where it is deposited within the pericellular matrix (PCM) surrounding chondrocytes and disappears nearly completely at the chondro-osseous junction (COJ) of developing long bones. Destruction of perlecan at the COJ converts an avascular cartilage compartment into one that permits blood vessel infiltration and osteogenesis. Mutations in perlecan are associated with chondrodysplasia with widespread musculoskeletal and joint defects. This study elucidated novel signaling roles of perlecan core protein in endochondral bone formation and chondrocyte behavior. Perlecan subdomains were tested for chondrogenic properties in ATDC5 cells, a model for early chondrogenesis. A region within domain IV of perlecan (HSPG2 IV-3) was found to promote rapid prechondrocyte clustering. Introduction of the mutation (R3452Q) associated with the human skeletal disorder Schwartz-Jampel syndrome limited HSPG2 IV-3-induced clustering. HSPG2 IV-3 activity was enhanced when thermally unfolded, likely because of increased exposure of the active motif(s). HSPG2 IV-3-induced clustering was accompanied by the deactivation of key components of the focal adhesion complex, FAK and Src, with increased messenger RNA (mRNA) levels of precartilage condensation markers Sox9 and N-cadherin ( Cdh2), and cartilage PCM components collagen II ( Col2a1) and aggrecan ( Acan). HSPG2 IV-3 reduced signaling through the ERK pathway, where loss of ERK1/2 phosphorylation coincided with reduced FoxM1 protein levels and increased mRNA levels cyclin-dependent kinase inhibitor 1C (Cdkn1c) and activating transcription factor 3 ( Atf3), reducing cell proliferation. These findings point to a critical role for perlecan domain IV in cartilage development through triggering chondrocyte condensation.

ObjectiveCD14 is a monocyte/macrophage pattern-recognition receptor that modulates innate inflammatory signaling. Soluble CD14 levels in knee OA synovial fluids are associated with symptoms and progression of disease. Here we investigate the role of this receptor in development of OA using a murine joint injury model of disease.Methods10-week-old Male C57BL/6 (WT) and CD14-deficient (CD14-/-) mice underwent destabilization of the medial meniscus (DMM) surgery to induce OA. Joint histopathology was used to examine cartilage damage, and microCT to evaluate subchondral bone (SCB) remodeling at 6 and 19 weeks after surgery. Synovial and fat pad expression of macrophage markers (F4/80, CD11c, CD68, iNOS, CCR7, CD163 and CD206) was assessed by flow cytometry and droplet digital (dd)PCR. Changes in locomotive activity indicative of joint pain were evaluated longitudinally up to 16 weeks by automated behavioral analysis.ResultsEarly cartilage damage scores 6 weeks post-DMM were similar in both strains (Mean score ±SEM WT: 4.667±1.38, CD14-/-: 4.6±0.6), but at 19 weeks were less severe in CD14-/- (6.0±0.46) than in WT mice (13.44±2.5, p = 0.0002). CD14-/- mice were protected from both age-related and post-surgical changes in SCB mineral density and trabecular thickness. In addition, CD14-/- mice were protected from decreases in climbing activity (p = 0.015 vs. WT, 8 weeks) observed after DMM. Changes in synovial/fat pad expression of CCR7, a marker of M1 macrophages, were slightly reduced post-DMM in the absence of CD14, while expression of CD68 (pan-macrophage marker) and CD163 (M2 marker) were unchanged.ConclusionCD14 plays an important role in progression of structural and functional features of OA in the DMM model, and may provide a new target for therapeutic development.

Objective We have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor–β3 [TGF-β3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone. Design Trochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-β3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. Results Treatment with TGF-β3 led to a marked increase in positive staining for collagen type II within defects ( P < 0.05), while delivery of MSCs did not ( P > 0.05). Neither condition had an impact on other histological semiquantitative scores ( P > 0.05), and inclusion of MSCs led to significantly less defect fill ( P < 0.05). For all measurements, no synergistic interaction was found between TGF-β3 and MSC treatment when they were delivered together ( P > 0.05). Conclusions At this early healing time point, treatment with TGF-β3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-β3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics, and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc.

Preclinical large animal models are essential for evaluating new tissue engineering (TE) technologies and refining surgical approaches for cartilage repair. Some preclinical animal studies, including the commonly used minipig model, have noted marked remodeling of the subchondral bone. However, the mechanisms underlying this response have not been well characterized. Thus, our objective was to compare in-vivo outcomes of chondral defects with varied injury depths and treatments.Trochlear chondral defects were created in 11 Yucatan minipigs (6 months old). Groups included an untreated partial-thickness defect (PTD), an untreated full-thickness defect (FTD), and FTDs treated with microfracture, autologous cartilage transfer (FTD-ACT), or an acellular hyaluronic acid hydrogel. Six weeks after surgery, micro-computed tomography (μCT) was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. A finite element model (FEM) was developed to assess load transmission.Using μCT, substantial bone remodeling was observed for all FTDs, but not for the PTD group. The best overall histological scores and greatest type II collagen staining was found for the FTD-ACT and PTD groups. The FEM confirmed that only the FTD-ACT group could initially restore appropriate transfer of compressive loads to the underlying bone.The bony remodeling observed in this model system appears to be a biological phenomena and not a result of altered mechanical loading, with the depth of the focal chondral defect (partial vs. full thickness) dictating the bony remodeling response. The type of cartilage injury should be carefully controlled in studies utilizing this model to evaluate TE approaches for cartilage repair.

Elevated chemokine receptor Ccr7 is observed in knee osteoarthritis (OA) and associated with severity of symptoms. In this study, we confirmed that CCR7 protein expression is elevated in synovial tissue from OA patients by immunohistochemical staining. We then investigated whether Ccr7 deficiency impacted structural and functional joint degeneration utilizing a murine model of OA. OA-like disease was induced in male C57BL/6 and Ccr7-deficient (Ccr7-/-) mice by destabilization of the medial meniscus (DMM). Functional deficits were measured by computer integrated monitoring of spontaneous activity every 4 weeks after DMM surgery up 16 weeks. Joint degeneration was evaluated at 6 and 19 weeks post-surgery by histopathology, and subchondral bone changes analyzed by microCT. Results showed reduction in locomotor activities in DMM-operated C57BL/6 mice by 8 weeks, while activity decreases in Ccr7-/- mice were delayed until 16 weeks. Histopathologic evaluation showed minimal protection from early cartilage degeneration (p = 0.06) and osteophytosis (p = 0.04) in Ccr7-/- mice 6 weeks post-DMM compared to C57BL/6 controls, but not at 19 weeks. However, subchondral bone mineral density (p = 0.03) and histologic sclerosis (p = 0.02) increased in response to surgery in C57BL/6 mice at 6 weeks, while Ccr7-/- mice were protected from these changes. Our results are the first to demonstrate a role for Ccr7 in early development of functional deficits and subchondral bone changes in the DMM model. Understanding the mechanism of Ccr7 receptor signaling in the initiation of joint pathology and disability will inform the development of innovative therapies to slow symptomatic OA development after injury. This article is protected by copyright. All rights reserved

The purpose of study was to investigate the maturation of mesenchymal stem cells (MSC) laden in HA constructs with various combinations of chemically defined medium (CM) components and determine the impact of dexamethasone and serum on construct properties. Constructs were cultured in CM with the addition or withdrawal of media components or were transferred to serum containing media that partially represents an in vivo-like condition where pro-inflammatory signals are present. Constructs cultured in CM+ (CM with TGF-β3) and DEX- (CM+ without dexamethasone) conditions produced robust matrix, while those in ITS/BSA/LA- (CM+ without ITS/BSA/LA) and Serum+ (10% FBS with TGF-β3) produced little matrix. While construct properties in DEX- were greater than those in CM+ at 4 weeks, properties in CM+ and DEX- reversed by 8 weeks. While construct properties in DEX- were greater than those in CM+ at 4 weeks, the continued absence or removal of dexamethasone resulted in marked GAG loss by 8 weeks. Conversely, the continued presence or new addition of dexamethasone at 4 weeks further improved or maintained construct properties through 8 weeks. Finally, when constructs were converted to Serum (in the continued presence of TGF-β3 with or without dexamethasone) after pre-culture in CM+ for 4 weeks, GAG loss was attenuated with addition of dexamethasone. Interestingly, however, collagen content and type was not impacted. In conclusion, dexamethasone influences the functional maturation of MSC-laden HA constructs, and may help to maintain properties during long-term culture or with in vivo translation by repressing pro-inflammatory signals. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1717-1727, 2018.

Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the field has progressed toward utilizing large animal models for proof of concept and preclinical studies. Mechanical testing of the regenerative tissue is an essential outcome for functional evaluation. However, testing modalities and constitutive frameworks used to evaluate in vitro grown samples differ substantially from those used to evaluate in vivo derived samples. To address this, we developed finite element (FE) models (using FEBio) of unconfined compression and indentation testing, modalities commonly used for such samples. We determined the model sensitivity to tissue radius and subchondral bone modulus, as well as its ability to estimate material parameters using the built-in parameter optimization tool in FEBio. We then sequentially tested agarose gels of 4%, 6%, 8%, and 10% weight/weight using a custom indentation platform, followed by unconfined compression. Similarly, we evaluated the ability of the model to generate material parameters for living constructs by evaluating engineered cartilage. Juvenile bovine mesenchymal stem cells were seeded (2 × 107 cells/mL) in 1% weight/volume hyaluronic acid hydrogels and cultured in a chondrogenic medium for 3, 6, and 9 weeks. Samples were planed and tested sequentially in indentation and unconfined compression. The model successfully completed parameter optimization routines for each testing modality for both acellular and cell-based constructs. Traditional outcome measures and the FE-derived outcomes showed significant changes in material properties during the maturation of engineered cartilage tissue, capturing dynamic changes in functional tissue mechanics. These outcomes were significantly correlated with one another, establishing this FE modeling approach as a singular method for the evaluation of functional engineered and native tissue regeneration, both in vitro and in vivo.

Objective To test different fixation methods of a 3-dimensionally woven poly(ϵ-caprolactone) (PCL) scaffold within chondral defects of a weightbearing large animal model. Methods Full thickness chondral defects were made in the femoral condyles of 15 adult male Yucatan mini-pigs. Two surgical approaches were compared including total arthrotomy (traditional) and a retinaculum-sparing, minimally invasive surgery (MIS) approach. Following microfracture (MFX), scaffolds were placed without fixation or were fixed with fibrin glue, suture, or subchondral anchor. Experimental endpoints were between 1 and 6 weeks. Micro–computed tomography and histology were used to assess samples. Results The MIS approach was superior as the traditional approach caused medial condyle cartilage wear. One of 13 (7.7%) of scaffolds without fixation, 4 of 11 (36.3%) fibrin scaffolds, 1 of 4 (25%) of sutured scaffolds, and 9 of 9 (100%) of anchor-fixed scaffolds remained in place. Histology demonstrated tissue filling with some overgrowth of PCL scaffolds. Conclusions Of the methods tested, the MIS approach coupled with subchondral anchor fixation provided the best scaffold retention in a mini-pig chondral defect model. This finding has implications for fixation strategies in future animal studies and potential future human use.

Summary Objective A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. Method We utilized a high-throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. Results Engineered cartilage response to injury was strain dependent, with a 2-fold increase in glycosaminoglycan (GAG) loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in lactate dehydrogenase (LDH) release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor [Z-VAD-FMK (ZVF)] was effective at reducing cell death, while the amphiphilic polymer [Poloxamer 188 (P188)] and the free-radical scavenger [ N -Acetyl-L-cysteine (NAC)] reduced GAG loss as compared to injury alone. Conclusions The injury response in this engineered cartilage model replicated key features of the response of cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA.

One of the most critical parameters in cartilage tissue engineering which influences the clinical success of a repair therapy is the ability to match the load-bearing capacity of the tissue as it functions in vivo. While mechanical forces are known to positively influence the development of cartilage matrix architecture, these same forces can induce long-term implant failure due to poor integration or structural deficiencies. As such, in the design of optimal repair strategies, it is critical to understand the timeline of construct maturation and how the elaboration of matrix correlates with the development of mechanical properties. We have previously characterized a scaffold-free method to engineer cartilage utilizing primary chondrocytes cultured at high density in hydrogel-coated culture vessels to promote the formation of a self-aggregating cell suspension that condenses to form a cartilage-like biomass, or cartilage tissue analog (CTA). Chondrocytes in these CTAs maintain their cellular phenotype and deposit extracellular matrix to form a construct that has characteristics similar to native cartilage; however, the mechanical integrity of CTAs had not yet been evaluated. In this study, we found that chondrocytes within CTAs produced a robust matrix of proteoglycans and collagen that correlated with increasing mechanical properties and decreasing cell-matrix ratios, leading to properties that approached that of native cartilage. These results demonstrate a unique approach to generating a cartilage-like tissue without the complicating factor of scaffold, while showing increased compressive properties and matrix characteristics consistent with other approaches, including scaffold-based constructs. To further improve the mechanics of CTAs, studies are currently underway to explore the effect of hydrodynamic loading and whether these changes would be reflective of in vivo maturation in animal models. The functional maturation of cartilage tissue analogs as described here support this engineered cartilage model for use in clinical and experimental applications for repair and regeneration in joint-related pathologies.

Given the limitations of current surgical approaches to treat articular cartilage injuries, tissue engineering (TE) approaches have been aggressively pursued. Despite reproduction of key mechanical attributes of native tissue, the ability of TE cartilage constructs to integrate with native tissue must also be optimized for clinical success. In this paper, we propose a "trajectory-based" tissue engineering (TB-TE) approach, based on the hypothesis that time-dependent increases in construct maturation in-vitro prior to implantation (i.e. positive rates) may provide a reliable predictor of in-vivo success. As an example TE system, we utilized hyaluronic acid hydrogels laden with mesenchymal stem cells. We first modeled the maturation of these constructs in-vitro to capture time-dependent changes. We then performed a sensitivity analysis of the model to optimize the timing and amount of data collection. Finally, we showed that integration to cartilage in-vitro is not correlated to the maturation state of TE constructs, but rather their maturation rate, providing a proof-of-concept for the use of TB-TE to enhance treatment outcomes following cartilage injury. This new approach challenges the traditional TE paradigm of matching only native state parameters of maturity and emphasizes the importance of also establishing an in-vitro trajectory in constructs in order to improve the chance of in-vivo success.

The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and may reflect an important adaptive response to its complex, multiaxial loading environment. However, these functional properties are associated with a location-dependent structure and composition that have not been fully elucidated. Therefore, the objective of this study was to determine the concentrations of types I, II and III collagen in six distinct regions of the SST and compare changes in collagen concentration across regions with local changes in mechanical properties. We hypothesized that type I collagen content would be high throughout the tendon, type II collagen would be restricted to regions of compressive loading and type III collagen content would be high in regions associated with damage. We further hypothesized that regions of high type III collagen content would correspond to regions with low tensile modulus and a low degree of collagen alignment. Although type III collagen content was not significantly higher in regions that are frequently damaged, all other hypotheses were supported by our results. In particular, type II collagen content was highest near the insertion while type III collagen was inversely correlated with tendon modulus and collagen alignment. The measured increase in type II collagen under the coracoacromial arch provides evidence of adaptation to compressive loading in the SST. Moreover, the structure-function relationship between type III collagen content and tendon mechanics established in this study demonstrates a mechanism for altered mechanical properties in pathological tendons and provides a guideline for identifying therapeutic targets and pathology-specific biomarkers.