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Introduction: Nipple reconstruction is the essential last step of breast reconstruction after total mastectomy, resulting in improved general and aesthetic satisfaction. However, most techniques are limited by secondary scar contracture and loss of neo-nipple projection leading to patient dissatisfaction. Approximately, 16,000 patients undergo autologous flap breast reconstruction annually, during which the excised costal cartilage (CC) is discarded. We propose utilizing processed CC placed within biocompatible 3D-printed external scaffolds to generate tissue cylinders that mimic the shape, size and biomechanical properties of native human nipple tissue while mitigating contracture and projection loss., Methods: External scaffolds were designed and then 3D-printed using polylactic acid (PLA). Patient-derived CC was processed by mincing or zesting, then packed into the scaffolds, implanted into nude rats and explanted after 3 months for volumetric, histologic and biomechanical analyses. Similar analyses were performed on native human nipple tissue and unprocessed CC., Results: After 3 months in vivo, gross analysis demonstrated significantly greater preservation of contour, projection and volume of the scaffolded nipples. Mechanical analysis demonstrated that processing of the cartilage resulted in implant equilibrium modulus values closer to that of the human nipple. Histologic analysis showed the presence of healthy and viable cartilage after 3 months in vivo, invested with fibrovascular tissue., Conclusions: Autologous CC can be processed intraoperatively and placed within biocompatible external scaffolds to mimic the shape and biomechanical properties of the native human nipple. This allows for custom design and fabrication of individualized engineered autologous implants tailored to patient desire, without the loss of projection seen with traditional approaches., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Multiple human tissue engineered cartilage constructs are showing promise in advanced clinical trials but identifying important measures of manufacturing reproducibility remains a challenge. FDA guidance suggests measuring multiple mechanical properties prior to implantation, because these properties could affect the long term success of the implant. Additionally, these engineered cartilage mechanics could be sensitive to the autologous chondrocyte source, an inherently irregular manufacturing starting material. If any mechanical properties are sensitive to changes in the autologous chondrocyte source, these properties may need to be measured prior to implantation to ensure manufacturing reproducibility and quality. Therefore, this study identified variability in the compressive, friction, and shear properties of a human tissue engineered cartilage constructs due to the chondrocyte source. Over 200 constructs were created from 7 different chondrocyte sources and tested using 3 distinct mechanical experiments. Under confined compression, the compressive properties (aggregate modulus and hydraulic permeability) varied by orders of magnitude due to the chondrocyte source. The friction coefficient changed by a factor of 5 due to the chondrocyte source and high intrapatient variability was noted. In contrast, the shear modulus was not affected by changes in the chondrocyte source. Finally, measurements on the local compressive and shear mechanics revealed variability in the depth dependent strain fields based on chondrocyte source. Since the chondrocyte source causes large amounts of variability in the compression and local mechanical properties of engineered cartilage, these mechanical properties may be important measures of manufacturing reproducibility. STATEMENT OF SIGNIFICANCE: Although the FDA recommends measuring mechanical properties of human tissue engineered cartilage constructs during manufacturing, the effect of manufacturing variability on construct mechanics is unknown. As one of the first studies to measure multiple mechanical properties on hundreds of human tissue engineered cartilage constructs, we found the compressive properties are most sensitive to changes in the autologous chondrocyte source, an inherently irregular manufacturing variable. This sensitivity to the autologous chondrocyte source reveals the compressive properties should be measured prior to implantation to assess manufacturing reproducibility., Competing Interests: Declaration of Competing Interest This research was partially funded through Histogenics. C.D. E.C. and S.K, were full time employees and stockholders of Histogenics Corp during the data collection. J.M., N.D., B.K., and L.B., were partially funded by an award from Histogenics to Cornell University., (Copyright © 2021. Published by Elsevier Ltd.)

Recapitulating the collagen fiber structure of native menisci is one of the major challenges in the development of tissue-engineered menisci. Native collagen fibers are developed by the complex interplay of biochemical and biomechanical signals. In this study, we optimized glucose and transforming growth factor-β1 (TGF-β1) concentrations in combination with mechanical anchoring to balance contributions of proteoglycan synthesis and contractile behavior in collagen fiber assembly. Glucose had a profound effect on the final dimensions of collagen-based constructs. TGF-β1 influenced construct contraction rate and glycosaminoglycan (GAG) production with two half-maximal effective concentration (EC 50 ) ranges, which are 0.23 to 0.28 and 0.53 to 1.71 ng/mL, respectively. At concentrations less than the EC 50 , for the GAG production and contraction rate, TGF-β1 treatment resulted in less organized collagen fibers. At concentrations greater than the EC 50 , TGF-β1 led to dense, disorganized collagen fibers. Between the two EC 50 values, collagen fiber diameter and length increased. The effects of TGF-β1 on fiber development were enhanced by mechanical anchoring, leading to peaks in fiber diameter, length, and alignment index. Fiber diameter and length increased from 7.9 ± 1.4 and 148.7 ± 16.4 to 17.5 ± 2.1 and 262.0 ± 13.0 μm, respectively. The alignment index reached 1.31, comparable to that of native tissue, 1.40. These enhancements in fiber architecture resulted in significant increases in tensile modulus and ultimate tensile stress (UTS) by 1.6- and 1.4-fold. Correlation analysis showed that tensile modulus and UTS strongly correlated with collagen fiber length, diameter, and alignment, while compressive modulus correlated with GAG content. These outcomes highlight the need for optimization of both biochemical and biomechanical cues in the culture environment for enhancing fiber development within tissue-engineered constructs.

The human intervertebral disc (IVD) is a complex organ composed of fibrous and cartilaginous connective tissues, and it serves as a boundary between 2 adjacent vertebrae. It provides a limited range of motion in the torso as well as stability during axial compression, rotation, and bending. Adult IVDs have poor innate healing potential due to low vascularity and cellularity. Degenerative disc disease (DDD) generally arises from the disruption of the homeostasis maintained by the structures of the IVD, and genetic and environmental factors can accelerate the progression of the disease. Impaired cell metabolism due to pH alteration and poor nutrition may lead to autophagy and disruption of the homeostasis within the IVD and thus plays a key role in DDD etiology. To develop regenerative therapies for degenerated discs, future studies must aim to restore both anatomical and biomechanical properties of the IVDs. The objective of this review is to give a detailed overview about anatomical, radiological, and biomechanical features of the IVDs as well as discuss the structural and functional changes that occur during the degeneration process., (This manuscript is generously published free of charge by ISASS, the International Society for the Advancement of Spine Surgery. Copyright © 2021 ISASS.)

Achieving lateral integration of articular cartilage repair tissue with surrounding native cartilage remains a clinical challenge. Histological and bulk mechanical studies have identified extracellular matrix components that correlate with superior failure strength, but it is unclear how local changes in geometry and composition at the repair interface affect tissue strains under physiologic loading. Here, we investigated the effects of local compositional and interface geometry on lateral cartilage repair integration by coupling microscale Raman spectroscopy and confocal elastography to measure tissue strains under compressive and shear loading. Histological integration assessments did not have significant relationships with interface strains under compressive loading (p > 0.083) and only the perimeter attachment score was trending towards statistical significance with the |E xy | strain tensor under shear loading (p = 0.050). Interface slope had a stronger correlation with local tissue strains under compressive and shear loading compared to compositional measures of GAG, collagen, or proteins (compressive loading |E yy | tensor: R 2  = 0.400 (interface slope), 0.005 (GAG), 0.024 (collagen), and 0.012 (protein); shear loading |E xy | tensor: R 2  = 0.457 (interface slope), 0.003 (GAG), 0.006 (collagen), and 0.000 (total protein)). These data support surgical publications detailing the need for vertical walls when debriding chondral defects. Current histological integration assessments and local compositional measures were insufficient for identifying the variation in interface strains under compressive and shear loading. Thus, our data points to the importance of controlling interface geometry at the time of surgery, which has implications for cartilage repair integration and long-term healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Inferior synovial lubrication is a hallmark of osteoarthritis (OA), and synovial fluid (SF) lubrication and composition are variable among OA patients. Hyaluronic acid (HA) viscosupplementation is a widely used therapy for improving SF viscoelasticity and lubrication, but it is unclear how the effectiveness of HA viscosupplements varies with arthritic endotype. The objective of this study was to investigate the effects of the HA viscosupplement, Hymovis®, on the lubricating properties of diseased SF from patients with noninflammatory OA and inflammatory arthritis (IA). The composition (cytokine, HA, and lubricin concentrations) of the SF was measured as well as the mechanical properties (rheology, tribology) of the SF alone and in a 1:1 mixture with the HA viscosupplement. Using rotational rheometry, no difference in SF viscosity was detected between disease types, and the addition of HA significantly increased all fluids' viscosities. In noninflammatory OA SF, friction coefficients followed a typical Stribeck pattern, and their magnitude was decreased by the addition of HA. While some of the IA SF also showed typical Stribeck behavior, a subset showed more erratic behavior with highly variable and larger friction coefficients. Interestingly, this aberrant behavior was not eliminated by the addition of HA, and it was associated with low concentrations of lubricin. Aberrant SF exhibited significantly lower effective viscosities compared to noninflammatory OA and IA SF with typical tribological behavior. Collectively, these results suggest that different endotypes of arthritis exist with respect to lubrication, which may impact the effectiveness of HA viscosupplements in reducing friction., (Copyright © 2020 by ASME.)

Alterations to the biochemical composition of the intervertebral disc (IVD) are hallmarks of aging and degeneration. Methods to assess biochemical content, such as histology, immunohistochemistry, and spectrophotometric assays, are limited in their ability to quantitatively analyze the spatial distribution of biochemical components. Fourier transform infrared (FTIR) microscopy is a biochemical analysis method that can yield both quantitative and high-resolution data about the spatial distribution of biochemical components. This technique has been largely unexplored for use with the IVD, and existing methods use complex analytical techniques that make results difficult to interpret. The objective of the present study is to describe an FTIR microscopy method that has been optimized for imaging the collagen and proteoglycan content of the IVD. The method was performed on intact and discectomized IVDs from the sheep lumbar spine after 6 weeks in vivo in order to validate FTIR microscopy in healthy and degenerated IVDs. FTIR microscopy quantified collagen and proteoglycan content across the entire IVD and showed local changes in biochemical content after discectomy that were not observed with traditional histological methods. Changes in collagen and proteoglycans content were found to have strong correlations with Pfirrmann grades of degeneration. This study demonstrates how FTIR microscopy is a valuable research tool that can be used to quantitatively assess the local biochemical composition of IVDs in development, degeneration, and repair., Competing Interests: Roger Härtl is a consultant for AO Spine, Brainlab, Depuy‐Synthes and Lanx, and received research funding from Baxter. Lawrence J. Bonassar is a consultant for Fidia Pharmaceuticals and 3DBio Corp., (© 2020 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Tissue-engineered cartilage has shown promising results in the repair of focal cartilage defects. However, current clinical techniques rely on an extra surgical procedure to biopsy healthy cartilage to obtain human chondrocytes. Alternatively, induced pluripotent stem cells (iPSCs) have the ability to differentiate into chondrocytes and produce cartilaginous matrix without the need to biopsy healthy cartilage. However, the mechanical properties of tissue-engineered cartilage with iPSCs are unknown and might be critical to long-term tissue function and health. This study used confined compression, cartilage on glass tribology, and shear testing on a confocal microscope to assess the macroscale and microscale mechanical properties of two constructs seeded with either chondrocyte-derived iPSCs (Ch-iPSCs) or native human chondrocytes. Macroscale properties of Ch-iPSC constructs provided similar or better mechanical properties than chondrocyte constructs. Under compression, Ch-iPSC constructs had an aggregate modulus that was two times larger than chondrocyte constructs and was closer to native tissue. No differences in the shear modulus and friction coefficients were observed between Ch-iPSC and chondrocyte constructs. On the microscale, Ch-iPSC and chondrocyte constructs had different depth-dependent mechanical properties, neither of which matches native tissue. These observed depth-dependent differences may be important to the function of the implant. Overall, this comparison of multiple mechanical properties of Ch-iPSC and chondrocyte constructs shows that using Ch-iPSCs can produce equivalent or better global mechanical properties to chondrocytes. Therefore, iPSC-seeded cartilage constructs could be a promising solution to repair focal cartilage defects. The chondrocyte constructs used in this study have been implanted into humans for clinical trials. Therefore, Ch-iPSC constructs could also be used clinically in place of the current chondrocyte construct., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Interfaces between soft tissue and bone are characterized by transitional gradients in composition and structure that mediate substantial changes in mechanical properties. For interfacial tissue engineering, scaffolds with mineral gradients have shown promise in controlling osteogenic behavior of seeded bone marrow stromal cells (bMSCs). Previously, we have demonstrated a 'top-down' method for creating monolithic bone-derived scaffolds with patterned mineral distributions similar to native tissue. In the present work, we evaluated the ability of these scaffolds to pattern osteogenic behavior in bMSCs in basic, osteogenic, and chondrogenic biochemical environments. Immunohistochemical (IHC) and histological stains were used to characterize cellular behavior as a function of local mineral content. Alkaline phosphatase, an early marker of osteogenesis, and osteocalcin, a late marker of osteogenesis, were positively correlated with mineral content in basic, osteogenic, and chondrogenic media. The difference in bMSC behavior between the mineralized and demineralized regions was most pronounced in an basic biochemical environment. In the mineralized regions of the scaffold, osteogenic markers were clearly present as early as 4 days in culture. In osteogenic media, osteogenic behavior was observed across the entire scaffold, whereas in chondrogenic media, there was an overall reduction in osteogenic biomarkers. Overall, these results indicate local mineral content of the scaffold plays a key role in spatially patterning bMSC behavior. Our results can be utilized for the development of interfacial tissue engineered scaffolds and understanding the role of local environment in determining bMSC behavior. STATEMENT OF SIGNIFICANCE: Soft tissue-to-bone interfaces, such as tendon-bone, ligament-bone, and cartilage-bone, are ubiquitous in mammalian musculoskeletal systems. These interfacial tissues have distinct, hierarchically-structured gradients of cellular, biochemical, and materials components. Given the complexity of the biological structures, interfacial tissues present unique challenges for tissue engineering. Here, we demonstrate that material-derived cues can spatially pattern osteogenic behavior in bone marrow stromal cells (bMSCs). Specifically, we observed that when the bMSCs are cultured on bone-derived scaffolds with mineral gradients, cells in contact with higher mineral content display osteogenic behavior at earlier times than those on the unmineralized substrate. The ability to pattern the cellular complexity found in native interfaces while maintaining biologically relevant structures is a key step towards creating engineered tissue interfaces., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Posttraumatic osteoarthritis (PTOA) involves the mechanical and biological deterioration of articular cartilage that occurs following joint injury. PTOA is a growing problem in health care due to the lack of effective therapies combined with an aging population with high activity levels. Recently, acute mitochondrial dysfunction and altered cellular respiration have been associated with cartilage degeneration after injury. This finding is particularly important because recently developed mitoprotective drugs, including SS peptides, can preserve mitochondrial structure and function after acute injury in other tissues. It is not known, however, if cartilage injury induces rapid structural changes in mitochondria, to what degree mitochondrial dysfunction in cartilage depends on the mechanics of injury or the time frame over which such dysfunction develops. Similarly, it is unknown if SS-peptide treatment can preserve mitochondrial structure and function after cartilage injury. Here, we combined fast camera elastography, longitudinal fluorescence assays, and computer vision techniques to track the fates of thousands of individual cells. Our results show that impact induces mechanically dependent mitochondrial depolarization within a few minutes after injury. Electron microscopy revealed that impact causes rapid structural changes in mitochondria that are related to reduced mitochondrial function, namely, fission and loss of cristae structure. We found that SS-peptide treatment prior to impact protects the mitochondrial structure and preserves mitochondrial function at levels comparable with that of unimpacted control samples. Overall, this study reveals the vital role of mitochondria in mediating cartilage's peracute (within minutes) response to traumatic injury and demonstrates mitoprotection as a promising therapeutic strategy for injury-induced cartilage damage., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Human tissue engineered cartilage is a promising solution for focal cartilage defects, but these constructs do not have the same local mechanical properties as native tissue. Most clinically relevant engineered cartilage constructs seed human chondrocytes onto a collagen scaffold, which buckles at low loads and strains. This buckling creates local regions of high strain that could cause cell death and damage the engineered tissue. Since human tissue engineered cartilage is commonly grown in-vivo prior to implantation, new matrix deposition could improve the local implant mechanics and prevent local tissue buckling. However, the relationship between local biochemical composition and the local mechanics or local buckling probability has never been quantified. Therefore, this study correlated the local biochemical composition of human tissue engineered cartilage constructs using Fourier transform infrared spectroscopy (FTIR) with the local shear modulus and local buckling probability. The local shear modulus and local buckling probability were obtained using a confocal elastography technique. The local shear modulus increased with increases in local aggrecan content in the interior region (inside the scaffold). A minimum amount of aggrecan was required to prevent local construct buckling at physiologic strains. Since the original scaffold was primarily composed of collagen, increases in collagen content due to new matrix deposition was minimal and had little effect on the mechanical properties. Thus, we concluded that aggrecan deposition inside the scaffold pores is the most effective way to improve the mechanical function and prevent local tissue damage in human tissue engineered cartilage constructs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Injection of hyaluronic acid (HA) viscosupplements is a prevalent treatment for patients suffering from mild to moderate osteoarthritis. The efficacy of these supplements is attributed to increased synovial fluid viscosity, which leads to improved lubrication and reduced pain. Therefore, viscosity is a key parameter to consider in the development of HA supplements. HA localizes near the cartilage surface, resulting in a viscosity gradient with heightened viscosity near the surface. Traditional rheological measurements confine HA between metal fixtures and therefore do not capture the effect of HA localization that occurs on cartilage. In these experiments, we investigate the effect of modifying rheometer fixtures with cartilage surface coatings on the effective viscosity of HA solutions. Our results demonstrate up to a 20-fold increase in effective viscosity when HA was confined between cartilage surfaces compared to steel surfaces. For low-molecular-weight HA, the effective viscosity was dependent on the gap height between the rheometer plates, which is consistent with the formation of a viscous boundary film. Together, these results indicate that this method for assessing HA viscosity may be more relevant to lubrication than traditional methods and may provide a more accurate method for predicting the viscosity of HA viscosupplements in vivo where HA is able to interact with the cartilage surface.

Objective: Viscosupplementation has been used for decades to treat mild to moderate osteoarthritis, yet it is unknown if the lubricating function of different pathological synovial fluids (SF) vary, or if they respond differentially to viscosupplementation. The objectives of this study were to (i) evaluate the friction coefficients and induced shear strains in articular cartilage when lubricated with pathological SF, (ii) identify the effect of hyaluronic acid (HA) supplementation on friction coefficients and shear strains, and (iii) identify SF biomarkers that correlate with lubricating function., Method: Human pathological SF was grouped by white blood cell count (inflammatory: >2000 cells/mm 3 , n = 6; non-inflammatory: <2000 cells/mm 3 , n = 6). Compositional analyses for lubricin and cytokines were performed. Friction coefficients and local tissue shear strain measurements were coupled using new, microscale rheological analyses by lubricating neonatal bovine cartilage explants with SF alone and in a 1:1 ratio with HA (Hymovis®)., Results: Friction coefficients were not significantly different between the inflammatory and non-inflammatory pathologies (p = 0.09), and were poorly correlated with peak tissue strains at the cartilage articular surface (R 2  = 0.34). A subset of inflammatory SF samples induced higher tissue strains, and HA supplementation was most effective at lowering friction and tissue strains in this inflammatory subset. Across all pathologies there were clear relationships between polymorphonuclear neutrophil (PMN), IL-8, and lubricin concentrations with cartilage tissue strains., Conclusion: These results suggest that pathological SF is characterized by distinct tribological endotypes where SF lubricating behaviors are differentially modified by viscosupplementation and are identifiable by biomarkers., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Tissue-engineered approaches for the treatment of early-stage intervertebral disc degeneration have shown promise in preclinical studies. However, none of these therapies has been approved for clinical use, in part because each therapy targets only one aspect of the intervertebral disc's composite structure. At present, there is no reliable method to prevent intervertebral disc degeneration after herniation and subsequent discectomy. Here, we demonstrate the prevention of degeneration and maintenance of mechanical function in the ovine lumbar spine after discectomy by combining strategies for nucleus pulposus augmentation using hyaluronic acid injection and repair of the annulus fibrosus using a photocrosslinked collagen patch. This combined approach healed annulus fibrosus defects, restored nucleus pulposus hydration, and maintained native torsional and compressive stiffness up to 6 weeks after injury. These data demonstrate the necessity of a combined strategy for arresting intervertebral disc degeneration and support further translation of combinatorial interventions to treat herniations in the human spine., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Early intervention with mesenchymal stem cells (MSCs) after articular trauma has the potential to limit progression of focal lesions and prevent ongoing cartilage degeneration by modulating the joint environment and/or contributing to repair. Integrin α10β1 is the main collagen type II binding receptor on chondrocytes, and MSCs that are selected for high expression of the α10 subunit have improved chondrogenic potential. The ability of α10β1-selected (integrin α10 high ) MSCs to protect cartilage after injury has not been investigated., Purpose: To investigate integrin α10 high MSCs to prevent posttraumatic osteoarthritis in an equine model of impact-induced talar injury., Study Design: Controlled laboratory study., Methods: Focal cartilage injuries were created on the tali of horses (2-5 years, n = 8) by using an impacting device equipped to measure impact stress. Joints were treated with 20 × 10 6 allogenic adipose-derived α10 high MSCs or saline vehicle (control) 4 days after injury. Synovial fluid was collected serially and analyzed for protein content, cell counts, markers of inflammation (prostaglandin E2, tumor necrosis factor α) and collagen homeostasis (procollagen II C-propeptide, collagen type II cleavage product), and glycosaminoglycan content. Second-look arthroscopy was performed at 6 weeks, and horses were euthanized at 6 months. Joints were imaged with radiographs and quantitative 3-T magnetic resonance imaging. Postmortem examinations were performed, and India ink was applied to the talar articular surface to identify areas of cartilage fibrillation. Synovial membrane and osteochondral histology was performed, and immunohistochemistry was used to assess type I and II collagen and lubricin. A mixed effect model with Tukey post hoc and linear contrasts or paired t tests were used, as appropriate., Results: Integrin α10 high MSC-treated joints had less subchondral bone sclerosis on radiographs ( P = .04) and histology ( P = .006) and less cartilage fibrillation ( P = .04) as compared with control joints. On gross pathology, less India ink adhered to impact sites in treated joints than in controls, which may be explained by the finding of more prominent lubricin immunostaining in treated joints. Prostaglandin E2 concentration in synovial fluid and mononuclear cell synovial infiltrate were increased in treated joints, suggesting possible immunomodulation by integrin α10 high MSCs., Conclusion: Intra-articular administration of integrin α10 high MSCs is safe, and evidence suggests that the cells mitigate the effects of joint trauma., Clinical Relevance: This preclinical study indicates that intra-articular therapy with integrin α10 high MSCs after joint trauma may be protective against posttraumatic osteoarthritis.

Articular cartilage is one of the most unique materials found in nature. This tissue's ability to provide low friction and low wear over decades of constant use is not surpassed, as of yet, by any synthetic materials. Lubrication of the body's joints is essential to mammalian locomotion, but breakdown and degeneration of cartilage is the leading cause of severe disability in the industrialized world. In this paper, we review how theories of cartilage lubrication have evolved over the past decades and connect how theories of cartilage lubrication have been translated to lubrication-based therapies. Here, we call upon these historical perspectives and highlight the open questions in cartilage lubrication research. Additionally, these open questions within the field's understanding of natural lubrication mechanisms reveal strategic directions for lubrication therapy., (Copyright © 2020 by ASME.)

Background: Lubricin, a mucinous glycoprotein, plays a chondroprotective role as a constituent of synovial fluid. Structural analogs have been synthesized to mimic the structure and function of native lubricin in an effort to recapitulate this effect with the goal of delaying progression of osteoarthritis (OA)., Purpose: To investigate the efficacy of intra-articular injections of lubricin mimetics in slowing or preventing the progression of posttraumatic OA by using a rat anterior cruciate ligament transection model., Study Design: Controlled laboratory design., Methods: Four lubricin mimetics were investigated, differing from one another in their binding orientations and steric interactions. Eighty skeletally mature Sprague-Dawley rats underwent bilateral anterior cruciate ligament transections and were randomly allocated to receive intra-articular injections (50 µL/injection) of 1 of the 4 mimetics in the right knee and equal volumes of saline injection in the contralateral knee (control). All rats were euthanized 8 weeks postoperatively and assessed via biomechanical analysis, which evaluated comparative friction coefficients across the 4 groups, and histological evaluation of articular cartilage, osteophytes, and synovitis. The Osteoarthritis Research Society International (OARSI) histopathological assessment system was used to evaluate the degree of articular cartilage degeneration and osteophytes, while synovitis was assessed through a semiquantitative scoring system. Binding efficacy of the 4 mimetics was assessed in vitro and in vivo through the immunohistochemical localization of polyethylene glycol. Articular cartilage degeneration and synovitis scoring data analyses were performed with generalized estimating equation modeling., Results: Injection of the group 3 mimetic (random 24 + 400 + 30) directly correlated with improved OARSI scores for femoral articular cartilage degeneration when compared with saline-injected contralateral control knees ( P = .0410). No lubricin mimetic group demonstrated statistically significant differences in OARSI scores for tibial articular cartilage degeneration. Injection of the group 4 mimetic (AB 24 + 400 + 30) led to a statistically significant difference in osteophyte OARSI score ( P = .0019). None of the 4 lubricin mimetics injections incited an additive synovial inflammatory response. Immunohistochemical staining substantiated the binding capacity of all 4 mimetics, while in vivo experimentation revealed that the group 1 and 3 mimetics were still retained within the joint 4 weeks after injection. There were no differences in friction coefficients between any pair of groups and no significant trends based on lubricin mimetic structure., Conclusion: We demonstrated that the tribosupplementation of a traumatically injured knee with a specific lubricin structural analog may attenuate the natural progression of OA., Clinical Relevance: The current lack of efficacious clinical options to counter the onset and subsequent development of OA suggests that further investigation into the synthesis and behavior of lubricin analogs could yield novel translational applications.

We report how the tribological properties of a class of diblock copolymers with architecture and function inspired by the lubricating glycoprotein lubricin correlate to chemical composition. This class of diblock copolymers, consisting of a cationic cartilage-binding block and a brush-lubricating block, demonstrates that boundary lubrication of articular cartilage more strongly depends on the cartilage-binding block than the lubrication block. Specifically, the cartilage-binding functional groups (tertiary or quaternary amines) and cartilage-binding block length significantly influence the degree of lubrication under boundary mode experimental conditions. An optimal number (∼24 in this case) of cartilage-binding groups led to the lowest coefficient of friction, and an increase or decrease in the number of cations in the binding block led to partial (>24, and between 12 and 24) or complete (=12) loss of lubricating ability. The length of the lubricating block (DP = 200 or 400) chosen in this study had no effect on the degree of lubrication. These results are put into context in terms of binding affinity to the cartilage and the spatial packing density of the polymer on the cartilage surface and can serve as a useful guide for future designs of synthetic lubricants that rival the efficacy of natural lubricants.

The synovial fluid (SF) that lubricates articular joints exhibits complex rheological and tribological properties due to the interactions and behaviors of its various molecular components. Under shear, SF films abruptly thicken by more than 300% and large, dense aggregates form within the fluid. In this study, we used the Surface Force Apparatus to elucidate which SF components are involved in this shear-induced transformation by (i) determining which (if any) of all major SF components replicate the behavior of SF under shear and (ii) observing the effect of removing implicated components from SF by enzymatic digestion. While most previous studies of SF have focused on the tribological roles of lubricin or hyaluronic acid, our results indicate that albumin is a key contributor to the formation of aggregates in SF under shear. Our results also suggest that SF aggregation is associated with efficient surface protection against wear. As our findings are based on experiments involving rigid, nonporous surfaces, they may be used to investigate shear-mediated aggregation mechanisms occurring during the lubrication of artificial joints, ultimately advancing our current vision of implant design.

Temporomandibular joint (TMJ) diseases such as osteoarthritis and disc displacement have no permanent treatment options, but lubrication therapies, used in other joints, could be an effective alternative. However, the healthy TMJ contains fibrocartilage, not hyaline cartilage as is found in other joints. As such, the effect of lubrication therapies in the TMJ is unknown. Additionally, only a few studies have characterized the friction coefficient of the healthy TMJ. Like other cartilaginous tissues, the mandibular condyles and discs are subject to changes in friction coefficient due to fluid pressurization. In addition, the friction coefficients of the inferior joint space of the TMJ are affected by the sliding direction and anatomic location. However, these previous findings have not been able to identify how all three of these parameters (anatomic location, sliding direction, and fluid pressurization) influence changes in friction coefficient. This study used Stribeck curves to identify differences in the friction coefficients of mandibular condyles and discs based on anatomic location, sliding direction, and amount of fluid pressurization (friction mode). Friction coefficients were measured using a cartilage on glass tribometer. Both mandibular condyle and disc friction coefficients were well described by Stribeck curves (R2 range 0.87-0.97; p < 0.0001). These curves changed based on anatomic location (Δμ ∼ 0.05), but very few differences in friction coefficients were observed based on sliding direction. Mandibular condyles had similar boundary mode and elastoviscous mode friction coefficients to the TMJ disc (μmin ∼ 0.009 to 0.19) and both were lower than hyaline cartilage in other joints (e.g., knee, ankle, etc.). The observed differences here indicate that the surface characteristics of each anatomic region cause differences in friction coefficients., (Copyright © 2019 by ASME.)

Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues and therefore commonly used as a biomaterial in tissue engineering applications. In the native environment, collagen fibers are arranged in a complex hierarchical structure that is often difficult to recreate in a tissue engineered construct. Small leucine rich proteoglycans as well as hyaluronan binding proteoglycans, aggrecan and versican, have been implicated in regulating fiber formation. In this study, we modified proteoglycan production in vitro by altering culture medium glucose concentrations (4500, 1000, 500, 250, and 125 mg/L), and evaluated its effect on the formation of collagen fibers inside tissue engineered meniscal constructs. Reduction of extracellular glucose resulted in a dose dependent decrease in total sulfated glycosaminoglycan (GAG) production, but minimal decreases of decorin and biglycan. However, fibromodulin doubled in production between 125 and 4500 mg/L glucose concentration. A peak in fiber formation was observed at 500 mg/L glucose concentration and corresponded with reductions in total GAG production. Fiber formation reduction at 125 and 250 mg/L glucose concentrations are likely due to changes in metabolic activity associated with a limited supply of glucose. These results point to proteoglycan production as a means to manipulate fiber architecture in tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues; however achieving appropriate assembly and organization of collagen fibers in engineered connective tissues is a persistent challenge. Proteoglycans have been implicated in regulating collagen fiber organization both in vivo and in vitro, however little is known about methods to control proteoglycan production and the subsequent fiber organization in tissue engineered menisci. Here, we show that media glucose content can be optimized to control proteoglycan production and collagen fiber assembly, with optimal collagen fiber assembly occurring at sub-physiologic levels of glucose., (Copyright © 2019. Published by Elsevier Ltd.)

Fibrin sealants are commonly used in cartilage repair surgeries to adhere cells or grafts into a cartilage defect. Both autologous and commercial allogeneic fibrin sealants are used in cartilage repair surgeries, yet there are no studies characterizing and comparing the mechanical properties of fibrin sealants from all-autologous sources. The objectives of this study were to investigate (i) the effect of fibrinogen and thrombin sources on failure mechanics of sealants, and (ii) how sealants affect the adhesion of particulated cartilage graft material (BioCartilage) to surrounding cartilage under physiological loading. Allogeneic thrombin and fibrinogen were purchased (Tisseel), and autologous sources were prepared from platelet-rich plasma (PRP) and platelet-poor plasma (PPP) generated from human blood. To compare failure characteristics, sealants were sandwiched between cartilage explants and pulled to failure. The effect of sealant on the adhesion of BioCartilage graft to cartilage was determined by quantifying microscale strains at the graft-cartilage interface using an in vitro cartilage defect model subjected to shear loading at physiological strains well below failure thresholds. Fibrinogen sources were not equivalent; PRP fibrinogen created sealants that were more brittle, failed at lower strains, and resulted in sustained higher strains through the graft-cartilage interface depth compared to PPP and allogeneic sources. PPP clotted slower compared to PRP, suggesting PPP may percolate deeper into the repair to provide more stability through the tissue depth. There was no difference in bulk failure properties or microscale strains at the graft-cartilage interface between the purely autologous sealant (autologous thrombin + PPP fibrinogen) and the commercial allogeneic sealant. Clinical Significance: All-autologous fibrin sealants fabricated with PPP have comparable adhesion strength as commercial allogeneic sealants in vitro, whereas PRP creates an inferior all-autologous sealant that sustains higher strains through the graft-cartilage interface depth., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Andrea Matuska is a full-time employee at Arthrex Inc., and Brian Cole and I are both consultants for Arthrex Inc. Arthrex Inc. partially funded this research. This does not alter our adherence to the PLOS ONE policies on sharing data and materials.

Intervertebral disc (IVD) herniations are currently treated with interventions that leave the IVD with persistent lesions prone to further herniations. Annulus fibrosus (AF) repair has become of interest as a method to seal defects in the IVD and prevent reherniation, but this requires strong adhesion of the implanted biomaterial to the native AF tissue. Our group has previously developed a high-density collagen (HDC) gel for AF repair and tested its efficacy in vivo, but its adhesion to the AF could be improved. Increased cell adhesion to cartilage has previously been reported through chondroitinase ABC (ChABC) digestion, which removes proteoglycans and increases access to cell binding motifs. Such approaches could also increase biomaterial adhesion to tissue, but the effects of ChABC digestion on AF have yet to be investigated. In this study, ovine AF tissue was digested with either 10 U/mL ChABC or saline for up to 10 min and the effect of this treatment on collagen adhesion between AF tissue samples was investigated by histology and mechanical testing in a lap-shear configuration. ChABC digestion removed proteoglycans within the AF in a time-dependent fashion and enhanced adhesion of the HDC gel to the AF. ChABC digestion increased the elastic toughness and total shear energy of the HDC gel-AF interface by 88% and 46% respectively. ChABC treatment enhanced the adhesion of the HDC gel to the AF without significantly decreasing native AF cell viability. Thus, ChABC digestion is a viable method to improve adhesion of biomaterials for AF repair. STATEMENT OF SIGNIFICANCE: Intervertebral disc herniations are currently treated with interventions that leave persistent lesions in the annulus fibrosus that are prone to further herniations. Annular repair is a promising method to seal lesions and prevent reherniation, but requires strong adhesion of the implanted biomaterial to native annulus fibrosus. Since large proteoglycans like aggrecan occupy regions of the extracellular matrix between collagen fibers in the annulus fibrosus, we hypothesized that removing proteoglycans via chondroitinase digestion would increase the adhesion of annular repair hydrogels. This investigation demonstrated that chondroitinase removed proteoglycans within annulus fibrosus tissue, enhanced the interaction of an injected collagen gel with the native tissue, and mechanically improved adhesion between the collagen gel and annulus fibrosus. This is the first study of its kind to evaluate the biochemical and mechanical effects of short-term chondroitinase digestion on annulus fibrosus tissue., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

An advantage of bioprinting is the ability to incorporate cells into the hydrogel bioink allowing for precise control over cell placement within a construct. Previous work found that the printability of collagen bioinks is highly dependent on their rheological properties. The effect of cell density on collagen rheological properties and, therefore, printability has not been assessed. Therefore, the objective of this study was to determine the effects of incorporating cells on the rheology and printability of collagen bioinks. Primary chondrocytes, at densities relevant to cartilage tissue engineering (up to 100 × 10 6 cells ml -1 ), were incorporated into 8 mg ml -1 collagen bioinks. Bioink rheological properties before, during, and after gelation as well as printability were assessed. Cell-laden printed constructs were also cultured for up to 14 d to assess longer-term cell behavior. The addition of cells resulted in an increase in the storage modulus and viscosity of the collagen before gelation. However, the storage modulus after gelation and the rate of gelation decreased with increasing cell density. Theoretical models were compared to the rheological data to suggest frameworks that could be used to predict the rheological properties of cell-laden bioinks. Printability testing showed that improved printability could be achieved with higher cell densities. Fourteen-day culture studies showed that the printing process had no adverse effects on the viability or function of printed cells. Overall, this study shows that collagen bioinks are conducive to bioprinting with a wide range of cell densities while maintaining high printability and chondrocyte viability and function.

Background: Our group has previously demonstrated in vivo annulus fibrosus repair in animal models using an acellular, riboflavin crosslinked, high-density collagen (HDC) gel., Objective: To assess if seeding allogenic mesenchymal stem cells (MSCs) into this gel yields improved histological and radiographic benefits in an in vivo sheep model of annular injury., Methods: Fifteen lumbar intervertebral discs (IVDs) were randomized into 4 groups: intact, injury only, injury + acellular gel treatment, or injury + MSC-seeded gel treatment. Sheep were sacrificed at 6 wk. Disc height index (DHI), Pfirrmann grade, nucleus pulposus area, and T2 relaxation time (T2-RT) were calculated for each IVD and standardized to healthy controls from the same sheep. Quantitative histological assessment was also performed using the Han scoring system., Results: All treated IVDs retained gel plugs on gross assessment and there were no adverse perioperative complications. The MSC-seeded gel treatment group demonstrated statistically significant improvement over other experimental groups in DHI (P = .002), Pfirrmann grade (P < .001), and T2-RT (P = .015). There was a trend for greater Han scores in the MSC-seeded gel-treated discs compared with injury only and acellular gel-treated IVDs (P = .246)., Conclusion: MSC-seeded HDC gel can be delivered into injured IVDs and maintained safely in live sheep to 6 wk. Compared with no treatment and acellular HDC gel, our data show that MSC-seeded HDC gel improves outcomes in DHI, Pfirrmann grade, and T2-RT. Histological analysis shows improved annulus fibrosus and nucleus pulposus reconstitution and organization over other experimental groups as well., (Copyright © 2018 by the Congress of Neurological Surgeons.)

Recently, the scientific community has shown considerable interest in engineering tissues with organized compositional and structural gradients to mimic hard-to-soft tissue interfaces. This effort is hindered by an incomplete understanding of the construction of native tissue interfaces. In this work, we combined Raman microscopy and confocal elastography to map compositional, structural, and mechanical features across the stiff-to-compliant interface of the attachments of the meniscus in the knee. This study provides new insight into the methods by which biology mediates multiple orders of magnitude changes in stiffness over tens of microns. We identified how the nano- to mesoscale architecture mediates complex microscale transitional regions across the interface: two regions defined by chemical composition, five distinguished by structural features, and three mechanically distinct regions. We identified three major components that lead to a robust interface between a soft tissue and bone: mobile collagen fiber units, a continuous interfacial region, and a local stiffness gradient. This tissue architecture allows for large displacements of collagen fibers in the attachments, enabling meniscal movement without localizing strains to the soft tissue-to-bone interface. The interplay of these regions reveals a method relying on hierarchical structuring across multiple length scales to minimize stress concentrators between highly dissimilar materials. These insights inspire new design strategies for synthetic soft tissue-to-bone attachments and biomimetic material interfaces.

We report the design of a diblock copolymer with architecture and function inspired by the lubricating glycoprotein lubricin. This diblock copolymer, synthesized by sequential reversible addition-fragmentation chain-transfer polymerization, consists of a cationic cartilage-binding domain and a brush-lubricating domain. It reduces the coefficient of friction of articular cartilage under boundary mode conditions (0.088 ± 0.039) to a level equivalent to that provided by lubricin (0.093 ± 0.011). Additionally, both the EC 50 (0.404 mg/mL) and cartilage-binding time constant (7.19 min) of the polymer are comparable to purified human and recombinant lubricin. Like lubricin, the tribological properties of this polymer are dependent on molecular architecture. When the same monomer composition was evaluated either as an AB diblock copolymer or as a random copolymer, the diblock effectively lubricated cartilage under boundary mode conditions whereas the random copolymer did not. Additionally, the individual polymer blocks did not lubricate independently, and lubrication could be competitively inhibited with an excess of binding domain. This diblock copolymer is an example of a synthetic polymer with lubrication properties equal to lubricin under boundary mode conditions, suggesting its potential utility as a therapy for joint pathologies like osteoarthritis., Competing Interests: Conflict of interest statement: Z.S., L.J.B, and D.P. are listed as inventors on a patent filing. The patent is not yet issued and the patent is not licensed to any entity.

Materials engineering can generally be divided into "bottom-up" and "top-down" approaches, where current state-of-the-art methodologies are bottom-up, relying on the advent of atomic-scale technologies. Applying bottom-up approaches to biological tissues is challenging due to the inherent complexity of these systems. Top-down methodologies provide many advantages over bottom-up approaches for biological tissues, given that some of the complexity is already built into the system. Here, we generate interfacial scaffolds by the spatially controlled removal of mineral content from trabecular bone using a chelating solution. We controlled the degree and location of the mineral interface, producing scaffolds that support cell growth, while maintaining the hierarchical structure of these tissues. We characterized the structural and compositional gradients across the scaffold using X-ray diffraction, microcomputed tomography (μCT), and Raman microscopy, revealing the presence of mineral gradients on the scale of 20 - 40 μm. Using these data, we generated a model showing the dependence of mineral removal as function of time in the chelating solution and initial bone morphology, specifically trabecular density. These scaffolds will be useful for interfacial tissue engineering, with application in the fields of orthopedics, developmental biology, and cancer metastasis to bone.

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or "synDNAs," for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems., (© 2019 Wiley Periodicals, Inc.)

Hyaluronic acid injections have been a mainstay of arthritis treatment for decades. However, much controversy remains about their clinical efficacy and their potential mechanism of action. This approach to arthritis therapy is often called viscosupplementation, a term which is rooted in the elevated viscosity of the injected solutions. This terminology also suggests a mechanical pathway of action and further implies that their efficacy is dependent on viscosity. Notably, previous studies of the relationship between viscous properties of hyaluronic acid solutions and their clinical efficacy have not been definitive. Recently we developed an experimental and analytical framework for studying cartilage lubrication that captures the Stribeck-like behavior of cartilage in an elastoviscous transition curve. Here we apply this framework to study the lubricating behavior of six hyaluronan products currently used for injectable arthritis therapy in the US. Despite the fact that the source and chemical modifications endow these products with a range of lubricating properties, we show that the lubricating effect of all of these materials can be described by this Stribeck-like elastoviscous transition. Fitting this data to the elastoviscous transition model enables the calculation of effective lubricating viscosities for each material, which differ substantially from the viscosities measured using standard rheometry. Further we show that while data from standard rheometry are poor predictors of clinical performance of these materials, measurements of friction coefficient and effective lubricating viscosity correlate well (R2 = 0.77; p < 0.005) with assessments of improved clinical function reported previously. This approach offers both a novel method that can be used to evaluate potential clinical efficacy of hyaluronic acid formulations and provide new insight on their mode of action., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Authors LJB and EDB are consultants for Fidia Pharma USA, LJB is a consultant for Fidia Farmaceutici SpA, and authors DG and CS are employees of Fidia Farmaceutici SpA. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The objective of this study was to examine temporal variations in synovial fluid composition and lubrication following articular fracture. Post-traumatic osteoarthritis (PTOA) was induced by creating an osteochondral fracture in the middle carpal joint of four horses while the contralateral limb served as a sham-operated control. Horses were exercised on a high-speed treadmill, and synovial fluid was collected pre-operatively and at serial timepoints until 75 days post-operatively. Lubricin and hyaluronic acid (HA) concentrations were measured using sandwich ELISAs, and the molecular weight distribution of HA was analyzed via gel electrophoresis. Synovial fluid viscosity and cartilage friction coefficients across all modes of lubrication were measured on days 0, 19, 33, and 61 using a commercial rheometer and a custom tribometer, respectively. HA concentrations were significantly decreased post-operatively, and high molecular weight HA (>6.1MDa) did not recover to pre-operative values by the study termination at day 75. Lubricin concentrations increased after surgery to a greater extent in the OA as compared to sham-operated limbs. Viscosity was significantly reduced after surgery. While boundary and elastoviscous mode friction coefficients did not vary, the transition number, representing the shift between these modes, was lower. Although more pronounced in the OA limbs, similar derangements in HA, HA molecular weight distribution, viscosity, and transition number were observed in the sham-operated limbs, which may be explained by synovial fluid washout during arthroscopy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tissue-engineered menisci hold promise as an alternative to allograft procedures but require a means of robust fixation to the native bone. The insertion of the meniscus into bone is critical for meniscal function and inclusion of a soft tissue-to-bone interface in a tissue engineered implant can aid in the fixation process. The native insertion is characterized by gradients in composition, tissue architecture, and cellular phenotype, which are all difficult to replicate. In this study, a soft tissue-to-bone interface is tissue engineered with a cellular gradient of fibrochondrocytes and mesenchymal stem cells and subjected to a biochemical gradient through a custom media diffusion bioreactor. These constructs, consisting of interpenetrating collagen and boney regions, display improved mechanical performance and collagen organization compared to controls without a cellular or chemical gradient. Media gradient exposure produces morphological features in the constructs that appear similar to the native tissue. Collectively, these data show that cellular and biochemical gradients improve integration between collagen and bone in a tissue engineered soft tissue-to-bone construct., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cancer metastasis is a physical process in which tumor cells break away from the primary tumor, enter, and then exit the blood or lymph vessels, and establish secondary tumors in distant organs. Current clinical studies report a higher risk of cancer metastasis for diabetics than non-diabetics. However, due to complex overlapping risk factors between diabetes and cancer, the mechanism underlying this correlation is largely unknown. Elevated lifetime blood sugar levels in diabetics are known to increase glycation of collagen, causing stiffening of the ECM and connective tissue. In this study, we explored the roles of glycation of 3D collagen matrices in tumor cell invasion and migration. Using time-lapse images, we quantitatively compared the motility behavior of malignant breast tumor cells (MDA-MB-231) and co-culture spheroids (1:1 ratio of MDA-MB-231 cells with normal epithelial MCF-10A cells) embedded in glycated and non-glycated collagen matrices of various concentrations. Experimental results demonstrated that glycation increased tumor invasion within collagen matrices. More specifically, the average speed of MDA-MB-231 cells was higher in glycated collagen gels than in non-glycated collagen gels for all three gel concentrations tested. Cell spreading characterized by its diffusion coefficient or the effective spheroid radii at various time points was significantly greater in glycated collagen than in non-glycated collagen at a concentration of 3.5 mg/mL. This enhancement was moderate and less evident at lower collagen concentrations of 1.0 and 2.0 mg/mL. These results suggest a possible biomechanical link that relates to the high blood sugar level in diabetic patients and the cancer metastatic outcome., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Developing effective therapeutics for osteoarthritis (OA) necessitates that such molecules can reach and target chondrocytes within articular cartilage. However, predicting how well very large therapeutic molecules diffuse through cartilage is often difficult, and the relationship between local transport mechanics for these molecules and tissue heterogeneities in the tissue is still unclear. In this study, a 150 kDa antibody diffused through the articular surface of healthy and enzymatically degraded cartilage, which enabled the calculation of local diffusion mechanics in tissue with large compositional variations. Local cartilage composition and structure was quantified with Fourier transform infrared (FTIR) spectroscopy and second harmonic generation (SHG) imaging techniques. Overall, both local concentrations of aggrecan and collagen were correlated to local diffusivities for both healthy and surface-degraded samples (0.3 > R 2  < 0.9). However, samples that underwent surface degradation by collagenase exhibited stronger correlations (R 2  > 0.75) compared to healthy samples (R 2  < 0.46), suggesting that the highly aligned collagen at the surface of cartilage acts as a barrier to macromolecular transport., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Molecular transport of osteoarthritis (OA) therapeutics within articular cartilage is influenced by many factors, such as solute charge, that have yet to be fully understood. This study characterizes how solute charge influences local diffusion and convective transport of antibodies within the heterogeneous cartilage matrix. Three fluorescently tagged solutes of varying isoelectric point (pI) (4.7-5.9) were tested in either cyclic or passive cartilage loading conditions. In each case, local diffusivities were calculated based on local fluorescence in the cartilage sample, as observed by confocal microscopy. In agreement with past research, local solute diffusivities within the heterogeneous cartilage matrix were highest around 200-275 μm from the articular surface, but 3-4 times lower at the articular surface and in the deeper zones of the tissue. Transport of all 150 kDa solutes was significantly increased by the application of mechanical loading at 1 Hz, but local transport enhancement was not significantly affected by changes in solute isoelectric point. More positively charged solutes (higher pI) had significantly higher local diffusivities 200-275 μm from the tissue surface, but no other differences were observed. This implies that there are certain regions of cartilage that are more sensitive to changes in solute charge than others, which could be useful for future development of OA therapeutics., (Copyright © 2019 by ASME.)

Objective: For the last half century, transport of nutrients and therapeutics in articular cartilage has been studied with various in vitro systems that attempt to model in vivo conditions. However, experimental technique, tissue species, and tissue storage condition (fresh/frozen) vary widely and there is debate on the most appropriate model system. Additionally, there is still no clear overarching framework with which to predict solute transport properties based on molecular characteristics. This review aims to develop such a framework, and to assess whether experimental procedure affects trends in transport data., Methods: Solute data from 31 published papers that investigated transport in healthy articular cartilage were obtained and analyzed for trends., Results: Here, we show that diffusivity of spherical and globular solutes in cartilage can be predicted by molecular weight (MW) and hydrodynamic radius via a power-law relationship. This relationship is robust for many solutes, spanning 5 orders of magnitude in MW and was not affected by variations in cartilage species, age, condition (fresh/frozen), and experimental technique. Traditional models of transport in porous media exhibited mixed effectiveness at predicting diffusivity in cartilage, but were good in predicting solute partition coefficient., Conclusion: Ultimately, these robust relationships can be used to accurately predict and improve transport of solutes in adult human cartilage and enable the development of better optimized arthritis therapeutics., (Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Children suffering from microtia have few options for auricular reconstruction. Tissue engineering approaches attempt to replicate the complex anatomy and structure of the ear with autologous cartilage but have been limited by access to clinically accessible cell sources. Here we present a full-scale, patient-based human ear generated by implantation of human auricular chondrocytes and human mesenchymal stem cells in a 1:1 ratio. Additional disc construct surrogates were generated with 1:0, 1:1, and 0:1 combinations of auricular chondrocytes and mesenchymal stem cells. After 3 months in vivo, monocellular auricular chondrocyte discs and 1:1 disc and ear constructs displayed bundled collagen fibers in a perichondrial layer, rich proteoglycan deposition, and elastin fiber network formation similar to native human auricular cartilage, with the protein composition and mechanical stiffness of native tissue. Full ear constructs with a 1:1 cell combination maintained gross ear structure and developed a cartilaginous appearance following implantation. These studies demonstrate the successful engineering of a patient-specific human auricle using exclusively human cell sources without extensive in vitro tissue culture prior to implantation, a critical step towards the clinical application of tissue engineering for auricular reconstruction., Competing Interests: LJB is a co-founder and equity holder in 3D BioCorp. Remaining authors declare no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Total disc replacement using tissue-engineered intervertebral discs (TE-IVDs) may offer a biological alternative to treat radiculopathy caused by disc degeneration. A composite TE-IVD was previously developed and evaluated in rat tail and beagle cervical spine models in vivo. Although cell viability and tissue integration into host tissue were promising, significant implant displacement occurred at multiple spinal levels. The goal of the present study was to assess the effects of a resorbable plating system on the stiffness of motion segments and stability of tissue-engineered implants subjected to axial compression. Canine motion segments from levels C2/C3 to C5/C6 were assessed as intact (CTRL), after discectomy (Dx), with an implanted TE-IVD only (PLATE-), and with a TE-IVD combined with an attached resorbable plate (PLATE+). Segments under PLATE+ conditions fully restored separation between endplates and showed significantly higher compressive stiffness than segments under PLATE- conditions. Plated segments partially restored more than 25% of the CTRL motion segment stiffness. Plate attachment also prevented implant extrusion from the disc space at 50% compressive strain, and this effect was more significant in segments from levels C3/C4 when compared to segments from level C5/C6. These results suggest that stabilization of motion segments via resorbable plating assists TE-IVD retention in the disc space while allowing the opportunity for implants to fully integrate into the host tissue and achieve optimal restoration of spine biomechanics., Competing Interests: L.J.B. is a co‐founder and holds equity in 3DBio Corp. R.H. is a consultant and holds equity in 3DBio Corp. R.H. has the following disclosures: Consulting fees: AOSpine, Brainlab, Depuy‐Synthes, Lanx, and Supported/Contracted research: Baxter.

Developing therapeutic molecules that target chondrocytes and locally produced inflammatory factors within arthritic cartilage is an active area of investigation. The extensive studies that have been conducted over the past 50 years have enabled the accurate prediction and reliable optimization of the transport of a wide variety of molecules into cartilage. In this Review, the factors that can be used to tune the transport kinetics of therapeutics are summarized. Overall, the most crucial factor when designing new therapeutic molecules is solute size. The diffusivity and partition coefficient of a solute both decrease with increasing solute size as indicated by molecular mass or by hydrodynamic radius. Surprisingly, despite having an effective pore size of ~6 nm, molecules of ~16 nm radius can diffuse through the cartilage matrix. Alteration of the shape or charge of a solute and the application of physiological loading to cartilage can be used to predictably improve solute transport kinetics, and this knowledge can be used to improve the development of therapeutic agents for osteoarthritis that target the cartilage.

Mounting evidence suggests that altered lubricant levels within synovial fluid have acute biological consequences on chondrocyte homeostasis. While these responses have been connected to increased friction, the mechanisms behind this response remain unknown. Here, we combine a frictional bioreactor with confocal elastography and image-based cellular assays to establish the link between cartilage friction, microscale shear strain, and acute, adverse cellular responses. Our incorporation of cell-scale strain measurements reveals that elevated friction generates high shear strains localized near the tissue surface, and that these elevated strains are closely associated with mitochondrial dysfunction, apoptosis, and cell death. Collectively, our data establish two pathways by which chondrocytes negatively respond to friction: an immediate necrotic response and a longer term pathway involving mitochondrial dysfunction and apoptosis. Specifically, in the surface region, where shear strains can exceed 0.07, cells are predisposed to acute death; however, below this surface region, cells exhibit a pathway consistent with apoptosis in a manner predicted by local shear strains. These data reveal a mechanism through which cellular damage in cartilage arises from compromised lubrication and show that in addition to boundary lubricants, there are opportunities upstream of apoptosis to preserve chondrocyte health in arthritis therapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Lower back pain, the leading cause of workplace absences and disability, is often attributed to intervertebral disc degeneration, in which nucleus pulposus (NP) herniates through lesions in the annulus fibrosus (AF) and impinges on the spinal cord and surrounding nerves. Surgeons remove extruded NP via discectomy when indicated by local/radicular pain supported by radiographic evidence; however, current interventions do not alter the underlying disease or seal the AF. The reported rates of recurrent herniation or pain following discectomy cases range from 5% to 25%, which has pushed spine research in recent years toward annular repair and closure strategies. Synthetic implants designed to mechanically seal the AF have been subject to large animal and clinical trials, with limited success in preventing recurrent herniation. Like gold standard interventions, purely mechanical devices fail to promote tissue integration, long-term healing, or restore native biomechanical function to the spine. Biological repair strategies utilizing principles of tissue engineering have demonstrated success in overcoming the inadequacies of current interventions and mechanical implants, yet, none has reached clinical or proof-of-concept trials in humans. In this review, we will discuss annular repair strategies promoting biological healing that have been implemented in small and large animal models in vivo, and ways to enhance the efficacy of these treatments.

Hyaluronic acid (HA) is widely injected as a viscosupplement in the treatment of osteoarthritis. Despite its extensive use, it is not currently known if cartilage degradation alters how HA-based solutions lubricate the articular surface. In this study we utilized a model of cartilage degradation by IL-1β along with a recently developed framework to study role of cartilage degradation on lubrication by clinically-approved HA-based lubricants with high viscosities. Cartilage explants were cultured up to 8 days with 10 ng/ml IL-1β. After culture, samples were examined histologically, immunohistochemically, biochemically, mechanically, topographically, and tribologically. The tribological testing analyzed both boundary and mixed lubrication modes to assess individual effects of viscosity and boundary lubricating ability. Friction testing was carried out using PBS and two clinically approved HA-based viscosupplements in a cartilage-glass configuration. After culture with IL-1β, boundary mode friction was elevated after both 4 and 8 days. Additionally, friction in mixed mode lubrication, where HA is most effective as a lubricant, was significantly elevated after 8 days of culture. As cartilage became rougher, softer, and more permeable after culture, the boundary mode plateau was extended, and as a result, significantly increased lubricant viscosities or sliding speeds were necessary to achieve effective mixed lubrication. Overall, this study revealed that lubrication of cartilage by HA is degradation-dependent and coincides with changes in mechanics and roughness. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1456-1464, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Cracks in articular cartilage are a common sign of joint damage, but failure properties of cartilage are poorly understood, especially for damage initiation. Cartilage failure may be further complicated by rate-dependent and depth-dependent properties, including the compliant surface layer. Existing blunt impact methods do not resolve local cartilage inhomogeneities and traditional fracture mechanics tests induce crack blunting and may violate underlying assumptions of linear elasticity. To address this knowledge gap, we developed and applied a method to indent cartilage explants with a sharp blade and initiate damage across a range of loading rates (strain rates 0.5%/s-500%/s), while recording local sample deformation and strain energy fields using confocal elastography. To investigate the importance of cartilage's compliant surface, we repeated the experiment for samples with the surface removed. Bulk data suggest a critical force at which the tissue cuts, but local strains reveals that the deformation the sample can sustain before reaching this force is significantly higher in the surface layer. Bulk and local results also showed significant rate dependence, such that samples were easier to cut at faster speeds. This result highlights the importance of rate for understanding cracks in cartilage and parallels recent studies of rate-dependent failure in hydrogels. Notably, local sample deformation fields were well fit by classical Hookean elasticity. Overall, this study illustrates how local and global measurements surrounding the initiation of damage in articular cartilage can be combined to reveal the importance of cartilage's zonal structure in protecting against failure across physiologically relevant loading rates., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cells receive mechanical cues from their extracellular matrix (ECM), which direct migration, differentiation, apoptosis, and in some cases, the transition to a cancerous phenotype. As a result, there has been significant research to develop methods to tune the mechanical properties of the ECM and understand cell-ECM dynamics more deeply. Here, we show that ionizing radiation can reduce the stiffness of an ex vivo tumor and an in vitro collagen matrix. When non-irradiated cancer cells were seeded in the irradiated matrix, adhesion, spreading, and migration were reduced. These data have ramifications for both in vitro and in vivo systems. In vitro , these data suggest that irradiation may be a method that could be used to create matrices with tailored mechanical properties. In vivo , these suggest that therapeutic doses of radiation may alter tissue mechanics directly.

Purpose: The significant shortcomings associated with current autologous reconstructive options for auricular deformities have inspired great interest in a tissue engineering solution. A major obstacle in the engineering of human auricular cartilage is the availability of sufficient autologous human chondrocytes. A clinically obtainable amount of auricular cartilage tissue (ie, 1 g) only yields approximately 10 million cells, where 25 times this amount is needed for the fabrication of a full-scale pediatric ear. It is thought that repeated passaging of chondrocytes leads to dedifferentiation and loss of the chondrogenic potential. However, little to no data exist regarding the ideal number of times that human auricular chondrocytes (HAuCs) can be passaged in a manner that maximizes the cellular expansion while minimizing dedifferentiation., Methods: Human auricular chondrocytes were isolated from discarded otoplasty specimens. The HAuCs were then expanded, and cells from passages 3, 4, and 5 were encapsulated into discs 8 mm in diameter made from type I collagen hydrogels with a cell density of 25 million cells/mL. The constructs were implanted subcutaneously in the dorsa of nude mice and harvested after 1 and 3 months for analysis., Results: Constructs containing passages 3, 4, and 5 chondrocytes all maintained their original cylindrical geometry. After 3 months in vivo, the diameters of the P3, P4, and P5 discs were 69 ± 9%, 67 ± 10%, and 73 ± 15% of their initial diameter, respectively. Regardless of the passage number, all constructs developed a glossy white cartilaginous appearance, similar to native auricular cartilage. Histologic analysis demonstrated development of an organized perichondrium composed of collagen, a rich proteoglycan matrix, cellular lacunae, and a dense elastin fibrin network by Safranin-O and Verhoeff stain. Biochemical analysis confirmed similar amounts of proteoglycan and hydroxyproline content in late passage constructs when compared with native auricular cartilage., Conclusions: These data indicate that late passage HAuCs (up to passage 5) form elastic cartilage that is histologically, biochemically, and biomechanically similar to native human elastic cartilage and have the potential to be used for auricular cartilage engineering.

No disease-modifying osteoarthritis (OA) drugs are available to prevent posttraumatic osteoarthritis (PTOA). Mitochondria (MT) mediate the pathogenesis of many degenerative diseases, and recent evidence indicates that MT dysfunction is a peracute (within minutes to hours) response of cartilage to mechanical injury. The goal of this study was to investigate cardiolipin-targeted mitoprotection as a new strategy to prevent chondrocyte death and cartilage degeneration after injury. Cartilage was harvested from bovine knee joints and subjected to a single, rapid impact injury (24.0 ±1.4 MPa, 53.8 ± 5.3 GPa/s). Explants were then treated with a mitoprotective peptide, SS-31 (1µM), immediately post-impact, or at 1, 6, or 12 h after injury, and then cultured for up to 7 days. Chondrocyte viability and apoptosis were quantified in situ using confocal microscopy. Cell membrane damage (lactate dehydrogenase activity) and cartilage matrix degradation (glycosaminoglycan loss) were quantified in cartilage-conditioned media. SS-31 treatment at all time points after impact resulted in chondrocyte viability similar to that of un-injured controls. This effect was sustained for up to a week in culture. Further, SS-31 prevented impact-induced chondrocyte apoptosis, cell membrane damage, and cartilage matrix degeneration., Clinical Significance: This study is the first investigation of cardiolipin-targeted mitoprotective therapy in cartilage. These results suggest that even when treatment is delayed by up to 12 h after injury, mitoprotection may be a useful strategy in the prevention of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-10, 2018., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Study Design: Ovine in vivo study., Objective: To perform lateral approach lumbar surgery in an ovine model to administer an injectable riboflavin cross-linked high-density collagen (HDC) gel and to assess its ability to mitigate intervertebral disc (IVD) degeneration after induced annulus fibrosus (AF) injury., Summary of Background Data: Biological-based injectable gels have shown efficacy in restoring biomechanical, radiographic, and histological parameters in IVD-injured animal models. Riboflavin cross-linked HDC gel has previously demonstrated retention of nucleus pulposus (NP) tissue, reduced loss of disc height, and prevention of terminal cellular degenerative changes in rat-tail spines. However, this biological therapy has never been tested in large animal models., Methods: Forty lumbar IVDs were accessed from eight sheep via lateral approach surgery. IVDs were randomly assigned to healthy control, injury and HDC treatment, or negative control with injury and no treatment. IVD injury was carried out using a drill-bit through the AF followed by needle puncture of the NP. Sheep were followed for 16 weeks and underwent qualitative/quantitative magnetic resonance imaging, x-ray, and histological analyses of collagen and proteoglycan content., Results: The lateral approach to the ovine lumbar spine to deliver HDC gel proved to be safe and reproducible. IVDs treated with the HDC gel revealed less degenerative changes at the microscopic level based on AF and NP histology. However, mean Pfirrmann grade, T2 relaxation time, NP voxel size, and disc height index were not significantly different between the two injury groups., Conclusion: Injectable HDC gel can be administered safely via lateral approach surgery in an ovine AF injury model. IVDs treated with HDC gel demonstrated less degeneration at the microscopic level though radiographic changes were slight when comparing treated to untreated IVDs. Future studies will need to elucidate the role of injury technique and time frame for follow-up in correlating histological and radiographical outcomes., Level of Evidence: N /A.

Mitochondrial (MT) dysfunction is known to occur in chondrocytes isolated from end-stage osteoarthritis (OA) patients, but the role of MT dysfunction in the initiation and early pathogenesis of post-traumatic OA (PTOA) remains unclear. The objective of this study was to investigate chondrocyte MT function immediately following mechanical injury in cartilage, and to determine if the response to injury differed between a weight bearing region (medial femoral condyle; MFC) and a non-weight bearing region (distal patellofemoral groove; PFG) of the same joint. Cartilage was harvested from the MFC and PFG of 10 neonatal bovids, and subjected to injurious compression at varying magnitudes (5-17 MPa, 5-34 GPa/s) using a rapid single-impact model. Chondrocyte MT respiratory function, MT membrane polarity, chondrocyte viability, and cell membrane damage were assessed in situ. Cartilage impact resulted in MT depolarization and impaired MT respiratory function within 2 h of injury. Cartilage from a non-weight bearing region of the joint (PFG) was more sensitive to impact-induced MT dysfunction and chondrocyte death than cartilage from a weight-bearing surface (MFC). Our findings suggest that MT dysfunction is an acute response of chondrocytes to cartilage injury, and that MT may play a key mechanobiological role in the initiation and early pathogenesis of PTOA., Clinical Significance: Direct therapeutic targeting of MT function in the early post-injury time frame may provide a strategy to block perpetuation of tissue damage and prevent the development of PTOA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:739-750, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)