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Collagen-based hydrogels have been widely used in biomedical applications due to their biocompatibility. Enhancing mechanical properties of collagen gels remains challenging while maintaining biocompatibility. Here, we demonstrate that gelation pH has profound effects on cellular activity, collagen fibril structure, and mechanical properties of the fibrochondrocyte-seeded collagen gels in both short- and long-terms. Acidic and basic gelation pH, below pH 7.0 and above 8.5, resulted in dramatic cell death. Gelation pH ranging from 7.0 to 8.5 showed a relatively high cell viability. Furthermore, physiologic gelation (pH 7.5) showed the greatest collagen deposition while glycosaminoglycan deposition appeared independent of gelation pH. Scanning electron microscopy showed that neutral and physiologic gelation pH, 7.0 and 7.5, exhibited well-aligned collagen fibril structure on day 0 and enhanced collagen fibril structure with laterally joined fibrils on day 30. However, basic pH, 8.0 and 8.5, displayed a densely packed collagen fibril structure on day 0, which was also persistent on day 30. Initial equilibrium modulus increased with increasing gelation pH. Notably, after 30 days of culture, gelation pH of 7.5 and 8.0 showed the highest equilibrium modulus, reaching 150 -160 kPa. While controlling gelation pH is simply achieved compared with other strategies to improve mechanical properties, its influences on biochemical and biomechanical properties of the collagen gel are long-lasting. As such, gelation pH is a useful means to modulate both biochemical and biomechanical properties of the collagen-based hydrogels and can be utilized for diverse types of tissue engineering due to its simple application.

There remains a need for stiffer collagen hydrogels for tissue engineering and disease modeling applications. Pre-glycation, or glycation of collagen in solution prior to gelation, has been shown to increase the mechanics of collagen hydrogels while maintaining high viability of encapsulated cells. The stiffness of glycated collagen gels can be increased by increasing the collagen concentration, sugar concentration, and glycation time. However, previous studies on pre-glycation of collagen have used low collagen concentrations and/or low sugar concentrations and have not investigated the effect of glycation time. Therefore, the objective of this study was to determine the effects of pre-glycation with high sugar concentrations (up to 500 mM) and extended glycation times (up to 21 days) on high concentration collagen (8 mg/ml). The addition of sugar to the collagen and the formation of advanced glycation end products (AGEs) were quantified. The ability to gel successfully and rheological properties were determined and correlated with biochemical characterizations. Successful collagen gelation and rheological properties of pre-glycated collagen were found to be strongly dependent on the ratio of added sugars to added AGEs with high ratios impairing gelation and low ratios resulting in optimal storage moduli. There is likely a competing effect during pre-glycation of the formation of AGEs resulting in crosslinking of collagen and the formation of Amadori intermediates acting to increase collagen solubility. Overall, this study shows that collagen glycation can be optimized by increasing the formation of AGEs while maintaining a low ratio of added sugar to added AGEs.

A major challenge to the clinical translation of tissue-engineered ear scaffolds for ear reconstruction is the limited auricular chondrocyte (hAuC) yield available from patients. Starting with a relatively small number of chondrocytes in culture results in dedifferentiation and loss of phenotype with subsequent expansion. To significantly decrease the number of chondrocytes required for human elastic cartilage engineering, we co-cultured human mesenchymal stem cells (hMSCs) with HAuCs to promote healthy elastic cartilage formation. HAuCs along with human bone marrow-derived hMSCs were encapsulated into 1% Type I collagen at 25 million/mL total cell density with different ratios (HAuCs/hMSCs: 10/90, 25/75, 50/50) and then injected into customized 3D-printed polylactic acid (PLA) ridged external scaffolds, which simulate the shape of the auricular helical rim, and implanted subcutaneously in nude rats for 1, 3 and 6 months. The explanted constructs demonstrated near complete volume preservation and topography maintenance of the ridged "helical" feature after 6 months with all ratios. Cartilaginous appearing tissue formed within scaffolds by 3 months, verified by histologic analysis demonstrating mature elastic cartilage within the constructs with chondrocytes seen in lacunae within a Type II collagen and proteoglycan-enriched matrix, and surrounded by a neoperichondrial external layer. Compressive mechanical properties comparable to human elastic cartilage were achieved after 6 months. Co-implantation of hAuCs and hMSCs in collagen within an external scaffold efficiently produced shaped human elastic cartilage without volume loss even when hAuC comprised only 10% of the implanted cell population, marking a crucial step toward the clinical translation of auricular tissue engineering.

Investigating the mechanobiology of chondrocytes is challenging due to the complex micromechanical environment of cartilagetissue. The innate zonal differences and poroelastic properties of the tissue combined with its heterogeneous composition create spatial- and temporal-dependent cell behavior, which further complicates the investigation. Despite the numerous challenges, understanding the mechanobiology of chondrocytes is crucial for developing strategies for treating cartilage related diseases as chondrocytes are the only cell type within the tissue. The effort to understand chondrocyte behavior under various mechanical stimuli has been ongoing over the last 50 years. Early studies examined global biosynthetic behavior under unidirectional mechanical stimulus. With the technological development in high-speed confocal imaging techniques, recent studies have focused on investigating real-time individual and collective cell responses to multiple / combined modes of mechanical stimuli. Such efforts have led to tremendous advances in understanding the influence of local physical stimuli on chondrocyte behavior. In addition, we highlight the wide variety of experimental techniques, spanning from static to impact loading, and analysis techniques, from biochemical assays to machine learning, that have been utilized to study chondrocyte behavior. Finally, we review the progression of hypotheses about chondrocyte mechanobiology and provide a perspective on the future outlook of chondrocyte mechanobiology.

Low back pain is the leading cause of work absences and years lived with disability, and it is often associated with degenerative disc disease. In recent years, biological treatment approaches such as the use of growth factors, cell injections, annulus fibrosus (AF) repair, nucleus pulposus replacement, and tissue-engineered discs have been explored as means for preventing or reversing degenerative disc disease. Both animal and clinical studies have shown promising results for cell-based therapy on the grounds of its regenerative potential. Clinical data also indicate that stem cell injection is safe when appropriately performed, albeit its long-term safety and efficacy are yet to be explored. Numerous challenges also remain to be overcome, such as isolating, differentiating, and preconditioning the disc cells, as well as managing the nutrient-deficient and oxygen-deficient micromilieu of the intervertebral disc (IVD). AF repair methods including devices used in clinical trials have shown success in decreasing reherniation rates and improving overall clinical outcomes. In addition, recent studies that combined AF repair and nucleus pulposus replacement have shown improved biomechanical stability in IVDs after the combined treatment. Tissue-engineered IVDs for total disc replacement are still being developed, and future studies are necessary to overcome the challenges in their delivery, efficacy, and safety.

Summary 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.

This paper describes the compositional, mechanical, and structural differences between collagen gels fabricated from different sources and processing methods. Despite extensive use of collagen in the manufacturing of biomaterials and implants, there is little information as to the variation in properties based on collagen source or processing methods. As such, differences in purity and composition may affect gel structure and mechanical performance. Using mass spectrometry, we assessed protein composition of collagen from seven different sources. The mechanics and gelation kinetics of each gel were assessed through oscillatory shear rheology. Scanning electron microscopy enabled visualization of distinct differences in fiber morphology. Mechanics and gelation kinetics differed with source and processing method and were found to correlate with differences in composition. Gels fabricated from telopeptide-containing collagens had higher storage modulus (144 vs. 54 Pa) and faster gelation (251 vs. 734 seconds) compared to atelocollagens, despite having lower purity (93.4 vs 99.8%). For telopeptide-containing collagens, as collagen purity increased, storage modulus increased, and fiber diameter decreased. As α1/α2 chain ratio increased, fiber diameter increased, and gelation slowed. As such, this study provides an examination of the effects of collagen processing on key quality attributes for use of collagen gels in biomedical contexts.

Avascular soft tissues of the skeletal system, including articular cartilage, have limited healing capacity, in part due to their low metabolic activity. No drugs are available that can prevent or slow the development of osteoarthritis (OA) after joint injury. Therefore, mesenchymal stromal cell (MSC)-based regenerative therapies are increasingly common in the treatment of OA, but questions regarding their clinical efficacy and mechanisms of action remain unanswered. Our group recently reported that mitochondrial dysfunction is one of the earliest responses of cartilage to injury, resulting in chondrocyte death, extracellular matrix degeneration, and ultimately OA. MSCs have been found to rescue injured cells and improve healing by donating healthy mitochondria in highly metabolic tissues, but mitochondrial transfer has not been investigated in cartilage. Here, we demonstrate that MSCs transfer mitochondria to stressed chondrocytes in cell culture and in injured cartilage tissue. Conditions known induce chondrocyte mitochondrial dysfunction, including stimulation with rotenone/antimycin and hyperoxia, increased transfer. Stressed chondrocytes increased expression of genes related to inflammation and senescence, further supporting the link between mitochondrial dysfunction and transfer. MSC-chondrocyte mitochondrial transfer was blocked by non-specific and specific (connexin-43) gap-junction inhibition. When MSCs were exposed to mechanically injured cartilage they localized to areas of matrix damage and extended cellular processes deep into microcracks, delivering mitochondria to chondrocytes. This work provides insights into the chemical, environmental, and mechanical conditions that can elicit MSC-chondrocyte mitochondrial transfer in vitro and in situ, and our findings suggest a new potential role for MSC-based therapeutics after cartilage injury.Significance StatementRecent evidence suggests that although articular cartilage is avascular and relatively metabolically quiescent, acute injury induces chondrocyte mitochondrial dysfunction, driving cartilage degradation and OA. We present the first evidence that MSCs donate mitochondria to articular chondrocytes undergoing mitochondrial dysfunction in vitro and in situ. These findings support a new role for MSCs in the context of cartilage injury and OA, and intercellular mitochondrial transfer may represent a new biological approach to augment mitochondrial capacity in injured chondrocytes. This work establishes multiple experimental models to study MSC mitochondrial donation for the treatment of OA and related degenerative diseases of avascular orthopedic tissues.

Intra-articular injections of hyaluronic acid have been a mainstay of osteoarthritis treatment for decades. However, controversy surrounds the mechanism of action and efficacy of this therapy. As such, there has been recent interest in developing synthetic lubricants that lubricate cartilage. Recently, a synthetic 4 wt% polyacrylamide (pAAm) hydrogel was shown to effectively decrease lameness in horses. However, its mechanism of action and ability to lubricate cartilage is unknown. The goal of this study was to characterize the lubricating ability of this hydrogel and determine its efficacy for healthy and degraded cartilage. The study utilized previously established IL-1β-induced biochemical degradation and mechanical impact injury models to degrade cartilage. The lubricating ability of the hydrogel was then characterized using a custom-built tribometer using a glass counterface and friction was evaluated using the Stribeck framework for articular cartilage. pAAm hydrogel was shown to significantly lower the friction coefficient of cartilage explants from both degradation models (30%-40% reduction in friction relative to controls). A striking finding from this study was the aggregation of the pAAm hydrogel at the articulating surface. The surface aggregation was observed in the histological sections of explants from all treatment groups after tribological evaluation. Using the Stribeck framework, the hydrogel was mapped to higher Sommerfeld numbers and was characterized as a viscous lubricant predominantly in the minimum friction mode. In summary, this study revealed that pAAm hydrogel lubricates native and degraded cartilage explants effectively and may have an affinity for the articulating surface of the cartilage.

Healthy cartilage is a remarkable tissue, able to withstand tens of millions of loading cycles with minimal damage. While much is known about the structural origins of its compressive mechanics, how composition determines its mechanical behavior in shear, a major mode of failure, is poorly understood. Through a combination of microscale structure-function experiments and modeling, we develop a rigidity percolation framework to explain the structural origins of cartilage shear mechanics. This framework provides a new quantitative understanding for how the known degradative events in osteoarthritis determine the mechanical changes that are a hallmark of this disease. As such, this work provides a road map for understanding disease progression and in combination with non-invasive techniques such as MRI will enable more effective diagnosis and treatment., 9 pages, 3 pages of SI, 5 figures

Summary 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: 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.

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.

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.

Tunable mechanics and fracture resistance are hallmarks of biological tissues whose properties arise from extracellular matrices comprised of double networks. To elucidate the origin of these desired properties, we study the shear modulus and fracture properties of a rigidly percolating double network model comprised of a primary network of stiff fibers and a secondary network of flexible fibers. We find that when the primary network density is just above its rigidity percolation threshold, the secondary network density can be tuned to facilitate stress relaxation via non-affine deformations and provide mechanical reinforcement. In contrast, when the primary network is far above its rigidity threshold, the double network is always stiff and brittle. These results highlight an important mechanism behind the tunability and resilience of biopolymer double networks: the secondary network can dramatically alter mechanical properties from compliant and ductile to stiff and brittle only when the primary network is marginally rigid.

Objective A major obstacle in the clinical translation of engineered auricular scaffolds is the significant contraction and loss of topography that occur during maturation of the soft collagen-chondrocyte matrix into elastic cartilage. We hypothesized that 3-dimensional-printed, biocompatible scaffolds would “protect” maturing hydrogel constructs from contraction and loss of topography. Design External disc-shaped and “ridged” scaffolds were designed and 3D-printed using polylactic acid (PLA). Acellular type I collagen constructs were cultured in vitro for up to 3 months. Collagen constructs seeded with bovine auricular chondrocytes (BAuCs) were prepared in 3 groups and implanted subcutaneously in vivo for 3 months: preformed discs with (“Scaffolded/S”) or without (“Naked/N”) an external scaffold and discs that were formed within an external scaffold via injection molding (“Injection Molded/SInj”). Results The presence of an external scaffold or use of injection molding methodology did not affect the acellular construct volume or base area loss. In vivo, the presence of an external scaffold significantly improved preservation of volume and base area at 3 months compared to the naked group ( P < 0.05). Construct contraction was mitigated even further in the injection molded group, and topography of the ridged constructs was maintained with greater fidelity ( P < 0.05). Histology verified the development of mature auricular cartilage in the constructs within external scaffolds after 3 months. Conclusion Custom-designed, 3D-printed, biocompatible external scaffolds significantly mitigate BAuC-seeded construct contraction and maintain complex topography. Further refinement and scaling of this approach in conjunction with construct fabrication utilizing injection molding may aid in the development of full-scale auricular scaffolds.

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.

Significance Osteoarthritis is a global health problem that affects load-bearing joints, causing loss of mobility and enormous healthcare costs. However, disease-modifying approaches are lacking. Here, we report a cellular mechanism of inflammatory signaling in chondrocytes, the cellular component of cartilage. We show how osteoarthritis-relevant levels of interleukin-1α reprogram articular chondrocytes so that they become more susceptible to mechanical trauma, which chondrocytes sense via Piezo1/2-mechanosensitive ion channels. We uncover that IL-1α enhances gene expression of Piezo1 in primary articular chondrocytes underlying Piezo1 enhanced function. We elucidate signaling from membrane to nucleus, including transcription factors that enhance Piezo1 expression. We also define consequences of increased expression of Piezo1, for mechanotransduction and at rest, that implicate this reprogramming mechanism in osteoarthritis pathogenesis., Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23–32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114–E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263–273 (2017)]. We therefore asked whether interleukin-1–mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.

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.

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.

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 > R2 0.75) compared to healthy samples (R2

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

Purpose: Proteoglycans, especially small leucine rich proteoglycans (SLRPs), play major roles in facilitating the development and regulation of collagen fibers and other extracellular matrix components. However, their roles in fibrocartilage have not been widely reviewed. Here, we discuss both SLRP and large aggregating proteoglycan's roles in collagen fibrillogenesis and extracellular matrix assembly in fibrocartilage tissues such as the meniscus, annulus fibrosus (AF), and TMJ disc. We also discuss their expression levels throughout development, aging and degeneration, as well as repair. Methods: A review of literature discussing proteoglycans and collagen fibrillogenesis in fibrocartilage was conducted and data from these manuscripts were analyzed and grouped to discuss trends throughout the tissue's architectural zones and developmental stage.Results: The spatial collagen architecture of these fibrocartilaginous tissues is reflected in the distribution of proteoglycans expressed, suggesting that each proteoglycan plays an important role in the type of architecture presented and associated mechanical function.Conclusion: The unique structure-function relationship of fibrocartilage makes the varied architectures throughout the tissues imperative for their success and understanding the functions of these proteoglycans in developing and maintaining the fiber structure could inform future work in fibrocartilage replacement using tissue engineered constructs.

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.

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 (EC50) ranges, which are 0.23 to 0.28 and 0.53 to 1.71 ng/mL, respectively. At concentrations less than the EC50, for the GAG production and contraction rate, TGF-β1 treatment resulted in less organized collagen fibers. At concentrations greater than the EC50, TGF-β1 led to dense, disorganized collagen fibers. Between the two EC50 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.

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.

Sensation of mechanical stimuli by chondrocytes is critical to cartilage homeostasis and osteoarthritis development. The earliest responses in chondrocyte mechanotransduction pathways involve calcium influx and changes in mitochondrial function, which occur in seconds to minutes. Deeper understanding of these events can elucidate new therapeutic targets for early intervention to prevent osteoarthritis.

Post-traumatic osteoarthritis (PTOA) is typically initiated by momentary supraphysiologic shear and compressive forces delivered to articular cartilage during acute joint injury and develops through subsequent degradation of cartilage matrix components and tissue remodeling. PTOA affects 12% of the population who experience osteoarthritis and is attributed to over $3 billion dollars annually in healthcare costs. It is currently unknown whether articulation of the joint post-injury helps tissue healing or exacerbates cellular dysfunction and eventual death. We hypothesize that post-injury cartilage articulation will lead to increased cartilage damage. Our objective was to test this hypothesis by mimicking the mechanical environment of the joint during and post-injury and determining if subsequent joint articulation exacerbates damage produced by initial injury. We use a model of PTOA that combines impact injury and repetitive sliding with confocal microscopy to quantify and track chondrocyte viability, apoptosis, and mitochondrial depolarization in a depth-dependent manner. Cartilage explants were harvested from neonatal bovine knee joints and subjected to either rapid impact injury (17.34 ± 0.99 MPa, 21.6 ± 2.45 GPa/s), sliding (60 min at 1 mm/s, under 15% axial compression), or rapid impact injury followed by sliding. Explants were then bisected and fluorescently stained for cell viability, caspase activity (apoptosis), and mitochondria polarization. Results show that compared to either impact or sliding alone, explants that were both impacted and slid experienced higher magnitudes of damage spanning greater tissue depths. This article is protected by copyright. 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.