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Although the sacroiliac (SI) joint can be a source of lower back and buttock pain, no comprehensive characterization studies on SI cartilage have been conducted. Using the minipig as a large animal model, this study conducted the first biomechanical, biochemical, and histological characterization of SI joint cartilage. Because previous literature has reported that sacral cartilage and iliac cartilage within the SI joint are histologically distinct, concomitantly it was expected that functional properties of the sacral cartilage would differ from those of the iliac cartilage. Creep indentation, uniaxial tension, biochemical, and histological analyses were conducted on the sacral and iliac cartilage of skeletally mature female Yucatan minipigs (n = 6-8 for all quantitative tests). Concurring with prior literature, the iliac cartilage appeared to be more fibrous than the sacral cartilage. Glycosaminoglycan content was 2.2 times higher in the sacral cartilage. The aggregate modulus of the sacral cartilage was 133 ± 62 kPa, significantly higher than iliac cartilage, which only had an aggregate modulus of 51 ± 61 kPa. Tensile testing was conducted in both cranial-caudal and ventral-dorsal axes, and Young's modulus values ranged from 2.5 ± 1.5 MPa to 13.6 ± 1.5 MPa, depending on anatomical structure (i.e., sacral vs. iliac) and orientation of the tensile test. The Young's modulus of sacral cartilage was 5.5 times higher in the cranial-caudal axis and 2.0 times higher in the ventral-dorsal axis than the iliac cartilage. The results indicate that the sacral and iliac cartilages are functionally distinct from each other. Understanding the distinct differences between sacral and iliac cartilage provides insight into the structure and function of the SI joint, which may inform future research aimed at repairing SI joint cartilage., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Drs. Athanasiou and Hu have an equity interest in Cartilage, Inc. Their relationship with Cartilage, Inc. has been reviewed and approved by the University of California, Irvine in accordance with its conflict-of-interest policies. Dr. Elder is a consultant for Depuy Synthes and SI Bone, is on the medical advisory board and has stock options in Injectsense, is the site PI for clinical trials for Stryker and SI Bone, and is on the executive board of the International Society for Hydrocephalus and CSF Disorders., (Published by Elsevier Ltd.)

The tunica albuginea (TA) of the penis is an elastic layer that serves a structural role in penile erection. Disorders affecting the TA cause pain, deformity, and erectile dysfunction. There is a substantial clinical need for engineered replacements of TA, but data are scarce on the material properties and biochemical composition of healthy TA. The objective of this study was to assess tissue organization, protein content, and mechanical properties of porcine TA to establish structure-function relationships and design criteria for tissue engineering efforts. TA was isolated from six pigs and subjected to histomorphometry, quantification of collagen content and pyridinoline crosslinks, bottom-up proteomics, and tensile mechanical testing. Collagen was 20 ± 2%/wet weight (WW) and 53 ± 4%/dry weight (DW). Pyridinoline content was 426 ±131 ng/mg WW, 1011 ± 190 ng/mg DW, and 45 ± 8 mmol/mol hydroxyproline. Bottom-up proteomics identified 14 proteins with an abundance of >0.1% of total protein. The most abundant collagen subtype was type I, representing 95.5 ± 1.5% of the total protein in the samples. Collagen types III, XII, and VI were quantified at 1.7 ± 1.0%, 0.8 ± 0.2%, and 0.4 ± 0.2%, respectively. Tensile testing revealed anisotropy: Young's modulus was significantly higher longitudinally than circumferentially (60 ± 18 MPa vs. 8 ± 5 MPa, p < 0.01), as was ultimate tensile strength (16 ± 4 MPa vs. 3 ± 3 MPa, p < 0.01). Taken together, the tissue mechanical and compositional data obtained in this study provide important benchmarks for the development of TA biomaterials. STATEMENT OF SIGNIFICANCE: The tunica albuginea of the penis serves an important structural role in physiologic penile erection. This tissue can become damaged by disease or trauma, leading to pain and deformity. Treatment options are limited. Little is known about the precise biochemical composition and biomechanical properties of healthy tunica albuginea. In this study, we characterize the tissue using proteomic analysis and tensile testing to establish design parameters for future tissue engineering efforts. To our knowledge, this is the first study to quantify tissue anisotropy and to use bottom-up proteomics to characterize the composition of penile tunica albuginea., 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 © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nose reconstruction often requires scarce cartilage grafts. Nasal cartilage properties must be determined to serve as design criteria for engineering grafts. Thus, mechanical and biochemical properties were obtained in multiple locations of human nasal septum, upper lateral cartilage (ULC), and lower lateral cartilage (LLC). Within each region, no statistical differences among locations were detected, but anisotropy at some septum locations was noted. In the LLC, the tensile modulus and ultimate tensile strength (UTS) in the inferior-superior direction were statistically greater than in the anterior-posterior direction. Cartilage from all regions exhibited hyperelasticity in tension, but regions varied in degree of hyalinicity (i.e., Col II:Col I ratio). The septum contained the most collagen II and least collagen I and III, making it more hyaline than the ULC and LLC. The septum had a greater aggregate modulus, UTS, and lower total collagen/wet weight (Col/WW) than the ULC and LLC. The ULC had greater tensile modulus, DNA/WW, and lower glycosaminoglycan/WW than the septum and LLC. The ULC had a greater pyridinoline/Col than the septum. Histological staining suggested the presence of chondrons in all regions. In the ULC and LLC, tensile modulus correlated with total collagen content, while aggregate modulus correlated with pyridinoline content and weakly with pentosidine content. However, future studies should be performed to validate these proposed structure-function relationships. This study of human nasal cartilage provides 1) crucial design criteria for nasal cartilage tissue engineering efforts, 2) quantification of major and minor collagen subtypes and crosslinks, and 3) structure-function relationships. Surprisingly, the large mechanical properties found, particularly in the septum, suggests that nasal cartilage may experience higher-than-expected mechanical loads. STATEMENT OF SIGNIFICANCE: While tissue engineering holds promise to generate much-needed cartilage grafts for nasal reconstruction, little is known about nasal cartilage from an engineering perspective. In this study, the mechanical and biochemical properties of the septum, upper lateral cartilage (ULC), and lower lateral cartilage (LLC) were evaluated using cartilage-specific methods. For the first time in this tissue, all major and minor collagens and collagen crosslinks were measured, demonstrating that the septum was more hyaline than the ULC and LLC. Additionally, new structure-function relationships in the ULC and LLC were identified. This study greatly expands upon the quantitative understanding of human nasal cartilage and provides crucial engineering design criteria for much-needed nasal cartilage tissue engineering efforts., Competing Interests: Declaration of Competing Interests 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 © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Signals that recapitulate in vitro the conditions found in vivo, such as hypoxia or mechanical forces, contribute to the generation of tissue-engineered hyaline-like tissues. The cell regulatory processes behind hypoxic and mechanical stimuli rely on ion concentration; iron is required to degrade the hypoxia inducible factor 1a (HIF1α) under normoxia, whereas the initiation of mechanotransduction requires the cytoplasmic increase of calcium concentration. In this work, we propose that ion modulation can be used to improve the biomechanical properties of self-assembled neocartilage constructs derived from rejuvenated expanded minipig rib chondrocytes. The objectives of this work were 1) to determine the effects of iron sequestration on self-assembled neocartilage constructs using two doses of the iron chelator deferoxamine (DFO), and 2) to evaluate the performance of the combined treatment of DFO and ionomycin, a calcium ionophore that triggers cytoplasmic calcium accumulation. This study employed a two-phase approach. In Phase I, constructs treated with a high dose of DFO (100 µM) exhibited an 87% increase in pyridinoline crosslinks, a 57% increase in the Young's modulus, and a 112% increase in the ultimate tensile strength (UTS) of the neotissue. In Phase II, the combined use of both ion modulators resulted in 150% and 176% significant increases in the Young's modulus and UTS of neocartilage constructs, respectively; for the first time, neocartilage constructs achieved a Young's modulus of 11.76±3.29 MPa and UTS of 4.20±1.24 MPa. The results of this work provide evidence that ion modulation can be employed to improve the biomechanical properties in engineered neotissues. STATEMENT OF SIGNIFICANCE: The translation of tissue-engineered products requires the development of strategies capable of producing biomimetic neotissues in a replicable, controllable, and cost-effective manner. Among other functions, Fe 2+ and Ca 2+ are involved in the control of the hypoxic response and mechanotransduction, respectively. Both stimuli, hypoxia and mechanical forces, are known to favor chondrogenesis. This study utilized ion modulators to improve the mechanical properties self-assembled neocartilage constructs derived from expanded and rejuvenated costal chondrocytes via Fe 2+ sequestration and Ca 2+ influx, alone or in combination. The results indicate that ion modulation induced tissue maturation and a significant improvement of the mechanical properties, and holds potential as a tool to mitigate the need for bioreactors and engineer hyaline-like tissues., 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 © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The objective of this work is to examine the development of porcine cartilage by analyzing its mechanical properties, biochemical content, and proteomics at different developmental stages. Cartilage from the knees of fetal, neonatal, juvenile, and mature pigs was analyzed using histology, mechanical testing, biochemical assays, fluorophore-assisted carbohydrate electrophoresis, and bottom-up proteomics. Mature cartilage has 2.2-times the collagen per dry weight of fetal cartilage, and fetal cartilage has 2.1-times and 17.9-times the glycosaminoglycan and DNA per dry weight of mature cartilage, respectively. Tensile and compressive properties peak in the juvenile stage, with a tensile modulus 4.7-times that of neonatal. Proteomics analysis reveals increases in collagen types II and III, while collagen types IX, XI, and XIV, and aggrecan decrease with age. For example, collagen types IX and XI decrease 9.4-times and 5.1-times, respectively from fetal to mature. Mechanical and biochemical measurements have their greatest developmental changes between the neonatal and juvenile stages, where mechanotransduction plays a major role. Bottom-up proteomics serves as a powerful tool for tissue characterization, showing results beyond those of routine biochemical analysis. For example, proteomic analysis shows significant drops in collagen types IX, XI, and XIV throughout development, which shows insight into the permanence of cartilage's matrix. Changes in overall glycosaminoglycan content compared to aggrecan and link protein indicate non-enzymatic degradation of aggrecan structures or hyaluronan in mature cartilage. In addition to tissue characterization, bottom-up proteomics techniques are critical in tissue engineering efforts toward repair or regeneration of cartilage in animal models. STATEMENT OF SIGNIFICANCE: In this study, the development of porcine articular cartilage is interrogated through biomechanical, biochemical, and proteomic techniques, to determine how mechanics and extracellular matrix composition change from fetal to mature cartilage. For the first time, a bottom-up proteomics approach is used to reveal a wide variety of protein changes through aging; for example, the collagen subtype composition of the cartilage increases in collagen types II and III, and decreases in collagen types IX, XI, and XIV. This analysis shows that bottom-up proteomics is a critical tool in tissue characterization, especially toward developing a deeper understanding of matrix composition and development in tissue engineering studies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kyriacos A. Athanasiou and Jerry C. Hu are scientific consultants at Cartilage Inc., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

To gain regulatory approval for the clinical use of knee biologics and devices in humans, translational large-animal studies are typically required. Animal models that permit second-look arthroscopy are valuable because they allow for longitudinal assessment of the treated tissue without needing to sacrifice the animal. The minipig is an ideal preclinical animal model for the investigation of therapies for the knee, in part because arthroscopy can be performed in its stifle (knee) joint with the use of standard surgical equipment used in humans. The purpose of this Technical Note is to describe a reproducible technique for diagnostic arthroscopy of the minipig stifle (knee) joint., (© 2020 by the Arthroscopy Association of North America. Published by Elsevier.)

A significant portion of equine lameness is localized to the stifle joint. Effective cartilage repair strategies are largely lacking, however, recent advances in surgical techniques, biomaterials, and cellular therapeutics have broadened the clinical strategies of cartilage repair. To date, no studies have been performed directly comparing neonatal and adult articular cartilage from the stifle across multiple sites. An understanding of the differences in properties between the therapeutic target cartilage (i.e., adult cartilage) as well as potential donor cartilage (i.e., neonatal cartilage) could aid in selection of optimal harvest sites within a donor joint as well as evaluation of the success of the grafted cells or tissues within the host. Given the dearth of characterization studies of the equine stifle joint, and in particular neonatal stifle cartilage, the goal of this study was to measure properties of both potential source tissue and host tissue. Articular cartilage of the distal femur and patella (P) was assessed in regards to two specific factors, age of the animal and specific site within the joint. Two age groups were considered: neonatal (<1 week) and adult (4-14 years). Cartilage samples were harvested from 17 sites across the distal femur and patella. It was hypothesized that properties would vary significantly between neonatal and adult horses as well as within age groups on a site-by-site basis. Adult thickness varied by site. With the exception of water content, there were no significant biochemical differences among sites within regions of the distal femur (condyles and trochlea) and the patella in either the adult or neonate. Neonatal cartilage had a significantly higher water content than adult. Surprisingly, biochemical measurements of cellularity did not differ significantly between neonatal and adult, however, adult cartilage had greater variance in cellularity than neonatal. Overall, there were no significant differences between neonatal and adult glycosaminoglycan content. Collagen per wet weight was found to be significantly higher in adult cartilage than neonatal when averaged across all levels. In terms of biomechanical properties, aggregate modulus varied significantly across the condyles of adult cartilage but not the neonate. Neonatal cartilage was significantly less permeable, and the Young's modulus of neonatal cartilage was significantly higher than the adult. The tensile strength did not vary in a statistically significant manner between age groups. An understanding of morphological, histological, biochemical, and biomechanical properties enhances the understanding of cartilage tissue physiology and structure-function relationships. This study revealed important differences in biomechanical and biochemical properties among the 17 sites and among the six joint regions, as well as age-related differences between neonatal and adult cartilage. These location and age-related variations are informative toward determining the donor tissue harvest site., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Knee meniscus injury is frequent, resulting in over 1 million surgeries annually in the United States and Europe. Because of the near-avascularity of this fibrocartilaginous tissue and its intrinsic lack of healing, tissue engineering has been proposed as a solution for meniscus repair and replacement. This study describes an approach employing bioactive stimuli to enhance both extracellular matrix content and organization of neomenisci toward augmenting their mechanical properties. Self-assembled fibrocartilages were treated with TGF-β1, chondroitinase ABC, and lysyl oxidase-like 2 (collectively termed TCL) in addition to lysophosphatidic acid (LPA). TCL + LPA treatment synergistically improved circumferential tensile stiffness and strength, significantly enhanced collagen and pyridinoline crosslink content per dry weight, and achieved tensile anisotropy (circumferential/radial) values of neomenisci close to 4. This study utilizes a combination of bioactive stimuli for use in tissue engineering studies, providing a promising path toward deploying these neomenisci as functional repair and replacement tissues. STATEMENT OF SIGNIFICANCE: This study utilizes a scaffold-free approach, which strays from the tissue engineering paradigm of using scaffolds with cells and bioactive factors to engineer neotissue. While self-assembled neomenisci have attained compressive properties akin to native tissue, tensile properties still require improvement before being able to deploy engineered neomenisci as functional tissue repair or replacement options. In order to augment tensile properties, this study utilized bioactive factors known to augment matrix content in combination with a soluble factor that enhances matrix organization and anisotropy via cell traction forces. Using a bioactive factor to enhance matrix organization mitigates the need for bioreactors used to apply mechanical stimuli or scaffolds to induce proper fiber alignment., 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 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Nasal cartilage pathologies are common; for example, up to 80% of people are afflicted by deviated nasal septum conditions. Because cartilage provides the supportive framework of the nose, afflicted patients suffer low quality of life. To correct pathologies, graft cartilage is often required. Grafts are currently sourced from the patient's septum, ear, or rib. However, their use yields donor site morbidity and is limited by tissue quantity and quality. Additionally, rhinoplasty revision rates exceed 15%, exacerbating the shortage of graft cartilage. Alternative grafts, such as irradiated allogeneic rib cartilage, are associated with complications. Tissue-engineered neocartilage holds promise to address the limitations of current grafts. The engineering design process may be used to create suitable graft tissues. This process begins by identifying the surgeon's needs. Second, nasal cartilages' properties must be understood to define engineering design criteria. Limited investigations have examined nasal cartilage properties; numerous additional studies need to be performed to examine topographical variations, for example. Third, tissue-engineering processes must be applied to achieve the engineering design criteria. Within the recent past, strategies have frequently utilized human septal chondrocytes. As autologous and allogeneic rib graft cartilage is used, its suitability as a cell source should also be examined. Fourth, quantitative verification of engineered neocartilage is critical to check for successful achievement of the engineering design criteria. Finally, following the FDA paradigm, engineered neocartilage must be orthotopically validated in animals. Together, these steps delineate a path to engineer functional nasal neocartilages that may, ultimately, be used to treat human patients. STATEMENT OF SIGNIFICANCE: Nasal cartilage pathologies are common and lead to greatly diminished quality of life. The ability to correct pathologies is limited by cartilage graft quality and quantity, as well as donor site morbidity and surgical complications, such as infection and resorption. Despite the significance of nasal cartilage pathologies and high rhinoplasty revision rates (15%), little characterization and tissue-engineering work has been performed compared to other cartilages, such as articular cartilage. Furthermore, most work is published in clinical journals, with little in biomedical engineering. Therefore, this review discusses what nasal cartilage properties are known, summarizes the current state of nasal cartilage tissue-engineering, and makes recommendations via the engineering design process toward engineering functional nasal neocartilage to address current limitations., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

It is crucial that the properties of engineered neocartilage match healthy native cartilage to promote the functional restoration of damaged cartilage. To accurately assess the quality of neocartilage and the degree of biomimicry achieved, its properties must be evaluated against native cartilage and tissue from which the cells for neocartilage formation were sourced. Fetal ovine cartilage is a promising and translationally relevant cell source with which to engineer neocartilage, yet, it is largely non-characterized. The influence of biomechanics during cartilage development, as well as their potential impact on structure-function relationships in utero motivates additional study of fetal cartilage. Toward providing tissue engineering design criteria and elucidating structure-function relationships, 11 locations across four regions of the fetal ovine stifle were characterized. Locational and regional differences were found to exist. Although differences in GAG content were observed, compressive stiffness did not vary or correlate with any biochemical component. Patellar cartilage tensile stiffness and strength were significantly greater than those of the medial condyle. Tensile modulus and UTS significantly correlated with pyridinoline content. More advanced zonal organization, more intense collagen II staining, and greater collagen and pyridinoline contents in the trochlear groove and patella suggest these regions exhibit a more advanced maturational state than others. Regional differences in functional properties and their correlations suggest that structure-function relationships emerge in utero. These data address the dearth of information of the fetal ovine stifle, may serve as a repository of information for cartilage engineering strategies, and may help elucidate functional adaptation in fetal articular cartilage. STATEMENT OF SIGNIFICANCE: Engineered neocartilage must be evaluated against healthy native cartilage and cell source tissue to determine its quality and degree of biomimicry. While fetal ovine cartilage has emerged as a promising and translationally relevant cell source with which to engineer neocartilage, it is largely non-characterized. Therefore, 11 locations across four regions (medial condyle, lateral condyle, trochlear groove, and patella) of the fetal ovine stifle were characterized. Importantly, locational and regional differences in functional properties were observed, and significant correlations of tensile properties to collagen and crosslink contents were detected, suggesting that functional adaptation begins in utero. This study provides a repository of quantitative information, clarifies the developmental order of cartilage functional properties, and informs future cartilage engineering efforts., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Advances in cartilage tissue engineering have led to constructs with mechanical integrity and biochemical composition increasingly resembling that of native tissues. In particular, collagen cross-linking with lysyl oxidase has been used to significantly enhance the mechanical properties of engineered neotissues. In this study, development of collagen cross-links over time, and correlations with tensile properties, were examined in self-assembling neotissues. Additionally, quantitative MRI metrics were examined in relation to construct mechanical properties as well as pyridinoline cross-link content and other engineered tissue components. Scaffold-free meniscus fibrocartilage was cultured in the presence of exogenous lysyl oxidase, and assessed at multiple time points over 8weeks starting from the first week of culture. Engineered constructs demonstrated a 9.9-fold increase in pyridinoline content, reaching 77% of native tissue values, after 8weeks of culture. Additionally, engineered tissues reached 66% of the Young's modulus in the radial direction of native tissues. Further, collagen cross-links were found to correlate with tensile properties, contributing 67% of the tensile strength of engineered neocartilages. Finally, examination of quantitative MRI metrics revealed several correlations with mechanical and biochemical properties of engineered constructs. This study displays the importance of culture duration for collagen cross-link formation, and demonstrates the potential of quantitative MRI in investigating properties of engineered cartilages., Statement of Significance: This is the first study to demonstrate near-native cross-link content in an engineered tissue, and the first study to quantify pyridinoline cross-link development over time in a self-assembling tissue. Additionally, this work shows the relative contributions of collagen and pyridinoline to the tensile properties of collagenous tissue for the first time. Furthermore, this is the first investigation to identify a relationship between qMRI metrics and the pyridinoline cross-link content of an engineered collagenous tissue., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The facet joint, a synovial joint located on the posterior-lateral spine, is highly susceptible to degenerative changes and plays a significant role in back-related morbidities. Despite its significance, the facet is rarely studied and thus current treatment strategies are lacking. This study aimed to characterize, for the first time, the properties of human, pig, monkey, and rabbit lumbar facet cartilage providing much-needed design criteria for tissue engineering approaches. In this study, where possible, the facet's morphological, histological, mechanical, and biochemical properties were evaluated. Comparisons between the properties of the inferior and superior facet surfaces, as well as among spinal levels were performed within each species. In addition, interspecies comparisons of the properties were determined. The human facet joint was found to be degenerated; 100% of joint surfaces showed signs of pathology and approximately 71% of these were considered to be grade 4. Joint morphology varied among species, demonstrating that despite the mini-pig facet being closest to the human in terms of width and length, it was far more curved than the human or any of the other species. No notable differences were found in the mini-pig, monkey, and rabbit mechanical and biochemical properties, suggesting that these species, despite morphological differences, may serve as suitable animal models for studying structure-function relationships of the human facet joint. The characterization data reported in this study may increase our understanding of this ill-described joint as well as provide the foundation for the development of new treatments such as tissue engineering., Statement of Significance: This work provides the first comprehensive description of the properties of lumbar facet joint cartilage. Importantly, this work establishes that histological, biochemical, and mechanical properties are comparable between bipedal and quadrupedal animals, helping to guide future selection of appropriate animal models. This work also suggests that the human facet joint is highly susceptible to pathology. The mechanical properties of facet cartilage, found to be inferior to those of other synovial joints, provide a greater understanding of the joint's structure-function relationships as well as the potential etiology of facet joint pathology. Lastly, this work will serve as the foundation for the development of much-needed facet joint treatments, especially those based on tissue engineering approaches., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Chondrocyte dedifferentiation presents a major barrier in engineering functional cartilage constructs. To mitigate the effects of dedifferentiation, this study employed a post-expansion aggregate culture step to enhance the chondrogenic phenotype of passaged articular chondrocytes (ACs) before their integration into self-assembled neocartilage constructs. The objective was twofold: (1) to explore how passage number (P2, P3, P4, P5, P6, and P7), with or without aggregate culture, affected construct properties; and (2) to determine the highest passage number that could form neocartilage with functional properties. Juvenile leporine ACs were passaged to P2-P7, with or without aggregate culture, and self-assembled into 5mm discs in non-adhesive agarose molds without using any exogenous scaffolds. Construct biochemical and biomechanical properties were assessed. With aggregate culture, neocartilage constructs had significantly higher collagen content, higher tensile properties, and flatter morphologies. These beneficial effects were most obvious at higher passage numbers. Specifically, collagen content, Young's modulus, and instantaneous compressive modulus in the P7, aggregate group were 53%, 116%, and 178% higher than those in the P7, non-aggregate group. Most interestingly, these extensively passaged P7 ACs (expansion factor of 85,000), which are typically highly dedifferentiated, were able to form constructs with properties similar to or higher than those formed by lower passage number cells. This study not only demonstrated that post-expansion aggregate culture could significantly improve the properties of self-assembled neocartilage, but also that chondrocytes of exceedingly high passage numbers, expanded using the methods in this study, could be used in cartilage engineering applications., Statement of Significance: This work demonstrated that extensively passaged chondrocytes (up to passage 7 (P7); expansion factor of 85,000) could potentially be used for cartilage tissue engineering applications. Specifically, an aggregate culture step, employed after cell expansion and before cell integration into a neocartilage construct, was shown to enhance the ability of the chondrocytes to form neocartilage with better biochemical and biomechanical properties. The beneficial effects of this aggregate culture step was especially noticeable at the high passage numbers. Most interestingly, P7 chondrocytes, which are typically highly dedifferentiated, were able to form neocartilage with properties similar to or higher than those formed by lower passage number cells. The ability to obtain high chondrocyte yields with an enhanced chondrogenic potential could have a broad, beneficial impact in improving current therapies (e.g., using higher cell seeding densities for repair) or developing new strategies that require high cell numbers, such as a scaffold-free approach in forming engineered cartilage., Competing Interests: The author declare that they have no conflict of interest., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The ability to repair damaged cartilage is a major goal of musculoskeletal tissue engineering. Allogeneic (same species, different individual) or xenogeneic (different species) sources can provide an attractive source of chondrocytes for cartilage tissue engineering, since autologous (same individual) cells are scarce. Immune rejection of non-autologous hyaline articular cartilage has seldom been considered due to the popular notion of "cartilage immunoprivilege". The objective of this study was to determine the suitability of allogeneic and xenogeneic engineered neocartilage tissue for cartilage repair. To address this, scaffold-free tissue engineered articular cartilage of syngeneic (same genetic background), allogeneic, and xenogeneic origin were implanted into two different locations of the rabbit knee (n=3 per group/location). Xenogeneic engineered cartilage and control xenogeneic chondral explants provoked profound innate inflammatory and adaptive cellular responses, regardless of transplant location. Cytological quantification of immune cells showed that, while allogeneic neocartilage elicited an immune response in the patella, negligible responses were observed when implanted into the trochlea; instead the responses were comparable to microfracture-treated empty defect controls. Allogeneic neocartilage survived within the trochlea implant site and demonstrated graft integration into the underlying bone. In conclusion, the knee joint cartilage does not represent an immune privileged site, strongly rejecting xenogeneic but not allogeneic chondrocytes in a location-dependent fashion. This difference in location-dependent survival of allogeneic tissue may be associated with proximity to the synovium., Statement of Significance: Through a series of in vivo studies this research demonstrates that articular cartilage is not fully immunoprivileged. In addition, we now show that anatomical location of the defect, even within the same joint compartment, strongly influences the degree of the resultant immune response. This is one of the first investigations to show that (1) immune tolerance to allogeneic tissue engineered cartilage and (2) subsequent implant survival are dependent on the implant location and proximity to the synovium., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A recent development in the field of tissue engineering is the rise of all-biologic, scaffold-free engineered tissues. Since these biomaterials rely primarily upon cells, investigation of initial seeding densities constitutes a particularly relevant aim for tissue engineers. In this study, a scaffold-free method was used to create fibrocartilage in the shape of the rabbit knee meniscus. The objectives of this study were to: (i) determine the minimum seeding density, normalized by an area of 44 mm(2), necessary for the self-assembling process of fibrocartilage to occur; (ii) examine relevant biomechanical properties of engineered fibrocartilage, such as tensile and compressive stiffness and strength, and their relationship to seeding density; and (iii) identify a reduced, or optimal, number of cells needed to produce this biomaterial. It was found that a decreased initial seeding density, normalized by the area of the construct, produced superior mechanical and biochemical properties. Collagen per wet weight, glycosaminoglycans per wet weight, tensile properties and compressive properties were all significantly greater in the 5 million cells per construct group as compared to the historical 20 million cells per construct group. Scanning electron microscopy demonstrated that a lower seeding density results in a denser tissue. Additionally, the translational potential of the self-assembling process for tissue engineering was improved though this investigation, as fewer cells may be used in the future. The results of this study underscore the potential for critical seeding densities to be investigated when researching scaffold-free engineered tissues., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The articulation of the temporomandibular joint (TMJ) is composed of the temporal bone dorsally, the mandibular condyle ventrally and a fibrous articular disc. The TMJ disc plays an essential role in distributing load between the two articular surfaces. Degeneration of the disc in the presence of joint pathology has been shown in man; however, TMJ pathology has not been documented previously in tigers (Panthera tigris). The mandibular condyle and TMJ disc of a Bengal tiger (P. tigris tigris) and a Siberian tiger (P. tigris altaica) were evaluated grossly and the TMJ disc was characterized biochemically and mechanically. Characterization of the TMJ disc verified region- and direction-dependent biochemical and mechanical properties, reflective of the functional demands on the joint. Degenerative joint disease was observed in both cases and this was more severe in the Siberian tiger. Simultaneous evaluation of joint pathology, biochemical composition and mechanical properties of the TMJ disc revealed a loss in functional properties (tensile anisotropy) of the disc as joint pathology advanced from moderate to severe. TMJ degeneration may compromise the ability of the animal to eat and thrive and may be a factor contributing to the endangered status of these species., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The ability of residual antigens on decellularized tissue to elicit the immune response upon implantation motivates development of a more rigorous antigen removal (AR) process for xenogeneic scaffold generation. Antigen removal strategies promoting solubilization of hydrophilic proteins (predominantly cytoplasmic) enhance the reduction of hydrophilic antigenicity in bovine pericardium (BP); however, the diversity of protein antigens within a tissue necessitates development of AR strategies capable of addressing a spectrum of protein antigen solubilities. In the present study, methods for promoting solubilization of lipophilic proteins (predominantly membrane) were investigated for their ability to reduce lipophilic antigenicity of BP when applied as a second AR step following our previously described hydrophilic AR method. Bovine pericardium following AR (BP-AR) was assessed for residual hydrophilic and lipophilic antigenicity, removal of known lipophilic xenoantigens, tensile properties, and extracellular matrix structure and composition. Facilitating hydrophile solubilization (using dithiothreitol and potassium chloride) followed by lipophile solubilization (using amidosulfobetaine-14 (ASB-14)), in a two-step sequential, differential AR strategy, significantly reduces residual hydrophilic and lipophilic antigenicity of BP-AR beyond that achieved with either one-step hydrophilic AR or decellularization using 1% (w/v) sodium dodecyl sulfate. Moreover, use of 1% (w/v) ASB-14 for lipophilic AR eliminates the two most critical known barriers to xenotransplantation (galactose-α(1,3)-galactose and major histocompatibility complex I)) from BP-AR without compromising the structure-function properties of the biomaterial. This study demonstrates the importance of a sequential, differential protein solubilization approach to reduce biomaterial antigenicity in the production of a xenogeneic scaffold for heart valve tissue engineering., (Published by Elsevier Ltd.)

Knee meniscus fibrocartilage is frequently injured, resulting in approximately 1 million procedures annually in the US and Europe. Its near-avascularity contributes heavily to its inability to heal, and places it as a prime candidate for replacement through regenerative medicine. Here, we describe a novel approach to increase extracellular matrix organization, rather than content, in order to augment the mechanical properties of engineered tissue. To synthesize fibrocartilage, we employ a self-assembling process, which is free of exogenous scaffolds and relies on cell-to-cell interactions to form all-biologic constructs. When treated with the signaling phospholipid lysophosphatidic acid (LPA), tissue constructs displayed increased tensile properties and collagen organization, while total collagen content remained unchanged. LPA-treated constructs exhibited greater DNA content, indicative that the molecule exerted a signaling effect. Furthermore, LPA-treated cells displayed significant cytoskeletal reorganization. We conclude that LPA induced cytoskeletal reorganization and cell-matrix traction, which resulted in matrix reorganization and increased tensile properties. This study emphasizes the potential of non-traditional stimuli, such as signaling phospholipids, for use in tissue development studies. The extension of these results to other collagen-rich tissues represents a promising avenue for future exploration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A persistent hurdle in the field of tissue regeneration is to produce tissues with biochemical and biomechanical properties robust enough to meet the aggressive physiological demands of the native milieu. In an effort to improve these properties tissues grown in vitro are often subjected to mechanical stimuli that aim to recapitulate the in vivo physiology. These mechanical stimuli are thought to produce downstream alterations in intracellular ion concentrations, which ultimately give rise to increased biosynthesis. There is mounting evidence that these perturbations in the cellular microenvironment are regulated by the Ca(2+)-permeable transient receptor potential vanilloid 4 (TRPV4) channel. In this study we examined the effects of targeted TRPV4 activation on self-assembled articular cartilage constructs. The objectives of this study were: (i) to determine whether TRPV4 activation would enhance self-assembled constructs; (ii) to identify an optimal treatment time window for TRPV4 activation; and (iii) to compare TRPV4 activation which Na(+)/K(+) pump inhibition, which has previously been shown to improve the construct tensile properties. This study employed a two phase approach. In Phase I self-assembled constructs were grown for 4weeks and subjected to treatment with the TRPV4 agonist 4α-phorbol-12,13-didecanoate (4α-PDD) during three treatment time windows: t=6-10, t=10-14, and t=14-18days. Treatment for t=10-14days produced an 88% increase in collagen and a 153% increase in tensile stiffness. This treatment window was carried forward to Phase II. In Phase II we performed a head to head comparison between TRPV4 activation using 4α-PDD and Na(+)/K(+) pump inhibition using ouabain. Treatment with 4α-PDD produced improvements on a par with ouabain (91-107% increases in tensile stiffness). The results of this study demonstrate the effectiveness of ion channel modulation as a strategy for improving engineered tissues. To our knowledge this is the first study to examine TRPV4 channel activation in tissue engineering., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The development of functionally equivalent fibrocartilage remains elusive despite efforts to engineer tissues such as knee meniscus, intervertebral disc and temporomandibular joint disc. Attempts to engineer these structures often fail to create tissues with mechanical properties on a par with native tissue, resulting in constructs unsuitable for clinical applications. The objective of this study was to engineer a spectrum of biomimetic fibrocartilages representative of the distinct functional properties found in native tissues. Using the self-assembly process, different co-cultures of meniscus cells and articular chondrocytes were seeded into agarose wells and treated with the catabolic agent chondroitinase-ABC (C-ABC) and the anabolic agent transforming growth factor-β1 (TGF-β1) via a two-factor (cell ratio and bioactive treatment), full factorial study design. Application of both C-ABC and TGF-β1 resulted in a beneficial or positive increase in the collagen content of treated constructs compared to controls. Significant increases in both the collagen density and fiber diameter were also seen with this treatment, increasing these values by 32 and 15%, respectively, over control values. Mechanical testing found the combined bioactive treatment to synergistically increase the Young's modulus and ultimate tensile strength of the engineered fibrocartilages compared to controls, with values reaching the lower spectrum of those found in native tissues. Together, these data demonstrate that C-ABC and TGF-β1 interact to develop a denser collagen matrix better able to withstand tensile loading. This study highlights a way to optimize the tensile properties of engineered fibrocartilage using a biochemical and a biophysical agent together to create distinct fibrocartilages with functional properties mimicking those of native tissue., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Articular cartilage cannot repair itself in response to degradation from injury or osteoarthritis. As such, there is a substantial clinical need for replacements of damaged cartilage. Tissue engineering aims to fulfill this need by developing replacement tissues in vitro. A major goal of cartilage tissue engineering is to produce tissues with robust biochemical and biomechanical properties. One technique that has been proposed to improve these properties in engineered tissue is the use of non-enzymatic glycation to induce collagen crosslinking, an attractive solution that may avoid the risks of cytotoxicity posed by conventional crosslinking agents such as glutaraldehyde. The objectives of this study were (1) to determine whether continuous application of ribose would enhance biochemical and biomechanical properties of self-assembled articular cartilage constructs, and (2) to identify an optimal time window for continuous ribose treatment. Self-assembled constructs were grown for 4 weeks using a previously established method and were subjected to continuous 7-day treatment with 30 mM ribose during culture weeks 1, 2, 3, or 4, or for the entire 4-week culture. Control constructs were grown in parallel, and all groups were evaluated for gross morphology, histology, cellularity, collagen and sulfated glycosaminoglycan (GAG) content, and compressive and tensile mechanical properties. Compared to control constructs, it was found that treatment with ribose during week 2 and for the entire duration of culture resulted in significant 62% and 40% increases in compressive stiffness, respectively; significant 66% and 44% increases in tensile stiffness; and significant 50% and 126% increases in tensile strength. Similar statistically significant trends were observed for collagen and GAG. In contrast, constructs treated with ribose during week 1 had poorer biochemical and biomechanical properties, although they were significantly larger and more cellular than all other groups. We conclude that non-enzymatic glycation with ribose is an effective method for improving tissue engineered cartilage and that specific temporal intervention windows exist to achieve optimal functional properties., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-β1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790 kPa), relaxed compressive moduli (120 kPa) and Young's moduli (1.87 MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-β1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young's modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-β1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Tissue donor scarcity is a major hindrance to articular cartilage tissue engineering. Previous research shows that dermal fibroblasts express chondrocytic markers after seeding on aggrecan-coated surfaces. Since cell roundness appears to correlate with chondrocytic behavior of dermal fibroblasts, this study quantified roundness by measuring cell height and surface area-volume ratio. In addition to aggrecan as a surface coating, collagen type II and decorin, two other major extracellular matrix components of articular cartilage, were examined. Aggrecan, collagen type II, and decorin were coated onto a glass substrate using three application techniques: static drying, airbrush, and painting. Vertical scanning interferometry (VSI) is a novel technique that allows for the expedient morphological determination of single cells. Interferometry was used for the characterization of protein-coated surfaces in addition to characterizing the morphology of single dermal fibroblasts after 24 h of seeding. Fibroblast height was found to vary from 1.0 to 4.0 microm and protein coating, application technique, and seeding position were significant factors (p < 0.002). The largest cell heights were observed on aggrecan and collagen type II coated surfaces using the air brush and static applications. Additionally, variations were observed for surface area-volume ratio, ranging from 1.75 to 11.94 microm(-1) with decorin resulting in the lowest ratio, followed by collagen type II and aggrecan. This study identifies optimal coating conditions for stimulating morphology in dermal fibroblasts that is characteristic of the chondrocytic phenotype. These conditions can be employed to attempt articular cartilage regeneration and bypass difficulties due to a paucity of donor tissue.

Characterization of the extracellular matrix of the temporomandibular joint (TMJ) disc is crucial to advancing efforts in tissue engineering the disc. However, the current literature is incomplete and often contradictory in its attempts to describe the nature of the TMJ disc matrix. The aim of this study was to identify the variation of key matrix components along the three axes of the porcine disc using ELISAs to quantify these matrix components, immunohistochemistry to identify their regional distribution, and SEM to examine collagen fiber diameter and orientation. The overall GAG content of the TMJ disc (including the dermatan sulfate proteoglycans) was 5.3+/-1.2% of the dry weight. Chondroitin sulfate, which comprised 74% of this total GAG content, was 4.4, 8.2, and 164 times more abundant than dermatan sulfate proteoglycan, keratan sulfate, and hyaluronic acid, respectively. In general, these GAGs were most concentrated in the intermediate zone of the TMJ disc, appearing in dense clusters, and least concentrated in the posterior band. Additionally, chondroitin sulfate was more abundant medially than laterally. Collagen II was discovered in trace amounts, with higher relative amounts in the intermediate zone. Collagen fibers were observed to run primarily in a ring-like fashion around the periphery of the disc and anteroposteriorly through the intermediate zone, with a mean fiber diameter of 18+/-9 mum. Characterization studies of the TMJ disc, including prior biomechanical and cell studies along with the current study of the extracellular matrix, collectively reveal a distinct character of the intermediate zone of the disc compared to its anterior and posterior bands.

As the ability to understand the peculiarities of successful healing of articular cartilage defects moves forward, it becomes clear that this complex orthopaedic problem soon will be successfully addressed. A multidisciplinary approach, combining clinical experience, cogent biomaterial designs, new cell biologic processes, biomechanical assessment, and modern molecular biology, clearly is leading toward clinically acceptable, viable, and consistent articular cartilage regeneration.