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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

The temporomandibular joint (TMJ) disc complex (i.e., the TMJ disc and its six attachments) is crucial to everyday functions such as mastication and speaking. The TMJ can be afflicted by many conditions, including disc displacement and defects. Pathologies of the TMJ disc complex most commonly present first as anterior disc displacement, which the field hypothesizes may implicate the two posterior attachments. As a result of anterior disc displacement, defects may develop in the lateral disc complex. Tissue engineering is poised to improve treatment paradigms for these indications of the TMJ disc complex by engineering biomimetic implants, but, first, gold-standard design criteria for such implants should be established through characterization studies. This study's objective was to characterize the structural, mechanical, biochemical, and crosslinking differences among the two posterior attachments and the lateral disc in the Yucatan minipig, a well-accepted TMJ animal model. In tension, it was found that the posterior inferior attachment (PIA) was significantly stiffer and stronger by 2.13 and 2.30 times, respectively, than the posterior superior attachment (PSA). It was found that collagen in both attachments was primarily aligned mediolaterally; however, the lateral disc was much more aligned and anisotropic than either attachment. Among the three locations, the PSA exhibited the greatest degree of heterogeneity and highest proportion of fat vacuoles. The PIA and lateral disc were 1.93 and 1.91 times more collagenous, respectively, by dry weight (DW) than the PSA. The PIA also exhibited 1.78 times higher crosslinking per DW than the PSA. Glycosaminoglycan per DW was significantly higher in the lateral disc by 1.48 and 5.39 times than the PIA and PSA, respectively. Together, these results establish design criteria for tissue-engineering of the TMJ disc complex and indicate that the attachments are less fibrocartilaginous than the disc, while still significantly contributing to the mechanical stability of the TMJ disc complex during articulation. These results also support the biomechanical function of the PIA and PSA, suggesting that the stiffer PIA anchors the disc to the mandibular condyle during articulation, while the softer PSA serves to allow translation over the articular eminence. Impact Statement Characterization of the temporomandibular joint (TMJ) disc complex (i.e., the disc and its attachments) has important implications for those aiming to tissue-engineer functional replacements and can help elucidate its biomechanical function. For example, the findings shown here suggest that the stiffer posterior inferior attachment anchors the disc during articulation, while the softer posterior superior attachment allows translation over the articular eminence.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe, dysregulated inflammation driven by the inability of T cells to clear an antigenic target. When associated with malignancy (mHLH), the HLH syndrome is typically associated with extremely poor survival. Here, we review the diagnosis of secondary HLH (sHLH) syndromes in adults, with emphasis on the appropriate workup and treatment of mHLH. At present, the management of HLH in adults, including most forms of mHLH, is based on the use of corticosteroids and etoposide following the HLH-94 regimen. In some cases, this therapeutic approach may be cohesively incorporated into malignancy-directed therapy, while in other cases, the decision about whether to treat HLH prior to initiating other therapies may be more complicated. Recent studies exploring the efficacy of other agents in HLH, in particular ruxolitinib, offer hope for better outcomes in the management of mHLH. Considerations for the management of lymphoma-associated mHLH, as well as other forms of mHLH and immunotherapy treatment-related HLH, are discussed.

BackgroundThe self-assembling process of cartilage tissue engineering is a promising technique to heal cartilage defects, preventing osteoarthritic changes. Given that chondrocytes dedifferentiate when expanded, it is not known if cellular expansion affects the development of self-assembled neocartilage. The objective of this study was to use proteomic, mechanical, and biochemical analyses to quantitatively investigate the development of self-assembled neocartilage derived from passaged, rejuvenated costal chondrocytes.MethodsYucatan minipig costal chondrocytes were used to create self-assembled neocartilage constructs. After 1, 4, 7, 14, 28, 56, or 84 days of self-assembly, constructs were analyzed through a variety of histological, biomechanical, biochemical, and proteomic techniques.ResultsIt was found that temporal trends in neocartilage formation are similar to those seen in native hyaline articular cartilage development. For example, between days 7 and 84 of culture, tensile Young's modulus increased 4.4-times, total collagen increased 2.7-times, DNA content decreased 69.3%, collagen type II increased 1.5-times, and aggrecan dropped 55.3%, mirroring trends shown in native knee cartilage. Importantly, collagen type X, which is associated with cartilage calcification, remained at low levels (≤ 0.05%) at all neocartilage developmental time points, similar to knee cartilage (

Facet joint arthrosis causes pain in approximately 7 % of the U.S. population, but current treatments are palliative. The objective of this study was to elucidate structure-function relationships and aid in the development of future treatments for the facet joint. This study characterized the articular surfaces of cervical, thoracic, and lumbar facet cartilage from skeletally mature (18-24 mo) Yucatan minipigs. The minipig was selected as the animal model because it is recognized by the U.S. Food and Drug Administration (FDA) and the American Society for Testing and Materials (ASTM) as a translationally relevant model for spine-related indications. It was found that the thoracic facets had a ∼2 times higher aspect ratio than lumbar and cervical facets. Lumbar facets had 6.9-9.6 times higher % depth than the cervical and thoracic facets. Aggregate modulus values ranged from 135 to 262 kPa, much lower than reported aggregate modulus in the human knee (reported to be 530-701 kPa). The tensile Young's modulus values ranged from 6.7 to 20.3 MPa, with the lumbar superior facet being 304 % and 286 % higher than the cervical inferior and thoracic superior facets, respectively. Moreover, 3D reconstructions of entire vertebral segments were generated. The results of this study imply that structure-function relationships in the facet cartilage are different from other joint cartilages because biochemical properties are analogous to other articular cartilage sources whereas mechanical properties are not. By providing functional properties and a 3D database of minipig facet geometries, this work may supply design criteria for future facet tissue engineering efforts.

Long-term clinical repair of articular cartilage remains elusive despite advances in cartilage tissue engineering. Only one cartilage repair therapy classified as a "cellular and gene therapy product" has obtained Food and Drug Administration (FDA) approval within the past decade although more than 200 large animal cartilage repair studies were published. Here, we identify the challenges impeding translation of strategies and technologies for cell-based cartilage repair, such as the disconnect between university funding and regulatory requirements. Understanding the barriers to translation and developing solutions to address them will be critical for advancing cell therapy products for cartilage repair to clinical use.

Ion signaling through Ca2+ and Na+ plays a key role in mechanotransduction and encourages a chondrogenic phenotype and tissue maturation. In this study, we propose that the pleiotropic effects of Ca2+ and Na+ modulation can be used to induce maturation and improvement of neocartilage derived from redifferentiated expanded chondrocytes from minipig rib cartilage. Three ion modulators were employed: (1) 4α-phorbol-12,13-didecanoate (4-αPDD), an agonist of the Ca2+-permeable transient receptor potential vanilloid 4 (TRPV4), (2) ouabain, an inhibitor of the Na+/K+ pump, and (3) ionomycin, a Ca2+ ionophore. These ion modulators were used individually or in combination. While no beneficial effects were observed when using combinations of the ion modulators, single treatment of constructs with the three ion modulators resulted in multiple effects in structure-function relationships. The most significant findings were related to ionomycin. Treatment of neocartilage with ionomycin produced 61% and 115% increases in glycosaminoglycan and pyridinoline crosslink content, respectively, compared with the control. Moreover, treatment with this Ca2+ ionophore resulted in a 45% increase of the aggregate modulus, and a 63% increase in the tensile Young's modulus, resulting in aggregate and Young's moduli of 567 kPa and 8.43 MPa, respectively. These results support the use of ion modulation to develop biomimetic neocartilage using expanded redifferentiated costal chondrocytes. Impact Statement New cost-effective, replicable, and highly controllable strategies are required to develop neocartilage with biomimetic properties akin to native tissue. Ion signaling plays a key role in mechanotransduction, promoting chondrogenic phenotype. Using rib cartilage, we proposed that Ca2+ and Na+ modulation could be used to induce maturation of neotissue derived from redifferentiated, expanded costal chondrocytes, improving its mechanical properties. Our results indicate that Ca2+ modulation with ionomycin, which stimulated extracellular matrix deposition and collagen crosslinking, improved morphological and mechanical features of neocartilage constructs, and holds potential as a powerful tool to engineer hyaline-like tissues.

IntroductionAlongside the improved survival of nasopharyngeal cancer (NPC), late radiation toxicities are alarmingly hampering survivors’ quality of life. A patient-reported symptom burden survey is lacking to address the unmet need for symptom management among local NPC survivors.MethodsA single-center cross-sectional survey was conducted on 211 NPC survivors who had completed radiation therapy for three to 120 months. We employed the Chinese version M. D. Anderson Symptom Inventory - Head & Neck Module (MDASI-HN-C), Functional Assessment of Cancer Therapy - Head & Neck (FACT-HN-C), and a question extracted from the Cancer Survivors’ Unmet Needs Measure (CaSUN).ResultsTwo hundred valid responses were collected. Participants suffered from at least four moderate to severe symptoms (mean = 4.84, SD = 4.99). The top five severe symptoms were dry mouth, mucus problems, difficulty swallowing or chewing, teeth or gum problems, and memory problems. MDASI-HN-C subscales were negatively correlated with the physical, emotional, functional, and HN-specific domains of the FACT-HN-C. The unmet need for symptom management was positively associated with symptom burden, either general symptoms (Adjusted odds ratio [ORadj] = 1.566, 95% CI = 1.282 – 1.914, p < 0.001) or top-5 symptoms (ORadj = 1.379, 95% CI = 1.185 – 1.604, p < 0.001), while negatively associated with post-RT time (ORadj = 0.981, 95% CI [0.972, 0.991], p < 0.001).ConclusionVirtually all NPC survivors suffer from late toxicities, which interplay with survivors’ perceptions intricately to affect their unmet needs for symptom management. Personalized supportive care strategies with regular assessments and stratifications are warranted.

Cartilage does not naturally heal, and cartilage lesions from trauma and wear-and-tear can lead to eventual osteoarthritis. To address long-term repair, tissue engineering of functional biologic implants to treat cartilage lesions is desirable, but the development of such implants is hindered by several limitations, including (1) donor tissue scarcity due to the presence of diseased tissues in joints, (2) dedifferentiation of chondrocytes during expansion, and (3) differences in functional output of cells dependent on donor age. Toward overcoming these challenges, (1) costal cartilage has been explored as a donor tissue, and (2) methods have been developed to rejuvenate the chondrogenic phenotype of passaged chondrocytes for generating self-assembled neocartilage. However, it remains unclear how the rejuvenation processes are influenced by donor age and, thus, how to develop strategies that specifically target age-related differences. Using histological, biochemical, proteomic, and mechanical assays, this study sought to determine the differences among neocartilage generated from neonatal, juvenile, and adult donors using the Yucatan minipig, a clinically relevant large animal model. Based on the literature, a relatively young adult population of animals was chosen due to a reduction in functional output of human articular chondrocytes after 40 years of age. After isolation, costal chondrocytes were expanded, rejuvenated, and self-assembled, and the neocartilages were assessed. The aggregate modulus values of neonatal constructs were at least 1.65-fold of those from the juvenile or adult constructs. Poisson's ratio also significantly differed among all groups, with neonatal constructs exhibiting values 49% higher than adult constructs. Surprisingly, other functional properties such as tensile modulus and glycosaminoglycan content did not significantly differ among groups. Total collagen content was slightly elevated in the adult constructs compared to neonatal and juvenile constructs. A more nuanced view using bottom-up mass spectrometry showed that Col2a1 protein was not significantly different among groups, but protein content of several other collagen subtypes (i.e., Col1a1, Col9a1, Col11a2, and Col12a1) was modulated by donor age. For example, Col12a1 protein content in adult constructs was found to be 102.9% higher than neonatal-derived constructs. Despite these differences, this study shows that different aged donors can be used to generate neocartilages of similar functional properties. Impact statement Tissue-engineered neocartilage can be generated with functional properties that mimic native cartilage tissue. However, cell sourcing challenges hinder clinical translation of tissue-engineered cartilage. Chondrocytes can be expanded and rejuvenated for the generation of functional self-assembled cartilage, making an allogeneic approach feasible. However, it is currently unclear if donor age impacts functional properties. In this study, using the Yucatan minipig as a clinically relevant large animal model, we demonstrate that functional properties of self-assembled neocartilage are relatively consistent regardless of donor age, suggesting that a wider range of donor ages may be used for cartilage tissue engineering than previously expected.

Neocartilage tissue engineering aims to address the shortcomings of current clinical treatments for articular cartilage indications. However, advancement is required toward neocartilage functionality (mechanical and biochemical properties) and translatability (construct size, gross morphology, passage number, cell source, and cell type). Using fluid-induced shear (FIS) stress, a potent mechanical stimulus, over four phases, this work investigates FIS stress' efficacy toward creating large neocartilage derived from highly passaged minipig costal chondrocytes, a species relevant to the preclinical regulatory process. In Phase I, FIS stress application timing was investigated in bovine articular chondrocytes and found to improve the aggregate modulus of neocartilage by 151% over unstimulated controls when stimulated during the maturation stage. In Phase II, FIS stress stimulation was translated from bovine articular chondrocytes to expanded minipig costal chondrocytes, yielding a 46% improvement in aggregate modulus over nonstimulated controls. In Phase III, bioactive factors were combined with FIS stress to improve the shear modulus by 115% over bioactive factor-only controls. The translatability of neocartilage was improved in Phase IV by utilizing highly passaged cells to form constructs more than 9-times larger in the area (11 × 17 mm), yielding an improved aggregate modulus by 134% and a flat morphology compared to free-floating, bioactive factor-only controls. Overall, this study represents a significant step toward generating mechanically robust, large constructs necessary for animal studies, and eventually, human clinical studies.

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.

Knee meniscus injuries are the most frequent causes of orthopedic surgical procedures in the U.S., motivating tissue engineering attempts and the need for suitable animal models. Despite extensive use in cardiovascular research and the existence of characterization data for the menisci of farm pigs, the farm pig may not be a desirable preclinical model for the meniscus due to rapid weight gain. Minipigs are conducive to in vivo experiments due to their slower growth rate than farm pigs and similarity in weight to humans. However, characterization of minipig knee menisci is lacking. The objective of this study was to extensively characterize structural and functional properties within different regions of both medial and lateral Yucatan minipig knee menisci to inform this model's suitability as a preclinical model for meniscal therapies. Menisci measured 23.2-24.8 mm in anteroposterior length (33-40 mm for human), 7.7-11.4 mm in width (8.3-14.8 mm for human), and 6.4-8.4 mm in peripheral height (5-7 mm for human). Per wet weight, biochemical evaluation revealed 23.9-31.3% collagen (COL; 22% for human) and 1.20-2.57% glycosaminoglycans (GAG; 0.8% for human). Also, per dry weight, pyridinoline crosslinks (PYR) were 0.12-0.16% (0.12% for human) and, when normalized to collagen content, reached as high as 1.45-1.96 ng/µg. Biomechanical testing revealed circumferential Young's modulus of 78.4-116.2 MPa (100-300 MPa for human), circumferential ultimate tensile strength (UTS) of 18.2-25.9 MPa (12-18 MPa for human), radial Young's modulus of 2.5-10.9 MPa (10-30 MPa for human), radial UTS of 2.5-4.2 MPa (1-4 MPa for human), aggregate modulus of 157-287 kPa (100-150 kPa for human), and shear modulus of 91-147 kPa (120 kPa for human). Anisotropy indices ranged from 11.2-49.4 and 6.3-11.2 for tensile stiffness and strength (approximately 10 for human), respectively. Regional differences in mechanical and biochemical properties within the minipig medial meniscus were observed; specifically, GAG, PYR, PYR/COL, radial stiffness, and Young's modulus anisotropy varied by region. The posterior region of the medial meniscus exhibited the lowest radial stiffness, which is also seen in humans and corresponds to the most prevalent location for meniscal lesions. Overall, similarities between minipig and human menisci support the use of minipigs for meniscus translational research.

ObjectiveTissue-engineered cartilage implants must withstand the potential inflammatory and joint loading environment for successful long-term repair of defects. The work's objectives were to develop a novel, direct cartilage-macrophage co-culture system and to characterize interactions between self-assembled neocartilage and differentially stimulated macrophages.DesignIn study 1, it was hypothesized that the proinflammatory response of macrophages would intensify with increasing construct stiffness; it was expected that the neocartilage would display a decrease in mechanical properties after co-culture. In study 2, it was hypothesized that bioactive factors would protect neocartilage properties during macrophage co-culture. Also, it was hypothesized that interleukin 10 (IL-10)-stimulated macrophages would improve neocartilage mechanical properties compared to lipopolysaccharide (LPS)-stimulated macrophages.ResultsAs hypothesized, stiffer neocartilage elicited a heightened proinflammatory macrophage response, increasing tumor necrosis factor alpha (TNF-α) secretion by 5.47 times when LPS-stimulated compared to construct-only controls. Interestingly, this response did not adversely affect construct properties for the stiffest neocartilage but did correspond to a significant decrease in aggregate modulus for soft and medium stiffness constructs. In addition, bioactive factor-treated constructs were protected from macrophage challenge compared to chondrogenic medium-treated constructs, but IL-10 did not improve neocartilage properties, although stiff constructs appeared to bolster the anti-inflammatory nature of IL-10-stimulated macrophages. However, co-culture of bioactive factor-treated constructs with LPS-treated macrophages reduced TNF-α secretion by over 4 times compared to macrophage-only controls.ConclusionsIn conclusion, neocartilage stiffness can mediate macrophage behavior, but stiffness and bioactive factors prevent macrophage-induced degradation. Ultimately, this co-culture system could be utilized for additional studies to develop the burgeoning field of cartilage mechano-immunology.

Regulatory guidelines for tissue engineered products require stringent characterization during production and necessitate the development of novel, non-destructive methods to quantify key functional parameters for clinical translation. Traditional assessments of engineered tissues are destructive, expensive, and time consuming. Here, we introduce a non-destructive, inexpensive, and rapid sampling and analysis system that can continuously monitor the mechanical, biochemical, and structural properties of a single sample over extended periods of time. The label-free system combines the imaging modalities of fluorescent lifetime imaging and ultrasound backscatter microscopy through a fiber-based interface for sterile monitoring of tissue quality. We tested the multimodal system using tissue engineered articular cartilage as an experimental model. We identified strong correlations between optical and destructive testing. Combining FLIm and UBM results, we created a novel statistical model of tissue homogeneity that can be applied to tissue engineered constructs prior to implantation. Continuous monitoring of engineered tissues with this non-destructive system has the potential for in-process monitoring of tissue engineered products, reducing costs and improving quality controls in research, manufacturing, and clinical applications.

Physiological loading of knee cartilage is highly dynamic and may contribute to the progression of osteoarthritis. Thus, an understanding of cartilage's dynamic mechanical properties is crucial in cartilage research. In this study, vibrometry was used as a fast (2 h), noncontact and novel alternative to the slower (30 h), traditional mechanical and biochemical assays for characterization of cartilage from the condyle, patella, trochlear groove and meniscus. Finite-element models predicted tissue resonant frequencies and bending modes, which strongly correlated with experiments (R2 = 0.93). Vibrometry-based viscoelastic properties significantly correlated with moduli from stress relaxation and creep tests, with correlation strengths reaching up to 0.78. Loss modulus also strongly correlated with glycosoaminoglycan (GAG) content. Dynamic properties measured by vibrometry significantly differed among various knee cartilages, ranging between 6.1 and 56.4 MPa. Interestingly, meniscus viscoelastic properties suggest that contrary to common belief, it may lack shock absorption abilities; instead, condylar hyaline cartilage may be a better shock absorber. These data demonstrate for the first time that vibrometry is a noncontact approach to dynamic mechanical characterization of hyaline and fibrocartilage cartilage with concrete relationships to standard quasi-static mechanical testing and biochemical composition. Thus, with a single tool, vibrometry greatly facilitates meeting multiple regulatory recommendations for mechanical characterization of cartilage replacements.

IntroductionThis study develops assays to quantify collagen subtypes and crosslinks with liquid chromatography-mass spectrometry (LC-MS) and characterizes the cartilages in the Yucatan minipig.MethodsFor collagen subtyping, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on tissues digested in trypsin. For collagen crosslinks, LC-MS analysis was performed on hydrolysates. Samples were also examined histologically and with bottom-up proteomics. Ten cartilages (femoral condyle, femoral head, facet joint, floating rib, true rib, auricular cartilage, annulus fibrosus, 2 meniscus locations, and temporomandibular joint disc) were analyzed.ResultsThe collagen subtyping assay quantified collagen types I and II. The collagen crosslinks assay quantified mature and immature crosslinks. Collagen subtyping revealed that collagen type I predominates in fibrocartilages and collagen type II in hyaline cartilages, as expected. Elastic cartilage and fibrocartilages had more mature collagen crosslink profiles than hyaline cartilages. Bottom-up proteomics revealed a spectrum of ratios between collagen types I and II, and quantified 42 proteins, including 24 collagen alpha-chains and 12 minor collagen types.DiscussionThe novel assays developed in this work are sensitive, inexpensive, and use a low operator time relative to other collagen analysis methods. Unlike the current collagen assays, these assays quantify collagen subtypes and crosslinks without an antibody-based approach or lengthy chromatography. They apply to any collagenous tissue, with broad applications in tissue characterization and tissue engineering. For example, a novel finding of this work was the presence of a large quantity of collagen type III in the white-white knee meniscus and a spectrum of hyaline and fibrous cartilages.

ObjectiveTo enhance the in vitro integration of self-assembled articular cartilage to native articular cartilage using chondroitinase ABC.DesignTo examine the hypothesis that chondroitinase ABC (C-ABC) integration treatment (C-ABCint) would enhance integration of neocartilage of different maturity levels, this study was conducted in 2 phases. In phase I, the impact on integration of 2 treatments, TCL (TGF-β1, C-ABC, and lysyl oxidase like 2) and C-ABCint, was examined via a 2-factor, full factorial design. In phase II, construct maturity (2 levels) and C-ABCint concentration (3 levels) were the factors in a full factorial design to determine whether the effective C-ABCint dose was dependent on neocartilage maturity level. Neocartilages formed or treated per the factors above were placed into native cartilage rings, cultured for 2 weeks, and, then, integration was studied histologically and mechanically. Prior to integration, in phase II, a set of treated constructs were also assayed to provide a baseline of properties.ResultsIn phase I, C-ABCint and TCL treatments synergistically enhanced interface Young's modulus by 6.2-fold (P = 0.004) and increased interface tensile strength by 3.8-fold (P = 0.02) compared with control. In phase II, the interaction of the factors C-ABCint and construct maturity was significant (P = 0.0004), indicating that the effective C-ABCint dose to improve interface Young's modulus is dependent on construct maturity. Construct mechanical properties were preserved regardless of C-ABCint dose.ConclusionsApplying C-ABCint to neocartilage is an effective integration strategy with translational potential, provided its dose is calibrated appropriately based on implant maturity, that also preserves implant biomechanical properties.

ObjectivePathology of the facet and sacroiliac (SI) joints contributes to 15% to 45% and 10% to 27% of lower back pain cases, respectively. Although tissue engineering may offer novel treatment options to patients suffering from cartilage degeneration in these joints, the tribological characteristics of the facet and SI joints have not been studied in either the human or relevant large animal models, which hinders the development of joint-specific cartilage implants.DesignCartilage was isolated from the knee, cervical facet, thoracic facet, lumbar facet, and SI joints of 6 skeletally mature Yucatan minipigs (Sus scrofa). Tribological characteristics were assessed via coefficient of friction testing, interferometry, and immunohistochemistry for lubricin organization.ResultsCompared with the knee, the coefficient of friction was higher by 43% in the cervical facet, 77% in the thoracic facet, 37% in the lumbar facet, and 28% in the SI joint. Likewise, topographical features of the facet and SI joints varied significantly, ranging from a 114% to 384% increase and a 48% to 107% increase in global and local surface roughness measures, respectively, compared with the knee. Additionally, the amount of lubricin in the SI joint was substantially greater than in the knee. Statistical correlations among the various tribological parameters revealed that there was a significant correlation between local roughness and coefficient of friction, but not global roughness or the presence of lubricin.ConclusionThese location-specific tribological characteristics of the articular cartilages of the spine will need to be taken into consideration during the development of physiologically relevant, functional, and durable tissue-engineered replacements for these joints.

Evaluating the host immune response to biomaterials is an essential step in the development of medical devices and tissue engineering strategies. To aid in this process, in vitro studies, whereby immune cells such as macrophages are cultured on biomaterials, can often expedite high throughput testing of many materials prior to implantation. While most studies to date utilize murine or human cells, the use of porcine macrophages has been less well described, despite the prevalent use of porcine models in medical device and tissue engineering development. In this study, we describe the isolation and characterization of porcine bone marrow- and peripheral blood-derived macrophages, and their interactions with biomaterials. We confirmed the expression of the macrophage surface markers CD68 and F4/80 and characterized the porcine macrophage response to the inflammatory stimulus, bacterial lipopolysaccharide. Finally, we investigated the inflammatory and fusion response of porcine macrophages cultured on different stiffness hydrogels, and we found that stiffer hydrogels enhanced inflammatory activation by more than two-fold and promoted fusion to form foreign body giant cells. Together, this study establishes the use of porcine macrophages in biomaterial testing and reveals a stiffness-dependent effect on biomaterial-induced giant cell formation.

Background/purpose: Artificial Intelligence (AI) can optimize treatment approaches in dental healthcare due to its high level of accuracy and wide range of applications. This study seeks to propose a new deep learning (DL) ensemble model based on deep Convolutional Neural Network (CNN) algorithms to predict tooth position, detect shape, detect remaining interproximal bone level, and detect radiographic bone loss (RBL) using periapical and bitewing radiographs. Materials and methods: 270 patients from January 2015 to December 2020, and all images were deidentified without private information for this study. A total of 8000 periapical radiographs with 27,964 teeth were included for our model. AI algorithms utilizing the YOLOv5 model and VIA labeling platform, including VGG-16 and U-Net architecture, were created as a novel ensemble model. Results of AI analysis were compared with clinicians' assessments. Results: DL-trained ensemble model accuracy was approximately 90% for periapical radiographs. Accuracy for tooth position detection was 88.8%, tooth shape detection 86.3%, periodontal bone level detection 92.61% and radiographic bone loss detection 97.0%. AI models were superior to mean accuracy values from 76% to 78% when detection was performed by dentists. Conclusion: The proposed DL-trained ensemble model provides a critical cornerstone for radiographic detection and a valuable adjunct to periodontal diagnosis. High accuracy and reliability indicate model's strong potential to enhance clinical professional performance and build more efficient dental health services.

Despite the prevalence of large (>5 cm2) articular cartilage defects involving underlying bone, current tissue-engineered therapies only address small defects. Tissue-engineered, anatomically shaped, native-like implants may address the need for off-the-shelf, tissue-repairing therapies for large cartilage lesions. This study fabricated an osteochondral construct of translationally relevant geometry with robust functional properties. Scaffold-free, self-assembled neocartilage served as the chondral phase, and porous hydroxyapatite served as the osseous phase of the osteochondral constructs. Constructs in the shape and size of an ovine femoral condyle (31 × 14 mm) were assembled at day 4 (early) or day 10 (late) of neocartilage maturation. Early osteochondral assembly increased the interfacial interdigitation depth by 244%, interdigitation frequency by 438%, interfacial shear modulus by 243-fold, and ultimate interfacial shear strength by 4.9-fold, compared to late assembly. Toward the development of a bioprosthesis for the repair of cartilage lesions encompassing up to an entire condylar surface, this study generated a large, anatomically shaped osteochondral construct with robust interfacial mechanical properties and native-like neocartilage interdigitation.

Human articular chondrocytes (hACs) are scarce and lose their chondrogenic potential during monolayer passaging, impeding their therapeutic use. This study investigated (a) the translatability of conservative chondrogenic passaging and aggregate rejuvenation on restoring chondrogenic properties of hACs passaged up to P9; and (b) the efficacy of a combined treatment of transforming growth factor-beta 1 (TGF-β1) (T), chondroitinase-ABC (C), and lysyl oxidase-like 2 (L), collectively termed TCL, on engineering functional human neocartilage via the self-assembling process, as a function of passage number up to P11. Here, we show that aggregate rejuvenation enhanced glycosaminoglycan (GAG) content and type II collagen staining at all passages and yielded human neocartilage with chondrogenic phenotype present up to P7. Addition of TCL extended the chondrogenic phenotype to P11 and significantly enhanced GAG content and type II collagen staining at all passages. Human neocartilage derived from high passages, treated with TCL, displayed mechanical properties that were on par with or greater than those derived from low passages. Conservative chondrogenic passaging and aggregate rejuvenation may be a viable new strategy (a) to address the perennial problem of chondrocyte scarcity and (b) to successfully rejuvenate the chondrogenic phenotype of extensively passaged cells (up to P11). Furthermore, tissue engineering human neocartilage via self-assembly in conjunction with TCL treatment advances the clinical use of extensively passaged human chondrocytes for cartilage repair.

Although the knee joint and temporomandibular joint (TMJ) experience similar incidence of cartilage ailments, the knee orthopedics field has greater funding and more effective end-stage treatment options. Translational research has resulted in the development of tissue-engineered products for knee cartilage repair, but the same is not true for TMJ cartilages. Here, we examine the anatomy and pathology of the joints, compare current treatments and products for cartilage afflictions, and explore ways to accelerate the TMJ field. We examine disparities, such as a 6-fold higher article count and 2,000-fold higher total joint replacement frequency in the knee compared to the TMJ, despite similarities in osteoarthritis incidence. Using knee orthopedics as a template, basic and translational research will drive the development and implementation of clinical products for the TMJ. With more funding opportunities, training programs, and federal guidance, millions of people afflicted with TMJ disorders could benefit from novel, life-changing therapeutics.

The surface of articular cartilage is integral to smooth, low-friction joint articulation. However, the majority of cartilage literature rarely includes measurements of surface characteristics and function. This may, in part, be due to a shortage of or unfamiliarity with fast, nondestructive, and, preferably, noncontact methods that can be applied to large cartilage surfaces for evaluating cartilage surface characteristics. A comprehensive methodology for characterizing cartilage surfaces is useful in determining changes in tissue function, as for example, in cases where the quality of cartilage grafts needs to be assessed. With cartilage storage conditions being an area of ongoing and active research, this study used interferometry and tribology methods as efficient and nondestructive ways of evaluating changes in cartilage surface topography, roughness, and coefficient of friction (CoF) resulting from various storage temperatures and durations. Standard, destructive testing for bulk mechanical and biochemical properties, as well as immunohistochemistry, were also performed. For the first time, interferometry was used to show cartilage topographical anisotropy through an anterior-posterior striated pattern in the same direction as joint articulation. Another novel observation enabled by tribology was frictional anisotropy, illustrated by a 53% increase in CoF in the medial-lateral direction compared to the anterior-posterior direction. Of the storage conditions examined, 37°C, 4°C, -20°C, and -80°C for 1 day, 1 week, and 1 month, a 49% decrease in CoF was observed at 1 week in -80°C. Interestingly, prolonged storage at 37°C resulted in up to an 83% increase in the compressive aggregate modulus by 1 month, with a corresponding increase in the glycosaminoglycan (GAG) bulk content. This study illustrates the differential effects of storage conditions on cartilage: freezing tends to target surface properties, while nonfreezing storage impacts the tissue bulk. These data show that a bulk-only analysis of cartilage function is not sufficient or representative. The nondestructive surface characterization assays described here enable improvement in cartilage functionality assessment by considering both surface and bulk cartilage properties; this methodology may thus provide a new angle to explore in future cartilage research and tissue engineering endeavors.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease defined by motor neuron (MN) loss. Multiple genetic risk factors have been identified, implicating RNA and protein metabolism and intracellular transport, among other biological mechanisms. To achieve a systems-level understanding of the mechanisms governing ALS pathophysiology, we built gene co-expression networks using RNA-sequencing data from control human spinal cord samples, identifying 13 gene co-expression modules, each of which represents a distinct biological process or cell type. Analysis of four RNA-seq datasets from a range of ALS disease-associated contexts reveal dysregulation in numerous modules related to ribosomal function, wound response, and leukocyte activation, implicating astrocytes, oligodendrocytes, endothelia, and microglia in ALS pathophysiology. To identify potentially causal processes, we partitioned heritability across the genome, finding that ALS common genetic risk is enriched within two specific modules, SC.M4, representing genes related to RNA processing and gene regulation, and SC.M2, representing genes related to intracellular transport and autophagy and enriched in oligodendrocyte markers. Top hub genes of this latter module include ALS-implicated risk genes such as KPNA3, TMED2, and NCOA4, the latter of which regulates ferritin autophagy, implicating this process in ALS pathophysiology. These unbiased, genome-wide analyses confirm the utility of a systems approach to understanding the causes and drivers of ALS.

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.

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 (

Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecan-coated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-β1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-β1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.

Collagen is a ubiquitous biomaterial in vertebrate animals. Although each of its 28 subtypes contributes to the functions of many different tissues in the body, most studies on collagen or collagenous tissues have focussed on only one or two subtypes. With recent developments in analytical chemistry, especially mass spectrometry, significant advances have been made toward quantifying the different collagen subtypes in various tissues; however, high-throughput and low-cost methods for collagen subtype quantification do not yet exist. In this Review, we introduce the roles of collagen subtypes and crosslinks, and describe modern assays that enable a deep understanding of tissue physiology and disease states. Using cartilage as a model tissue, we describe the roles of major and minor collagen subtypes in detail; discuss known and unknown structure-function relationships; and show how tissue engineers may harness the functional characteristics of collagen to engineer robust neotissues.

Diarthrodial joints, found at the ends of long bones, function to dissipate load and allow for effortless articulation. Essential to these functions are cartilages, soft hydrated tissues such as hyaline articular cartilage and the knee meniscus, as well as lubricating synovial fluid. Maintaining adequate lubrication protects cartilages from wear, but a decrease in this function leads to tissue degeneration and pathologies such as osteoarthritis. To study cartilage physiology, articular cartilage researchers have employed tribology, the study of lubrication and wear between two opposing surfaces, to characterize both native and engineered tissues. The biochemical components of synovial fluid allow it to function as an effective lubricant that exhibits shear-thinning behavior. Although tribological properties are recognized to be essential to native tissue function and a critical characteristic for translational tissue engineering, tribology is vastly understudied when compared to other mechanical properties such as compressive moduli. Further, tribometer configurations and testing modalities vary greatly across laboratories. This review aims to define commonly examined tribological characteristics and discuss the structure-function relationships of biochemical constituents known to contribute to tribological properties in native tissue, address the variations in experimental set-ups by suggesting a move toward standard testing practices, and describe how tissue-engineered cartilages may be augmented to improve their tribological properties.

Autism spectrum disorder (ASD) is a phenotypically and genetically heterogeneous neurodevelopmental disorder. Despite this heterogeneity, previous studies have shown patterns of molecular convergence in post-mortem brain tissue from autistic subjects. Here, we integrate genome-wide measures of mRNA expression, miRNA expression, DNA methylation, and histone acetylation from ASD and control brains to identify a convergent molecular subtype of ASD with shared dysregulation across both the epigenome and transcriptome. Focusing on this convergent subtype, we substantially expand the repertoire of differentially expressed genes in ASD and identify a component of upregulated immune processes that are associated with hypomethylation. We utilize eQTL and chromosome conformation datasets to link differentially acetylated regions with their cognate genes and identify an enrichment of ASD genetic risk variants in hyperacetylated noncoding regulatory regions linked to neuronal genes. These findings help elucidate how diverse genetic risk factors converge onto specific molecular processes in ASD.

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.

OBJECTIVE:To enhance the in vitro integration of self-assembled articular cartilage to native articular cartilage using chondroitinase ABC. DESIGN:To examine the hypothesis that chondroitinase ABC (C-ABC) integration treatment (C-ABCint) would enhance integration of neocartilage of different maturity levels, this study was conducted in 2 phases. In phase I, the impact on integration of 2 treatments, TCL (TGF-β1, C-ABC, and lysyl oxidase like 2) and C-ABCint, was examined via a 2-factor, full factorial design. In phase II, construct maturity (2 levels) and C-ABCint concentration (3 levels) were the factors in a full factorial design to determine whether the effective C-ABCint dose was dependent on neocartilage maturity level. Neocartilages formed or treated per the factors above were placed into native cartilage rings, cultured for 2 weeks, and, then, integration was studied histologically and mechanically. Prior to integration, in phase II, a set of treated constructs were also assayed to provide a baseline of properties. RESULTS:In phase I, C-ABCint and TCL treatments synergistically enhanced interface Young's modulus by 6.2-fold (P = 0.004) and increased interface tensile strength by 3.8-fold (P = 0.02) compared with control. In phase II, the interaction of the factors C-ABCint and construct maturity was significant (P = 0.0004), indicating that the effective C-ABCint dose to improve interface Young's modulus is dependent on construct maturity. Construct mechanical properties were preserved regardless of C-ABCint dose. CONCLUSIONS:Applying C-ABCint to neocartilage is an effective integration strategy with translational potential, provided its dose is calibrated appropriately based on implant maturity, that also preserves implant biomechanical properties.