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Traditional destructive tests are used for quality assurance and control within manufacturing workflows. Their applicability to biomanufacturing is limited due to inherent constraints of the biomanufacturing process. To address this, photo- and acoustic-based nondestructive testing has risen in prominence to interrogate not only structure and function, but also to integrate quantitative measurements of biochemical composition to cross-correlate structural, compositional, and functional variances. We survey relevant literature related to single-mode and multimodal nondestructive testing of soft tissues, which adds numbers (quantitative measurements) to pictures (qualitative data). Native and tissue-engineered articular cartilage is highlighted because active biomanufacturing processes are being developed. Included are recent efforts and prominent trends focused on technologies for clinical and in-process biomanufacturing applications.

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.)

Toward the translation of allogeneic cell therapy products, cell banks are needed not only to manufacture the final human product but also during the preclinical evaluation of an animal-based analogous cellular product (ACP). These cell banks need to be established at both the master cell bank (MCB) level and the working cell bank (WCB) level. Inasmuch as most of the development of cell therapy products is at academic centers, it is imperative that academic researchers understand how to establish MCBs and WCBs within an academic environment. To illustrate this process, using articular cartilage as the model, a cell bank for an ACP was developed (MCBs at passage 2, WCBs at passage 5) to produce self-assembled neocartilage for preclinical evaluation (constructs at passage 7). The cell bank system is estimated to be able to produce between 160 000 and 400 000 constructs for each of the six MCBs. Overall, the ACP cell bank yielded constructs that are analogous to the intended human product, which is critical toward conducting preclinical evaluations of the ACP for inclusion in an Investigational New Drug application to the FDA., (© 2024 IOP Publishing Ltd.)

Objective: Fibrin sealants are routinely used for intra-articular surgical fixation of cartilage fragments and implants. However, the mechanical properties of fibrin sealants in the context of cartilage repair are unknown. The purpose of this study was to characterize the adhesive and frictional properties of fibrin sealants using an ex vivo model., Design: Native bovine cartilage-bone composites were assembled with a single application of Tisseel or Vistaseal. Composites were tested in tension and lap shear. In addition, the coefficient of friction (COF) was measured in a native cartilage annulus model alone and with minced cartilage. Finally, the effect of a double application of fibrin sealant was evaluated., Results: There were no significant differences in tensile modulus, ultimate tensile strength (UTS), shear modulus, or ultimate shear strength (USS) between the 2 fibrin sealants. Both fibrin sealants demonstrated a UTS and USS of <8 and <30 kPa, respectively. There were no differences in COF between the sealants when tested alone or with minced cartilage. A double application of fibrin sealant did not alter the mechanical properties compared with a single application of fibrin sealant., Conclusions: Fibrin sealant adhesive properties are not affected by the sealant type studied or the number of applications in a bovine cartilage-bone model. Fibrin sealant tribological properties are not affected by sealant type or the addition of minced cartilage. The adhesive properties of Tisseel and Vistaseal were less than those desired for the in vivo fixation of cartilage repair implants. These findings motivate the development of an improved cartilage-specific adhesive for cartilage repair applications., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Articular cartilage was expected to be one of the first successfully engineered tissues, but today, cartilage repair products are few and they exhibit considerable limitations. For example, of the cell-based products that are available globally, only one is marketed for non-knee indications, none are indicated for severe osteoarthritis or rheumatoid arthritis, and only one is approved for marketing in the USA. However, advances in cartilage tissue engineering might now finally lead to the development of new cartilage repair products. To understand the potential in this field, it helps to consider the current landscape of tissue-engineered products for articular cartilage repair and particularly cell-based therapies. Advances relating to cell sources, bioactive stimuli and scaffold or scaffold-free approaches should now contribute to progress in therapeutic development. Engineering for an inflammatory environment is required because of the need for implants to withstand immune challenge within joints affected by osteoarthritis or rheumatoid arthritis. Bringing additional cartilage repair products to the market will require an understanding of the translational vector for their commercialization. Advances thus far can facilitate the future translation of engineered cartilage products to benefit the millions of patients who suffer from cartilage injuries and arthritides., (© 2024. Springer Nature Limited.)

Objective: The purpose of this study was to determine the in vitro effects of a single exposure of bupivacaine on the mechanical properties of bovine cartilage explants at 3 weeks., Design: Femoral condyle articular cartilage explants were aseptically harvested from juvenile bovine stifle joints before being exposed to chondrogenic medium containing 0.50% (wt/vol) bupivacaine, 0.25% (wt/vol) bupivacaine, or no medication (control) for 1 hour. Explants were then washed and maintained in culture in vitro for 3 weeks before testing. Cell viability, tensile and compressive mechanical properties, histological properties, and biochemical properties were then assessed., Results: Explants exhibited a dose-dependent decrease in mean tensile Young's modulus with increasing bupivacaine concentration (9.86 MPa in the controls, 6.48 MPa in the 0.25% bupivacaine group [ P = 0.048], and 4.72 MPa in the 0.50% bupivacaine group [ P = 0.005]). Consistent with these results, collagen content and collagen crosslinking decreased with bupivacaine exposure as measured by mass spectrometry. Compressive properties of the explants were unaffected by bupivacaine exposure. Explants also exhibited a trend toward dose-dependent decreases in viability (51.2% for the controls, 47.3% for the 0.25% bupivacaine-exposed group, and 37.0% for the 0.50% bupivacaine-exposed group [ P = 0.072])., Conclusions: Three weeks after 1-hour bupivacaine exposure, the tensile properties of bovine cartilage explants were significantly decreased, while the compressive properties remained unaffected. These decreases in tensile properties corresponded with reductions in collagen content and crosslinking of collagen fibers. Physicians should be judicious regarding the intra-articular administration of bupivacaine in native joints., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: KTC: Stock in Abbott Laboratories. GO: Nothing to disclose. WEB: Research support: L’Oreal/AAAS, NIH, NSF; Cofounder: Cartilage Inc. KAA: Research support: NIH, NSF; Cofounder: Cartilage Inc. DW: Research support: NIH, NSF, Vericel; Consultant: Newclip Technics, Mitek Sports Medicine, Vericel; Cofounder: Cartilage Inc.

Microtia and anotia are congenital auricular anomalies that negatively impact the psychosocial development of those affected. Because auricular cartilage is a type of elastic cartilage that lacks regenerative capacity, any notable defect in its structure requires a surgical approach to reconstructing the auricle. While there are several reconstructive options available between alloplastic and prosthetic implants, autologous rib cartilage grafts remain the most commonly used treatment modality. Still, this widely used technique is accompanied by significant patient discomfort in a young child and carries additional risks secondary to the traumatic process of rib cartilage extraction, such as pneumothorax and chest wall deformities, and the final esthetic results may not be ideal. To circumvent these limitations, tissue engineering approaches have been used to create a realistic-looking ear that mirrors the complex anatomy of the normal ear. This article reviews the biochemical and biomechanical properties of human auricular cartilage as they relate to design criteria. In addition, a variety of cell sources, biocompatible scaffolds, scaffold-free techniques, and mechanical and biological stimuli are discussed. This review aims to identify knowledge gaps in the literature related to auricular cartilage characteristics and make recommendations to drive the field of auricular tissue engineering., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 by Mutaz B. Habal, MD.)

Background: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown., Purpose: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants., Study Design: Controlled laboratory study., Methods: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed., Results: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020)., Conclusion: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure., Clinical Relevance: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: J.A.S. has received speaking fees from Arthrex and support for education from Micromed Inc. D.W. has received hospitality payments from Saxum Surgical, Arthrex, Stryker, Linvatec, and Newclip; consulting fees from Medical Device Business Services, Newclip, and Ipsen Biosciences; research support from Immunis and Vericel; support for education from Micromed, Arthrex, and Smith + Nephew; and a grant from Arthrex. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Objective: The use of porcine animal models for cartilage injury has increased recently due to their similarity with humans with regard to cartilage thickness, limited intrinsic healing of chondral defects, and joint loading biomechanics. However, variations in the mechanical and biochemical properties of porcine hip articular cartilage among various tissue ages and weightbearing (WB) regions are still unknown. This study's aim was to characterize the mechanical and biochemical properties of porcine hip articular cartilage across various ages and WB regions., Methods: Articular cartilage explants were harvested from WB and non-weightbearing (NWB) surfaces of the femoral head and acetabulum of domesticated pigs ( Sus scrofa domesticus ) at fetal (gestational age: 80 days), juvenile (6 months), and adult (2 years) ages. Explants underwent compressive stress-relaxation mechanical testing, biochemical analysis for total collagen and glycosaminoglycan (GAG) content, and histological staining., Results: Juvenile animals consistently had the highest mechanical properties, with 2.2- to 7.6-time increases in relaxation modulus, 1.3- to 2.3-time increases in instantaneous modulus, and 4.1- to 14.2-time increases in viscosity compared with fetal cartilage. Mechanical properties did not significantly differ between the WB and NWB regions. Collagen content was highest in the NWB regions of the juvenile acetabulum (65.3%/dry weight [DW]) and femoral head (75.4%/DW) cartilages. GAG content was highest in the WB region of the juvenile acetabulum (23.7%/DW) and the WB region of the fetal femoral head (27.5%/DW) cartilages. Histological staining for GAG and total collagen content followed the trends from the quantitative biochemical assays., Conclusion: This study provides a benchmark for the development and validation of preclinical porcine models for hip cartilage pathologies., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dean Wang is an orthopedic surgery consultant for Cartilage Inc, Vericel Corp, Newclip Technics, and Mitek Sports Medicine. Ryan P. Donahue, Kyriacos A. Athanasiou, and Jerry C. Hu are scientific consultants for Cartilage Inc.

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.)

Objective: The medial femoral condyle of the knee exhibits some of the highest incidences of chondral degeneration. However, a dearth of healthy human tissues has rendered it difficult to ascertain whether cartilage in this compartment possesses properties that predispose it to injuries. Assessment of young, healthy tissue would be most representative of the tissue's intrinsic properties., Design: This work examined the topographical differences in tribological, tensile, and compressive properties of young (n = 5, 26.2 ± 5.6 years old), healthy, human medial femoral condyles, obtained from viable allograft specimens. Corresponding to clinical incidences of pathology, it was hypothesized that the lowest mechanical properties would be found in the posterior region of the medial condyle, and that tissue composition would correspond to the established structure-function relationships of cartilage., Results: Young's modulus, ultimate tensile strength, aggregate modulus, and shear modulus in the posterior region were 1.0-, 2.8-, 1.1-, and 1.0-fold less than the values in the anterior region, respectively. Surprisingly, although glycosaminoglycan content is thought to correlate with compressive properties, in this study, the aggregate and shear moduli correlated more robustly to the amount of pyridinoline crosslinks per collagen. Also, the coefficient of friction was anisotropic and ranged 0.22-0.26 throughout the condyle., Conclusion: This work showed that the posteromedial condyle displays lower tensile and compressive properties, which correlate to collagen crosslinks and may play a role in this region's predisposition to injuries. Furthermore, new structure-function relationships may need to be developed to account for the role of collagen crosslinks in compressive properties.

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.

Background: The chondrotoxic effects of methylprednisolone acetate (MP) and triamcinolone acetonide (TA) have been well described. However, the mechanical effects of these commonly used steroids on native cartilage are largely unknown., Purpose: To investigate the in vitro effects of a single 1-hour MP or TA exposure on the viability, mechanics, and biochemical content of native articular cartilage explants., Study Design: Controlled laboratory study., Methods: Articular cartilage explants (n = 6 per group) were harvested from the femoral condyles of bovine stifles. Explants were exposed to chondrogenic medium containing a clinical dose of MP or TA for 1 hour, followed by fresh medium wash and exchange. Explants in the control group underwent the same treatment with chondrogenic medium alone. At 24 hours after treatment, samples were assessed for viability (live/dead), mechanical properties (creep indentation and Instron tensile testing), biochemical (collagen and glycosaminoglycan) content, and pyridinoline crosslinking via mass spectrometry., Results: Mean cell viability was significantly decreased in native explants exposed to MP (35.5%) compared with the control (49.8%; P < .001) and TA (45.7%; P = .01) specimens. Significant decreases were seen in the mechanical properties of steroid-treated native explants when compared with controls, with decreases in aggregate modulus (646.3 vs 312.8 kPa [MP] and 257.0 kPa [TA]; P < .001), shear modulus (370.1 vs 191.2 kPa [MP] and 157.4 kPa [TA]; P < .001), and ultimate tensile strength (9.650 vs 5.648 MPa [MP; P = .021] and 6.065 MPa [TA; P = .0403]). No significant differences in collagen and glycosaminoglycan content were found in the steroid-treated groups. Pyridinoline crosslinking was significantly decreased in explants exposed to TA compared with controls ( P = .027)., Conclusion: Exposure of MP to articular cartilage explants was chondrotoxic, and exposure of articular cartilage explants to MP or TA resulted in significant decreases in mechanical properties of articular cartilage explants compared with controls. Clinicians should be judicious regarding use of intra-articular steroids, particularly in patients with intact healthy articular cartilage.

Background: Posterolateral corner (PLC) reconstruction of the knee involves precise drilling of a single tunnel from anterolateral to posteromedial in the fibular head (FH) to ensure adequate graft fixation. Misplacement of the tunnel in a too lateral or too superior trajectory can lead to cortical breach and inadequate graft fixation., Purpose: To (1) determine the mechanical consequence of a lateralized FH tunnel in PLC reconstruction and (2) compare the mechanical strength of 3 salvage fixation techniques for a lateralized FH tunnel., Study Design: Controlled laboratory study., Methods: Sawbones models of a uniform density were utilized. FH tunnels (7-mm diameter) were drilled from anterolateral to posteromedial in positive controls (lateral cortex thickness, 7.6 ± 0.7 mm) to represent an improperly placed FH tunnel at risk of lateral cortical breach. For negative controls and salvage experimental groups, FH tunnels were drilled from anterolateral to posterolateral (lateral cortex thickness, 2.7 ± 0.9 mm). Three salvage fixation techniques were compared: suture anchor fixation, tunnel redrilling, and nitinol staple fixation. Samples (n = 5 per group) underwent uniaxial tension testing, and the ultimate tensile strength (UTS) and mode of failure were recorded. Data were analyzed using the 1-sample t test and nonparametric 1-sample Wilcoxon signed-rank test., Results: The negative control group had a 4-fold lower mean UTS relative to the positive control group (1.49 ± 0.17 vs 6.25 ± 1.98 MPa; P < .01) and exhibited failure through the lateral cortex and tunnel. Nitinol staple fixation improved the mean UTS by >16 times compared with the negative control group (24.06 ± 6.49 vs 1.49 ± 0.17 MPa; P < .01). Suture anchors and tunnel redrilling exhibited similar UTS and mode of failure to those of negative controls., Conclusion: Reinforcement of a thinned lateral FH cortex with a single nitinol staple improved graft fixation strength in a sawbones model., Clinical Relevance: A lateralized FH tunnel can be a common intraoperative pitfall during PLC reconstruction. Salvage of a thinned lateral FH cortex with a single nitinol staple may reduce the risk of cortical breach and graft failure., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: D.W. has received education payments from Arthrex and Smith & Nephew; consulting fees from DePuy, Ipsen Bioscience, Newclip, and Vericel; and hospitality payments from Linvatec and Stryker. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2022.)

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.)

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., (© 2022. The Author(s).)

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., 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

Background: The 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., Methods: Yucatan 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., Results: It 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 (< 0.01%) and unlike donor rib cartilage (0.98%)., Conclusions: In this work, bottom-up proteomics, a powerful tool to interrogate tissue composition, was used for the first time to quantify and compare the proteome of a developing engineered tissue to a recipient tissue. Furthermore, it was shown that self-assembled, costal chondrocyte-derived neocartilage is suitable for a non-homologous approach in the knee., (© 2022. The Author(s).)

Ion signaling through Ca 2+ 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 Ca 2+ 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 Ca 2+ -permeable transient receptor potential vanilloid 4 (TRPV4), (2) ouabain, an inhibitor of the Na + /K + pump, and (3) ionomycin, a Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.

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.

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.)

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., (© 2022 Federation of American Societies for Experimental Biology.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gonzalez-Leon, Hu and Athanasiou.)

Objective: Tissue-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., Design: In 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., Results: As 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., Conclusions: In 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.

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-ABC int ) 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-ABC int , was examined via a 2-factor, full factorial design. In phase II, construct maturity (2 levels) and C-ABC int concentration (3 levels) were the factors in a full factorial design to determine whether the effective C-ABC int 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-ABC int 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-ABC int and construct maturity was significant ( P = 0.0004), indicating that the effective C-ABC int dose to improve interface Young's modulus is dependent on construct maturity. Construct mechanical properties were preserved regardless of C-ABC int dose., Conclusions: Applying C-ABC int 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.

Introduction: This study develops assays to quantify collagen subtypes and crosslinks with liquid chromatography-mass spectrometry (LC-MS) and characterizes the cartilages in the Yucatan minipig., Methods: For 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., Results: The 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., Discussion: The 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.

Objective: Pathology 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., Design: Cartilage 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., Results: Compared 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., Conclusion: These 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.

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 ( R 2 = 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.

Meniscus tissue deficiency resulting from primary meniscectomy or meniscectomy after failed repair is a clinical challenge because the meniscus has little to no capacity for regeneration. Loss of meniscus tissue has been associated with early-onset knee osteoarthritis due to an increase in joint contact pressures in meniscectomized knees. Clinically available replacement strategies range from allograft transplantation to synthetic implants, including the collagen meniscus implant, ACTIfit, and NUSurface. Although short-term efficacy has been demonstrated with some of these treatments, factors such as long-term durability, chondroprotective efficacy, and return to sport activities in young patients remain unpredictable. Investigations of cell-based and tissue-engineered strategies to treat meniscus tissue deficiency are ongoing.

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.