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

Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell types for tissue engineering applications. However, their use in tissue engineering may be hindered through contamination by undesirable cell types. These include blood-associated cells as well as unwanted resident cell types found both in healthy and pathologic donor tissues. Ammonium-chloride-potassium (ACK) lysing buffer is used to lyse red blood cells (RBCs) during the isolation of stem cell populations, but has not been explored for the purification of fully differentiated cells. This study sought to investigate the effect of ACK buffer treatment of freshly isolated, fully differentiated cells to increase cell purity and enhance the formation of biofunctional engineered neotissues; this was tested in the well-established cartilage tissue engineering model of the self-assembling process using fetal ovine articular chondrocytes (foACs) and juvenile bovine articular chondrocytes (jbACs). ACK buffer treatment of foACs and jbACs decreased the number of contaminating RBCs by over 60% and additionally reduced the number of apoptotic chondrocytes in the cell isolates. Reducing the number of contaminating RBCs removed cellular detractors to the self-assembling process and eliminated an apoptotic stimulus, thus improving neocartilage homogeneity, chondrocyte distribution, and extracellular matrix deposition within the neotissues. For example, in foAC neocartilage, ACK buffer treatment ultimately led to a 170% increase in compressive aggregate modulus, a 130% increase in shear modulus, an 80% increase in tensile modulus, and a 130% increase in ultimate tensile strength of the neocartilage. This work represents the first time that ACK buffer has been used to purify fully differentiated cells and subsequently increase the functional properties of neotissue., Competing Interests: Statement No competing financial interests exist.

One of the most important issues facing cartilage tissue engineering is the inability to move technologies into the clinic. Despite the multitude of current research in the field, it is known that 90% of new drugs that advance past animal studies fail clinical trials. The objective of this review is to provide readers with an understanding of the scientific details of tissue engineered cartilage products that have demonstrated a certain level of efficacy in humans, so that newer technologies may be developed upon this foundation. Compared to existing treatments, such as microfracture or autologous chondrocyte implantation, a tissue engineered product can potentially provide more consistent clinical results in forming hyaline repair tissue and in filling the entirety of the defect. The various tissue engineering strategies (e.g., cell expansion, scaffold material, media formulations, biomimetic stimuli, etc.) used in forming these products, as collected from published literature, company websites, and relevant patents, are critically discussed. The authors note that many details about these products remain proprietary, not all information is made public, and that advancements to the products are continuously made. Nevertheless, by understanding the design and production processes of these emerging technologies, one can gain tremendous insight into how to best use them and also how to design the next generation of tissue engineered cartilage products., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Previous work has shown that increasing the production of boundary lubricant, superficial zone protein (SZP), did not reduce the friction coefficient of self-assembled articular cartilage constructs and was possibly due to poor retention of the lubricant. The aim of this investigation was to reduce the friction coefficient of self-assembled articular cartilage constructs through enhancing SZP retention by the exogenous addition of extracellular matrix (ECM) extracted from the superficial zone of native articular cartilage., Design: Superficial zone cartilage was shaved from juvenile bovine femoral condyles using a dermatome, minced finely with razor blades, extracted with 4 M guanidine-hydrochloride, buffer exchanged with culture medium, and added directly to the culture medium of self-assembled articular cartilage constructs at low (10 µg/mL) and high (100 µg/mL) concentrations for 4 weeks. Biochemical and biomechanical properties were determined at the conclusion of 4 weeks culture., Results: ECM treatment increased compressive and tensile stiffness of self-assembled articular cartilage constructs and decreased the friction coefficient. Glycosaminoglycan content decreased and collagen content increased significantly in self-assembled constructs by the ECM treatment., Conclusions: Friction coefficients of self-assembled articular cartilage constructs were reduced by adding extracted superficial zone ECM into the culture medium of self-assembled articular cartilage constructs.

Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell-cell and cell-matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation.

Joint injury often leads to post-traumatic osteoarthritis (PTOA). Acute injury responses to trauma induce production of pro-inflammatory cytokines and catabolic enzymes, which promote chondrocyte apoptosis and degrade cartilage to potentiate PTOA development. Recent studies show that the rate-limiting step for transcriptional activation of injury response genes is controlled by cyclin-dependent kinase 9 (CDK9), and thus it is an attractive target for limiting the injury response. Here, we determined the effects of CDK9 inhibition in suppressing the injury response in mechanically-injured cartilage explants. Bovine cartilage explants were injured by a single compressive load of 30 % strain at 100 %/s, and then treated with the CDK9 inhibitor Flavopiridol. To assess acute injury responses, we measured the mRNA expression of pro-inflammatory cytokines, catabolic enzymes, and apoptotic genes by RT-PCR, and chondrocyte viability and apoptosis by TUNEL staining. For long-term outcome, cartilage matrix degradation was assessed by soluble glycosaminoglycan release, and by determining the mechanical properties with instantaneous and relaxation moduli. Our data showed CDK9 inhibitor markedly reduced injury-induced inflammatory cytokine and catabolic gene expression. CDK9 inhibitor also attenuated chondrocyte apoptosis and reduced cartilage matrix degradation. Lastly, the mechanical properties of the injured explants were preserved by CDK9 inhibitor. Our results provide a temporal profile connecting the chain of events from mechanical impact, acute injury responses, to the subsequent induction of chondrocyte apoptosis and cartilage matrix deterioration. Thus, CDK9 is a potential disease-modifying agent for injury response after knee trauma to prevent or delay PTOA development.

Knowledge and resources derived from veterinary medicine represent an underused resource that could serve as a bridge between data obtained from diseases models in laboratory animals and human clinical trials. Naturally occurring disease in companion animals that display the defining attributes of similar, if not identical, diseases in humans hold promise for providing predictive proof of concept in the evaluation of new therapeutics and devices. Here we outline comparative aspects of naturally occurring diseases in companion animals and discuss their current uses in translational medicine, benefits, and shortcomings. Last, we envision how these natural models of disease might ultimately decrease the failure rate in human clinical trials and accelerate the delivery of effective treatments to the human clinical market., (Copyright © 2015, American Association for the Advancement of Science.)

Unlabelled: The exciting potential for regenerating organs from autologous stem cells is on the near horizon, and adult dermis stem cells (DSCs) are particularly appealing because of the ease and relative minimal invasiveness of skin collection. A substantial number of reports have described DSCs and their potential for regenerating tissues from mesenchymal, ectodermal, and endodermal lineages; however, the exact niches of these stem cells in various skin types and their antigenic surface makeup are not yet clearly defined. The multilineage potential of DSCs appears to be similar, despite great variability in isolation and in vitro propagation methods. Despite this great potential, only limited amounts of tissues and clinical applications for organ regeneration have been developed from DSCs. This review summarizes the literature on DSCs regarding their niches and the specific markers they express. The concept of the niches and the differentiation capacity of cells residing in them along particular lineages is discussed. Furthermore, the advantages and disadvantages of widely used methods to demonstrate lineage differentiation are considered. In addition, safety considerations and the most recent advancements in the field of tissue engineering and regeneration using DSCs are discussed. This review concludes with thoughts on how to prospectively approach engineering of tissues and organ regeneration using DSCs. Our expectation is that implementation of the major points highlighted in this review will lead to major advancements in the fields of regenerative medicine and tissue engineering., Significance: Autologous dermis-derived stem cells are generating great excitement and efforts in the field of regenerative medicine and tissue engineering. The substantial impact of this review lies in its critical coverage of the available literature and in providing insight regarding niches, characteristics, and isolation methods of stem cells derived from the human dermis. Furthermore, it provides analysis of the current state-of-the-art regenerative approaches using human-derived dermal stem cells, with consideration of current guidelines, to assist translation toward therapeutic use., (©AlphaMed Press.)

The diarthrodial, knee joint is a remarkably efficient bearing system; articulating cartilage surfaces provide nearly frictionless performance with minimal wear. The low friction properties of the cartilage surfaces are due in part to the boundary lubricant, superficial zone protein (SZP); also known as lubricin or proteoglycan 4 (PRG4). In previous work, SZP localization and cartilage friction were examined across the femoral condyles. Studies in the literature have also individually investigated the other tissues that comprise the human knee and four-legged animal stifle joint, such as the meniscus or patella. However, comparisons between individual studies are limited due to the variable testing conditions employed. Friction is a system property that is dependent on the opposing articulating surface, entraining speed, and loading. A cross-comparison of the frictional properties and SZP localization across the knee/stifle joint tissues utilizing a common testing configuration is therefore needed. The objective of this investigation was to determine the friction coefficient and SZP localization of the tissues comprising the three compartments of the bovine stifle joint: patella, patellofemoral groove, femoral condyles, meniscus, tibial plateau, and anterior cruciate ligament. The boundary mode coefficient of friction was greater in tissues of the patellofemoral compartment than the lateral and medial tibiofemoral compartments. SZP immunolocalization followed this trend with reduced depth of staining and intensity in the patella and patellofemoral groove compared to the femoral condyles and tibial plateau. These results illustrate the important role of SZP in reducing friction in the tissues and compartments of the knee/stifle joint., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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

Objective: To describe a surgical technique using a regenerative approach and internal fixation for immediate reconstruction of critical size bone defects after segmental mandibulectomy in dogs., Study Design: Prospective case series., Animals: Dogs (n = 4) that had reconstruction after segmental mandibulectomy for treatment of malignant or benign tumors., Methods: Using a combination of extraoral and intraoral approaches, a locking titanium plate was contoured to match the native mandible. After segmental mandibulectomy, the plate was secured and a compression resistant matrix (CRM) infused with rhBMP-2, implanted in the defect. The implant was then covered with a soft tissue envelope followed by intraoral and extraoral closure., Results: All dogs that had mandibular reconstruction healed with intact gingival covering over the mandibular defect and had immediate return to normal function and occlusion. Mineralized tissue formation was observed clinically within 2 weeks and solid cortical bone formation within 3 months. CT findings at 3 months showed that the newly regenerated mandibular bone had ∼50% of the bone density and porosity compared to the contralateral side. No significant complications occurred., Conclusion: Mandibular reconstruction using internal fixation and CRM infused with rhBMP-2 is an excellent solution for immediate reconstruction of segmental mandibulectomy defects in dogs., (© Copyright 2014 by The American College of Veterinary Surgeons.)

Objective: To describe a surgical technique using a regenerative approach and internal fixation for reconstruction of critical size bone defect non-union mandibular fractures., Study Design: Case series., Animals: Dogs (n = 6) that had internal fixation of defect non-union mandibular fracture., Methods: In 5 dogs, the repair was staged and extraction of teeth performed during the initial procedure. After 21-98 days (mean, 27 days) pharyngotomy intubation and temporary maxillomandibular fixation were performed. Using an extraoral approach, a locking titanium miniplate was contoured and secured to the mandible. A compression resistant matrix (CRM) infused with rhBMP-2 was implanted in the defect. The implant was then covered with a soft tissue envelope followed by surgical wound closure., Results: All dogs healed with intact gingival covering over the mandibular fracture site defect and had immediate return to normal function and correct occlusion. Hard-tissue formation was observed clinically within 2 weeks and solid cortical bone formation within 3 months. CT findings in 1 dog at 3 months postoperatively demonstrated that the newly regenerated mandibular bone had 92% of the bone density and porosity compared to the contralateral side. Long-term follow-up revealed excellent outcome., Conclusion: Mandibular reconstruction using internal fixation and CRM infused with rhBMP-2 is an excellent solution for the treatment of critical size defect non-union fractures in dogs., (© Copyright 2014 by The American College of Veterinary Surgeons.)

The objective of this study was to identify ERK 1/2 involvement in the changes in compressive and tensile mechanical properties associated with hydrostatic pressure treatment of self-assembled cartilage constructs. In study 1, ERK 1/2 phosphorylation was detected by immunoblot, following application of hydrostatic pressure (1 h of static 10 MPa) applied at days 10-14 of self-assembly culture. In study 2, ERK 1/2 activation was blocked during hydrostatic pressure application on days 10-14. With pharmacological inhibition of the ERK pathway by the MEK1/ERK inhibitor U0126 during hydrostatic pressure application on days 10-14, the increase in Young's modulus induced by hydrostatic pressure was blocked. Furthermore, this reduction in Young's modulus with U0126 treatment during hydrostatic pressure application corresponded to a decrease in total collagen expression. However, U0126 did not inhibit the increase in aggregate modulus or GAG induced by hydrostatic pressure. These findings demonstrate a link between hydrostatic pressure application, ERK signalling and changes in the biomechanical properties of a tissue-engineered construct., (Copyright © 2012 John Wiley & Sons, Ltd.)

Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca(2+)-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca(2+) modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10-14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52-110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca(2+) imaging experiments revealed that both digoxin and ATP were able to increase Ca(2+) oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca(2+) modulators.

Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs., (© 2014 AlphaMed Press.)

Introduction: The aim of this study was to investigate the effects of thyroid hormones tri-iodothyronine (T3), thyroxine (T4), and parathyroid hormone (PTH) from the parathyroid glands, known to regulate the developing limb and growth plate, on articular cartilage tissue regeneration using a scaffold-free in vitro model., Methods: In Phase 1, T3, T4, or PTH was applied during weeks 1 or 3 of a 4-week neocartilage culture. Phase 2 employed T3 during week 1, followed by PTH during week 2, 3, or weeks 2 to 4, to further enhance tissue properties. Resultant neotissues were evaluated biochemically, mechanically, and histologically., Results: In Phase 1, T3 and T4 treatment during week 1 resulted in significantly enhanced collagen production; 1.4- and 1.3-times untreated neocartilage. Compressive and tensile properties were also significantly increased, as compared to untreated and PTH groups. PTH treatment did not result in notable tissue changes. As T3 induces hypertrophy, in Phase 2, PTH (known to suppress hypertrophy) was applied sequentially after T3. Excitingly, sequential treatment with T3 and PTH reduced expression of hypertrophic marker collagen X, while yielding neocartilage with significantly enhanced functional properties. Specifically, in comparison to no hormone application, these hormones increased compressive and tensile moduli 4.0-fold and 3.1-fold, respectively., Conclusions: This study demonstrated that T3, together with PTH, when applied in a scaffold-free model of cartilage formation, significantly enhanced functional properties. The novel use of these thyroid hormones generates mechanically robust neocartilage via the use of a scaffold-free tissue engineering model.

Integration of engineered musculoskeletal tissues with adjacent native tissues presents a significant challenge to the field. Specifically, the avascularity and low cellularity of cartilage elicit the need for additional efforts in improving integration of neocartilage within native cartilage. Self-assembled neocartilage holds significant potential in replacing degenerated cartilage, though its stabilization and integration in native cartilage require further efforts. Physical and enzymatic stabilization methods were investigated in an in vitro model for temporomandibular joint (TMJ) disc degeneration. First, in phase 1, suture, glue and press-fit constructs were compared in TMJ disc intermediate zone defects. In phase 1, suturing enhanced interfacial shear stiffness and strength immediately; after four weeks, a 15-fold increase in stiffness and a ninefold increase in strength persisted over press-fit. Neither suture nor glue significantly altered neocartilage properties. In phase 2, the effects of the enzymatic stabilization regimen composed of lysyl oxidase, CuSO4 and hydroxylysine were investigated. A full factorial design was employed, carrying forward the best physical method from phase 1, suturing. Enzymatic stabilization significantly increased interfacial shear stiffness after eight weeks. Combined enzymatic stabilization and suturing led to a fourfold increase in shear stiffness and threefold increase in strength over press-fit. Histological analysis confirmed the presence of a collagen-rich interface. Enzymatic treatment additionally enhanced neocartilage mechanical properties, yielding a tensile modulus over 6 MPa and compressive instantaneous modulus over 1200 kPa at eight weeks. Suturing enhances stabilization of neocartilage, and enzymatic treatment enhances functional properties and integration of neocartilage in the TMJ disc. Methods developed here are applicable to other orthopaedic soft tissues, including knee meniscus and hyaline articular cartilage., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Lissner Medal, it primarily discusses work toward cartilage regeneration performed in Dr. Kyriacos A. Athanasiou's laboratory over the past 25 years. The prevalence and severity of degeneration of articular cartilage, a tissue whose main function is largely biomechanical, have motivated the development of cartilage tissue engineering approaches informed by biomechanics. This article provides a review of important steps toward regeneration of articular cartilage with suitable biomechanical properties. As a first step, biomechanical and biochemical characterization studies at the tissue level were used to provide design criteria for engineering neotissues. Extending this work to the single cell and subcellular levels has helped to develop biochemical and mechanical stimuli for tissue engineering studies. This strong mechanobiological foundation guided studies on regenerating hyaline articular cartilage, the knee meniscus, and temporomandibular joint (TMJ) fibrocartilage. Initial tissue engineering efforts centered on developing biodegradable scaffolds for cartilage regeneration. After many years of studying scaffold-based cartilage engineering, scaffoldless approaches were developed to address deficiencies of scaffold-based systems, resulting in the self-assembling process. This process was further improved by employing exogenous stimuli, such as hydrostatic pressure, growth factors, and matrix-modifying and catabolic agents, both singly and in synergistic combination to enhance neocartilage functional properties. Due to the high cell needs for tissue engineering and the limited supply of native articular chondrocytes, costochondral cells are emerging as a suitable cell source. Looking forward, additional cell sources are investigated to render these technologies more translatable. For example, dermis isolated adult stem (DIAS) cells show potential as a source of chondrogenic cells. The challenging problem of enhanced integration of engineered cartilage with native cartilage is approached with both familiar and novel methods, such as lysyl oxidase (LOX). These diverse tissue engineering strategies all aim to build upon thorough biomechanical characterizations to produce functional neotissue that ultimately will help combat the pressing problem of cartilage degeneration. As our prior research is reviewed, we look to establish new pathways to comprehensively and effectively address the complex problems of musculoskeletal cartilage regeneration.

Chondral and osteochondral lesions due to injury or other pathology commonly result in the development of osteoarthritis, eventually leading to progressive total joint destruction. Although current progress suggests that biologic agents can delay the advancement of deterioration, such drugs are incapable of promoting tissue restoration. The limited ability of articular cartilage to regenerate renders joint arthroplasty an unavoidable surgical intervention. This Review describes current, widely used clinical repair techniques for resurfacing articular cartilage defects; short-term and long-term clinical outcomes of these techniques are discussed. Also reviewed is a developmental pipeline of acellular and cellular regenerative products and techniques that could revolutionize joint care over the next decade by promoting the development of functional articular cartilage. Acellular products typically consist of collagen or hyaluronic-acid-based materials, whereas cellular techniques use either primary cells or stem cells, with or without scaffolds. Central to these efforts is the prominent role that tissue engineering has in translating biological technology into clinical products; therefore, concomitant regulatory processes are also discussed.

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

Expanded costochondral cells provide a clinically relevant cell source for engineering both fibrous and hyaline articular cartilage. Expanding chondrocytes in a monolayer results in a shift toward a proliferative, fibroblastic phenotype. Three-dimensional aggregate culture may, however, be used to recover chondrogenic matrix production. This study sought to engineer a spectrum of fibrous to hyaline neocartilage from a single cell source by varying the duration of three-dimensional culture following expansion. In third passage porcine costochondral cells, the effects of aggregate culture duration were assessed after 0, 8, 11, 14, and 21 days of aggregate culture and after 4 subsequent weeks of neocartilage formation. Varying the duration of aggregate redifferentiation generated a spectrum of fibrous to hyaline neocartilage. Within 8 days of aggregation, proliferation ceased, and collagen and glycosaminoglycan production increased, compared with monolayer cells. In self-assembled neocartilage, type II-to-I collagen ratio increased with increasing aggregate duration, yet glycosaminoglycan content varied minimally. Notably, 14 days of aggregate redifferentiation increased collagen content by 25%, tensile modulus by over 110%, and compressive moduli by over 50%, compared with tissue formed in the absence of redifferentiation. A spectrum of fibrous to hyaline cartilage was generated using a single, clinically relevant cell source, improving the translational potential of engineered cartilage.

The central hypothesis of functional tissue engineering is that an engineered construct can serve as a viable replacement tissue in vivo by replicating the structure and function of native tissue. In the case of articular cartilage, this requires the reproduction of the bulk mechanical and surface lubrication properties of native hyaline cartilage. Cartilage tissue engineering has primarily focused on achieving the bulk mechanical properties of native cartilage such as the compressive aggregate modulus and tensile strength. A scaffold-free self-assembling process has been developed that produces engineered cartilage with compressive properties approaching native tissue levels. Thus, the next step in this process is to begin addressing the friction coefficient and wear properties of these engineered constructs. The superficial zone protein (SZP), also known as lubricin or PRG4, is a boundary mode lubricant that is synthesized by surface zone (SZ) articular chondrocytes. Under conditions of high loading and low sliding speeds, SZP reduces friction and wear at the articular surface. The objective of this investigation was to determine whether increasing the proportion of SZ chondrocytes in cartilage constructs, in the absence of external stimuli such as growth factors and mechanical loading, would enhance the secretion of SZP and improve their frictional properties. In this study, cartilage constructs were engineered through a self-assembling process with varying ratios of SZ and middle zone (MZ) chondrocytes (SZ:MZ): 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs containing different ratios of SZ and MZ chondrocytes did not significantly differ in the glycosaminoglycan composition or compressive aggregate modulus. In contrast, tensile properties and collagen content were enhanced in nearly all constructs containing greater amounts of SZ chondrocytes. Increasing the proportion of SZ chondrocytes had the hypothesized effect of improving the synthesis and secretion of SZP. However, increasing the SZ chondrocyte fraction did not significantly reduce the friction coefficient. These results demonstrate that additional factors, such as SZP-binding macromolecules, surface roughness, and adhesion, need to be examined to modulate the lubrication properties of engineered cartilage.

Tissue-engineered musculoskeletal soft tissues typically lack the appropriate mechanical robustness of their native counterparts, hindering their clinical applicability. With structure and function being intimately linked, efforts to capture the anatomical shape and matrix organization of native tissues are imperative to engineer functionally robust and anisotropic tissues capable of withstanding the biomechanically complex in vivo joint environment. The present study sought to tailor the use of passive axial compressive loading to drive matrix synthesis and reorganization within self-assembled, shape-specific fibrocartilaginous constructs, with the goal of developing functionally anisotropic neotissues. Specifically, shape-specific fibrocartilaginous neotissues were subjected to 0, 0.01, 0.05, or 0.1 N axial loads early during tissue culture. Results found the 0.1-N load to significantly increase both collagen and glycosaminoglycan synthesis by 27% and 67%, respectively, and to concurrently reorganize the matrix by promoting greater matrix alignment, compaction, and collagen crosslinking compared with all other loading levels. These structural enhancements translated into improved functional properties, with the 0.1-N load significantly increasing both the relaxation modulus and Young's modulus by 96% and 255%, respectively, over controls. Finite element analysis further revealed the 0.1-N uniaxial load to induce multiaxial tensile and compressive strain gradients within the shape-specific neotissues, with maxima of 10.1%, 18.3%, and -21.8% in the XX-, YY-, and ZZ-directions, respectively. This indicates that strains created in different directions in response to a single axis load drove the observed anisotropic functional properties. Together, results of this study suggest that strain thresholds exist within each axis to promote matrix synthesis, alignment, and compaction within the shape-specific neotissues. Tailoring of passive axial loading, thus, presents as a simple, yet effective way to drive in vitro matrix development in shape-specific neotissues toward more closely achieving native structural and functional properties.

The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼ 16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

Purpose: The purposes of this study were to identify differences in the biomechanical and biochemical properties among the articulating surfaces of the ankle joint and to evaluate the functional and biological properties of engineered neocartilage generated using chondrocytes from different locations in the ankle joint., Methods: The properties of the different topographies within the ankle joint (tibial plafond, talar dome, and distal fibula) were evaluated in 28 specimens using 7 bovine ankles; the femoral condyle was used as a control. Chondrocytes from the same locations were used to form 28 neocartilage constructs by tissue engineering using an additional 7 bovine ankles. The functional properties of neocartilage were compared with native tissue values., Results: Articular cartilage from the tibial plafond, distal fibula, talar dome, and femoral condyle exhibited Young modulus values of 4.8 ± 0.5 MPa, 3.9 ± 0.1 MPa, 1.7 ± 0.2 MPa, and 4.0 ± 0.5 MPa, respectively. The compressive properties of the corresponding tissues were 370 ± 22 kPa, 242 ± 18 kPa, 255 ± 26 kPa, and 274 ± 18 kPa, respectively. The tibial plafond exhibited 3-fold higher tensile properties and 2-fold higher compressive and shear moduli compared with its articulating talar dome; the same disparity was observed in neocartilage. Similar trends were detected in biochemical data for both native and engineered tissues., Conclusions: The cartilage properties of the various topographic locations within the ankle are significantly different. In particular, the opposing articulating surfaces of the ankle have significantly different biomechanical and biochemical properties. The disparity between tibial plafond and talar dome cartilage and chondrocytes warrants further evaluation in clinical studies to evaluate their exact role in the pathogenesis of ankle lesions., Clinical Relevance: Therapeutic modalities for cartilage lesions need to consider the exact topographic source of the cells or cartilage grafts used. Furthermore, the capacity of generating neocartilage implants from location-specific chondrocytes of the ankle joint may be used in the future as a tool for the treatment of chondral lesions., (Copyright © 2014 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Xenogeneic tissues are derived from other animal species and provide a source of material for engineering mechanically functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. Xenogeneic tissues, however, contain molecules, known as antigens, which invoke an immune reaction following implantation into a patient. Therefore, it is necessary to remove the antigens from a xenogeneic tissue to prevent immune rejection of the graft. Antigen removal can be accomplished by treating a tissue with solutions and/or physical processes that disrupt cells and solubilize, degrade, or mask antigens. However, processes used for cell and antigen removal from tissues often have deleterious effects on the extracellular matrix (ECM) of the tissue, rendering the tissue unsuitable for implantation due to poor mechanical properties. Thus, the goal of an antigen removal process should be to reduce the antigen content of a xenogeneic tissue while preserving its mechanical functionality. To expand the clinical use of antigen-removed xenogeneic tissues as biomechanically functional grafts, it is essential that researchers examine tissue antigen content, ECM composition and architecture, and mechanical properties as new antigen removal processes are developed., (© 2013 Published by Elsevier Ltd.)

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O2) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O2) or normoxic (20% O2) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.

The limited regenerative capacity of articular cartilage and deficiencies of current treatments have motivated the investigation of new repair technologies. In vitro cartilage generation using primary cell sources is limited by cell availability and expansion potential. Pluripotent stem cells possess the capacity for chondrocytic differentiation and extended expansion, providing a potential future solution to cell-based cartilage regeneration. However, despite successes in producing cartilage using adult and embryonic stem cells, the translation of these technologies to the clinic has been severely limited. This review discusses recent advances in stem cell-based cartilage tissue engineering and the major current limitations to clinical translation of these products. Concerns regarding appropriate animal models and studies, stem cell manufacturing, and relevant regulatory processes and guidelines will be addressed. Understanding the significant hurdles limiting the clinical use of stem cell-based cartilage may guide future developments in the fields of tissue engineering and regenerative medicine.

The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. Whereas dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP, with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and transforming growth factor β (TGFβ) 1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFβ1. A decrease in TGFβ1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified using paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFβ-induced SZP accumulation. TGFβ-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1), and Y27632 (Rho effector Rho Kinase). On the other hand, lysophosphatidic acid, an upstream activator of Rho, increased SZP synthesis in response to TGFβ1. These results suggest that SZP production is dependent on the functional cytoskeleton, and Rho GTPases contribute to SZP accumulation by modulating the actions of TGFβ.

Toward addressing the difficult problems of knee meniscus regeneration, a self-assembling process has been used to re-create the native morphology and matrix properties. A significant problem in such attempts is the recapitulation of the distinct zones of the meniscus, the inner, more cartilaginous and the outer, more fibrocartilaginous zones. In this study, an anisotropic and zonally variant meniscus was produced by self-assembly of the inner meniscus (100% chondrocytes) followed by cell seeding the outer meniscus (coculture of chondrocytes and meniscus cells). After 4 weeks in culture, the engineered, inner meniscus exhibited a 42% increase in both instantaneous and relaxation moduli and a 62% increase in GAG/DW, as compared to the outer meniscus. In contrast, the circumferential tensile modulus and collagen/DW of the outer zone was 101% and 129% higher, respectively, than the values measured for the inner zone. Furthermore, there was no difference in the radial tensile modulus between the control and zonal engineered menisci, suggesting that the inner and outer zones of the engineered zonal menisci successfully integrated. These data demonstrate that not only can biomechanical and biochemical properties be engineered to differ by the zone, but they can also recapitulate the anisotropic behavior of the knee meniscus.

The knee meniscus, intervertebral disc, and temporomandibular joint (TMJ) disc all possess complex geometric shapes and anisotropic matrix organization. While these characteristics are imperative for proper tissue function, they are seldom recapitulated following injury or disease. Thus, this study's objective was to engineer fibrocartilages that capture both gross and molecular structural features of native tissues. Self-assembled TMJ discs were selected as the model system, as the disc exhibits a unique biconcave shape and functional anisotropy. To drive anisotropy, 50:50 co-cultures of meniscus cells and articular chondrocytes were grown in biconcave, TMJ-shaped molds and treated with two exogenous stimuli: biomechanical (BM) stimulation via passive axial compression and bioactive agent (BA) stimulation via chondroitinase-ABC and transforming growth factor-β1. BM + BA synergistically increased Col/WW, Young's modulus, and ultimate tensile strength 5.8-fold, 14.7-fold, and 13.8-fold that of controls, respectively; it also promoted collagen fibril alignment akin to native tissue. Finite element analysis found BM stimulation to create direction-dependent strains within the neotissue, suggesting shape plays an essential role toward driving in vitro anisotropic neotissue development. Methods used in this study offer insight on the ability to achieve physiologic anisotropy in biomaterials through the strategic application of spatial, biomechanical, and biochemical cues., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Temporomandibular disorders (TMD) are a class of degenerative musculoskeletal conditions associated with morphologic and functional deformities that affect up to 25% of the population, but their etiology and progression are poorly understood and, as a result, treatment options are limited. In up to 70% of cases, TMD are accompanied by malpositioning of the temporomandibular joint (TMJ) disc, termed "internal derangement." Although the onset is not well characterized, correlations between internal derangement and osteoarthritic change have been identified. Because of the complex and unique nature of each TMD case, diagnosis requires patient-specific analysis accompanied by various diagnostic modalities. Likewise, treatment requires customized plans to address the specific characteristics of each patient's disease. In the mechanically demanding and biochemically active environment of the TMJ, therapeutic approaches that can restore joint functionality while responding to changes in the joint have become a necessity. One such approach, tissue engineering, which may be capable of integration and adaptation in the TMJ, carries significant potential for the development of repair and replacement tissues. The following review presents a synopsis of etiology, current treatment methods, and the future of tissue engineering for repairing and/or replacing diseased joint components, specifically the mandibular condyle and TMJ disc. An analysis of native tissue characterization to assist clinicians in identifying tissue engineering objectives and validation metrics for restoring healthy and functional structures of the TMJ is followed by a discussion of current trends in tissue engineering.

The frequency and impact of temporomandibular joint (TMJ) disorders necessitate research in characterizing the joint's function. The 6 discal attachments have not yet been systematically characterized under tension. Understanding their role in joint function may guide our study of TMJ pathologies, including disc displacement. In the present study, a porcine model was used to characterize the attachments in tension anteroposteriorly and mediolaterally, based on previously identified similarities in the porcine and human masticatory behaviors and discal properties. Tensile stiffness, strength, toughness, and maximum strain were quantified. Collagen alignment was characterized via polarized light and scanning electron microscopy. Anisotropy was demonstrated in all attachments, with the exception of the anterior inferior attachment. Anteroposteriorly, the lateral attachment was stiffest (8.3 MPa) and the anterior superior was least stiff (1.4 MPa). Mediolaterally, the posterior superior attachment was stiffest (16.3 MPa) and the medial was least stiff (1.4 MPa). The greatest strain was observed in the lateral attachment in the mediolateral direction and the posterior superior attachment in the anteroposterior direction. With greatest strains in the most commonly observed directions of disc displacement, it is suggested that compromise in the posterior and lateral attachments contributes to partial lateral and anterior disc displacement.

The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross-links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross-links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self-assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2-factor, full-factorial study design. Following a 6-wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross-links in all copper sulfate-containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10-fold increase in PYR content over controls. This increase in PYR cross-links manifested in a 3.3-fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross-linking enhancement.

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

Objective: The focus of tissue engineering of neocartilage has traditionally been on enhancing extracellular matrix and thus biomechanical properties. Emphasis has been placed on the enhancement of collagen type and quantity, and, concomitantly, tensile properties. The objective of this study was to improve crosslinking of the collagen network by testing the hypothesis that hypoxia could promote pyridinoline (PYR) crosslinks and, thus, improve neocartilage's tensile properties., Methods: Chondrocyte expression of lysyl oxidase (LOX), an enzyme responsible for the formation of collagen PYR crosslinks, was first assessed pre- and post- hypoxia application. Then, the mechanical properties of self-assembled neocartilage constructs were measured, after 4 weeks of culture, for groups exposed to 4% O2 at different initiation times and durations, i.e., during the 1st and 3rd weeks, 3rd and 4th weeks, 4th week only, continuously after cell seeding, or never., Results: Results showed that LOX gene expression was upregulated ∼20-fold in chondrocytes in response to hypoxia. Hypoxia applied during the 3rd and 4th weeks significantly increased PYR crosslinks without affecting collagen content. Excitingly, neocartilage tensile properties were increased ∼2-fold. It should be noted that these properties exhibited a distinct temporal dependence to hypoxia exposure, since upregulation of these properties was due to hypoxia applied only during the 3rd and 4th weeks., Conclusion: These data elucidate the role of hypoxia-mediated upregulation of LOX and subsequent increases in PYR crosslinks in engineered cartilage. These results hold promise toward applying hypoxia at precise time points to promote tensile integrity and direct construct maturation., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

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

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

In recent years, the tissue engineering paradigm has shifted to include a new and growing subfield of scaffoldless techniques that generate self-organizing and self-assembling tissues. This review aims to cogently describe this relatively new research area, with special focus on applications toward clinical use and research models. Particular emphasis is placed on providing clear definitions of self-organization and the self-assembling process, as delineated from other scaffoldless techniques in tissue engineering and regenerative medicine. Significantly, during formation, self-organizing and self-assembling tissues display biological processes similar to those that occur in vivo. These processes help lead to the recapitulation of native tissue morphological structure and organization. Notably, functional properties of these engineered tissues, some of which are already in clinical trials, also approach native tissue values. This review endeavors to provide a cohesive summary of work in this field and to highlight the potential of self-organization and the self-assembling process for providing cogent solutions to currently intractable problems in tissue engineering.

When expanded through passage, chondrocytes lose their ability to produce high-quality cartilaginous matrix. This study attempts to improve the properties of constructs formed with expanded chondrocytes through alterations in the expansion protocol and the ratio of primary to expanded chondrocytes used to form cartilage constructs. A chondrogenically tuned expansion protocol provided similar monolayer growth rates as those obtained using serum-containing medium and enhanced cartilaginous properties of resultant constructs. Various ratios of primary to chondrogenically expanded chondrocytes were then self-assembled to form neocartilage. Biochemical analysis showed that constructs formed with only expanded cells had twice the GAG per wet weight and collagen II/collagen I ratio compared to constructs formed with primary chondrocytes. Biomechanically, compressive properties of constructs formed with only passaged cells matched the instantaneous modulus and exceeded the relaxation modulus of constructs formed with only primary cells. These counterintuitive results show that, by applying proper expansion and three-dimensional culture techniques, the cartilage-forming potential of adult chondrocytes expanded through passage can be enhanced over that of primary cells.

The insufficient healing capacity of articular cartilage necessitates mechanically functional biologic tissue replacements. Using cells to form biomimetic cartilage implants is met with the challenges of cell scarcity and donor site morbidity, requiring expanded cells that possess the ability to generate robust neocartilage. To address this, this study assesses the effects of expansion medium supplementation (bFGF, TFP, FBS) and self-assembled construct seeding density (2, 3, 4 million cells/5 mm dia. construct) on the ability of costochondral cells to generate biochemically and biomechanically robust neocartilage. Results show TFP (1 ng/mL TGF-β1, 5 ng/mL bFGF, 10 ng/mL PDGF) supplementation of serum-free chondrogenic expansion medium enhances the post-expansion chondrogenic potential of costochondral cells, evidenced by increased glycosaminoglycan content, decreased type I/II collagen ratio, and enhanced compressive properties. Low density (2 million cells/construct) enhances matrix synthesis and tensile and compressive mechanical properties. Combined, TFP and Low density interact to further enhance construct properties. That is, with TFP, Low density increases type II collagen content by over 100%, tensile stiffness by over 300%, and compressive moduli by over 140%, compared with High density. In conclusion, the interaction of TFP and Low density seeding enhances construct material properties, allowing for a mechanically functional, biomimetic cartilage to be formed using clinically relevant costochondral cells.

Objective: To describe CT findings in dogs and cats with temporomandibular joint (TMJ) disorders., Design: Retrospective case series., Animals: 41 dogs and 17 cats., Procedures: Medical records and CT images of the skull were reviewed for dogs and cats that were examined at a dentistry and oral surgery specialty practice between 2006 and 2011., Results: Of 142 dogs and 42 cats evaluated, 41 dogs and 17 cats had CT findings consistent with a TMJ disorder. In dogs, the most common TMJ disorder was osteoarthritis; however, in most cases, there were other TMJ disorders present in addition to osteoarthritis. Osteoarthritis was more frequently identified at the medial aspect rather than the lateral aspect of the TMJ, whereas the frequency of osteoarthritic involvement of the dorsal and ventral compartments did not differ significantly. In cats, fractures were the most common TMJ disorder, followed by osteoarthritis. Clinical signs were observed in all dogs and cats with TMJ fractures, dysplasia, ankylosis, luxation, and tumors; however, only 4 of 15 dogs and 2 of 4 cats with osteoarthritis alone had clinical signs., Conclusions and Clinical Relevance: Results indicated that TMJ disorders were frequently present in combination. Osteoarthritis was the most common TMJ disorder in dogs and the second most common TMJ disorder in cats. Computed tomography should be considered as a tool for the diagnosis of TMJ disorders in dogs and cats with suspected orofacial disorders and signs of pain. (J Am Vet Med Assoc 2013;242:69-75).

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

Articular cartilage and the menisci of the knee joint lack intrinsic repair capacity; thus, injuries to these tissues result in eventual osteoarthrotic changes to the joint. Tissue engineering offers the potential to replace damaged cartilage and mitigate long-term debilitating changes to the joint. In an attempt to enhance the ability of adult articular chondrocytes (ACs) and meniscus cells (MCs) to produce robust scaffoldless neocartilage, the effects of passage number, cryopreservation, and redifferentiation prior to construct formation were studied. By increasing passage number, smaller donor biopsies could be used to generate sufficient cells for tissue engineering and, in this study, no detrimental effects were observed when employing passage-4 versus passage-3 cells. Cryopreservation of cells would enable the generation of a cell bank thus reducing lead time and enhancing consistency of cell-based therapies. Interestingly, cryopreservation was shown to enhance the biomechanical properties of the resultant self-assembled constructs. With regard to redifferentiation prior to construct formation, aggregate redifferentiation was shown to enhance the biochemical and biomechanical properties of self-assembled constructs. By increasing passaging number, cryopreserving cells, and applying aggregate redifferentiation prior to neotissue formation, the utility of ACs and MCs in tissue engineering can be enhanced., (Copyright © 2013 S. Karger AG, Basel.)

This study determined the effects of exogenous hyaluronic acid (HA) on the biomechanical and biochemical properties of self-assembled bovine chondrocytes, and investigated biophysical and genetic mechanisms underlying these effects. The effects of HA commencement time, concentration, application duration and molecular weight were examined using histology, biomechanics and biochemistry. Additionally, the effects of HA application on sulphated glycosaminoglycan (GAG) retention were assessed. To investigate the influence of HA on gene expression, microarray analysis was conducted. HA treatment of developing neocartilage increased compressive stiffness onefold and increased sulphated GAG content by 35 per cent. These effects were dependent on HA molecular weight, concentration and application commencement time. Additionally, applying HA increased sulphated GAG retention within self-assembled neotissue. HA administration also upregulated 503 genes, including multiple genes associated with TGF-β1 signalling. Increased sulphated GAG retention indicated that HA could enhance compressive stiffness by increasing the osmotic pressure that negatively charged GAGs create. The gene expression data demonstrate that HA treatment differentially regulates genes related to TGF-β1 signalling, revealing a potential mechanism for altering matrix composition. These results illustrate the potential use of HA to improve cartilage regeneration efforts and better understand cartilage development.

The biomechanisms that govern the response of chondrocytes to mechanical stimuli are poorly understood. In this study, a series of in vitro tests are performed, in which single chondrocytes are subjected to shear deformation by a horizontally moving probe. Dramatically different probe force-indentation curves are obtained for untreated cells and for cells in which the actin cytoskeleton has been disrupted. Untreated cells exhibit a rapid increase in force upon probe contact followed by yielding behaviour. Cells in which the contractile actin cytoskeleton was removed exhibit a linear force-indentation response. In order to investigate the mechanisms underlying this behaviour, a three-dimensional active modelling framework incorporating stress fibre (SF) remodelling and contractility is used to simulate the in vitro tests. Simulations reveal that the characteristic force-indentation curve observed for untreated chondrocytes occurs as a result of two factors: (i) yielding of SFs due to stretching of the cytoplasm near the probe and (ii) dissociation of SFs due to reduced cytoplasm tension at the front of the cell. In contrast, a passive hyperelastic model predicts a linear force-indentation curve similar to that observed for cells in which the actin cytoskeleton has been disrupted. This combined modelling-experimental study offers a novel insight into the role of the active contractility and remodelling of the actin cytoskeleton in the response of chondrocytes to mechanical loading.

Articular cartilage was predicted to be one of the first tissues to successfully be regenerated, but this proved incorrect. In contrast, bone (but also vasculature and cardiac tissues) has seen numerous successful reparative approaches, despite consisting of multiple cell and tissue types and, thus, possessing more complex design requirements. Here, we use bone-regeneration successes to highlight cartilage-regeneration challenges: such as selecting appropriate cell sources and scaffolds, creating biomechanically suitable tissues, and integrating to native tissue. We also discuss technologies that can address the hurdles of engineering a tissue possessing mechanical properties that are unmatched in human-made materials and functioning in environments unfavorable to neotissue growth.

Central to understanding mechanotransduction in the knee meniscus is the characterization of meniscus cell mechanics. In addition to biochemical and geometric differences, the inner and outer regions of the meniscus contain cells that are distinct in morphology and phenotype. This study investigated the regional variation in meniscus cell mechanics in comparison with articular chondrocytes and ligament cells. It was found that the meniscus contains two biomechanically distinct cell populations, with outer meniscus cells being stiffer (1.59 ± 0.19 kPa) than inner meniscus cells (1.07 ± 0.14 kPa). Additionally, it was found that both outer and inner meniscus cell stiffnesses were similar to ligament cells (1.32 ± 0.20 kPa), and articular chondrocytes showed the highest stiffness overall (2.51 ± 0.20 kPa). Comparison of compressibility characteristics of the cells showed similarities between articular chondrocytes and inner meniscus cells, as well as between outer meniscus cells and ligament cells. These results show that cellular biomechanics vary regionally in the knee meniscus and that meniscus cells are biomechanically similar to ligament cells. The mechanical properties of musculoskeletal cells determined in this study may be useful for the development of mathematical models or the design of experiments studying mechanotransduction in a variety of soft tissues.

Biomechanics plays a pivotal role in articular cartilage development, pathophysiology, and regeneration. During embryogenesis and cartilage maturation, mechanical stimuli promote chondrogenesis and limb formation. Mechanical loading, which has been characterized using computer modeling and in vivo studies, is crucial for maintaining the phenotype of cartilage. However, excessive or insufficient loading has deleterious effects and promotes the onset of cartilage degeneration. Informed by the prominent role of biomechanics, mechanical stimuli have been harnessed to enhance redifferentiation of chondrocytes and chondroinduction of other cell types, thus providing new chondrocyte cell sources. Biomechanical stimuli, such as hydrostatic pressure or compression, have been used to enhance the functional properties of neocartilage. By identifying pathways involved in mechanical stimulation, chemical equivalents that mimic mechanical signaling are beginning to offer exciting new methods for improving neocartilage. Harnessing biomechanics to improve differentiation, maintenance, and regeneration is emerging as pivotal toward producing functional neocartilage that could eventually be used to treat cartilage degeneration.