Andrea R. Tan

Differences in interleukin-1 response between engineered and native cartilage.

Unlike native cartilage explants that are used in autologous tissue transfer procedures, engineered cartilage constructs are typically highly fragile when first formed and must rely on cellular activity to develop over time. However, inflammatory cytokines such as interleukin-1alpha (IL-1alpha) are often present in target joints and may interfere with this development process. Herein we examine to what extent nascent engineered tissue is susceptible to chemical perturbations by IL-1alpha (10 ng/mL), especially when compared to native explants, and whether in vitro preconditioning may promote sufficient integrity to lessen this impact. The studies were carried out using a chemically defined medium supplemented with or without the antiinflammatory steroid dexamethasone. We find that engineered tissue (bovine chondrocytes in agarose hydrogel) at early time points (days 0 and 14) does not grow when exposed to the cytokine even temporarily, but both bovine explants and more developed engineered tissue (day 28) are able to withstand the same exposure without degradation of properties. We argue therefore that some in vitro preconditioning may be necessary to promote both sufficient mechanical integrity and the chemical fortitude without which insufficiently developed engineered constructs will not survive the harsh mechanochemical environment within the joint.

Genipin enhances the mechanical properties of tissue-engineered cartilage and protects against inflammatory degradation when used as a medium supplement.

Genipin is a naturally-derived biocompatible cross-linking agent commonly used to generate three dimensional tissue-engineered scaffolds or to fix biologically derived scaffolds prior to implantation. Here we propose a novel use for genipin as a long-term culture medium supplement to promote cross-linking of de novo cell products that are produced in engineered cartilage. We hypothesize that the application of genipin will stabilize the extracellular matrix components and increase the mechanical properties of developing engineered cartilage. Chondrocytes encapsulated in agarose hydrogel (a neutrally charged polysaccharide scaffold that is unaffected by genipin cross-linking) were cultured in a chemically-defined growth medium that was supplemented with varying concentrations of genipin (22 microM, 220 microM, 2200 microM) for various durations (continuous or intermittent). Tissues developed significantly higher mechanical properties (+28% dynamic modulus and +20% Youngs modulus) by day 42 with genipin treatment compared to untreated controls. These increases were not immediate, but presented over culture time after genipin treatment. The genipin treated groups were also more resistant to cytokine-induced degradation with interleukin-1alpha; maintaining an E(Y) (+218%), G* (+390%) and glycosaminoglycan (GAG) content (+477%) over genipin-untreated constructs subjected to interleukin. We hypothesize two mechanisms through which the physical enhancement of tissue properties may be fostered: (1) by cross-link mediated reorganization and enhanced retention of cell-elaborated extracellular matrix components, and (2) through reduction of the loss of extracellular matrix components by increasing their resilience to catabolic degradation. These studies demonstrate a potential use of genipin as a medium supplement to develop enhanced engineered cartilage.

Physiologic deformational loading does not counteract the catabolic effects of interleukin-1 in long-term culture of chondrocyte-seeded agarose constructs

An interplay of mechanical and chemical factors is integral to cartilage maintenance and/or degeneration. Interleukin-1 (IL-1) is a pro-inflammatory cytokine that is present at elevated concentrations in osteoarthritic joints and initiates the rapid degradation of cartilage when cultured in vitro. Several short-term studies have suggested that applied dynamic deformational loading may have a protective effect against the catabolic actions of IL-1. In the current study, we examine whether the long-term (42 days) application of dynamic deformational loading on chondrocyte-seeded agarose constructs can mitigate these catabolic effects. Three studies were carried out using two IL-1 isoforms (IL-1alpha and IL-1beta) in chemically defined medium supplemented with a broad range of cytokine concentrations and durations. Physiologic loading was unable to counteract the long-term catabolic effects of IL-1 under any of the conditions tested, and in some cases led to further degeneration over unloaded controls.

Response of engineered cartilage to mechanical insult depends on construct maturity

UNLABELLED Injury to articular cartilage leads to degenerative changes resulting in a loss of mechanical and biochemical properties. In engineered cartilage, the injury response of developing constructs is unclear. OBJECTIVE To characterize the cellular response of tissue-engineered constructs cultured in chemically-defined medium after mechanical insult, either by compression-induced cracking, or by cutting, as a function of construct maturity. METHODS Primary immature bovine articular chondrocytes (4-6 weeks) were encapsulated in agarose hydrogel (2%, 30 millioncells/mL) and cultured in chemically-defined medium supplemented with Transforming growth factor (TGF)-β3 (10ng/mL, first 2 weeks). At early (5 days) and late (35 days) times in culture, subsets of constructs were exposed to mechanical overload to produce a crack in the tissue or were exposed to a sharp wound with a perpendicular cut. Constructs were returned to culture and allowed to recover in static conditions. Mechanical and biochemical properties were evaluated at 2-week intervals to day 70, and cellular viability was assessed at 2-week intervals to day 85. RESULTS Constructs injured early in culture recovered their mechanical stiffness back to control values, regardless of the mode of injury. Later in culture, when constructs exhibited properties similar to those of native cartilage, compression-induced cracking catastrophically damaged the bulk matrix of the tissue and resulted in permanent mechanical failure with persistent cell death. No such detrimental outcomes were observed with cutting. Biochemical content was similar across all groups irrespective of mode or time of injury. CONCLUSIONS Unlike native cartilage, engineered cartilage constructs exhibit a reparative capacity when the bulk integrity of the developing tissue is preserved after injury.

Human chondrocyte migration behaviour to guide the development of engineered cartilage

Tissue‐engineering techniques have been successful in developing cartilage‐like tissues in vitro using cells from animal sources. The successful translation of these strategies to the clinic will likely require cell expansion to achieve sufficient cell numbers. Using a two‐dimensional (2D) cell migration assay to first identify the passage at which chondrocytes exhibited their greatest chondrogenic potential, the objective of this study was to determine a more optimal culture medium for developing three‐dimensional (3D) cartilage‐like tissues using human cells. We evaluated combinations of commonly used growth factors that have been shown to promote chondrogenic growth and development. Human articular chondrocytes (AC) from osteoarthritic (OA) joints were cultured in 3D environments, either in pellets or encapsulated in agarose. The effect of growth factor supplementation was dependent on the environment, such that matrix deposition differed between the two culture systems. ACs in pellet culture were more responsive to bone morphogenetic protein (BMP2) alone or combinations containing BMP2 (i.e. BMP2 with PDGF or FGF). However, engineered cartilage development within agarose was better for constructs cultured with TGFβ3. These results with agarose and pellet culture studies set the stage for the development of conditions appropriate for culturing 3D functional engineered cartilage for eventual use in human therapies. Copyright

Coculture of Engineered Cartilage With Primary Chondrocytes Induces Expedited Growth

BackgroundSoluble factors released from chondrocytes can both enhance and induce chondrocyte-like behavior in cocultured dedifferentiated cells. The ability to similarly prime and modulate biosynthetic activity of differentiated cells encapsulated in a three-dimensional environment is unknown.Questions/purposesTo understand the effect of coculture on engineered cartilage, we posed three hypotheses: (1) coculturing with a monolayer of chondrocytes (“chondrocyte feeder layer”) expedites and increases engineered tissue growth; (2) expedited growth arises from paracrine effects; and (3) these effects are dependent on the specific morphology and expression of the two-dimensional feeder cells.MethodsIn three separate studies, chondrocyte-laden hydrogels were cocultured with chondrocyte feeder layers. Mechanical properties and biochemical content were quantified to evaluate tissue properties. Histology and immunohistochemistry stains were observed to visualize each constituent’s distribution and organization.ResultsCoculture with a chondrocyte feeder layer led to stiffer tissue constructs (Young’s modulus and dynamic modulus) with greater amounts of glycosaminoglycan and collagen. This was dependent on paracrine signaling between the two populations of cells and was directly modulated by the rounded morphology and expression of the feeder cell monolayer.ConclusionsThese findings suggest a potential need to prime and modulate tissues before implantation and present novel strategies for enhancing medium formulations using soluble factors released by feeder cells.Clinical RelevanceDetermining the soluble factors present in the coculture system can enhance a chondrogenic medium formulation for improved growth of cartilage substitutes. The feeder layer strategy described here may also be used to prime donor cartilage allografts before implantation to increase their success in vivo.

Fabrication of tissue engineered osteochondral grafts for restoring the articular surface of diarthrodial joints.

Osteochondral allograft implantation is an effective cartilage restoration technique for large defects (>10 cm(2)), though the demand far exceeds the supply of available quality donor tissue. Large bilayered engineered cartilage tissue constructs with accurate anatomical features (i.e. contours, thickness, architecture) could be beneficial in replacing damaged tissue. When creating these osteochondral constructs, however, it is pertinent to maintain biofidelity to restore functionality. Here, we describe a step-by-step framework for the fabrication of a large osteochondral construct with correct anatomical architecture and topology through a combination of high-resolution imaging, rapid prototyping, impression molding, and injection molding.

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Youngs moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

Concise Review: Mesenchymal Stem Cells for Functional Cartilage Tissue Engineering: Taking Cues from Chondrocyte-Based Constructs

Osteoarthritis, the most prevalent form of joint disease, afflicts 9% of the U.S. population over the age of 30 and costs the economy nearly

Engineering Functional Cartilage Grafts

100 billion annually in healthcare and socioeconomic costs. It is characterized by joint pain and dysfunction, though the pathophysiology remains largely unknown. Due to its avascular nature and limited cellularity, articular cartilage exhibits a poor intrinsic healing response following injury. As such, significant research efforts are aimed at producing engineered cartilage as a cell‐based approach for articular cartilage repair. However, the knee joint is mechanically demanding, and during injury, also a milieu of harsh inflammatory agents. The unforgiving mechano‐chemical environment requires tissue replacements that are capable of bearing such burdens. The use of mesenchymal stem cells (MSCs) for cartilage tissue engineering has emerged as a promising cell source due to their ease of isolation, capacity to readily expand in culture, and ability to undergo lineage‐specific differentiation into chondrocytes. However, to date, very few studies utilizing MSCs have successfully recapitulated the structural and functional properties of native cartilage, exposing the difficult process of uniformly differentiating stem cells into desired cell fates and maintaining the phenotype during in vitro culture and after in vivo implantation. To address these shortcomings, here, we present a concise review on modulating stem cell behavior, tissue development and function using well‐developed techniques from chondrocyte‐based cartilage tissue engineering. Stem Cells Translational Medicine 2017;6:1295–1303