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Articular cartilage injuries are a common source of joint pain and dysfunction. We hypothesized that pulsed electromagnetic fields (PEMFs) would improve growth and healing of tissue-engineered cartilage grafts in a direction-dependent manner. PEMF stimulation of engineered cartilage constructs was first evaluated in vitro using passaged adult canine chondrocytes embedded in an agarose hydrogel scaffold. PEMF coils oriented parallel to the articular surface induced superior repair stiffness compared to both perpendicular PEMF (p = .026) and control (p = .012). This was correlated with increased glycosaminoglycan deposition in both parallel and perpendicular PEMF orientations compared to control (p = .010 and .028, respectively). Following in vitro optimization, the potential clinical translation of PEMF was evaluated in a preliminary in vivo preclinical adult canine model. Engineered osteochondral constructs (∅ 6 mm × 6 mm thick, devitalized bone base) were cultured to maturity and implanted into focal defects created in the stifle (knee) joint. To assess expedited early repair, animals were assessed after a 3-month recovery period, with microfracture repairs serving as an additional clinical control. In vivo, PEMF led to a greater likelihood of normal chondrocyte (odds ratio [OR]: 2.5, p = .051) and proteoglycan (OR: 5.0, p = .013) histological scores in engineered constructs. Interestingly, engineered constructs outperformed microfracture in clinical scoring, regardless of PEMF treatment (p < .05). Overall, the studies provided evidence that PEMF stimulation enhanced engineered cartilage growth and repair, demonstrating a potential low-cost, low-risk, noninvasive treatment modality for expediting early cartilage repair.

Articular cartilage defects are a common source of joint pain and dysfunction. We hypothesized that sustained low-dose dexamethasone (DEX) delivery via an acellular osteochondral implant would have a dual pro-anabolic and anti-catabolic effect, both supporting the functional integrity of adjacent graft and host tissue while also attenuating inflammation caused by iatrogenic injury. An acellular agarose hydrogel carrier with embedded DEX-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres (DLMS) was developed to provide sustained release for at least 99 days. The DLMS implant was first evaluated in an in vitro pro-inflammatory model of cartilage degradation. The implant was chondroprotective, as indicated by maintenance of Young's modulus (EY) (p = 0.92) and GAG content (p = 1.0) in the presence of interleukin-1β insult. In a subsequent preliminary in vivo experiment, an osteochondral autograft transfer was performed using a pre-clinical canine model. DLMS implants were press-fit into the autograft donor site and compared to intra-articular DEX injection (INJ) or no DEX (CTL). Functional scores for DLMS animals returned to baseline (p = 0.39), whereas CTL and INJ remained significantly worse at 6 months (p Statement of significance Articular cartilage defects are a common source of joint pain and dysfunction. Effective treatment of these injuries may prevent the progression of osteoarthritis and reduce the need for total joint replacement. Dexamethasone, a potent glucocorticoid with concomitant anti-catabolic and pro-anabolic effects on cartilage, may serve as an adjuvant for a variety of repair strategies. Utilizing a dexamethasone-loaded osteochondral implant with controlled release characteristics, we demonstrated in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes following osteochondral repair. These improved outcomes were correlated with superior histological cartilage scores and minimal-to-no comorbidity, which is a risk with high dose dexamethasone injections. Using this model of cartilage restoration, we have for the first time shown the application of targeted, low-dose dexamethasone for improved healing in a preclinical model of focal defect repair.

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 Ø3–4 mm × 2.3 mm, with cell seeding densities ranging from 15 to 90 × 106 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 Young’s moduli of up to ~250 kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950 kPa (0.01 Hz), and contained 5.7 percent 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.

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.

Fibroblast-like synoviocytes (FLS) reside in the synovial membrane of diarthrodial joints and are exposed to a dynamic fluid environment that presents both physical and chemical stimuli. The ability of FLS to sense and respond to these stimuli plays a key role in their normal function, and is implicated in the alterations to function that occur in osteoarthritis (OA). The present work characterizes the response of FLS to fluid flow-induced shear stress via real-time calcium imaging, and tests the hypothesis that this response is modulated by interleukin-1α (IL-1α), a cytokine elevated in OA. FLS demonstrated a robust calcium signaling response to fluid shear that was dose dependent upon stress level and required both external and internal calcium sources. Preconditioning with 10 ng/mL IL-1α for 24 hrs heightened this shear stress response by significantly increasing the percent of responding cells and peak magnitude, while significantly decreasing the time for a peak to occur. Intercellular communication via gap junctions was found to account for a portion of the FLS population response in normal conditions, and was significantly increased by IL-1α preconditioning. IL-1α was also found to significantly increase average length and incidence of the primary cilia, an organelle commonly implicated in shear mechanosensing. These findings suggest that the elevated levels of IL-1α found in the OA environment heighten FLS sensitivity to fluid shear by altering both intercellular communication and individual cell sensitivity, which could affect downstream functions and contribute to progression of the disease state.

Summary 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.

Osteoarthritis (OA) is a common and painful disorder characterized by the degeneration of the articular surfaces of diarthrodial joints. One of the most promising approaches to treat OA is the design of engineered tissue that reproduces the functional properties of healthy cartilage. This tissue engineering approach typically entails the use of living cells embedded in a three-dimensional scaffold that is cultured over time in a growth medium supplemented with various physical or biological stimulants to encourage development. Recently our laboratory has been able to cultivate engineered cartilage with an equilibrium modulus (EY) and an glycosaminoglycan (GAG) content that match the native tissue using the temporal application of growth factors over a six-week culture period1. While these findings are encouraging, the dynamic modulus (G*) and the collagen content for these constructs amount to less than a quarter of those of native articular cartilage. Of the two mechanical measurements, G*, which is correlated with the collagen content, is considered to be the more physiologically relevant as it measures the behavior of the tissue during the application of cyclic loads and better captures fluid pressurization within the biphasic tissue. No group has currently been able to reproduce native values of collagen regardless of the tissue engineering strategy employed. Obtaining native values of G* and collagen therefore remains an important but elusive challenge. Cross-linking agents such as glutaraldehyde, formaldehyde, or epoxy are typically used to chemically-treat (or fix) devitalized xenografts and allografts to reduce the immune rejection or the enzymatic degradation that is typical of transplanted bioprostheses2,3. Typically, however, cross-linking agents are lethal to cells. The use of genipin, a naturally occurring crosslinker with toxicity levels ten thousand fold less than glutaraldehyde4 as a biocompatible and stable cross-linker has been established by other groups5,6 and provides a promising new avenue to explore for tissue-engineering. Genipin cross-linking works by forming intra- and intermolecular cross-links of the amino residues on tropocollagen or proteoglycan molecules. A modified cyclic form of genipin can reside stably within the extracellular network, adding bridges across adjacent fibers. As such, previous groups have explored the use of genipin as a onetime treatment to fix tissue prior to implantation7, for the assembly of tissue-engineering scaffolds prior to cell seeding8,9, or in order to modulate the release of growth factors from degradable scaffolds or beads5. Genipin has also been studied for its anti-inflammatory effects, either administered directly to different cell lines10,11, or administered orally to animals12,13. Moreover, genipin cross-linking has been shown to affect the mechanical properties of biological tissues, increasing the tensile strength of bovine pericardium14, porcine tendon15, and type I collagen gels16. Our laboratory uses agarose hydrogel as a scaffold system for cartilage tissue engineering. This gel has been used extensively in chondrocyte biology studies and has shown some promise for tissue engineering applications. Agarose is a neutrally charged polysaccharide and as such is unaffected by genipin. However the chondrocytes embedded in the gel elaborate an extracellular matrix over time in culture that exhibits amine groups, which are subject to genipin cross-linking. In this set of studies we propose a novel use for genipin: not as a scaffold cross-linker, but as a medium supplement to promote cross-linking of de novo cell products as they are produced. We hypothesize that the application of genipin will stabilize the extracellular matrix components and increase the mechanical properties of developing cartilaginous tissue in our agarose hydrogels. We hypothesize two mechanisms through which the physical enhancement of tissue properties is fostered: (1) by reorganization and enhanced retention of cell synthesized extracellular matrix components, and (2) through reduction of the loss of extracellular matrix components by increasing their resilience to catabolic degradation.

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.

Electrospun fibrous scaffolds are being developed for the engineering of numerous tissues. Advantages of electrospun scaffolds include the similarity in fiber diameter to elements of the native extracellular matrix and the ability to align fibers within the scaffold to control and direct cellular interactions and matrix deposition. To further expand the range of properties available in fibrous scaffolds, we developed a process to electrospin photocrosslinkable macromers from a library of multifunctional poly(beta-amino ester)s. In this study, we utilized one macromer (A6) from this library for initial examination of fibrous scaffold formation. A carrier polymer [poly(ethylene oxide) (PEO)] was used for fiber formation because of limitations in electrospinning A6 alone. Various ratios of A6 and PEO were successfully electrospun and influenced the scaffold fiber diameter and appearance. When electrospun with a photoinitiator and exposed to light, the macromers crosslinked rapidly to high double bond conversions and fibrous scaffolds displayed higher elastic moduli compared to uncrosslinked scaffolds. When these fibers were deposited onto a rotating mandrel and crosslinked, organized fibrous scaffolds were obtained, which possessed higher moduli (approximately 4-fold) in the fiber direction than perpendicular to the fiber direction, as well as higher moduli (approximately 12-fold) than that of nonaligned crosslinked scaffolds. With exposure to water, a significant mass loss and a decrease in mechanical properties were observed, correlating to a rapid initial loss of PEO which reached an equilibrium after 7 days. Overall, these results present a process that allows for formation of fibrous scaffolds from a wide variety of possible photocrosslinkable macromers, increasing the diversity and range of properties achievable in fibrous scaffolds for tissue regeneration.

Summary Objective Galvanotaxis, the migratory response of cells in response to electrical stimulation, has been implicated in development and wound healing. The use of mesenchymal stem cells (MSCs) from the synovium (synovium-derived stem cells, SDSCs) has been investigated for repair strategies. Expansion of SDSCs is necessary to achieve clinically relevant cell numbers; however, the effects of culture passage on their subsequent cartilaginous extracellular matrix production are not well understood. Methods Over four passages of SDSCs, we measured the expression of cell surface markers (CD31, CD34, CD49c, CD73) and assessed their migratory potential in response to applied direct current (DC) electric field. Cells from each passage were also used to form micropellets to assess the degree of cartilage-like tissue formation. Results Expression of CD31, CD34, and CD49c remained constant throughout cell expansion; CD73 showed a transient increase through the first two passages. Correspondingly, we observed that early passage SDSCs exhibit anodal migration when subjected to applied DC electric field strength of 6 V/cm. By passage 3, CD73 expression significantly decreased; these cells exhibited cell migration toward the cathode, as previously observed for terminally differentiated chondrocytes. Only late passage cells (P4) were capable of developing cartilage-like tissue in micropellet culture. Conclusions Our results show cell priming protocols carried out for four passages selectively differentiate stem cells to behave like chondrocytes, both in their motility response to applied electric field and their production of cartilaginous tissue.