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-ß 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 ~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-ß 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.

Dexamethasone Release from Within Engineered Cartilage as a Chondroprotective Strategy Against Interleukin-1α.

While significant progress has been made toward engineering functional cartilage constructs with mechanical properties suitable for in vivo loading, the impact on these grafts of inflammatory cytokines, chemical factors that are elevated with trauma or osteoarthritis, is poorly understood. Previous work has shown dexamethasone to be a critical compound for cultivating cartilage with functional properties, while also providing chondroprotection from proinflammatory cytokines. This study tested the hypothesis that the incorporation of poly(lactic-co-glycolic acid) (PLGA) (75:25) microspheres that release dexamethasone from within chondrocyte-seeded agarose hydrogel constructs would promote development of constructs with functional properties and protect constructs from the deleterious effects of interleukin-1α (IL-1α). After 28 days of growth culture, experimental groups were treated with IL-1α (10 ng/mL) for 7 days. Reaching native equilibrium moduli and proteoglycan levels, dexamethasone-loaded microsphere constructs exhibited tissue properties similar to microsphere-free control constructs cultured in dexamethasone-supplemented culture media and were insensitive to IL-1α exposure. These findings are in stark contrast to constructs containing dexamethasone-free microspheres or no microspheres, cultured without dexamethasone, where IL-1α exposure led to significant tissue degradation. These results support the use of dexamethasone delivery from within engineered cartilage, through biodegradable microspheres, as a strategy to produce mechanically functional tissues that can also combat the deleterious effects of local proinflammatory cytokine exposure.

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.

Tissue-engineered articular cartilage exhibits tension-compression nonlinearity reminiscent of the native cartilage.

The tensile modulus of articular cartilage is much larger than its compressive modulus. This tension-compression nonlinearity enhances interstitial fluid pressurization and decreases the frictional coefficient. The current set of studies examines the tensile and compressive properties of cylindrical chondrocyte-seeded agarose constructs over different developmental stages through a novel method that combines osmotic loading, video microscopy, and uniaxial unconfined compression testing. This method was previously used to examine tension-compression nonlinearity in native cartilage. Engineered cartilage, cultured under free-swelling (FS) or dynamically loaded (DL) conditions, was tested in unconfined compression in hypertonic and hypotonic salt solutions. The apparent equilibrium modulus decreased with increasing salt concentration, indicating that increasing the bath solution osmolarity shielded the fixed charges within the tissue, shifting the measured moduli along the tension-compression curve and revealing the intrinsic properties of the tissue. With this method, we were able to measure the tensile (401±83kPa for FS and 678±473kPa for DL) and compressive (161±33kPa for FS and 348±203kPa for DL) moduli of the same engineered cartilage specimens. These moduli are comparable to values obtained from traditional methods, validating this technique for measuring the tensile and compressive properties of hydrogel-based constructs. This study shows that engineered cartilage exhibits tension-compression nonlinearity reminiscent of the native tissue, and that dynamic deformational loading can yield significantly higher tensile properties.

Bi-unicondylar knee replacement laxity with changes to simulated soft tissue constraints.

Unicondylar knee replacement systems have been shown to perform comparably to total knee replacements, while being much less surgically invasive. Proper ligament balancing, as well as knee laxity, has been shown to play an important role in optimizing kinematic behavior of these implant systems and improving long-term survival of the implant. This study investigates the effect of different simulated ligament laxity conditions of the anterior cruciate ligament and the posterior cruciate ligament on the resulting anteroposterior and mediolateral contact kinematics for medial and lateral pairs of UKR implants with flat and symmetric ultrahigh-molecular-weight polyethylene inserts during force-controlled ISO-14243-1 knee testing simulation. A novel method of capturing the tibiofemoral lowest point contact path was used to calculate the shear plane lowest point contact path kinematics in both the anteroposterior and the mediolateral directions. The results illustrated that multiple clinically relevant soft tissue configurations produce statistically different measured knee kinematics in unicondylar knee replacement systems than is seen in accepted "standard" knee simulator protocols with 95% confidence interval. The observed kinematic differences in anteroposterior and mediolateral movement from what was observed using standard wear testing protocols could aid in the development of unicondylar knee replacement design enhancements that are resistant to varying soft tissue deficiencies.