OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model

Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients

Self-healing hydrogels formed by complexation between calcium ions and bisphosphonate-functionalized star-shaped polymers

Star-shaped poly(ethylene glycol) (PEG) chain termini were functionalized with alendronate to create transient networks with reversible crosslinks upon addition of calcium ions. The gelation ability of alendronate-functionalized PEG was greatly dependent on the number of arms and arm molecular weight. After mixing polymer and calcium solutions, the formed hydrogels could be cut and then brought back together without any visible interface. After 2 minutes of contact, their connection was strong enough to allow for stretching without tearing through the previous fracture surface. Oscillatory rheology showed that the hydrogels recovered between 70 and 100% of the original storage and loss modulus after rupture. Frequency sweep measurements revealed a liquid-like behavior at lower frequencies and solid-like at high frequencies. Shifting frequency curves obtained at different calcium and polymer concentrations, all data collapsed in a single common master curve. This time-concentration superposition reveals a common relaxation mechanism intrinsically connected to the calcium-bisphosphonate complexation equilibrium