Osteochondral (OC) disorders like osteoarthritis (OA) and rheumatoid arthritis (RA) damage the joint's cartilage and subchondral bone. Their treatment remains a significant challenge for both researchers and orthopedics. In vitro models of OC tissue have become an essential tool to help investigate pathogenesis, develop drug screening, and test potential therapeutic approaches. This study aims to create a bio-printed OC construct recapitulating the bone and cartilage compartment as drugs testing platforms.
Two different hydrogels including a blend composed of gelatin methacrylate (GelMA) with nanosized hydroxyapatite (nHA) and tyramine-modified hyaluronic acid (THA) were selected for the bioprinting of bone and cartilage tissue mimics. The composition of GelMA hydrogel (10% w/v) with different concentrations of nHA (1-10% w/v) and THA with concentrations of 2.5-5% w/v were characterized by rheology and their cytotoxicity was assessed via live-dead assay. Later, the pre-differentiated osteoblast and endothelial cells were encapsulated into GelMA-nHA and micropellet chondrocytes into THA hydrogels for bioprinting osteochondral construct. After 2 weeks of culturing, the successful generation of OC tissue was confirmed by real-time RT-PCR and histology.
The storage modulus (G') of all GelMA/nHA hydrogels was significantly higher than GelMA, however, there was no significant difference in G' values for the GelMA/nHA as a function of added nHA. Due to the know temperature sensitivity of GelMA, a rheological temperature sweep and series of printing tests were performed to establish a suitable printing temperature, which was confirmed to be 20°C, independent of the addition of nHA. Calcein-AM (Ca-AM) and Ethidium Homodimer-1 (EthD-1) staining for GelMA (10% w/v) with three concentrations of nHA (1, 3, and 5% w/v) at 2, 24, 72, and 168 h after printing showed the percentage of living cells after 72h in GelMA containing 3 and 5% (w/v) nHA was less than 50%, while in GelMA with 1% (w/v) nHA it remained high (>95%) even after 168h. Therefore, this formulation was chosen for the subsequent generation of bone tissue mimic.
Shear flow curves of THA hydrogels showed an increase in viscosity as a function of THA concentration. The damping factor, which is a ratio between the loss modulus G'' and the storage modulus G', has been shown to be directly related to the extrudability. The calculated damping factors for each concentration of THA (%w/v) (THA 2.5%= 0.4947 ± 0.038, THA3.5%= 0.5935 ± 0.012, and THA5%= 0.7391 ± 0.039), indicated that THA3.5%(w/v) was in the printable range. Cell viability assays for THA hydrogels showed a high percentage of living cells for THA 3.5% (w/v) compared to THA 5% (w/v) after 168 h. Based on cell viability assay, viscosity, and printability, a 3.5%(w/v) concentration of THA was selected for generating cartilage tissue mimic part.
We developed GelMA-nHA and THA hydrogels for bone and cartilage parts respectively. We also optimized printing parameters based on printability and shape fidelity and cell density according to cell viability for bioprinting OC constructs.
Keywords: Osteochondral tissue, Bioprinting, Hydrogels, Rheology, Drug discovery
 Petta et.al, 2018, ACS Biomater. Sci. Eng, DOI: 10.1021/acsbiomaterials.8b00416.