Introduction

The regeneration of cartilage tissue in vitro is often dependent on a scaffold material with the biomimetic properties. Ideally the scaffold should mimic the properties of native extracellular matrix: it should allow the cells to divide, secrete phenotypic matrix molecules and also to remodeling the matrix in response to microinjuries. Hydrogels are often used as a scaffold material due to their high water content and inherent biocompatibility. As a polymer backbone, hyaluronan is one of the most common materials used as it is a component of the native cartilage extracellular matrix and has multiple groups for chemical functionalization. In this study we have explored the use of a hyaluronic acid hydrogel (HATG) in the presence and absence of alginate. The hydrogel undergoes crosslinking in the presence of activated Factor XIII. We studied the maturation of these chondrocyte-containing constructs using mechanical testing (compression and bioindentation measurements) and histological evaluations.

In order to reproduce more complex structures which could be used to treat patient specific defects, it is possible to render HA-TG processible in a bioprinter. To acheive this, nanofibers were added to the HATG/HATG-alg solutions and the rheological properties of the bioink measured. We able to bioprint HATG into unique shapes and investigate their properties in vitro and in vivo in subcutaneous nude rat models . In future work, we will also explore the challenges of cultivating large tissue and potential benefits of bioreactors and gene editing of the cells to improve the mechanical stability of the printed cartilage.

Content

Human chondrocytes were isolated from biopsies taken with informed patient consent. Chondrocytes were expanded to passage 3 in media containing fetal bovine serum, TGFb3 and FGF2. Cells were trypsinized and encapsulated in the HATG or HATG-Alg materials at a cell density of 15mio cells/ml and the constructs were cultured for up to 9 weeks in TGFb3 containing DMEM. The stability of the cartilage constructs in vivo was explored by transplanted the discs in a subcutaneous nude rat model.

The chondrocytes in HATG and HATG-Alg discs showed increase in modulus approaching 1 MPa after 9 week in vitro culture, with maturation of the cartilage more efficient in HATG-Alg. By day 21 of culture, the chondrocyte viability in both HATG and HATG-alg approached over 90%. Safranin O staining likewise increased over time in culture, approaching the staining intensity of native auricular cartilage. Immunohistochemistry analyses revealed copious deposition of both collagen II and collagen I types as well as elastin staining. Large, bioprinted HATG and HATG-alg constructs developed slower and showed poor structural stability in vivo, perhaps due to inadequate diffusion of nutrients.

In summary, HATG and in particular HATG-Alg provide a 3D environment highly conducive to the formation of robust cartilage tissue in vitro. Methods to improve the in vivo tissue stability of large constructs are being explored and include the addition of porosity in the scaffold, enhanced crosslinking of the scaffold, the use of bioreactors and increasing the stability of the cells through gene editing.

References

Fisch et al, Advanced Functional Materials, https://doi.org/10.1002/adfm.202008261

Acknowledgments

Funding from the Swiss National Science Foundation CRSII5_173868 is gratefully acknowledged.