StarPEG/heparin-hydrogel based in vivo engineering of stable bizonal cartilage with a calcified bottom layer

Abstract

Repaired cartilage tissue lacks the typical zonal structure of healthy native cartilage needed for appropriate function. Current grafts for treatment of full thickness cartilage defects focus primarily on a nonzonal design and this may be a reason why inferior nonzonal regeneration tissue developed in vivo. No biomaterial-based solutions have been developed so far to induce a proper zonal architecture into a non-mineralized and a calcified cartilage layer. The objective was to grow bizonal cartilage with a calcified cartilage bottom zone wherein main tissue development is occurring in vivo. We hypothesized that starPEG/heparin-hydrogel owing to the glycosaminoglycan heparin contained as a building-block would prevent mineralization of the upper cartilage zone and be beneficial in inhibiting long-term progression of calcified cartilage into bone. MSCs were pre-cultured as self-assembling non-mineralized cell discs before a chondrocyte-seeded fibrin- or starPEG/heparin-hydrogel layer was cast on top directly before ectopic implantation. Bizonal cartilage with a calcified bottom-layer developed in vivo showing stronger mineralization compared to in vitro samples, but the hydrogel strongly determined outcome. Zonal fibrin-constructs lost volume and allowed non-organized expansion of collagen type X, ALP-activity and mineralization from the bottom-layer into upper regions, whereas zonal starPEG/heparin-constructs were of stable architecture. While non-zonal MSCs-derived discs formed bone over 12 weeks, the starPEG/heparin-chondrocyte layer prevented further progression of calcified cartilage into bone tissue. Conclusively, starPEG/heparin-hydrogel-controlled and cell-type mediated spatiotemporal regulation allowed in vivo growth of bizonal cartilage with a stable calcified cartilage layer. Altogether our work is an important milestone encouraging direct in vivo growth of organized cartilage after biofabrication.

Treatment of Focal Cartilage Defects in Minipigs with Zonal Chondrocyte/Mesenchymal Progenitor Cell Constructs

“…Zonal designs mimicking the vertical organization of cells and matrix in a joint are promising tools to enhance cartilage regeneration. Several studies have shown that not only are such designs technically feasible [17,18] but that they can improve integration into osteochondral defects [14,19,20]. The importance of the calcified cartilage has already been depicted in 1975 [21].…”

“…In this study, we compared a zonal construct consisting of two layers with two different cell types to a non-zonal design for cartilage regeneration. We hypothesized that the zonal approach would facilitate integration [18,24,25,26] into the defects and would result in a more natural tissue when compared to a non-zonal design and to empty control defects. After six months in vivo, zonal and non-zonal constructs showed a significantly increased bone loss in the defects when compared to controls.…”

Differential Production of Cartilage ECM in 3D Agarose Constructs by Equine Articular Cartilage Progenitor Cells and Mesenchymal Stromal Cells

“…In the field of cartilage tissue engineering, several different approaches exist that try to produce cartilage constructs with zones similar to the native structure. Such zones are, for example, defined by the use of different cells (zone-specific chondrocytes, MSCs) [ 26 , 27 , 28 ] or different material properties (stiffness gradients, different hydrogels) [ 29 , 30 , 31 , 32 ]. The combination of ACPCs in a superficial layer and MSCs in a bottom layer has been tested in other hydrogels with encouraging results [ 18 , 20 , 21 ].…”

Significance of MEF2C and RUNX3 Regulation for Endochondral Differentiation of Human Mesenchymal Progenitor Cells

“…Of course, strong promotors may be able to drive overexpression also in the pellet center, in spite of an inappropriate microenvironment, but this would not add to our understanding of the natural mechanisms of MPC hypertrophy we are searching for, since expression of downstream targets like IHH and IBSP may also depend on the richer microenvironment. Thus, we propose that further understanding of the here-described 3D phenomena of chondrocyte hypertrophy requires new models and techniques that combine forced target gene expression with exposure to critical microenvironmental cues like this is possible by layered cartilage tissue design and layered tissue mineralization in transwells (Kunisch et al, 2018).…”