The development of a biomimetic cell-based construct for osteochondral regeneration using 3D bioprinting technology.

The edges of long bones that meet in a joint are covered with articular cartilage, a highly specialized tissue that protects the epiphyses from the friction and compression forces exerted in a joint. The joint surface cartilage can be damaged due to injury, disease or chronic conditions. Unfortunately, the articular cartilage has limited healing potential and defects in the articular surface can progress into the underlying subchondral bone. The resulting osteochondral defects cause pain and mobility impairment for the patient, and when left untreated, further damage to the articular cartilage, post-traumatic osteoarthritis and deformation of the subchondral bone can occur. Treatments like joint debridement and microfracture focus on temporary symptom relief and will typically result in the formation of mechanically inferior fibrocartilage. The presence of fibrocartilage is a factor that predisposes the joint for osteoarthritis, and in severe cases of osteoarthritis, a total knee replacement is required. However, as prosthetics have limited life spans, they are not a good solution for younger patients. Therefore, it is important that osteochondral defects are treated and that hyaline cartilage at the joint surface is reestablished. Current restorative treatments for deep osteochondral defects imply the transfer of healthy osteochondral tissue plugs from a donor or from a non-load bearing region of the joint to the defect site. Since healthy tissue is not always available and there are risks such as donor site morbidity, alternative treatment strategies are investigated.

Tissue engineering aims to develop a sustainable strategy to regenerate functional joint surface tissue, while avoiding secondary damage, as caused by large grafts. In cell-based approaches, a tissue engineered construct with tissue-forming cells from an allogeneic or autologous source is implanted with the goal to regenerate the joint surface. Incorporating biomaterials, and hydrogels in particular, as a supportive matrix for the tissue-forming cells can enhance tissue formation and allows easy handling of the tissue engineered construct. Moreover, cell-laden hydrogel constructs are suitable for use in various processing techniques, including automated fabrication.

Our strategy in this PhD study was to encapsulate potent matrix-forming cells, that are able to robustly form different skeletal tissues in vivo, in a biocompatible hydrogel. Gelatin methacryloyl (GelMA) is a semi-synthetic hydrogel based on hydrolyzed collagen. GelMA combines a natural polymer with properties similar to the native extracellular matrix, thus ensuring biocompatibility and biodegradability, with the advantages of a synthetic hydrogel allowing tunability of its mechanical properties. For articular cartilage tissue engineering, chondrocytes derived from the joint are the optimal cell type. Yet, obtaining the required quantity of healthy articular chondrocytes is challenging due to donor site morbidity, the lack of healthy chondrocytes in a diseased joint and dedifferentiation upon expansion in vitro. The solution we propose for these problems is the use of iPSC-derived chondrocytes, i.e. chondrocytes obtained from the differentiation of induced pluripotent stem cells (iPSCs). iPSCs are somatic cells that have been reprogrammed to their pluripotent state. These cells display limited immunogenicity and can be expanded before differentiation, allowing to obtain large numbers of functional chondrocytes.

First, we investigated different cell encapsulation densities and evaluated the in vitro behavior and matrix formation of iPSC-derived chondrocytes in GelMA hydrogel constructs. We found that the cells were able to produce cartilaginous matrix, displaying high expression of chondrogenic markers. Next, we evaluated these constructs in vivo in an ectopic setting, with as well as without an in vitro matrix formation period prior to implantation. We found that 3-week pre-cultured constructs generated hyaline-like cartilage after 4 weeks in vivo in the dorsal subcutaneous pockets of nude mice. Uncultured constructs were also able to generate abundant cartilage-like matrix in vivo, albeit less mature. To make the transition between tissue engineered cartilage and the subchondral bone at the bottom of the defect, we selected skeletal progenitors derived from the periosteum as potent candidates. These multipotent human periosteum-derived cells (hPDCs) play an important role in bone development as well as in fracture healing. We tested hPDCs in the same GelMA-based constructs and found that the cartilaginous matrix formed after in vitro culture in serum-free differentiation medium undergoes endochondral ossification in vivo. After 4 weeks, mineralized cartilage and cortical bone was formed ectopically. With the aim of using the iPSC-derived chondrocytes-laden GelMA and hPDC-laden GelMA in a dual approach, we repeated the experiments with iPSC-derived chondrocytes in the same serum-free differentiation medium. Using a single differentiation medium drastically simplifies the set-up of culturing a dual construct in vitro. We observed that also in these medium conditions, the iPSC-derived chondrocytes were able to form cartilaginous matrix in vivo, with the maturity of the tissue depending on the length of the in vitro pre-culture. Finally, we evaluated the combination of iPSC-derived chondrocyte- and hPDC-laden GelMA hydrogel in vivo in an orthotopic setting. Constructs with and without in vitro pre-culture were implanted in critically-sized osteochondral defects in the knee joint of nude rats, providing a proof-of-concept of tissue regeneration using iPSC-derived chondrocytes and hPDCs encapsulated in GelMA. However, there was room for improvement in terms of construct integration, possibly due to limitations to the construct fabrication method. As a follow-up, the two cell-laden hydrogels were applied as biological inks in an extrusion-based bioprinting set-up, to investigate the feasibility of automated fabrication of dual constructs. We concluded that, according to our in vitro studies, bioprinted individual iPSC-derived chondrocytes and hPDCs in GelMA were biologically functional, and could be tested in an orthotopic environment.

Taken together, we demonstrated the potential of the encapsulation of individual cells in a biocompatible and bioprintable hydrogel for cartilage and osteochondral tissue engineering. This approach has been evaluated in vitro and in vivo in small animal models, demonstrating the potential of this strategy to fabricate tissue engineered constructs for the treatment of joint surface defects.