Biofabrication of osteochondral tissue by stem cell spheroids engineered with biomimetic fibers and 3D printed micro-chambers

Abstract

Osteochondral tissue is composed of cartilage and subchondral bone, which are tightly connected by extracellular matrix (ECM) molecules and collagen fibrils. The distinct biological and mechanical property of these tissues support and enable the body movement. Despite this importance, diseases such as bone fracture, osteoporosis, arthritis, and other trauma condition occurring at osteochondral tissue may not be inevitable because of aging and wearing out. Current surgical treatments such as allograft, autograft, and implanting metallic substrates might be often limited because of unsuitable size, immune response, and less regenerative effect. These limitations have led to the investigation and development of new therapeutic methods based on tissue engineering. Notably delivering stem cells has been widely investigated because of its high therapeutic potential. For example, micro- or nano-scale cells-aggregates such as cells sheets and spheroids, which showed advanced in vitro differentiation than single cells, were developed to deliver the therapeutic cells to patients. Nevertheless, the method showed several limitations such as limited cells-extracellular matrix (ECM) interactions, limited viability, uncontrolled differentiation, limited in vivo regeneration and vascularization, unstable localization, and difficulty to manufacturing complex hierarchical structure of osteochondral tissue. In here, I developed a spheroid that incorporate ECM mimicking poly(ι-lactic acid) fragmented fibers (length: ~100 m) within human adipose-derived stem cells (hADSCs) spheroid to enhance the viability and control the differentiation of stem cells. The fibers were homogenously distributed in the spheroid and supported increased cells viability than cells-only spheroids which showed many apoptotic signals after culturing 7 days. Furthermore, the largest size of spheroid showed the greatest release of angiogenic factors, while the smallest one showed the greater effects in osteogenic differentiation. However, the effects on in vivo regeneration and engineering the vascularized structure of the natural tissue remain a challenge. So I advanced the osteogenic inductivities of the fragmented fibers by coating platelet-derived growth factor (PDGF) and bio-minerals, which were then assembled with the hADSCs to form a spheroid. The PDGF incorporation within the spheroid dramatically increased the proliferation and endothelial differentiation of hADSCs, and also the combination of PDGF and bio-minerals synergistically enhanced the osteogenic differentiation of cells. Finally, at two months following transplantation of PDGF and bio-minerals incorporated spheroids onto in vivo mouse calvarial defect, the bone regeneration was significantly enhanced (42.5 ± 10.8% of regenerated bone area) and the greatest number of capillaries and arterioles were observed. Incorporation of biomolecules or biomaterials within a stem cells spheroid showed the enhanced therapeutic effects than cells-only spheroid, however, transplantation of the engineered spheroids on in vivo cartilage or bone defect often showed unstable localization because of continuous movement of the joints. To address this issue, three-dimensional (3D) printed scaffolds have developed to entrap and deliver the growth factors and stem cells without loss. However, the stem cells within spheroids tend to suffer from low viability in the 3D printed scaffold because of diffusion limitation, and revealed limited tissue regeneration. Here in, I prepared a 3D printed micro-chamber that could deliver the spheroids by positioning them into the wells of chamber, which was not entrapped and fused into the strands of scaffold. The in vitro culture of cells from spheroids in the micro-chamber exhibited greater viability and proliferation compared with the cells cultured without chamber. Furthermore, the surface of chamber was coated with 500 ng of PDGF, and the fragmented fibers within a spheroid loaded 50 ng of bone morphogenetic protein 2 (BMP-2) as a new platform for dual-growth factors delivery system. The in vitro investigation of osteogenic differentiation proved that the cells from spheroids in the engineered micro-chamber showed enhanced osteogenic differentiation than those in micro-chamber without growth factors. In vivo transplantation of the chambers with dual growth factors into mouse calvarial defects showed dramatic increase in bone regeneration, which resulted in 77.0 ± 15.9% of new bone area. Finally, I designed biphasic construct using the micro-chambers to mimic hierarchical structure of natural osteochondral tissue. The two types of hADSCs spheroids; osteogenic or chondrogenic, were developed by incorporating each BMP-2 or transforming growth factor beta 3 (TGF-3) immobilized fragmented fibers within the spheroids. The in vitro assays proved that the growth factors immobilized fibers automatically induced each stem cells differentiation without supporting of differentiation medium. The spheroids were then positioned on micro-chambers to form each cartilage phase construct or bone phase construct. The stem cells from the spheroids loaded on the micro-chamber showed that the advanced in vitro differentiation than the cells just within a spheroid. Furthermore, the micro-chambers were integrated and cultured together for 21 days. As a result, the two phases were perfectly fused together by cells-cells interaction from the cells in each spheroid loaded in the chambers. The in vitro investigation of osteochondral construct demonstrated that the cells within BMP-2 incorporated spheroid successfully retained osteogenic differentiation and the cells within TGF-b3 incorporated spheroid showed chondrogenic characteristics even if they were integrated together. Taken together, I proposed the therapeutic approaches for osteochondral defect by using a tissue engineering method combining stem cells spheroids, ECM mimicking fibers, growth factors, bio-minerals, and 3D-printed micro-chamber. The harmonized system increasing therapeutic effects of stem cells and stable delivery using 3D printed micro-chamber might help successful in vivo regeneration of osteochondral defect.