Osteoarthritis (OA) is considered to be one of the leading causes of disability in the western countries and it represents an enormous financial burden on healthcare systems. OA can arise as the result of cartilage tissue degeneration in response to an acute injury or idiopathic degeneration. In both cases degeneration reflects the inherent limited self-repair capacity of articular cartilage. Despite significant and broad research efforts targeting cartilage defect repair via marrow stimulation, osteochondral graft transplantation, in vitro expanded chondrocyte implantation, and/or the use of various biomaterials as scaffolds, successful and robust repair remains elusive. Recent studies suggest that cell-based therapies do promote the regeneration of articular cartilage, however their modest efficacy suggests that further optimization and even a change in approach is needed.

Previous research from our group demonstrated that the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (MSC) was enhanced when the cells were assembled into micropellets, rather than traditional macroscopic pellets. Based on this critical finding, I investigated opportunities to exploit this phenomenon in cartilage tissue engineering applications. Through my Thesis, I addressed the following three specific questions:​

Q1. Does the micropellet approach also enhance the redifferentiation of monolayer expanded chondrocytes?

Q2. Can the matrix content and defect filling capacity of engineered cartilage be enhanced through the inclusion of donor matrix particles in MSC micropellets?

Q3. Can chondrogenic and osteogenic micropellets be used as building blocks and assembled into macroscopic osteochondral-like tissue?

High-throughput manufacture of micropellets was facilitated via a custom-made microwell surface originally generated as a silica wafer and then replicated with soft lithography method. According to this manufacturing process, single cells were seeded on a polydimethylsiloxane (PDMS) surface containing the microwell impression. When the cells were centrifuged or settled on this microwell surface, they were divided into small groups of 100-200 cells, which then formed micropellets within the first hours of culture. With this robust micropellet manufacturing technique, three independent but closely related studies were conducted, each addressing one of the previously mentioned questions. These three studies were reported as separate research articles in this Thesis by publication.

Chapter 3 addresses the first question and reports on the chondrogenic redifferentiation of expanded chondrocytes in macro- or micropellets and under normoxic or hypoxic atmospheres. It is a well-known phenomenon that the articular chondrocytes dedifferentiate during monolayer expansion. Similar to MSC chondrogenesis, pellet culture promotes redifferentiation, however these traditional pellets have 1-2 mm diameter, which causes diffusion gradients and mass transport issues leading to a heterogenic tissue structure. In order to overcome this problem, I tested the chondrocyte redifferentiation in micropellets that were approximately ten times smaller than the traditional pellets. According to chondrogenic assays, gene expression analyses and histological assessments, the redifferentiation of chondrocytes in micropellets was enhanced and yielded homogenous tissue structure. This work also demonstrated that micropellets could be assembled in order to engineer macroscopic cartilaginous tissue constructs.

Chapter 4 addresses the second question and reports on the effects of incorporation of microscopic cartilage pieces in macro- or micropellets of human bone marrow MSC. The fact that larger cartilage defects require a greater number of cells limits the applicability of cell-based therapies. As an alternative to articular chondrocytes, bone marrow-derived MSC can be harnessed because of their proliferation capacity, chondrogenic differentiation potential and ease of harvest. Additionally, use of xenogeneic cartilage pieces can increase the volume of engineered tissue graft in order to fill the larger defects more effectively. By combining two approaches, I tested the incorporation of microscopic bovine cartilage particles, termed as “cartilage dust”, into MSC micropellets. Chondrogenic characteristics, gene expression profile and histological structure assessment indicated that incorporation of cartilage dust into micropellets had significantly increased the size and cartilaginous matrix content, however it did not enhance the chondrogenic differentiation of MSC. It is also demonstrated that micropellets with or without cartilage dust can be assembled in order to engineer macroscopic cartilaginous tissue constructs.

Chapter 5 addresses the third question and reports on the assembly of osteogenic and chondrogenic micropellets into a biphasic tissue. The main purpose of building an osteochondral graft is to exploit more efficient bone-bone integration that takes place at the foundation of the defect and stabilizes the cartilaginous graft in place. The method of utilizing a scaffold with biphasic characteristics in order to generate an osteochondral graft is frequently trialed. In this study, I first tested the chondrogenic and osteogenic phenotype change in MSC micropellets, and then constructed the first scaffold-free biphasic tissue utilizing micropellets as building blocks.

In conclusion, this Thesis describes the characteristics of micropellets with different cell types (chondrocytes or MSC) and compositions (with or without the cartilage dust) under different conditions (hypoxic, normoxic, chondrogenic, osteogenic). The results presented in this Thesis introduce a novel intermediate component, micropellets, between the single cells and the tissue to be engineered. Utilization of micropellets may improve tissue engineering applications and cell-based therapies, ultimately leading to development of novel approaches in cartilage defect repair.