Microsphere-Based Scaffolds Carrying Opposing Gradients of Chondroitin Sulfate and Tricalcium Phosphate

Extracellular matrix (ECM) components, such as chondroitin sulfate (CS) and tricalcium phosphate, serve as raw materials, and thus spatial patterning of these raw materials may be leveraged to mimic the smooth transition of physical, chemical, and mechanical properties at the bone-cartilage interface. We hypothesized that encapsulation of opposing gradients of these raw materials in high molecular weight poly(d,l-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds would enhance differentiation of rat bone marrow–derived stromal cells. The raw material encapsulation altered the microstructure of the microspheres and also influenced the cellular morphology that depended on the type of material encapsulated. Moreover, the mechanical properties of the raw material encapsulating microsphere-based scaffolds initially relied on the composition of the scaffolds and later on were primarily governed by the degradation of the polymer phase and newly synthesized ECM by the seeded cells. Furthermore, raw materials had a mitogenic effect on the seeded cells and led to increased glycosaminoglycan (GAG), collagen, and calcium content. Interestingly, the initial effects of raw material encapsulation on a per-cell basis might have been overshadowed by medium-regulated environment that appeared to favor osteogenesis. However, it is to be noted that in vivo, differentiation of the cells would be governed by the surrounding native environment. Thus, the results of this study demonstrated the potential of the raw materials in facilitating neo-tissue synthesis in microsphere-based scaffolds and perhaps in combination with bioactive signals, these raw materials may be able to achieve intricate cell differentiation profiles required for regenerating the osteochondral interface

Investigating the deposition of the extracellular matrix and bone tissue development in vitro using 2D and 3D cell culture methods

The study of bone tissue development in vitro has received much attention over the years due to the increasing incidences of bone disorders worldwide. This has resulted in a growing need for improving the knowledge of bone cell physiology in order to investigate the causes and to develop potential treatments for bone diseases. One approach to this is by modelling osteoblast differentiation in vitro which usually involves culturing bone forming cells such as mesenchymal stem cells (MSCs) and bone derived cell lines such as MG-63 on two-dimensional (2D) culture plasticware. However, this culture environment results in alterations to the cell morphology, leading to changes in cell fate and differentiation potential as the microenvironment does not reflect the natural three-dimensional (3D) extracellular matrix in which cells reside in vivo. To overcome this, the development of 3D cell culture techniques has been widely investigated to better recapitulate the native environments of bone forming cells, thereby helping to enhance bone formation. This project describes the culture of bone forming cells on a 3D scaffold to investigate whether osteoblast differentiation and thereby bone formation will be enhanced when compared to 2D. Two types of bone forming cells were used in this study, primary rat MSCs and MG-63s. They were induced to undergo osteogenic differentiation via the use of osteogenic morphogens in both 2D and 3D culture conditions. Results indicated the increase in expression of osteoblast markers such as Collagen I and Alkaline Phosphatase in 3D cultures of MSCs and MG-63s. Additionally, matrix mineralisation was also suggested to be enhanced in 3D cultures. These results suggest bone formation is enhanced in 3D cell culture when compared to 2D methods and reveal the advantages of using a 3D cell culture system to model bone tissue development in vitro. However, more data and additional work is required to confirm these findings

Raw Material Encapsulating Microsphere-Based Scaffolds For Osteochondral Regeneration

The current thesis describes the evaluation of a microsphere-based scaffold that may be used as an early intervention therapy for treating focal cartilage and bone-cartilage interface defects. This scaffold is comprised of extracellular matrix materials, which serve as ‘raw materials,’ encapsulated in a biodegradable polymer for differentiation of progenitor or resident cells into bone and cartilage. The work in the thesis initially evaluated the in vitro performance of raw material encapsulating microsphere-based scaffolds fabricated using a high molecular weight polymer as a first step to establish their clinical efficacy. Subsequently, concentrations of raw materials were increased in microsphere-based scaffolds to stimulate in vitro osteogenesis and chondrogenesis in stem cells. Lastly, a novel combination of raw materials, demineralized bone matrix (DBM) and decellularized cartilage (DCC), encapsulated in a continuously graded scaffold design was tested for in vivo regeneration potential in a rabbit model. Results from the body of in vitro studies suggested that raw material encapsulation in microsphere-based scaffolds can potentially facilitate neo-tissue synthesis. The encapsulated raw materials readily enhanced biochemical production, stimulated gene expression, and tissue synthesis. Additionally, biochemical and gene expression evidence highlighted the benefits of using gradient-based strategies for regenerating bone and cartilage. The in vivo study demonstrated the feasibility and applicability of DBM and DCC gradient microsphere-based scaffolds in the New Zealand White rabbit knee defect. The results of the study indicated toward some benefits of using DCC and DBM and emphasized on the need to further refine the technology. The important next steps would be to investigate polymer degradation rate and its effect on tissue regeneration, and further attune raw material concentrations to augment osteochondral regeneration. Ultimately, this thesis demonstrated the benefits of raw material encapsulation in microsphere-based scaffolds, in addition to opening new areas of investigation with regard to transitioning this technology for clinical use