Tissue regeneration in porous structures for bone engineering applications

Abstract

Scaffold design for bone regeneration is currently widely investigated as pore architecture can dramatically impact tissue formation in porous biomaterials used in regenerative medicine. A wide variety of 3D structures is used for this purpose, which has become even more important given the geometric freedom offered by emerging rapid prototyping techniques. Therefore, optimal design of pore architecture to maximize tissue formation and ingrowth is required. Tissue formation is frequently assessed in certain established scaffold structures, produced mainly as the end result of a particular fabrication process and its limitations. However, instead, scaffold architecture design should be based on the knowledge of how tissue actually forms in porous structures in the first place.

Tissue formation within porous structures can be dependent on several parameters, such as cell generated forces, cell division, cytoskeleton and extracellular matrix arrangement. Tissue differentiation is also an important aspect as once cells commit to a lineage, the proliferation could decrease. These aspects have been extensively shown to be modulated biochemically. However, the impact of different 3D structures is still largely unclear.

Therefore, in this thesis, it is aimed to characterise 3D tissue formation within different structures. For this purpose, an in vitro system with well-defined open pore slots of 1cm length, 1mm thickness and varying width (hundreds of micrometres) was used to characterise tissue growth solely as a function of pore geometry. This system provided a 3D environment for neo-tissue formation while minimizing nutrient limitations associated with full 3D constructs.

This thesis is the outcome of three studies. The first was focused on tissue formation kinetics in four different pore widths of 200, 300, 400 and 500 ?m. For this purpose, a unique system made of calcium phosphate cement with open pores was designed and fabricated. Several types of microscopy were used such as optical microscopy, time-lapse microscopy, epi-fluorescence microscopy and confocal microscopy. Results demonstrated that the material was biocompatible with Human Bone Marrow Stromal Cells and that tissue formation was strongly influenced by pore geometry. Both velocity of tissue invasion and area of tissue formed increased as pores became narrower. This was associated with distinct patterns of actin cytoskeleton organisation depending on pore width, indicating the role of active cell generated forces.

The second study is a more detailed characterisation of the type of tissue regenerated and its organisation. The neo-tissue was seen to display an osteoid-like collagen matrix. The main elements constituting a tissue i.e. cells, actin cytoskeleton and collagen matrix were imaged and their organisation was quantified with various image analysis methods. Results showed a significantly higher alignment with the longitudinal pore axis in the 200 ?m compared to the 500 ?m pores for all the tissue components analysed. By relating tissue orientation with its expansion rate, the results suggested that increased tissue alignment could be an important factor enhancing tissue formation.

In the third study, tissue differentiation was assessed as a function of pore size. Expression of intermediate and late bone markers was assessed, Alkaline Phosphatase and Osteopontin respectively. Results showed that both markers were expressed, indicating that the neo-tissue regenerated reached late state of differentiation and is prepared for mineralization. The expression of these markers was semi-quantitatively evaluated. ALP expression was expressed increasingly as tissue “age” increased. A gradient was observed with increasing staining intensity towards the starting point of tissue formation. Thus, the results revealed presence of distinct zones in which cells are in different states associated with various functions (proliferation or differentiation). Additionally, the expression of Osteopontin assessed semi quantitatively did not show any notable differences between pore widths. However, the results obtained displayed high variability between replicates. Therefore, it was only concluded that neo-tissue formed in both structures was able to express early (second study, chapter 4), intermediate and late osteogenic markers, although no significant differences were found between the different pore widths. This demonstrated that the tissue regenerated had committed to the osteogenic pathway, with the potential for full differentiation into mineralized tissue, which needs to be confirmed in future studies.

Overall, the results presented in this thesis provide evidence for the hypothesis that pore geometry affects tissue growth capacity by modulating tissue organisation. Key factors governing tissue formation in vitro were elucidated, highlighting the importance of the interplay between cell division, cell mechanics, cytoskeleton dynamics, tissue spatial organisation, matrix deposition and differentiation in relation to porous structure.

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Identifiers

ORCID for B.G. Sengers: orcid.org/0000-0001-5859-6984

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