ABSTRACT
Synthetic hydroxyapatite (HA) is a widely studied bioceramic for bone tissue engineering (BTE) due to its similarity to the mineral component of bone. As bone mineral contains various ionic substitutions that play a crucial role in bone metabolism, the bioactivity of HA can be improved by adding small amounts of physiologically relevant ions into its crystal structure, with silicate-substituted HA (Si-HA) showing particularly promising results. Nevertheless, it remains unclear how distinct material characteristics influence the bioactivity due to the intertwined nature of surface properties. A coculture methodology was optimized and applied for in vitro quantification of the biological response. Initially, HA and Si-HA samples were produced and characterized. To compare the bioactivity of the samples, a method was developed to measure interactions in an increasingly complex environment, first including fibronectin (FN) adsorption and subsequently cell adhesion in mono and coculture using primary human osteoblasts (hOBs) and human dermal microvascular endothelial cells (HDMECs), with and without FN precoating. An experimental set-up was designed to assess to what extent different surface features of the samples contribute to the induced biological response. An 8-nm gold sputter coating was applied to eradicate the electrochemical differences and polishing and abrading was used to reduce the differences in surface topographies. Overall, 1.25 wt% Si-HA exhibited most nanoscale variations in surface potential. In terms of bioactivity, 1.25 wt% Si-HA samples induced the highest osteoblast attachment and vessel formation. Additionally, in vitro vessel formation was established on Si-HA surfaces using a hOB:HDMEC cell ratio of 70:30 and a methodology was established that enabled the assessment of the relative effect of topographical and electrochemical features induced by silicon substitution in the HA lattice on their bioactivity. It was found that the difference in the amount of protein attached to HA and 1.25 wt% Si-HA after 2 h was affected by topographical differences. Conversely, electrochemical differences induced different vessel-like structure formation in coculture with a FN precoating. Without an FN precoating, both topographical and electrochemical differences dictated the differences in angiogenic response. Overall, 1.25 wt% Si-HA surface features appear to induce the most favorable protein adsorption and cell adhesion in mono and coculture with and without FN precoating.
ABSTRACT
Bone tissue engineering (BTE) aims to improve the healing of bone fractures using scaffolds that mimic the native extracellular matrix. For successful bone regeneration, scaffolds should promote simultaneous bone tissue formation and blood vessel growth for nutrient and waste exchange. However, a significant challenge in regenerative medicine remains the development of grafts that can be vascularized successfully. Amongst other things, optimization of physicochemical conditions of scaffolds is key to achieving appropriate angiogenesis in the period immediately following implantation. Calcium phosphates and collagen scaffolds are two of the most widely studied biomaterials for BTE, due to their close resemblance to inorganic and organic components of bone, respectively, and their bioactivity, tunable biodegradability and the ability to produce tailored architectures. While various strategies exist to enhance vascularization of these scaffolds in vivo, further in vitro assessment is crucial to understand the relation between physicochemical properties of a biomaterial and its ability to induce angiogenesis. While mono-culture studies can provide evidence regarding cell-material interaction of a single cell type, a co-culture procedure is crucial for assessing the complex mechanisms involved in angiogenesis. A co-culture more closely resembles the natural tissue both physically and biologically by stimulating natural intercellular interactions and mimicking the organization of the in vivo environment. Nevertheless, a co-culture is a complex system requiring optimization of various parameters including cell types, cell ratio, culture medium and seeding logistics. Gaining fundamental knowledge of the mechanism behind the bioactivity of biomaterials and understanding the contribution of surface and architectural features to the vascularization of scaffolds, and the biological response in general, can provide an invaluable basis for future optimization studies. This review gives an overview of the available literature on scaffolds for BTE, and trends are extracted on the relationship between architectural features, biochemical properties, co-culture parameters and angiogenesis.
ABSTRACT
Fluorescent nanodiamonds (FNDs) are promising nanoprobes, owing to their stable and magnetosensitive fluorescence. Therefore they can probe properties as magnetic resonances, pressure, temperature or strain. The unprecedented sensitivity of diamond defects can detect the faint magnetic resonance of a single electron or even a few nuclear spins. However, these sensitivities are only achieved if the diamond probe is close to the molecules that need to be detected. In order to utilize its full potential for biological applications, the diamond particle has to enter the cell. Some model systems, like HeLa cells, readily ingest particles. However, most cells do not show this behavior. In this article we show for the first time generally applicable methods, which are able to transport fluorescent nanodiamonds into cells with a thick cell wall. Yeast cells, in particular Saccharomyces cerevisiae, are a favored model organism to study intracellular processes including aging on a cellular level. In order to introduce FNDs in these cells, we evaluated electrical transformation and conditions of chemical permeabilization for uptake efficiency and viability. 5% DMSO (dimethyl sulfoxide) in combination with optimized chemical transformation mix leads to high uptake efficiency in combination with low impact on cell biology. We have evaluated all steps in the procedure.
