Effects of Surface Characteristics of Hydroxyapatite and Substituted Hydroxyapatites on Protein Attachment and Angiogenesis

Autor: Ellermann, Else
Rok vydání: 2022
Předmět:
DOI: 10.17863/cam.89162
Popis: 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 through angiogenesis which is crucial for nutrient and waste exchange. Syn- thetic hydroxyapatite (HA) is one of the most widely studied bioceramics for BTE, due to its close resemblance to the mineral component of bone. As bone mineral contains various ionic substitutions that play a crucial role in bone metabolism, attempts to improve the bioactivity of HA have focussed on adding small amounts of physio- logically relevant ions into its crystal structure, with silicate-substituted HA (Si-HA) showing particularly promising results. Natural bone substitutes, which often contain ionic substitutions in the apatite crystal lattice, have also been assessed and Bio-Oss® has been applied widely in dentistry. While the biological performance of these materials has been of interest for many years, it remains unclear how distinct material characteristics influence the bioactivity due to the intertwined nature of physical and electrochemical surface properties. The main objective of this work was to develop a methodology that enables deconvolution of topographical and electrochemical surface features, to assess how differences in the chemical composition alter the bioactivity. A co-culture methodology was optimised and applied for in vitro quantification of the biological response in an environment that mimics the crucial simultaneous bone cell growth and blood vessel formation for bone healing. Initially, sintered discs of HA, Si-HA and Bio-Oss® discs were produced, and their surface characteristics were assessed and compared. The Si-HA discs were found to contain 1.25 wt% silicate (1.25wt%Si-HA), while Bio-Oss® contained multiple ionic substitutions. 1.25wt%Si-HA displayed the smallest grain size followed by HA and Bio-Oss®, with grain boundaries inducing nanoscale variations in surface potential. Lastly, Bio-Oss® and 1.25wt%Si-HA were significantly more hydrophilic than HA. To compare the bioactivity of the samples, a systematic method was developed to measure interactions at different scales, including fibronectin (FN) attachment and cell adhesion in mono- and co-culture using primary human osteoblasts (hOBs) and human dermal microvascular endothelial cells (HDMECs), with- and without FN pre-coating. A hOB:HDMEC cell ratio of 70:30 was found to promote vessel formation on all biomaterials. 1.25wt%Si-HA and Bio-Oss® discs were the most and least bioactive of all three samples, respectively, and 1.25wt%Si-HA induced the most complex vessel-like network. However, from the data obtained, it was not clear which of the physicochemical properties of the surfaces was causing the major difference in behaviour observed. A method was designed to separate the variables to assess to what extent different surface features of the discs contribute to the induced biological response. An 8 nm gold-sputter coating eradicated the electrochemical differences and polishing and abrading reduced the differences in surface topographies. This was evidenced by the similar biological response on gold-coated and polished and scratched (C-PS) control samples combined with a distinct biological response observed on gold-coated (C) surfaces without polishing and abrading. This methodology was, therefore, taken forward to assess the isolated effect of physical and electrochemical properties on the bioactivity of the biomaterials. A further study revealed that the difference in the amount of protein attached to HA and 1.25wt%Si-HA after 2 hours was affected by topographical differences. Conversely, electrochemical differences induced different vessel-like structure formation in co-culture with a FN pre-coating. Without a FN pre-coating, both topographical and electrochemical differences dictated the differences in cell response in co-culture. A comparison between Bio-Oss® and HA revealed similar vessel-like structure formation in co-culture without a FN pre-coating. While a FN pre-coating improved the angiogenic potential of HA, it did not affect the outcome on Bio-Oss®. The difference in vessel density found on HA and Bio-Oss® with a FN pre-coating was most strongly influenced by electrochemical differences. Overall, 1.25wt%Si-HA surface features appear to induce the most favourable protein attachment and cell adhesion in mono- and co-culture with- and without FN pre-coating. Taken together, this work provided a methodology that enabled the assessment of the relative effect of topographical and electrochemical features on the bioactivity of calcium phosphates. Additionally, a hOB and HDMEC co-culture methodology was designed to allow in vitro assessment of bone cell attachment and angiogenesis in a physiologically relevant environment.
Engineering and Physical Sciences Research Council and Geistlich Pharma AG
Databáze: OpenAIRE
Externí odkaz: