Incipient stages of collagen mineralization monitored by atomic force microscopy

Stetco, Ionela Alina (2015) Incipient stages of collagen mineralization monitored by atomic force microscopy. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The process of collagen mineralization represents a current challenge in tissue engineering and is extensively studied, since collagen and calcium phosphate are the main constituents of natural human bone. In vitro biomineralization of collagen can lead to biomaterials which could resemble the bone structure and may be appropriate for use as bone substitutes and implants in medical fields. Due to the fact that in vitro mineralization of collagen contributes to the understanding of mechanisms of mineralization in vivo, our research focused on the in situ monitoring of the incipient stages of collagen/calcium phosphate composite formation as it happened, with the help of atomic force microscopy (AFM). Since AFM is an appropriate tool for characterizing the morphologic features of a surface and is able to show the modification of the surface by depositions, the effect of calcium and phosphate ion concentrations upon the mineralization process of a collagen matrix in aqueous medium has been studied and the stimulation of mineral deposition by collagen has been established. Raman spectroscopy enabled us to identify the mineralization products as well as obtain a qualitative measure of mineralization efficiency. According to literature, glycosaminoglycans may enhance the efficiency of the collagen mineralization. Glucuronic acid, a major component of glycosaminoglycans (e.g. hyaluronic acid) in the extracellular matrix, has been previously shown to stimulate collagen mineralization in vitro and to lead to the formation of the desired calcium phosphate phase, i.e. hydroxyapatite. We performed a comparison of glucuronic acid with galacturonic acid (an epimer) which reveals differences in their effects on collagen fibrillogenesis. Our approach shows that while the presence of uronic acids in the collagen matrix may build an efficient scaffold for collagen calcification, it also brings modifications to the collagen matrix assembly. Using a surface-induced process for collagen alignment and fibril formation, we can observe changes in fibril and film structure with AFM. In order to gain insight on how the glucuronic acid may potentially affect the collagen matrix during fibrillogenesis, we studied the behaviour of glucuronic acid in alkaline and acidic media using AFM. In addition to AFM visualization of the various pH-dependent aggregates formed by glucuronic acid in solution, our experiments led to the synthesis of α-polyglucuronic acid starting from glucuronic acid monomers in an acidic solution. The α-polyglucuronic acid fibers were observed by AFM and its structure was confirmed by ¹³C solution NMR.

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