Intrafibrillar Mineralization of Type I Collagen: Biomimetic Methods and Microscopic Characterization
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Date
2024-12
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The Ohio State University
Abstract
Bone is a connective tissue that provides structural support throughout the body. Depending on location and external loading, bone adjusts its material properties to meet functional demands of the body. This is due to collagen in the bone acting as a soft organic material that can be strengthened through intrafibrillar and extrafibrillar mineralization. While intrafibrillar mineralization is known to occur in bone, the multiple factors which mediate the process are not clearly understood.
The overarching goal of this research is to enhance understanding of how collagen mineralization strengthens bone, with a particular focus on the role of collagen piezoelectricity in the intrafibrillar mineralization of collagen. Achieving biomimetic intrafibrillar mineralization in vitro is a critical step toward this goal. To evaluate and confirm the effectiveness of this process, three consistent and repeatable biomimetic mineralization processes were applied to collagen sponges and self-assemblies, and their outcomes were analyzed using advanced imaging techniques.
Three mineralization solutions were investigated, each using polyaspartic acid as the polyanionic peptide to create a polymer-induced-liquid-precursor (PILP) that crystallizes into hydroxyapatite. The first solution used rac-glycerol 1-phosphate and alkaline phosphatase, the second used a tris-saline buffer, and the third used crushed ammonium carbonate for vapor diffusion. Atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were performed to understand the extent of intrafibrillar mineralization for each process.
Results from AFM, SEM, and TEM showed a clear picture of the control collagen samples with identified D-spacing for comparison with mineralized samples. AFM, SEM, and TEM results of the collagen sponge and self-assemblies for each mineralization method showed clear evidence of mineralization. Each of the three methods eliminated or reduced the appearance of D-spacing within the collagen fibrils, which is the main indication of mineralization. Of the three solutions investigated, the rac-glycerol 1-phosphate solution was the most effective at inducing intrafibrillar mineralization of the collagen self-assembly within 12 days, as shown by mineral crystals that formed within the collagen fibril.
With continued work, this research will further the understanding of natural bone mineralization by providing the most effective standard and consistent process for biomimetic mineralization of collagen. Now that the rac-glycerol 1-phosphate solution has been proven to effectively mineralize collagen samples, this method can be used to produce different degrees of mineralization for further investigation into the effect of collagen piezoelectricity on intrafibrillar mineralization.
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Keywords
Biomimetic Mineralization, Intrafibrillar Mineralization, Type I Collagen Mineralization, Mineralized Collagen Imaging, Collagen Self-Assembly Mineralization, Collagen Sponge Mineralization