Scanning Electron Microscopic Analysis of the Mineralization of Type I Collagen via a Polymer-Induced Liquid-Precursor (PILP) Process

Abstract

We have put forth the hypothesis that collagen is mineralized during bone formation by means of a polymer-induced liquid-precursor (PILP) process, in which a liquid-phase mineral precursor could be drawn into the gaps and grooves of the collagen fibrils by capillary action, and upon solidification, leave the collagenous matrix embedded with nanoscopic crystallites of hydroxyapatite. This hypothesis is based upon our observations of capillarity seen for liquid-phase mineral precursors generated with calcium carbonate. Here, we demonstrate proof-of-concept of this mechanism by mineralizing Cellagen sponges (type I reconstituted bovine collagen) in the presence of a liquid-precursor phase to calcium carbonate. Scanning electron microscopy (SEM) was used to examine the mineralized collagen, which in combination with selective etching studies, revealed the extent to which the mineral phase infiltrated the collagenous matrix. A roughly periodic array of disk-like crystals was found to be embedded within the collagen fibers, demonstrating that the mineral phase spans across the diameter of the fibers. Some of the morphological features of the mineralized fibers in our in vitro model system are similar to those seen in natural bone (albeit of a different mineral phase), lending support to our hypothesis that these non-equilibrium morphologies might be generated by a PILP process. SEM provides a different perspective on the morphology of bone, and has been useful here for examining the extent of mineralization in composite structures generated via the PILP process. However, further investigation is needed to examine the nanostructural arrangement of the crystallites embedded within the collagenous matrix.

CaCO3/chitin-whisker hybrids: formation of CaCO3 crystals in chitin-based liquid-crystalline suspension

“…32 It was reported that colloidal ACC nanoparticles were generated in the solution by using the ammonium carbonate diffusion method in the presence of highly concentrated acidic polymers and calcium ion. 33 The ACC nanoparticle is an effective vehicle for transportation of CaCO 3 to construct three-dimensional interpenetrated hybrids. 33 In recent years, it is widely recognized that the aggregation-mediated pathway of mineralization is important to understand biomineralization.…”

“…33 The ACC nanoparticle is an effective vehicle for transportation of CaCO 3 to construct three-dimensional interpenetrated hybrids. 33 In recent years, it is widely recognized that the aggregation-mediated pathway of mineralization is important to understand biomineralization. 34,35 It has been proposed that living organisms also use ACC nanoparticles for storage, transportation and mineralization of CaCO 3 in their systems.…”

“…36,37 In our present system, the colloidal ACC nanoparticles may be formed in the crystallization solution as previously reported. 33 The ACC nanoparticles might diffuse into the gel matrix because of the capillary force, eventually transforming into calcite in the gel matrix to form three-dimensional interpenetrated hybrid materials. In contrast, for the alternate soaking method, it was insufficient to transport CaCO 3 because only cation or anion was supplied to the gel matrix.…”

Crystalline, Porous Microspheres Made from Amino Acids by Using Polymer‐Induced Liquid Precursor Phases

“…A special feature of the PILP phases is that they allow easy morphosynthesis of novel, biorelevant mineral structures through the shaping of a liquid on the micrometer scale so that crystallization occurs without requiring mass transport over longer distances. [14,15] Herein we extend this approach to polar organic molecules by studying the formation of PILP phases consisting of charged amino acids and oppositely charged polyelectrolytes in ethanol/water mixtures. While addition of ethanol to an aqueous solution of the pure amino acid leads to well shaped single crystals, stable PILP microdroplets are formed spontaneously by adding an oppositely charged polyelectrolyte to the starting solution.…”

On Mineral Retrosynthesis of a Complex Biogenic Scaffold

“…For instance, mineralization of collagen in the presence of poly(acrylic acid) (PAA), a synthetic anionic polymer, leads to a hierarchical composite structure similar to natural bone tissue [37]. The polymer-induced liquid precursor (PILP) phase stabilized by PAA exhibits liquid-like properties and is suggested to be drawn into the gaps within collagen fibrils by capillary effects.…”