David J. Fink

Biomineralization and Eggshells: Cell-Mediated Acellular Compartments of Mineralized Extracellular Matrix

Publisher Summary This chapter summarizes cell biology, morphological organization, crystallography, chemical composition, process of mineralization, and biological function of avian eggshells. It also discusses emerging concepts of biomineralization and speculates about the mechanism of eggshell assembly and its implications for fabrication of polymer-ceramic composites. The eggshell is a microenvironmental compartment for housing developing embryos of a number of species. This unique microenvironment provides physical protection to the embryo and regulates gas, water, and ionic exchange. The avian eggshell can be characterized as a multilayered, polymer–ceramic composite. The three main layers include an outer mucous layer, an intermediate calcified zone, and an inner fibrous membrane layer. The shell membranes are the most internal layer of the eggshell and are formed by two nonmineralized fibrillar sublayers, the outer membrana testae externa, and the inner membrana testae interna or putaminis. The most external layer of the eggshell is referred to as the cuticle. This proteinaceous layer covers the entire calcified portion of the shell to a depth of about 10 μ m. The complementary use of biological, chemical, and crystallographic approaches demonstrates that the avian eggshell is a very promising model for the study of biomineralization. Avian eggshell is one of the most rapidly mineralizing biological systems known.

The Avian Eggshell Extracellular Matrix as a Model for Biomineralization

The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest that the dermatan sulfate proteoglycans may be important in modulating crystal morphology in the hen eggshell and correlate with mineralization-modulating biomolecules from other calcified tissue, which are generally anionic.

Crystallization studies on avian eggshell membranes: Implications for the molecular factors controlling eggshell formation

The avian eggshell is a natural biopolymer and mineral composite. It is a very useful model for biomimetic mineralization, since it is among the fastest forming hard tissues known. Isolated eggshell membranes, which were demineralized in vitro, were used to investigate the in vitro modulation of CaCO3 crystal deposition by organic matrix materials. Crystallization on the demineralized eggshell membrane occurred almost exclusively at the peripheries of residual calcium reserve assemblies, which contain a high concentration of sulfur. Similar structures are observed for eggshell membranes after natural demineralization. The characteristic rhombohedral crystal morphologies of the calcite crystals grown in this in vitro system are much less regular when grown in the presence of organic matrix or partially purified dermatan sulfate proteoglycans obtained from the eggshell. The effect of these macromolecules on the morphology and size of CaCO3 crystals is concentration-dependent. These studies indicate the complexity of the molecular and ionic interactions involved in the initiation and formation of the eggshell, with the focus on the role of the organic matrix.

Development of a peptide-targeted, myocardial ischemia-homing, mesenchymal stem cell

Directing stem cells to the heart is critical in producing an effective cell therapy for myocardial infarction (MI). Mesenchymal stem cells (MSCs) offer an exquisite drug delivery platform with environment-sensing cytokine release and MSCs have shown therapeutic potential in MI. Peptide-based targeting offers a novel method to increase cell homing, wherein MI-specific peptides, identified by phage display, are synthesized with a palmitic acid tail to facilitate cell membrane integration. Phage-peptides were screened in a mouse MI model and four peptides (CRPPR, CRKDKC, KSTRKS, and CARSKNKDC) were selected and synthesized as palmitated derivatives for further investigation. Cell coating was optimized and coating persistence and cytotoxicity were evaluated. MSCs were coated with peptides, injected into mice with MI, and MSCs in the heart quantified. Greater numbers of MSCs were found in heart of animals treated with the peptide-coated MSCs compared to uncoated controls. MSC numbers had positive correlation with MI severity in peptide-coated cells but a negative correlation in MSCs alone. A transient cell coating (“painting”) method has been developed that labels cells efficiently, non-toxically and increases cell localization in MI hearts.

The Effect of Organic Macromolecules on the Vateritet to Calcite Transformation

Small quantities of acidic macromolecules were added to crystallizing calcium carbonate in an attempt to engineer crystal growth. The additives are simple analogs to those molecules found in close association with the mineral phase in biological ceramics. When 5 or 10 ppm of the four additives (poly-L-glutamate, poly-L-aspartate, polyacrylate and polymaleate) were added to aqueous suspensions of metastable vaterite, the transformation to calcite was markedly retarded, and the morphology of the final crystal product was altered. The two polypeptides were most effective in inhibiting calcite nucleation and growth; they also promoted vaterite aggregation, and caused the formation of large calcite crystals.

Oriented Collagen Matrices: the Control of Biomineralizaton in Bone

Bone-forming cells fabricate a highly ordered collagen matrix (osteoid) which subsequently mineralizes. A variety of cell culture systems exist for osteogenic cells, yet none of these is optimal for the organized formation of a mineralized matrix. We have generated collagen substrates which have different degrees of fibrillar orientation, and have cultured osteogenic cells on these matrices. In this format, von Kossa-stained sections show that highly oriented collagen matrix starts to calcify in 6–7 days, while a random fibrillar matrix does not mineralize even after 21 days. Mineral has been detected only within the collagen matrix with a narrow, unmineralized region between the cells and the mineral.