THE AVIAN EGGSHELL EXTRACELLULAR-MATRIX AS A MODEL FOR BIOMINERALIZATION

The avian eggshell is a complex, extracellularly assembled structure w hich contains both mineralized and non-mineralized regions, The compos ition of the hen eggshell organic matrix was examined by immunohistoch emistry with antibodies to different extracellular matrix molecules, T ype I collagen is found in the shell membranes, but only after treatme nt of the tissue sections with pepsin, When incomplete eggshells are r emoved from the oviduct and immunostained, type I collagen can be dete cted in the shell membranes without pepsin treatment. The shell membra nes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of t ype X collagen in the shell membranes is surprising, since this collag en has not been found in any tissue other than hypertrophic cartilage, Immunostaining for various glycosaminoglycans shows the presence of k eratan sulfate and dermatan sulfate, Several different antibodies to k eratan sulfate stain different regions of the eggshell; one keratan su lfate epitope is prominent in the calcium reserve assemblies, Dermatan sulfate staining is very intense in the palisade region, Demineralize d matrix from the palisade region was extracted with guanidine and fra ctionated by ion exchange chromatography, A similar to 200-kDa dermata n 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 demine ralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteogly can preparation were added to the solution from which the crystals wer e formed, This material causes a concentration-dependent change in cry stal morphology to one in which the crystals are smaller and more roun ded, which more closely approximates the crystals normally observed in eggshells, These results suggest that the dermatan sulfate proteoglyc ans may be important in modulating crystal morphology in the hen eggsh ell and correlate with mineralization-modulating biomolecules from oth er calcified tissue, which are generally anionic.