Ectopic Bone Matrix Mineralization: Unveiling the Osteoinductive Nature of Crab Cuticle.

摘要

Large bone defects do not heal spontaneously and often require substitute materials. Ideally, a bone replacement material should mimic bone tissue from a mechanical, chemical, biological and functional point of view, and facilitate new bone formation. No single existing synthetic material possesses all the necessary properties required in an ideal bone implant. Using biomimetic principles and the kinship among biologically derived hard tissues, crustacean exoskeleton emerged as a natural material for bone implant because of its similarities to bone in composition, structure, and function. The purpose of this work is to serve as a preliminary investigation of the role in which crab shell, from Callinectes sapidus or Chesapeake blue claw crab, can play in bone healing. Soft tissue implantation studies, in rats, were used to investigate the osteoinductive potential of the crab cuticle.;Crushed crab cuticle was subcutaneously implanted in the abdominal region of 28-day-old Sprague-Dawley rats and aged for time periods ranging from 1-30 days by our collaborators at Howard University. Tissue samples which grew in in the region of the crushed crab shell implant were harvested and processed for microscopy and transmission electron microscopy (TEM) analysis. This work focuses on characterizing the crystalline nature and physical characteristics of the mineral phase which formed in the implant samples. Fascinating structures and architectures were observed in TEM mode---collagen fibers with the characteristic 67 nm banding pattern, collagen bundles, fibroblasts, dark regions of crystal-like particles, and 20 x 40 nm nanocrystals. X-ray microanalysis of 20 x 40 nm nanocrystals showed an average calcium:phosphorus ratio of 1.81 +/- 0.37. Selected area diffraction (SAD) was initially used to determine the degree of crystallinity of mineral phases. Dark electron-dense regions found around collagen produced diffraction patterns indicative of amorphous solids. Upon further inspection using high resolution transmission electron microscopy (HRTEM), approximately 2-4 nm crystalline-nano-building-blocks with lattice spacings of 0.95 nm were revealed. Nanodiffraction was employed to investigate these 2-4 nm nano-structures with lattice spacings of 0.95 nm in more detail. Nanodiffraction clearly indicated the particle was a single crystal. Both the END pattern of the crystalline-nano-building-blocks and the SAD pattern of the 20 x 40 nm nanocrystal were both indexed and found to be of the apatite family. Compellingly, the SAD pattern of the 20 x 40 nm nanocrystals displayed speckled rings made up of discrete spots. This suggest that there are many oriented single crystals and that the larger crystals are made up of an assembly of smaller single crystals. This gives evidence for the mesocrystal model of crystallization for biologically derived hydroxyapatite (HAP). Arguably, our study is the first of its kind to find biologically produced HAP crystals approximately 2-4 nm in size with evidence they assemble to make larger HAP crystals based on the mesocrystal model.