Chitosan paste from crawfish shells as a synthetic bone graft material: Preparation, characterization and animal study

Bone loss associated with musculoskeletal trauma or metabolic diseases often requires bone grafts. Autograft or allograft bones are limited in supply. Therefore, development of synthetic bone graft materials is an active area of research. For several years the biomaterials group at LSUHSC-Shreveport has been investigating the use of chitosan purified from crawfish shells using a process patented by my research advisor, Dr. Debi Mukherjee. Chitin is a polysaccharide that exists in fungi, exoskeleton of insects and outer shell of crustaceans. It is biocompatible, osteoconductive, antimicrobial, biodegradable, non-toxic, haemostatic, fungicidal, and the second most abundant natural polysaccharide on earth. Removal of the acetyl group from chitin forms chitosan. Chitosan is more useful due to the presence of amino groups that impart a positive charge to the molecule. This positive charge interacts with cells or can act as a binding site for other functional groups thereby expanding the role of chitosan molecules. The objectives of this study are: (1) Test for the presence of bone in grafted and ungrafted defects and compare the results among the different groups. (2) Test and compare the differences in stiffnesses and fracture loads among the different groups. (3) Test, via microcomputed tomography, defect reduction in samples treated with the graft. (4) Test for the amorphous nature of crawfish chitosan. (5) Compare the purity of crawfish chitosan extracted using Dr. Debi Mukherjee\u27s procedure with commercially available chitosan. The first portion of this experiment was characterization of crawfish chitosan, extracted using a method patented by Dr. Debi Mukherjee, to determine the individual chemical fingerprint of crawfish chitosan. Tests established that crawfish chitosan contains identical functional groups as chitosan monomer and chitosan glutamate and possessing an amorphous structure. In the second portion of this study, purified chitosan was mixed with cultured osteoblasts and compounded with a calcium salt creating a bone graft paste. Eighteen male Lewis rats underwent the surgical procedure. A critical size defect was created in rat femurs following a published protocol and repaired through use of a metal plate with and without crawfish chitosan bone graft paste. Histological examination and bone graft analysis of defected areas demonstrated the presence of bone in defect areas that were filled with experimental paste material and reduced or no bone growth on control femurs. Chitosan possesses the necessary properties, when combined with plaster of Paris and osteoblasts, to create a bone graft that is osteoconductive, osteoinductive and osteogenic

Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves

The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (nﾠ=ﾠ14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small ﾓprenodulesﾔ (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-? stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease