Deacetylation of Chitosan: Material Characterization and in vitro Evaluation via Albumin Adsorption and Pre-Osteoblastic Cell Cultures.

Degree of deacetylation (DDA) and molecular weight (MW) of chitosans are important to their physical and biological properties. In this study, two chitosans, HS (DDA = 73.3%) and AT (DDA = 76.8%), were deacetylated with 45% sodium hydroxide under nitrogen atmosphere at 80 °C or 90 °C for up to 120 min, to obtain two series of chitosans. The polymers produced were characterized for MW by gel permeation chromatography, DDA by titration and UV-vis methods, and crystallinity, hydrophilicity and thermal stability by X-ray diffraction, water contact angle and differential scanning calorimetry respectively. Films, made by solution casting in dilute acetic acid at ambient conditions, were evaluated for biological activity by albumin adsorption and the attachment and growth of a pre-osteoblast cell line. Chitosans with between 80-93% DDA's (based on titration) were reproducibly obtained. Even though deacetylation under nitrogen was supposed to limit chain degradation during decetylation, MW decreased (by maximum of 37.4% of HS and 63.0% for AT) with increasing deacetylation reaction time and temperature. Crystallinity and decomposition temperature increased and water contact angles decreased with processing to increase DDA. Significantly less albumin was absorbed on films made with 93% DDA chitosans as compared with the original materials and the AT chitosans absorbed less than the HS chitosans. The cells on higher DDA chitosan films grew faster than those on lower DDA films. In conclusion, processing conditions increased DDA and influenced physicochemical and biological properties. However, additional studies are needed to unambiguously determine the influence of DDA or MW on in vitro and in vivo performance of chitosan materials for bone/implant applications.

Composite chitosan/nano-hydroxyapatite scaffolds induce osteocalcin production by osteoblasts in vitro and support bone formation in vivo.

There is a significant clinical need to develop alternatives to autografts and allografts for bone grafting procedures. Porous, biodegradable scaffolds based on the biopolymer chitosan have been investigated as bone graft substitutes, and the addition of calcium phosphate to these scaffolds has been shown to improve the mechanical properties of the scaffold and may increase osteoconductivity. In this study, in vitro mineralization was examined for osteoblasts seeded in a porous scaffold composed of fused chitosan/nano-hydroxyapatite microspheres. Human fetal osteoblasts were cultured on composite and chitosan scaffolds for 21 days. On days 1, 4, 7, 14, and 21, total dsDNA, alkaline phosphatase, type I collagen, and osteocalcin production were measured. Total cellularity (measured by dsDNA), alkaline phosphatase, and type I collagen production were similar between the two scaffold groups. However, osteocalcin production occurred significantly earlier (day 7 vs. day 21) and was more than three times greater (0.0022 vs. 0.0068 ng/mL/ng DNA) on day 21 when osteoblasts were cultured on composite scaffolds. Osteocalcin is a marker of late osteoblastic differentiation and mineralized bone matrix formation. Therefore, the increase in osteocalcin production seen when cells were cultured on composite scaffolds may indicate that these scaffolds were superior to chitosan-only scaffolds in facilitating osteoblast mineralization. Composite scaffolds were also shown to be biocompatible and osteoconductive in a preliminary critical size rat calvarial defect study. These results demonstrate the potential of composite chitosan/nano-hydroxyapatite scaffolds to be used in bone tissue engineering.

Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration.

To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.

Effects of material property and heat treatment on nanomechanical properties of chitosan films.

The influence of degree of deacetylation (DDA), molecular weight (MW), and heat treatment on nanomechanical properties of three different chitosan coatings was assessed. Chitosans (2.5 wt % in 2% acetic acid) were solution cast and bonded to glass slides, subjected to 40, 70, or 90 degrees C heat treatments for 1 h, and then allowed to air dry. Non-heat treated films were used as controls. The nanomechanical and structural properties were evaluated using quasi-static nanoindentation, X-ray diffraction (XRD), and atomic force microscopy (AFM). Nanoindentation results showed that the coating with the lowest MW and highest DDA (95.6% DDA and MW = 2.43 x 10(6) Da) had higher reduced modulus of elasticity, (E = 4.02 +/- 0.85 GPa) as compared to the other chitosans; 76.1% DDA/MW = 3.20 x 10(6) Da, E = 3.66 +/- 0.68 GPa, and 92.3% DDA/MW = 7.52 x 10(6) Da, E = 3.56 +/- 0.25 GPa; (n = 75, p < 0.05). Crystallinity index, estimated via XRD, ranged from 36.4% +/- 1% to 49.7% +/- 6.5%. AFM revealed the presence of different crystalline morphologies such as needle-like crystals, sheet-like crystals, and small spherulites. The heat treatments applied during solution casting of the chitosan coatings did not affect morphology or nanomechanical properties. In conclusion, nanomechanical properties of chitosan coatings varied with DDA and MW and may be important to cell/tissue interactions.

Mechanical property, degradation rate, and bone cell growth of chitosan coated titanium influenced by degree of deacetylation of chitosan.

Chitosan has shown promise as a coating for dental/craniofacial and orthopaedic implants. However, the effects of degree of deacetylation (DDA) of chitosan on coating bond strength, degradation, and biological performance is not known. The aim of this project was to evaluate bonding, degradation, and bone cell growth on titanium coated with chitosans of different DDA and from different manufacturers. Three different chitosans, 80.6%, 81.7%, and 92.3% DDA were covalently bonded to titanium coupons via silane-glutaraldehyde molecules. Bond strengths were evaluated in mechanical tensile tests, and degradation, over 5 weeks, was conducted in cell culture medium with and without 100 microg/mL lysozyme. Cytocompatibility was evaluated for 10 days using UMR 106 osteoblastic cells. Results showed that mean chitosan coating bond strengths ranged from 2.2-3.8 MPa, and that there was minimal affect of DDA on coating bond strengths. The coatings exhibited little dissolution over 5 weeks in medium with or without lysozyme. However, the molecular weight (MW) of the chitosan coatings remaining on the titanium samples after 5 weeks decreased by 69-85% with the higher DDA chitosan coatings exhibiting less percent change in MW than the lower DDA materials. The growth of the UMR 106 osteoblast cells on the 81.7% DDA chitosan coating was lower on days 3 and 5, as compared with the other two coatings, but by day 10, there were no differences in growth among three coatings or to the uncoated titanium controls. Differences in growth were attributed to differences in manufacturer source material, though all coatings were judged to be osteocompatible in vitro.

The integration of chitosan-coated titanium in bone: an in vivo study in rabbits.

PROCEDURE: Much research is directed at surface modifications to enhance osseointegration of implants. A new potential coating is the biopolymer, chitosan, the deacetylated derivative of the natural polysaccharide, chitin. Chitosan is biocompatible, degradable, nontoxic, and exhibits osteogenic properties. The aim of this research was to investigate the hypothesis that chitosan-coated titanium supports bone formation and osseointegration. MATERIALS AND METHODS: Chitosan (1 wt% of 92.3% deacetylated chitosan in 1% acetic acid) was solution cast and bonded to rough ground titanium pins (2-mm diameterx4-mm long) via silane reactions. Calcium phosphate sputter-coated titanium and uncoated titanium pins were used as controls. Two chitosan-coated pins, and 1 each of calcium phosphate coated and uncoated pins were implanted unilaterally in the tibia of 16 adult male New Zealand white rabbits. At 2, 4, 8, and 12 weeks, undecalcified sections were histologically evaluated for healing and bone formation. RESULTS: Histological evaluations of tissues in contact with the chitosan-coated pins indicated minimal inflammatory response and a typical healing sequence of fibrous, woven bone formation, followed by development of lamellar bone. These observations were similar to those for tissues interfacing the control calcium phosphate-coated and uncoated titanium implants. Quantitative comparisons of the bone-implant interface were not possible since 31% of the implants migrated into the tibial marrow space after implantation due to insufficient cortical bone thickness to hold pins in place during healing. CONCLUSION: These data support the hypothesis that chitosan-coatings are able to develop a close bony apposition or the osseointegration of dental/craniofacial and orthopedic implants.

Polyurethane vascular catheter surface grafted with zwitterionic sulfobetaine monomer activated by ozone.

Polyurethane (PU) is a conventional biomedical material with favorable biocompatibility and excellent mechanical properties and widely used in making vascular catheter, but its antithrombogenic property is not good enough to make it as a more demanding applicable biomaterial. Surface modification is an effective way to improve the hemocompatibility for biomaterials. The purpose of present study was to use ozonization method to modify the surface of PU vascular catheter slice to improve its antithrombogenicity by grafting N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium (DMMSA), a zwitterionic sulfobetaine monomer. PU vascular catheter (PUVC) grafted with DMMSA (PUVC-g-PDMMSA) was characterized by ATR-FTIR and XPS. ATR-FTIR and XPS investigation confirmed the graft polymerization. The blood compatibility of the grafted films was evaluated by platelet rich plasma (PRP) platelet adhesion study and scanning electron microscopy (SEM) was used to observe the morphology of platelet using PU vascular catheter (PUVC) as the reference. No platelet adhesion was observed for the grafted PUVC slice incubated with PRP at 37 degrees C for 120 min. It is significant that this new zwitterionic sulfobetaine grafted PUVC have improved antithrobogenicity. It is effective that the inner surface of vascular catheter with inner diameter in only 3mm can be grafted with PDMMSA by using ozonization method.

Platelet adhesive resistance of segmented polyurethane film surface-grafted with vinyl benzyl sulfo monomer of ammonium zwitterions.

Platelet from human plasma adhered on the segmented poly(ether urethane) (SPEU) film grafted with N,N-dimethyl-N-(p-vinylbenyl)-N-(3-sulfopropyl) ammonium (DMVSA) was studied. SPEU films were hydroxylated by potassium peroxosulfate (KPS) and then grafted with DMVSA using ceric ammonium nitrate (CAN) as initiator. The mixing time of hydroxylated SPEU/CAN and the monomer concentration effect on graft polymerization yield were determined by ATR-FTIR. Surface analysis of the grafted films by ATR-FTIR and ESCA confirmed that DMVSA was successfully grafted onto the SPEU film surface. The grafted film possessed a relatively hydrophilic surface, as revealed by water contact angle measurement. The improved blood compatibility of the grafted films was preliminarily evaluated by a platelet-rich plasma adhesion study and scanning electron microscopy, using original SPEU and hydroxylated SPEU films as the controls. The results showed that platelet attachment was decreased greatly on the segmented polyurethane films grafted with DMVSA. This kind of new biomaterials grafted with sulfo ammonium zwitterionic monomers might have potential for biomedical applications.

Cell adhesion behavior of chitosan surface modified by bonding 2-methacryloyloxyethyl phosphorylcholine.

2-Methacryloyloxyethyl phosphorylcholine (MPC)-bonded chitosan was prepared by Michael addition of MPC to the amino groups of chitosan. The modified surfaces were characterized by static contact angle and electron spectroscopy for chemical analysis (ESCA). The water contact angle of chitosan decreased with the MPC bonding and the rate of decrease depended on the amount of MPC bonding. ESCA analysis results proved that MPC had been bonded on the chitosan surface and the chitosan modified directly by MPC had a much higher concentration of MPC on the surface compared with that of MPC on chitosan modified indirectly by MPC. Cell adhesion tests indicated that a low concentration of MPC bonded chitosan was more favorable to cell adhesion while a high concentration of MPC bonded chitosan inhibited cell attachment.