Joel D. Bumgardner

Chitosan: potential use as a bioactive coating for orthopaedic and craniofacial/dental implants

Chitosan is a biopolymer that exhibits osteoconductive, enhanced wound healing and antimicrobial properties which make it attractive for use as a bioactive coating to improve osseointegration of orthopaedic and craniofacial implant devices. Coatings made from 91.2% de-acetylated chitosan were chemically bonded to titanium coupons via silane-glutaraldehyde molecules. The bond strength of the coatings was evaluated in mechanical tensile tests, and their dissolution and cyto-compatibility were evaluated in vitro using cell-culture medium and UMR 106 osteoblastic cells, respectively. The results showed that the chitosan coatings were chemically bonded to the titanium substrate and that the bond strengths (1.5-1.8 MPa) were not affected by gas sterilization. However, the chitosan bond strengths were less than those reported for calcium-phosphate coatings. The gas-sterilized coatings exhibited little dissolution over 8 weeks in cell-culture solution, and the attachment and growth of the UMR 106 osteoblast cells was greater on the chitosan-coated samples than on the uncoated titanium. These results indicated that chitosan has the potential to be used as a biocompatible, bioactive coating for orthopaedic and craniofacial implant devices.

Biomaterial and antibiotic strategies for peri-implantitis: a review.

Dental implants have 89% plus survival rates at 10-15 years, but peri-implantitis or dental implant infections may be as high as 14%. Peri-implantitis can limit clinical success and impose health and financial burdens to patients and health providers. The pathogenic species associated with periodontitis (e.g., Fusobacterium ssp, A. actinomycetemcomitans, P. gingivalis) are also associated with peri-implantitis. Incidence of peri-implantitis is highest within the first 12 months after implantation, and is higher in patients who smoke or have poor oral health as well as with calcium-phosphate-coated or surface-roughened implants. Biomaterial therapies using fibers, gels, and beads to deliver antibiotics have been used in the treatment of Peri-implantitis though clinical efficacy is not well documented. Guided tissue regeneration membranes (e.g., collagen, poly-lactic/glycolic acid, chitosan, ePTFE) loaded with antimicrobials have shown success in reosseointegrating infected implants in animal models but have not been proven in humans. Experimental approaches include the development of anti-bioadhesion coatings, coating surfaces with antimicrobial agents (e.g., vancomycin, Ag, Zn) or antimicrobial releasing coatings (e.g., calcium phosphate, polylactic acid, chitosan). Future strategies include the development of surfaces that become antibacterial in response to infection, and improvements in the permucosal seal. Research is still needed to identify strategies to prevent bacterial attachment and enhance normal cell/tissue attachment to implant surfaces.

A chitosan/β-glycerophosphate thermo-sensitive gel for the delivery of ellagic acid for the treatment of brain cancer

We report here the development of a chitosan/beta-glycerophosphate(Ch/beta-GP) thermo-sensitive gel to deliver ellagic acid (EA) for cancer treatment. The properties of the Ch/beta-GP gels were characterized regarding chemical structure, surface morphology, and viscoelasticity. In vitro EA release rate from the EA loaded Ch/beta-GP gel and chitosan degradation rate were investigated. The anti-tumor effect of the EA loaded Ch/beta-GP gel on brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was evaluated by examining cell viability. Cell number and activity were monitored by the MTS assay. The Ch/beta-GP solution formed a heat-induced gel at body temperature, and the gelation temperature and time were affected by the final pH of the Ch/beta-GP solution. The lysozyme increased the EA release rate by 2.5 times higher than that in the absence of lysozyme. Dialyzed chitosan solution with final pH 6.3 greatly reduced the beta-GP needed for gelation, thereby significantly improving the biocompatibility of gel (p < 0.001). The chitosan gels containing 1% (w/v) of ellagic acid significantly reduced viability of U87 cells and C6 cells compared with the chitosan gels at 3 days incubation (p < 0.01, and p < 0.001, respectively).

Contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium.

Chitosan, a derivative of the bio-polysaccharide chitin, has shown promise as a bioactive material for implant, tissue engineering and drug-delivery applications. The aim of this study was to evaluate the contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium. Rough ground titanium (Ti) coupons were solution cast and bonded to 91.2% de-acetylated chitosan (1 wt% chitosan in 0.2% acetic acid) coatings via silane reactions. Non-coated Ti was used as controls. Samples were sterilized by ethylene oxide gas prior to experiments. Contact angles on all surfaces were measured using water. 5 × 104 cells/ml of ATCC CRL 1486 human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, were used for the cell attachment study. SEM evaluations were performed on cells attached to all surfaces. Contact angles and cell attachment on all surfaces were statistically analyzed using ANOVA. The chitosan-coated surfaces (76.4 ± 5.1°) exhibited a significantly greater contact angle compared to control Ti surfaces (32.2±6.1°). Similarly, chitosan-coated surfaces exhibited significantly greater (P < 0.001) albumin adsorption, fibronectin adsorption and cell attachment, as compared to the control Ti surfaces. Coating chitosan on Ti surfaces decreased the wettability of the Ti, but increased protein adsorption and cell attachment. Increased protein absorption and cell attachment on the chitosan-coated Ti may be of benefit in enhancing osseointegration of implant devices.

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.

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.

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.

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 (1wt% of 92.3% deacetylated chitosan in 1% acetic acid) was solution cast and bonded to rough ground titanium pins (2-mm diameter × 4-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.

Osteoblast Precursor Cell Attachment on Heat-treated Calcium Phosphate Coatings

The influence of properties of calcium phosphate (CaP) coatings on bone cell activity and bone-implant osseointegration is not well-established. This study investigated the effects of characterized CaP coatings of various heat treatments on osteoblast response. It was hypothesized that heat treatments of CaP coatings alter the initial osteoblast attachment. The 400°C heat-treated coatings were observed to exhibit poor crystallinity and significantly greater phosphate or apatite species compared with as-sputtered and 600°C heat-treated coatings. Similarly, human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, seeded on 400°C heat-treated coatings, exhibited significantly greater cell attachment compared with Ti surfaces, as-sputtered coatings, and 600°C heat-treated coatings. The HEPM cells on Ti surfaces and heat-treated coatings were observed to attach through filopodia, and underwent cell division, whereas the cells on as-sputtered coatings displayed fewer filopodia extensions and cell damage. Analysis of the data suggested that heat treatment of CaP coatings affects cell attachment.

An overview of chitin or chitosan/nano ceramic composite scaffolds for bone tissue engineering.

Chitin and chitosan based nanocomposite scaffolds have been widely used for bone tissue engineering. These chitin and chitosan based scaffolds were reinforced with nanocomponents viz Hydroxyapatite (HAp), Bioglass ceramic (BGC), Silicon dioxide (SiO2), Titanium dioxide (TiO2) and Zirconium oxide (ZrO2) to develop nanocomposite scaffolds. Plenty of works have been reported on the applications and characteristics of the nanoceramic composites however, compiling the work done in this field and presenting it in a single article is a thrust area. This review is written with an aim to fill this gap and focus on the preparations and applications of chitin or chitosan/nHAp, chitin or chitosan/nBGC, chitin or chitosan/nSiO2, chitin or chitosan/nTiO2 and chitin or chitosan/nZrO2 in the field of bone tissue engineering in detail. Many reports so far exemplify the importance of ceramics in bone regeneration. The effect of nanoceramics over native ceramics in developing composites, its role in osteogenesis etc. are the gist of this review.