In Vitro Evaluation of Electrospun Chitosan Mats Crosslinked with Genipin as Guided Tissue Regeneration Barrier Membranes

Guided tissue regeneration (GTR) is a surgical technique commonly used to exclude bacteria and soft tissues from bone graft sites in oral/maxillofacial bone graft sites by using a barrier membrane to maintain the graft contour and space. Current clinical barrier membrane materials based on expanded polytetrafluoroethylene (ePTFE) and bovine type 1 collagen are non-ideal and experience a number of disadvantages including membrane exposure, bacterial colonization/biofilm formation and premature degradation, all of which result in increased surgical intervention and poor bone regeneration. These materials do not actively participate in tissue regeneration, however bioactive materials, such as chitosan, may provide advantages such as the ability to stimulate wound healing and de novo bone formation. Our hypothesis is that electrospun chitosan GTR membranes will support cell attachment and growth but prevent cell infiltration/penetration of membrane, demonstrate in vitro degradation predictive of 4-6 month in vivo functionality, and will deliver antibiotics locally to prevent/inhibit periopathogenic complications. To test this hypothesis a series of chitosan membranes were electrospun, in the presence or absence of genipin, a natural crosslinking agent, at concentrations of 5 and 10 mM. These membranes were characterized by scanning electron microscopy, tensile testing, suture pullout testing, Fourier transform infrared spectroscopy, X-ray diffraction, and gel permeation chromatography, and in vitro biodegradation for diameter/morphology of fibers, membrane strengths, degree of crosslinking, crystallinity, molecular weight, and degradation kinetics, respectively. Cytocompability of membranes was evaluated in osteoblastic, fibroblastic and monocyte cultures. The activity of minocycline loaded and released from the membranes was determined in zone of inhibition tests using P. gingivalis microbe. The results demonstrated that genipin crosslinking extended the in vitro degradation timeframe, extended the release of minocycline, and increased the tensile strength of the resultant membranes while cytocompatibility, swelling, and tear strength were unaffected. In conclusion, electrospun chitosan membranes crosslinked with genipin are a suitable material for guided tissue regeneration and may help reduce bacterial infection and bacteria-induced host inflammatory response

Chitosan coatings deliver antimicrobials from titanium implants: A preliminary study

OBJECTIVE: Chitosan was investigated as a coating for local delivery of antimicrobials for prevention of acute implant infection. The objectives of this study were to (1) measure the release of 2 antimicrobials from chitosan coatings, (2) determine efficacy of eluted antimicrobials against bacteria, in vitro, and (3) evaluate toxicity of eluted drugs to host cells/tissues. METHODS: Chitosan coatings (80.7% deacetylated, 108 kDa) containing 20% tetracycline or 0.02% chlorhexidine digluconate were bonded to titanium via silane reactions. After elution in culture medium for 7 days, eluates were tested against model pathogens Actinobacillus actinomycetemcomitans and Staphylococcus epidermidis in turbidity tests and in 24-hour cytotoxicity tests using human osteoblasts and fibroblasts. Finally, antibiotic-loaded chitosan-coated titanium pins were implanted for 7 days in muscle of Sprague-Dawley rats to evaluate the initial tissue response. RESULTS: Coatings released 89% of tetracycline in 7 days and 100% chlorhexidine in 2 days. Released tetracycline inhibited growth (95%-99.9%) of pathogens for up to 7 days with no cytotoxicity to human cells. Released chlorhexidine was active against pathogens for 1 to 2 days (56%-99.5% inhibition) but was toxic to cells on the first day of elution. Typical acute inflammatory response was observed to antimicrobial-loaded chitosan coatings similar to unloaded coatings. CONCLUSION: These preliminary data support the hypothesis that chitosan coatings have the potential to locally deliver antimicrobials to inhibit bacteria without being toxic to host cells/tissues and warrant additional studies to evaluate the ability of the coatings to prevent/resist infection and promote osseointegration. © 2011 Lippincott Williams & Wilkins, Inc

Novel naturally crosslinked electrospun nanofibrous chitosan mats for guided bone regeneration membranes: Material characterization and cytocompatibility

Guided bone regeneration (GBR) barrier membranes are used to prevent soft tissue infiltration into the graft space during dental procedures that involve bone grafting. Chitosan materials have shown promise as GBR barrier membranes, due to their biocompatibility and predictable biodegradability, but degradation rates may still be too high for clinical applications. In this study, chitosan GBR membranes were electrospun using chitosan (70% deacetylated, 312kDa, 5.5w/v%), with or without the addition of 5 or 10mm genipin, a natural crosslinking agent, in order to extend the degradation to meet the clinical target time frame of 4-6months. Membranes were evaluated for fibre diameter, tensile strength, biodegradation rate, bond structure and cytocompatibility. Genipin addition, at 5 or 10mm, resulted in median fibre diameters 184, 144 and 154nm for uncrosslinked, 5mm and 10mm crosslinked, respectively. Crosslinking, examined by Fourier transform infrared spectroscopy, showed a decrease in N-H stretch as genipin levels were increased. Genipin-crosslinked mats exhibited only 22% degradation based on mass loss, as compared to 34% for uncrosslinked mats at 16weeks in vitro. The ultimate tensile strength of the mats was increased by 165% to 32MPa with 10mm crosslinking as compared to the uncrosslinked mats. Finally, genipin-crosslinked mats supported the proliferation of SAOS-2 cells in a 5day growth study, similar to uncrosslinked mats. These results suggest that electrospun chitosan mats may benefit from genipin crosslinking and have the potential to meet clinical degradation time frames for GBR applications