Suture pullout strength and in vitro fibroblast and RAW 264.7 monocyte biocompatibility of genipin crosslinked nanofibrous chitosan mats for guided tissue regeneration
ABSTRACT Guided tissue regeneration (GTR) is a surgical technique used to direct the formation of bone in the graft space by protecting it with a barrier membrane used to exclude soft tissues during healing. Chitosan has been advocated for GTR applications because of its biocompatibility, degradability, wound healing, and osteogenic properties. In this study, electrospun chitosan membranes, crosslinked with 5 mM or 10 mM geinipin, a natural crosslinker extracted from the gardenia plant, were evaluated for suture pullout strength, crystallinity, and cytocompatibility with normal human dermal fibroblast and TIB 71(™) RAW 264.7 monocyte cells. Ultimate suture pullout strength was significantly lower (51-67%) than that of commercially available collagen membranes. Crystallinity of the electrospun chitosan mats decreased upon crosslinking by 14-17% (p = 0.013). The molecular weight of the chitosan polymer was decreased by 75% during the electrospinning process. Uncrosslinked and genipin-crosslinked chitosan mats were cytocompatible and supported fibroblast cell proliferation for 9 days. Uncrosslinked and genipin-crosslinked membranes did not activate monocytes to produce nitric oxide (NO) in vitro in the absence of lipopolysaccharide (LPS). Finally, chitosan membranes inhibited LPS-induced NO production of RAW 264.7 cells by 59-67% as compared to tissue culture plastic and collagen membrane. Improvements are needed in the tear strength of electrospun chitosan membranes for clinical application. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:2890-2896, 2012.
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- "Genipin-crosslinking has recently been shown to increase the mechanical strength of electrospun chitosan fibers, as inferred from suture pullout strength tests . While the mechanism of how genipin crosslinks chitosan is still under investigation, a recent study suggests that this process entails a spontaneous reaction between genipin and the NH 2 subunits on the chitosan chain, which might lead to the observed increase in the scaffold stiffness. "
ABSTRACT: Reconstruction of large bone defects remains problematic in orthopedic and craniofacial clinical practice. Autografts are limited in supply and are associated with donor site morbidity while other materials show poor integration with the host's own bone. This lack of integration is often due to the absence of periosteum, the outer layer of bone that contains osteoprogenitor cells and is critical for the growth and remodeling of bone tissue. In this study we developed a one-step platform to electrospin nanofibrous scaffolds from chitosan, which also contain hydroxyapatite nanoparticles and are crosslinked with genipin. We hypothesized that the resulting composite scaffolds represent a microenvironment that emulates the physical, mineralized structure and mechanical properties of non-weight bearing bone extracellular matrix while promoting osteoblast differentiation and maturation similar to the periosteum. The ultrastructure and physicochemical properties of the scaffolds were studied using scanning electron microscopy and spectroscopic techniques. The average fiber diameters of the electrospun scaffolds were 227 ± 154 nm as spun, and increased to 335 ± 119 nm after crosslinking with genipin. Analysis by X-ray diffraction, Fourier transformed infrared spectroscopy and energy dispersive spectroscopy confirmed the presence of characteristic features of hydroxyapatite in the composite chitosan fibers. The Young's modulus of the composite fibrous scaffolds was 142 ± 13 MPa, which is similar to that of the natural periosteum. Both pure chitosan scaffolds and composite hydroxyapatite-containing chitosan scaffolds supported adhesion, proliferation and osteogenic differentiation of mouse 7F2 osteoblast-like cells. Expression and enzymatic activity of alkaline phosphatase, an early osteogenic marker, were higher in cells cultured on the composite scaffolds as compared to pure chitosan scaffolds, reaching a significant, 2.4 fold, difference by day 14 (p < 0.05). Similarly, cells cultured on hydroxyapatite-containing scaffolds had the highest rate of osteonectin mRNA expression over 2 weeks, indicating enhanced osteoinductivity of the composite scaffolds. Our results suggest that crosslinking electrospun hydroxyapatite-containing chitosan with genipin yields bio-composite scaffolds, which combine non-weight-bearing bone mechanical properties with a periosteum-like environment. Such scaffolds will facilitate the proliferation, differentiation and maturation of osteoblast-like cells. We propose that these scaffolds might be useful for the repair and regeneration of maxillofacial defects and injuries.Biomaterials 09/2012; 33(36):9167-78. DOI:10.1016/j.biomaterials.2012.09.009 · 8.31 Impact Factor
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ABSTRACT: Chitosan, a natural polysaccharide, has demonstrated potential as a degradable biocompatible guided bone regeneration membrane. This study aimed to evaluate the in vivo biocompatibility and degradation of chitosan nanofiber membranes, with and without genipin crosslinking as compared with a commercial collagen membrane in rat model. Chitosan nanofiber membranes, with and without genipin crosslinking, and collagen membrane (control) were implanted subcutaneously in the backs of 30 rats. The membranes were analyzed histologically at 2, 4, 8, 12, 16, and 20 weeks. Sections were viewed and graded by a blinded pathologist using a 4-point scoring system (0 = absent, 1 = mild, 2 = moderate, and 3 = severe) to determine the tissue reaction to the membranes and to observe membrane degradation. There was no statistically significant difference in histological scores among chitosan and collagen membranes at different time points. Absence or minimal inflammation was observed in 57-74% of the membranes across all groups. Most chitosan membranes persisted for 16-20 weeks, whereas most collagen membranes disappeared by resorption at 12-16 weeks. The general tissue response to chitosan nanofiber membranes with and without genipin crosslinking, was similar to that of control commercial collagen membrane. However, the chitosan membranes exhibited slower degradation rates than collagen membranes. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.Journal of Biomedical Materials Research Part B Applied Biomaterials 07/2014; 102(5). DOI:10.1002/jbm.b.33090 · 2.33 Impact Factor
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ABSTRACT: Chitosan nanofibers fabricated by electrospinning are contaminated by acidic anions from the acid spinning solution, leading to instability of the nanofibers in aqueous solutions, and the traditional fiber treatment method will also lead to the deterioration of the nanostructure. Here we demonstrate a novel approach to removing the acidic anions with full preservation of the nanofibrous structure. The as-spun nanofibers are first protected (stabilized) by reversible acylation. Second, contaminants are then eliminated by hydrolysis; finally, acylation is reversed. Chemical analysis showed the removal of the acidic anions and the graft and removal of acyl groups. Morphological analysis showed that the reversibly acylated fibers had diametersCellulose 08/2014; 21(4):2549-2556. DOI:10.1007/s10570-014-0306-3 · 3.03 Impact Factor