Jessica Amber Jennings

Transcriptional response of dermal fibroblasts in direct current electric fields.

During the course of normal wound healing, fibroblasts at the wound edge are exposed to electric fields (EFs) ranging from 40 to 200 mV/mm. Various forms of EFs influence fibroblast migration, proliferation, and protein synthesis. Thus, EFs may contribute to fibroblast activation during wound repair. To elucidate the role of EFs during the normal progression of healing, this study compares gene expression in normal adult dermal fibroblasts exposed to a 100 mV/mm EF for 1 h to non-stimulated controls. Significantly increased expression of 162 transcripts and decreased expression of 302 transcripts was detected using microarrays, with 126 transcripts above the level of 1.4-fold increases or decreases compared to the controls. Above the level of twofold, only 11 genes were significantly increased or decreased compared to controls. Many of these significantly regulated genes are associated with wound repair through the processes of matrix production, cellular signaling, and growth. Activity within specific cellular signaling pathways is noted, including TGF-beta, G-proteins, and inhibition of apoptosis. In addition, RT-PCR analysis of the expression of KLF6, FN1, RGS2, and JMJD1C over continued stimulation and at different field strengths suggests that there are specific windows of field characteristics for maximum induction of these genes. EFs thus appear to have an important role in controlling fibroblast activity in the process of wound healing.

Physical properties and in vitro evaluation of collagen–chitosan–calcium phosphate microparticle-based scaffolds for bone tissue regeneration:

Due to limitations of bone autografts and allografts, synthetic bone grafts using osteoconductive biomaterials have been designed. In this study, collagen–chitosan–calcium phosphate microparticle-based scaffolds fused with glycolic acid were compared to their counterparts without collagen in terms of degradation, cytocompatibility, porosity, and Young’s modulus. It was found that 26–30% collagen was incorporated and that hydroxyapatite was present. Moreover, there were no differences between control and collagen scaffolds in degradation, cytocompatibility, porosity, and Young’s modulus. In general, scaffolds exhibited 23% porosity, 0.6–1.2 MPa Young’s modulus, 23% degradation over 4 weeks, and supported a four to seven fold increase in osteoblast cell number over 7 days in culture. Collagen can be incorporated into these bone graft substitute scaffolds, which show an improved degradation profile.

Preliminary investigation of crosslinked chitosan sponges for tailorable drug delivery and infection control

Local versus systemic antibiotic delivery may be an effective strategy for treating musculoskeletal infections, especially when antibiotic-resistant bacteria are present. Lyophilized uncrosslinked, genipin crosslinked, and genipin crosslinked with poly(N-isopropylacrylamide) (PNIPAM) chitosan sponges were analyzed for their in vitro degradation rate, chemical crosslinking, antibiotic uptake, elution, biologic activity, and cytotoxicity. These evaluations were pursued to determine if crosslinking with genipin could be used to create a tailorable point of care loaded sponge for local infection control. Crosslinking the chitosan sponges decreased degradation in phosphate-buffered saline from 4.48 ± 2.28 wt % remaining of the uncrosslinked sponges to 78.82 ± 1.15 and 73.87 ± 1.27 wt % remaining at week 1 for the genipin and PNIPAM/genipin crosslinked sponges, respectively. The PNIPAM/genipin crosslinked sponges exhibited the most sustained release of biologically active antibiotics, with an average antibiotic release 63% higher than uncrosslinked and 37% higher than genipin crosslinked sponges, after 96 h. No significant cytotoxic effects from sponges or eluates were exhibited with NIH 3T3 fibroblasts. These preliminary results indicate that genipin crosslinked chitosan sponges, with or without PNIPAM, have potential as local delivery systems for adjunctive therapy for infection control, especially when longer degradation periods and higher antibiotic elutions are desired.

Chitosan coating to enhance the therapeutic efficacy of calcium sulfate-based antibiotic therapy in the treatment of chronic osteomyelitis

We demonstrate that coating calcium sulfate with deacetylated chitosan enhances the elution profile of daptomycin by prolonging the period during which high concentrations of antibiotic are released. Coatings reduced initial bolus release of daptomycin by a factor of 10 to approximately 1000 µg/ml, and levels remained above 100 µg/ml for up to 10 days. Chitosan-coated and uncoated calcium sulfate implants with and without 15% daptomycin were evaluated in an experimental model of staphylococcal osteomyelitis through bacteriology scores, radiology, histopathology, and Gram staining. Significant reduction in bacteriology scores was observed for implants containing daptomycin and coated with chitosan compared with all the other groups. We confirm that the use of chitosan-coated calcium sulfate beads for local antibiotic delivery can be correlated with an improved therapeutic outcome following surgical debridement in the treatment of chronic osteomyelitis.

Characterization of local delivery with amphotericin B and vancomycin from modified chitosan sponges and functional biofilm prevention evaluation

Polymicrobial musculoskeletal wound infections are troublesome complications and can be difficult to treat when caused by invasive fungi or bacteria. However, few local antifungal delivery systems have been studied. Chitosan and polyethylene glycol (PEG) sponge local antifungal delivery systems have been developed for adjunctive therapy to reduce musculoskeletal wound contamination. This study evaluated the effects of blending PEG, at 6,000 or 8,000 g/mol, with chitosan in sponge form on in vitro amphotericin B and vancomycin elution, eluate activity, cytocompatibility, and in vivo prevention of a bacterial biofilm. Blended chitosan sponges released both amphotericin B and vancomycin in vitro. All tested amphotericin B eluates remained active against Candida albicans, and vancomycin eluates from blended sponges maintained activity against Staphylococcus aureus. Amphotericin B eluates obtained after 1 h from blended sponges elicited 62–95% losses in fibroblast viability, but 3 h eluates only caused 22–60% decreases in viability. In a Staphylococcus aureus infected mouse catheter biofilm prevention model, vancomycin loaded chitosan/PEG 6000 sponge cleared bacteria from 100% of the catheters, with reduced clearance rate observed in other sponges. These results indicate that the chitosan/PEG blended sponges have potential for local antifungal and/or antibiotic combination delivery as an adjunctive therapy to prevent wound infections.

Preparation and Functional Assessment of Composite Chitosan-Nano-Hydroxyapatite Scaffolds for Bone Regeneration

Composite chitosan-nano-hydroxyapatite microspheres and scaffolds prepared using a co-precipitation method have shown potential for use in bone regeneration. The goal of this research was to improve the functional properties of the composite scaffolds by modifying the fabrication parameters. The effects of degree of deacetylation (DDA), drying method, hydroxyapatite content and an acid wash on scaffold properties were investigated. Freeze-dried 61% DDA scaffolds degraded faster (3.5 ± 0.5% mass loss) than air-dried 61% DDA scaffolds and 80% DDA scaffolds, but had a lower compressive modulus of 0.12 ± 0.01 MPa. Air-dried 80% DDA scaffolds displayed the highest compressive modulus (3.79 ± 0.51 MPa) and these scaffolds were chosen as the best candidate for use in bone regeneration. Increasing the amount of hydroxyapatite in the air-dried 80% DDA scaffolds did not further increase the compressive modulus of the scaffolds. An acid wash procedure at pH 6.1 was found to increase the degradation of air-dried 80% DDA scaffolds from 1.3 ± 0.1% to 4.4 ± 0.4%. All of the formulations tested supported the proliferation of SAOS-2 cells.

Preliminary evaluation of local drug delivery of amphotericin B and in vivo degradation of chitosan and polyethylene glycol blended sponges

This research investigated the combination of polyethylene glycol with chitosan in point-of-care loaded sponges made by one or two lyophilizations for adjunctive local antifungal delivery in musculoskeletal wounds. Blended and control chitosan sponges were evaluated in vitro for antifungal release and activity, degradation, cytocompatibility, and characterized for spectroscopic, crystallinity, thermal, and morphologic material properties. In vivo biocompatibility and degradation of sponges were also evaluated in a rat intramuscular pouch model 4 and 10 days after implantation. Blended sponges released amphotericin B active against Candida albicans (>0.25 µg/mL) over 72 h and did not elicit cytotoxicity response of fibroblasts. Blended sponges exhibited decreases in surface roughness, decreased thermal decomposition temperatures, as well as small Fourier transform infrared spectroscopy and crystallinity differences, compared with chitosan-only sponges. Three of the four blended sponge formulations exhibited 31%-94% increases in in vitro degradation from the chitosan sponges after 10 days, but did not demonstrate the same increase in in vivo degradation. Low inflammatory in vivo tissue response to blended and chitosan-only sponges was similar over 10 days. These results demonstrated that adding polyethylene glycol to chitosan sponges does improve local antifungal release, cytocompatibility, and in vitro degradation, but does not increase in vivo degradation.

Osteoinductivity Assessment of BMP-2 Loaded Composite Chitosan-Nano-Hydroxyapatite Scaffolds in a Rat Muscle Pouch

The objective of this study was to evaluate the osteoinductivity of composite chitosan-nano-hydroxyapatite scaffolds in a rat muscle pouch model. Previous in vitro characterization demonstrated the ability of the scaffolds to promote bone regeneration and as a carrier for local delivery of BMP-2. Composite microspheres were prepared using a co-precipitation method, and scaffolds were fabricated using an acid wash to adhere beads together. To determine the in vivo osteoinductivity of the scaffolds, the following groups (n = 6) were implanted into muscle pouches created in the latissimus dorsi of Sprague Dawley rats: (A) lyophilized scaffolds without rhBMP-2, (B) lyophilized scaffolds with rhBMP-2, (C) non-lyophilized scaffolds with rhBMP-2, and (D) absorbable collagen sponge with rhBMP-2 (control). Groups B, C, and D were loaded with 4 mL of a 9.0 μg/mL solution of rhBMP-2 for 48 h. The rats were sacrificed after one month and samples were analyzed for amount of residual implant material, new bone, and osteoid. Although the experimental groups displayed minimal degradation after one month, all of the scaffolds contained small amounts of woven bone and considerable amounts of osteoid. Approximately thirty percent of the open space available for tissue ingrowth in the scaffolds contained new bone or osteoid in the process of mineralization. The ability of the composite scaffolds (with and without BMP-2) to promote ectopic bone growth in vivo was demonstrated.

Chitosan coatings to control release and target tissues for therapeutic delivery.

The natural biopolymer chitosan has versatile applications in therapeutic delivery. Coating drug delivery matrices or biomaterials with chitosan offers several advantages in drug delivery, including control of drug release, slowing degradation rate and improving biocompatibility. Advanced uses of chitosan in coating form include targeting drug delivery vehicles to specific tissue as well as providing a stimulus-controlled release response. The present review summarizes the current applications of chitosan coatings in the context of different biomaterial delivery technologies, as well as future directions of chitosan coatings for drug delivery technologies under development.

Effects of VEGF-loaded chitosan coatings

Vascular endothelial growth factor (VEGF) is a powerful growth factor that promotes vascularization as well as osteoblastic differentiation and bone regeneration, all of which are key processes in the osseointegration of dental implants. Strategies to increase vascularization through delivery of VEGF may improve osseointegration, especially in patients with reduced bone healing potential. The aim of this study was to determine the potential of chitosan coatings on titanium to deliver VEGF and to support growth and matrix production of osteoblastic cells in vitro. Chitosan was chemically bonded to titanium coupons via silane-glutaraldehyde linker molecules and loaded with 0, 20, 50, or 100 ng of VEGF. Protein was released during a three day period with around 75% of VEGF (4.44, 11.37, and 22.10 ng/mL/cm(2) from the 20, 50, and 100 ng loaded levels, respectively) released during the first 12 h, and 90-95% of the VEGF released from the coatings by day 3. Saos-2 bone cells continued to proliferate over the 28-day period on the VEGF-loaded chitosan coatings in contrast to cells seeded on uncoated titanium, which plateaued after 14 days. Cells on uncoated titanium exhibited a peak in alkaline phosphatase expression at approximately 14 days, concomitant with the plateau in growth. While osteoblast-like cells on all chitosan coatings exhibited up to a 2-fold enhancement of the alkaline phosphatase activity and 10-fold increase in calcium deposition compared to uncoated controls, the incorporation of VEGF into the coatings did not enhance osteoblast matrix production over plain chitosan coatings throughout this study.