Jaydee D. Cabral

Antimicrobial Properties of a Chitosan Dextran-Based Hydrogel for Surgical Use

ABSTRACT A chitosan dextran-based (CD) hydrogel, developed for use in endoscopic sinus surgery, was tested for antimicrobial activity in vitro against a range of pathogenic microorganisms. The microdilution technique was used to determine minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations. In addition, the time-kill efficacy of CD hydrogel was determined for two bacterial species. Scanning and transmission electron microscopy were carried out to elucidate the antimicrobial mechanism of this compound. CD hydrogel was found to be effective against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Clostridium perfringens at its surgical concentration of 50,000 mg/liter. Minimum bactericidal concentrations ranged from 2,000 to 50,000 mg/liter. Dextran aldehyde (DA) was found to be the antimicrobial component of the CD hydrogel with MBC ranging from 2,000 to 32,000 mg/liter. S. aureus appeared to be killed at a slightly faster rate than E. coli. Candida albicans and Pseudomonas aeruginosa were more resistant to CD hydrogel and DA. Scanning and transmission electron microscopy of E. coli and S. aureus incubated with CD hydrogel and DA alone revealed morphological damage, disrupted cell walls, and loss of cytosolic contents, compatible with the proposed mode of action involving binding to cell wall proteins and disruption of peptide bonds. Motility and chemotaxis tests showed E. coli to be inhibited when incubated with DA. The antibacterial activity of CD hydrogel may make it a useful postsurgical aid at other body sites, especially where there is a risk of Gram-positive infections.

The efficacy of hemostatic techniques in the sheep model of carotid artery injury

The most dramatic complication in endonasal surgery is inadvertent injury to the internal carotid artery (ICA) with massive bleeding. Nasal packing is the favored technique for control; however, this often causes complete carotid occlusion or carotid stenosis, contributing to the morbidity and mortality of the patient. The aim of this study is to compare the efficacy of endoscopically applied hemostatic techniques that maintain vascular flow in an animal model of carotid artery injury.

Hydrogels for biomedical applications

Hydrogels are swollen, crosslinked networks that have great potential for use in biomedicine. Their softness, biocompatibility and ability for rapid diffusion of molecules make them useful for drug delivery, cell culture, wound healing and sensing applications. The chemical functionality of the gels can be easily modified to provide signalling and growth factors for cell proliferation. To allow the ingress of large cells, either porosity of the substrate can be controlled, or the gel can be made biodegradable. One ultimate goal is the growth of entire organs in the laboratory for eventual transplantation. Gels can be used as drug-delivery vehicles, either as implantable depots, or as microgels in blood-based delivery systems. One expanding area is the use of gels as surgical aides to prevent bleeding, infection and post-operative complications.

In vitro biocompatibility and cellular interactions of a chitosan/dextran‐based hydrogel for postsurgical adhesion prevention

In this paper, we report the in vitro biocompatibility and cellular interactions of a chitosan/dextran-based (CD) hydrogel and its components as determined by mutagenicity, cytotoxicity, cytokine/chemokine response, and wound healing assays. The CD hydrogel, developed for postsurgical adhesion prevention in ear, nose, and throat surgeries, was shown by previously published experiments in animal and human trials to be effective. The hydrogel was synthesized from the reaction between succinyl chitosan (SC) and oxidized dextran (DA). Cytotoxicity was assessed in an xCELLigence system and cytokine/chemokine responses were measured by ELISA in human macrophage, nasopharyngeal epithelial, and dermal fibroblast cells. A wound healing model utilized nasopharyngeal epithelial cells. CD hydrogel and DA were nonmutagenic in the Ames test. CD hydrogel showed moderate cytotoxicity for the cell lines, DA being the cytotoxic component. Some inhibition of wound healing occurred due to the cytotoxic nature of DA. Cells cultured with CD hydrogel showed no increase in TNF-α, IL-10, and IL-8 levels. It is hypothesized that the cytotoxicity of DA is moderated when reacted with SC and that CD hydrogel inhibits unwanted fibroblastic invasion preventing scarring and adhesions. Together with the previously published human and animal trial data, the results indicate CD hydrogel is biocompatible in the setting of endoscopic sinus surgery. This work represents the first study of CD hydrogel with human cell lines and provides essential information for its future application in biomedicine.

Cyclodextrin-polyhydrazine degradable gels for hydrophobic drug delivery

An injectable and biocompatible hydrogel system was designed for hydrophobic drug delivery. This hydrogel consisted of degradable polymers with cyclodextrin (CD) moieties. CD groups were used to increase the solubility of a hydrophobic molecule (nicardipine) in an aqueous solution through the formation of the inclusion complex. Two sets of gels were prepared by mixing oxidized dextran (DA) and CD functionalized polyhydrazine (PH) at physiological conditions and different level of crosslinking via hydrazone bonds. Cytotoxicity studies on the gels and their components confirmed the biocompatibility of these materials. Gel-30 with higher crosslinking density showed a two week degradation period whereas this period was 10days for gel-10, with lower crosslinking density, to complete degradation. The results from swelling tests and rheological measurements were also found to be dependent on crosslinking density of the hydrogels. Release profile of the hydrogel displayed a sustained release of nicardipin up to 6days for gel-30 and a 4day release with initial burst release for gel-10.

Strong poly(ethylene oxide) based gel adhesives via oxime cross-linking.

UNLABELLED There is a demand for materials to replace or augment the use of sutures and staples in surgical procedures. Currently available commercial surgical adhesives provide either high bond strength with biological toxicity or polymer and protein-based products that are biologically acceptable (though with potential sensitizing potential) but have much reduced bond strength. It is desirable to provide novel biocompatible and biodegradable surgical adhesives/sealants capable of high strength with minimal immune or inflammatory response. In this work, we report the end group derivatization of 8-arm star PEOs with aldehyde and amine end groups. Gels were prepared employing the Schiff-base chemistry between the aldehydes and the amines. Gel setting times, swelling behavior and rheological characterization were carried out for these gels. The mechanical-viscoelastic properties were found to be directly proportional to the crosslinking density of the gels, the 10K PEO gel was stiffer in comparison to the 20K PEO gel. The adhesive properties of these gels were tested using porcine skin and showed excellent adhesion properties. Cytotoxicity studies were carried out for the individual gel components using two different methods: (a) Crystal Violet Staining assay (CVS assay) and (b) impedance and cell index measurement by the xCELLigence system at concentrations >5%. Gels prepared by mixing 20% w/w solutions were also tested for cytotoxicity. The results revealed that the individual gel components as well as the prepared gels and their leachables were non-cytotoxic at these concentrations. STATEMENT OF SIGNIFICANCE This work presents a new type of glue that is aimed at surgery applications using a water soluble star shaped polymer. It show excellent adhesion to skin and is tough and easy to use. We show that it is very biocompatible based on tests on live human cells, and could therefore in principle be used for internal surgery. Comparison with other reported and commercial glues shows that it is stronger than most, and does not swell in water to the same degree as many other water based bioadhesives.

Characterization of the in vivo host response to a bi-labeled chitosan-dextran based hydrogel for postsurgical adhesion prevention

In developing a chitosan/dextran-based (CD) hydrogel as an adhesion prevention postsurgical aid, the in vivo biodegradation rate, biodistribution, and inflammatory response are important parameters to the biomedical device design. Herein, for the first time, a CD hydrogel was prepared by mixing aqueous solutions of a near infrared (NIR) labeled succinylated chitosan (SC) and tritiated [(3) H] oxidized dextran (DA). The biodegradation and biodistribution of the NIR/[(3) H]-CD hydrogel was tracked noninvasively using NIR fluorescence imaging, and by liquid scintillation counting (LSC) of organs/tissues after subcutaneous injection in BALB/c mice. The inflammatory response was assessed by measuring serum cytokine levels using a Bio-plex assay and by histological examination of injection site tissue. Fluorescence imaging showed the hydrogel to degrade in under a week. LSC revealed the hydrogel to reside mainly at the injection site, and excreted primarily via the urine within the first 48 h. The CD hydrogel showed a mild inflammatory response as cytokine levels were comparable to saline injected controls. Histological examination of injection site tissue confirmed the cytokine results. In summary, the CD hydrogels in vivo biodegradation rate, biodistribution, and inflammatory response was determined. Our results indicate that the CD hydrogel has an appropriate biocompatibility after s.c. administration.

Reducing the Oxidation Level of Dextran Aldehyde in a Chitosan/Dextran-Based Surgical Hydrogel Increases Biocompatibility and Decreases Antimicrobial Efficacy

A highly oxidized form of a chitosan/dextran-based hydrogel (CD-100) containing 80% oxidized dextran aldehyde (DA-100) was developed as a post-operative aid, and found to significantly prevent adhesion formation in endoscopic sinus surgery (ESS). However, the CD-100 hydrogel showed moderate in vitro cytotoxicity to mammalian cell lines, with the DA-100 found to be the cytotoxic component. In order to extend the use of the hydrogel to abdominal surgeries, reformulation using a lower oxidized DA (DA-25) was pursued. The aim of the present study was to compare the antimicrobial efficacy, in vitro biocompatibility and wound healing capacity of the highly oxidized CD-100 hydrogel with the CD-25 hydrogel. Antimicrobial studies were performed against a range of clinically relevant abdominal microorganisms using the micro-broth dilution method. Biocompatibility testing using human dermal fibroblasts was assessed via a tetrazolium reduction assay (MTT) and a wound healing model. In contrast to the original DA-100 formulation, DA-25 was found to be non-cytotoxic, and showed no overall impairment of cell migration, with wound closure occurring at 72 h. However, the lower oxidation level negatively affected the antimicrobial efficacy of the hydrogel (CD-25). Although the CD-25 hydrogel’s antimicrobial efficacy and anti-fibroblast activity is decreased when compared to the original CD-100 hydrogel formulation, previous in vivo studies show that the CD-25 hydrogel remains an effective, biocompatible barrier agent in the prevention of postoperative adhesions.

A chitosan/dextran-based hydrogel as a delivery vehicle of human bone-marrow derived mesenchymal stem cells

A chitosan/dextran-based (CD) injectable, surgical hydrogel has been developed and shown to be an effective post-operative aid in prevention of scar tissue formation in vivo. The CD hydrogels effectiveness in a surgical setting prompted an investigation into its capacity as a potential delivery vehicle for bone marrow derived mesenchymal stem cells (BM-MSCs) for regenerative wound healing applications. By housing BM-MSCs within a biocompatible, injectable, hydrogel matrix, viability and protection in cultivation, as well as direct delivery to the damaged site in the host tissue may be achieved. In vitro BM-MSC cell viability in the presence of CD hydrogel was determined by LIVE/DEAD® fluoresence staining. Flow cytometry studies revealed expression of a conventional BM-MSC surface marker profile. A colony forming cell assay showed a slight statistically significant decrease in the number of colonies grown in CD hydrogel as compared to control cells. In addition, BM-MSCs in the CD hydrogel were able to successfully differentiate into adipocytes and osteocytes. In summary, the CD hydrogel supports MSC growth and differentiation; and therefore, may be used as a potential stem cell delivery vehicle for regenerative medicine and tissue engineering applications.

3D-printed chitosan-based scaffolds: An in vitro study of human skin cell growth and an in-vivo wound healing evaluation in experimental diabetes in rats

The fabrication of porous 3D printed chitosan (CH) scaffolds for skin tissue regeneration and their behavior in terms of biocompatibility, cytocompatibility and toxicity toward human fibroblasts (Nhdf) and keratinocytes (HaCaT), are presented and discussed. 3D cell cultures achieved after 20 and 35 days of incubation showed significant in vitro qualitative and quantitative cell growth as measured by neutral red staining and MTT assays and confirmed by scanning electron microphotographs. The best cell growth was obtained after 35 days on 3D scaffolds when the Nhdf and HaCaT cells, seeded together, filled the pores in the scaffolds. An early skin-like layer consisting of a mass of fibroblast and keratinocyte cells growing together was observed. The tests of 3D printed scaffolds in wound healing carried out on streptozotocin-induced diabetic rats demonstrate that 3D printed scaffolds improve the quality of the restored tissue with respect to both commercial patch and spontaneous healing.