Integrating Fused Deposition Modeling and Melt Electrowriting for Engineering Branched Vasculature.

We demonstrate for the first time the combination of two additive manufacturing technologies used in tandem, fused deposition modelling (FDM) and melt electrowriting (MEW), to increase the range of possible MEW structures, with a focus on creating branched, hollow scaffolds for vascularization. First, computer-aided design (CAD) was used to design branched mold halves which were then used to FDM print conductive polylactic acid (cPLA) molds. Next, MEW was performed over the top of these FDM cPLA molds using polycaprolactone (PCL), an FDA-approved biomaterial. After the removal of the newly constructed MEW scaffolds from the FDM molds, complementary MEW scaffold halves were heat-melded together by placing the flat surfaces of each half onto a temperature-controlled platform, then pressing the heated halves together, and finally allowing them to cool to create branched, hollow constructs. This hybrid technique permitted the direct fabrication of hollow MEW structures that would otherwise not be possible to achieve using MEW alone. The scaffolds then underwent in vitro physical and biological testing. Specifically, dynamic mechanical analysis showed the scaffolds had an anisotropic stiffness of 1 MPa or 5 MPa, depending on the direction of the applied stress. After a month of incubation, normal human dermal fibroblasts (NHDFs) were seen growing on the scaffolds, which demonstrated that no deleterious effects were exerted by the MEW scaffolds constructed using FDM cPLA molds. The significant potential of our hybrid additive manufacturing approach to fabricate complex MEW scaffolds could be applied to a variety of tissue engineering applications, particularly in the field of vascularization.

Macrocystis pyrifera Lipids Reduce Cytokine-Induced Pro-Inflammatory Signalling and Barrier Dysfunction in Human Keratinocyte Models.

Atopic dermatitis is a chronic condition where epidermal barrier dysfunction and cytokine production by infiltrating immune cells exacerbate skin inflammation and damage. A total lipid extract from Macrocystis pyrifera, a brown seaweed, was previously reported to suppress inflammatory responses in monocytes. Here, treatment of human HaCaT keratinocytes with M. pyrifera lipids inhibited tumour necrosis factor (TNF)-α induced TNF receptor-associated factor 2 and monocyte chemoattractant protein (MCP)-1 protein production. HaCaT cells stimulated with TNF-α, interleukin (IL)-4, and IL-13 showed loss of claudin-1 tight junctions, but little improvement was observed following lipid pre-treatment. Three-dimensional cultures of HaCaT cells differentiated at the air-liquid interface showed increased MCP-1 production, loss of claudin-1 tight junctions, and trans-epidermal leakage with TNF-α, IL-4, and IL-13 stimulation, with all parameters reduced by lipid pre-treatment. These findings suggest that M. pyrifera lipids have anti-inflammatory and barrier-protective effects on keratinocytes, which may be beneficial for the treatment of atopic dermatitis or other skin conditions.

Development and optimisation of hydroxyapatite-polyethylene glycol diacrylate hydrogel inks for 3D printing of bone tissue engineered scaffolds.

In the event of excessive damage to bone tissue, the self-healing process alone is not sufficient to restore bone integrity. Three-dimensional (3D) printing, as an advanced additive manufacturing technology, can create implantable bone scaffolds with accurate geometry and internal architecture, facilitating bone regeneration. This study aims to develop and optimise hydroxyapatite-polyethylene glycol diacrylate (HA-PEGDA) hydrogel inks for extrusion 3D printing of bone tissue scaffolds. Different concentrations of HA were mixed with PEGDA, and further incorporated with pluronic F127 (PF127) as a sacrificial carrier. PF127 provided good distribution of HA nanoparticle within the scaffolds and improved the rheological requirements of HA-PEGDA inks for extrusion 3D printing without significant reduction in the HA content after its removal. Higher printing pressures and printing rates were needed to generate the same strand diameter when using a higher HA content compared to a lower HA content. Scaffolds with excellent shape fidelity up to 75-layers and high resolution (∼200 µm) with uniform strands were fabricated. Increasing the HA content enhanced the compression strength and decreased the swelling degree and degradation rate of 3D printed HA-PEGDA scaffolds. In addition, the incorporation of HA improved the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) onto the scaffolds. 3D printed scaffolds with 2 wt% HA promoted osteogenic differentiation of hBMSCs as confirmed by the expression of alkaline phosphatase activity and calcium deposition. Altogether, the developed HA-PEGDA hydrogel ink has promising potential as a scaffold material for bone tissue regeneration, with excellent shape fidelity and the ability to promote osteogenic differentiation of hBMSCs.

3D printing of chitooligosaccharide-polyethylene glycol diacrylate hydrogel inks for bone tissue regeneration.

To date, lack of functional hydrogel inks has limited 3D printing applications in tissue engineering. This study developed a series of photocurable hydrogel inks based on chitooligosaccharide (COS)-polyethylene glycol diacrylate (PEGDA) for extrusion-based 3D printing of bone tissue scaffolds. The scaffolds were prepared by aza-Michael addition of COS and PEGDA followed by photopolymerisation of unreacted PEGDA. The hydrogel inks showed sufficient shear thinning properties required for extrusion 3D printing. The printed scaffolds exhibited excellent shape fidelity and fine microstructure with a resolution of 250 µm. By increasing the COS content, the swelling ratio of the scaffolds decreased, while the compressive strength increased. 3D printed COS-PEGDA scaffolds showed high viability of human bone mesenchymal stem cells in vitro. In addition, scaffolds containing 2 wt% COS showed significantly higher alkaline phosphatase activity, calcium deposition, and bioactivity in simulated body fluid compared to the control (PEGDA). Altogether, 3D printed COS-PEGDA scaffolds represent promising candidates for bone tissue regeneration.

3D Living Dressing Improves Healing and Modulates Immune Response in a Thermal Injury Model.

Thermal injury trauma can induce a state of immunosuppression, causing wounds to become chronic in nature. Stem cell-based therapies represent a promising new approach to treat such wounds due to their capacity to self-renew and their multi-lineage potential. Mesenchymal stem cells (MSCs) are known to secrete endogenous factors that stimulate wound healing by promoting angiogenesis, extracellular matrix remodeling, skin regeneration, and by dampening down inflammation. MSC delivery in a biomaterial construct can augment their wound-healing capacity by concentrating cells at the burn site and upregulating trophic factor secretion. The work presented is the first to evaluate repair in an in vitro raft thermal injury model using a regenerative, dual cell delivery three-dimensional (3D) core/shell (c/s) "living dressing" construct. This previously characterized 3D c/s bioprinted construct, which delivers both MSCs and endothelial cells, was used to treat an in vitro 3D raft skin thermal injury wound model. The mesenchymal stromal cell line (T0523) was encapsulated within a gelatin-based shell bioink, and human umbilical vein endothelial cells within a chitosan-based core bioink to biofabricate a living dressing for enhanced thermal injury repair and regeneration. We hypothesized that the cell-laden c/s tissue engineered construct (TEC) would strengthen the wound's proangiogenic, anti-inflammatory, and skin regeneration potential. An in vitro thermal injury in a 3D raft skin model showed a slight delay in wound closure in the presence of the c/s TEC but was augmented by corresponding increases in the release of wound-healing factors, epidermal growth factor, matrix metalloproteinases-9, transforming growth factor-α, platelet-derived growth factor; a decrease in pro-inflammatory factor interleukin-6, and evidence of neovascularization.

A comparison between ß-tricalcium phosphate and chitosan poly-caprolactone-based 3D melt extruded composite scaffolds.

Melt extrusion 3D printing has become an attractive additive manufacturing technology to construct degradable scaffolds as tissue precursors in order to create clinically relevant medical devices. Towards this end, a commonly used synthetic polyester, poly-caprolactone (PCL), was used to make scaffolds composed of different biomaterial compositions to increase bioactivity using 3D melt pneumatic extrusion technology. Varying ratios of the natural biopolymer, chitosan, or the bioceramic, ß-tricalcium phosphate (TCP) were blended with PCL to fabricate support scaffolds with three-dimensional (3D) architecture for human bone-marrow derived mesenchymal stem cell (hBMSC) growth for potential bone regeneration application. In this study, basic printing requirements as well as biomaterial dynamic mechanical (DMA), elemental, and thermogravimetric (TGA) analysis results demonstrate material homogeneity as well as thermal stability. Scaffold morphology and microarchitecture were assessed using scanning electron microscopy (SEM) alongside in vitro scaffold degradation and biological characterisation. Human BMSC proliferation was assessed using fluorescence imaging, and quantitated via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay. These in vitro cell viability studies revealed that the highest chitosan concentration blend of 20% favoured the most hBMSC growth, exhibited the most swelling, and showed minimal degradation after 28 days. The 20% TCP blend had the second highest hBMSC growth, exhibited moderate swelling, and the fastest degradation rate. Overall, this study demonstrates the first direct comparison of a natural biopolymer-based, that is, chitosan, 3D melt extruded PCL composite with that of a bioceramic-based, that is, ß-TCP, PCL composite and their effects on hBMSC 3D proliferation. 3D melt extruded PCL-based composite scaffolds methodology offers a straightforward way to print scaffolds with good shape fidelity, interconnected porosities and enhanced bioactivity; and demonstrates their potential use for regenerative, bone repair applications.

Three-Dimensional Melt-Electrowritten Polycaprolactone/Chitosan Scaffolds Enhance Mesenchymal Stem Cell Behavior.

Melt electrowriting (MEW) is an emerging technique that precisely fabricates microfibrous scaffolds, ideal for tissue engineering, where biomimetic microarchitectural detail is required. Polycaprolactone (PCL), a synthetic polymer, was selected as the scaffold material due to its biocompatibility, biodegradability, mechanical strength, and melt processability. To increase PCL bioactivity, a natural polymer, chitosan, was added to construct MEW fibrous composite scaffolds. To date, this is the first study of its kind detailing the effects of stem cell behavior on PCL containing chitosan MEW scaffolds. The aim of this study was to melt electrowrite a range of PCL/chitosan tissue-engineered constructs (TECs) and assess their suitability to promote the growth of human bone-marrow-derived mesenchymal stem cells (hBMSCs). In vitro physical and biological characterizations of melt-electrowritten TECs were performed. Physical characterization showed that reproducible, layered micron-range scaffolds could be successfully fabricated. As well, cell migration and proliferation were assessed via an assay to monitor cell infiltration throughout the three-dimensional (3D) melt-electrowritten scaffold structure. A statistically significant increase (∼140%) in hBMSC proliferation in 1 wt % chitosan PCL blends in comparison to PCL-only scaffolds was found when monitored over two weeks. Overall, our study demonstrates the fabrication of melt-electrowritten PCL/chitosan composite scaffolds with controlled microarchitecture and their potential use for regenerative, tissue engineering applications.

Melt Electrowritten Sandwich Scaffold Technique Using Sulforhodamine B to Monitor Stem Cell Behavior.

Background: Three-dimensional (3D) printing using melt electrowriting (MEW) technology is a recently developed technique to produce biocompatible micron-level mesh scaffolds layer-by-layer that can be seeded with cells for tissue engineering. Examining cell behavior, such as growth rate and migration, can be problematic in these opaque 3D scaffolds. A straightforward and quantitative method was developed to examine these cellular parameters on poly-ɛ-caprolactone (PCL) multilayered MEW scaffolds developed as components of the annulus fibrosus region of bioengineered intervertebral discs. Experiment: The anti-adhesion protein, bovine serum albumin (BSA), was used to coat plasticware to improve mesenchymal stem cell (T0523) adhesion to MEW scaffolds. Cells were seeded on circular MEW (cMEW) discs as defined growth starting points sandwiched between two test template scaffolds investigated at varying pore sizes. Cell expansion, growth, and migration were quantitated utilizing the protein-specific dye sulforhodamine B (SRB). Live cell imaging combined with image analysis were used to examine cell motility and expansion on 3D scaffolds. Results: After one coating of BSA, cells remained nonadherent for the duration of the study with cell spheroids formed and enlarging over 21 days and becoming entangled in MEW scaffold pores. Cells grown on the 250 µm pore size scaffolds exhibited a doubling time of 7 days, whereas the 400 µm pore size scaffolds time was 11.5 days. Conclusions: BSA coating of tissue culture dishes prevented surface adhesion of cells to vessel surfaces and promoted spheroid formation that encouraged attachment to the PCL scaffolds. Batch-printed cMEW scaffolds were useful as a defined starting point for quantitative assays that successfully measured cell migration, expansion and proliferation on test scaffolds. The SRB assay was shown to be a useful and straightforward way to quantitate cell numbers in multilayered MEW scaffolds. A pore size of 250 µm exhibited the fastest cell growth, spread, and expansion. Impact statement In this article, a new, useful, and straightforward method to quantitate cell numbers on three-dimensional (3D) melt electrowritten (MEW) scaffolds is presented. By using the sulforhodamine B assay on bovine serum albumin-coated dishes cell migration, expansion and proliferation in 3D printed MEW test scaffolds were quantitatively measured. Printed circular MEW (cMEW) scaffolds sandwiched between two MEW test scaffolds (Fig. 3) were used as defined cellular growth starting points with a particular pore size of 250 µm displaying the fastest cell growth and migration. This MEW sandwich technique could potentially be used to quantitate cell numbers and migration in other 3D multilayered MEW scaffold systems.

Peptide Chitosan/Dextran Core/Shell Vascularized 3D Constructs for Wound Healing.

Three-dimensional (3D) bioprinting has emerged to create novel cell-based therapies for regenerative medicine applications. Vascularized networks within engineered constructs are required, and toward this end, we report a promising strategy using core-shell (c/s) extrusion 3D-bioprinting technology that employs biomimetic biomaterials to construct regenerative, prevascularized scaffolds for wound care. A custom-designed cell-responsive bioink consisting of a 13% (w/v) cell-laden gelatin methacryloyl (GelMA) shell surrounding a peptide-functionalized, succinylated chitosan (C)/dextran aldehyde (D) cell-laden core was successfully bioprinted resulting in organized microdesigns exhibiting excellent cell viability and subsequent vessel formation. Our templating strategy takes advantage of GelMA's intrinsic thermoreversible properties of low degree of acryloyl functionalization used in combination with a lightly, chemically cross-linked peptide-CD core to serve as temporal structural supports that stabilize during extrusion onto a cooled platform. Mechanical integrity was further strengthened layer-by-layer via GelMA UV photo-cross-linking. We report the first example of GelMA used in combination with a peptide-CD bioink to c/s 3D-bioprint regenerative, prevascularized constructs for wound care. Particular cell adhesion and proteolytic peptide-CD functionalized pair combinations, P15/MMP-2 and P15/cRGD, were found to significantly increase growth of human bone-marrow-derived mesenchymal stems cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs). The constructs delivered two cell types: hBMSCs in the shell bioink and HUVECs within the core bioink. Cord-like, natural microvascularization was shown with endothelial cell marker expression as confirmed by immunofluorescence (IF) staining exhibiting tubelike structures. In addition, in vitro skin wound healing activity of the construct showed a ∼twofold rate of wound closure. Overall, c/s 3D-bioprinted, peptide-CD/GelMA constructs provided the appropriate microenvironment for in vitro stem and endothelial cell viability, delivery, and differentiation. We foresee these custom constructs as representing a fundamental step toward engineering larger scale regenerative, prevascularized tissues.

Melt-electrowriting with novel milk protein/PCL biomaterials for skin regeneration.

Demand for skin replacements is rapidly increasing as burn and full-thickness wounds are difficult to repair due to the low regeneration capability of innate tissues, as well as the physical drawbacks associated with currently available substitutes. To address this need, an emerging 3D printing technique, melt-electrowriting (MEW) was used to create novel bioactive scaffolds to promote skin regeneration. Polycaprolactone (PCL), a bioresorbable and biocompatible, synthetic polymer with Food and Drug Administration approval for use in the human body was selected as scaffold material due to its mechanical stability, flexibility, and superior melt processing properties. In order to increase PCL's biological functionality bioactive milk proteins (MPs) were blended with PCL. To date, this is the first study of its kind detailing the tissue regenerative capacity of PCL containing MPs as bioactive additives for skin regeneration using MEW. The aim of this study was to MEW MP/PCL tissue engineered constructs (TEC) and assess their suitability for generating tissue in vitro. The MPs, lactoferrin (LF) and whey protein (WP), were mixed with PCL individually at varying concentrations (0.05%, 0.1%, 0.25%), and in combination (COMB) at concentrations of 0.25% each. TECs were characterised chemically, physically, and their biological activity assessed in vitro. Physical characterisation of MEW MP/PCL scaffolds showed that reproducible, layered micron range scaffolds could be fabricated; displaying high porosity, low degradation, and rapid protein release. Biological activity, determined via an in vitro skin model using human keratinocytes (HaCaTs) and normal human dermal fibroblasts cells, showed significantly increased cell growth, spreading, and infiltration into LF (0.25%) containing scaffolds and COMB scaffolds when compared to PCL alone (p ≤ 0.05). These findings demonstrated that the combined addition of LF and WP increased the biological activity of MEW PCL scaffolds and could be potentially used as a TEC for deep tissue dermal regeneration.

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 hydrogel's 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.

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

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.

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.

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 hydrogel's 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.

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.

Synthesis, physiochemical characterization, and biocompatibility of a chitosan/dextran-based hydrogel for postsurgical adhesion prevention.

An amine-functionalized succinyl chitosan and an oxidized dextran were synthesized and mixed in aqueous solution to form an in situ chitosan/dextran injectable, surgical hydrogel for adhesion prevention. Rheological characterization showed that the rate of gelation and moduli were tunable based on amine and aldehyde levels, as well as polymer concentrations. The CD hydrogels have been shown to be effective post-operative aids in prevention of adhesions in ear, nose, and throat surgeries and abdominal surgeries in vivo. In vitro biocompatibility testing was performed on CD hydrogels containing one of two oxidized dextrans, an 80 % oxidized (CD-100) or 25 % (CD-25) oxidized dextran. However, the CD-100 hydrogel showed moderate cytotoxicity in vitro to Vero cells. SC component of the CD hydrogel, however, showed no cytotoxic effect. In order to increase the biocompatibility of the hydrogel, a lower aldehyde level hydrogel was developed. CD-25 was found to be non-cytotoxic to L929 fibroblasts. The in vivo pro-inflammatory response of the CD-25 hydrogel, after intraperitoneal injection in BALB/c mice, was also determined by measuring serum TNF-α levels and by histological analysis of tissues. TNF-α levels were similar in mice injected with CD-25 hydrogel as compared to the negative saline injected control; and were significantly different (P < 0.05) as compared to the positive, lipopolysaccharide, injected control. Histological examination revealed no inflammation seen in CD hydrogel injected mice. The results of these in vitro and in vivo studies demonstrate the biocompatibility of the CD hydrogel as a post-operative aid for adhesion prevention.

Antimicrobial properties of a chitosan dextran-based hydrogel for surgical use.

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.