Zwitterionic Chitosan Derivative, a New Biocompatible Pharmaceutical Excipient, Prevents Endotoxin-Mediated Cytokine Release

Chitosan is a cationic polymer of natural origin and has been widely explored as a pharmaceutical excipient for a broad range of biomedical applications. While generally considered safe and biocompatible, chitosan has the ability to induce inflammatory reactions, which varies with the physical and chemical properties. We hypothesized that the previously reported zwitterionic chitosan (ZWC) derivative had relatively low pro-inflammatory potential because of the aqueous solubility and reduced amine content. To test this, we compared various chitosans with different aqueous solubilities or primary amine contents with respect to the intraperitoneal (IP) biocompatibility and the propensity to induce pro-inflammatory cytokine production from macrophages. ZWC was relatively well tolerated in ICR mice after IP administration and had no pro-inflammatory effect on naïve macrophages. Comparison with other chitosans indicates that these properties are mainly due to the aqueous solubility at neutral pH and relatively low molecular weight of ZWC. Interestingly, ZWC had a unique ability to suppress cytokine/chemokine production in macrophages challenged with lipopolysaccharide (LPS). This effect is likely due to the strong affinity of ZWC to LPS, which inactivates the pro-inflammatory function of LPS, and appears to be related to the reduced amine content. Our finding warrants further investigation of ZWC as a functional biomaterial

Transcatheter placement of a low-profile biodegradable pulmonary valve made of small intestinal submucosa: A long-term study in a swine model

ObjectiveWe sought to investigate a placement of a percutaneous low-profile prosthetic valve constructed of small intestinal submucosa in the pulmonary position in a swine model.MethodsTwelve female farm pigs were stented at the native pulmonary valve to induce pulmonary insufficiency. Once right ventricular dilation occurred, the small intestinal submucosa valve was implanted. The pigs were followed up with transthoracic echocardiographic Doppler scanning. One animal died of heart failure before valve replacement. Animals were euthanized at 1 day, 1 month, 3 months, 6 months, and 12 months after valve implantation.ResultsThe small intestinal submucosa pulmonary valve showed effective reversal of pulmonary regurgitation. There were no misplacements during deployment. There were no embolizations. One-year echocardiographic follow-up showed minimal regurgitation and no stenosis for a valve/vessel ratio of 0.78 or greater. Histologic examination demonstrated intensive remodeling of the small intestinal submucosal valve. Within 1 month, the surface was covered by endothelium, and fibroblasts invaded the interior. Over the following months, the small intestinal submucosal valve remodeled without apparent graft rejection.ConclusionThe small intestinal submucosa valve has the potential for graft longevity without the need for anticoagulation or immunosuppression. Histologic remodeling of the valve tissue provides a replacement capable of resembling a native valve that can be placed percutaneously with low-profile delivery systems

Novel patch biomaterial treatment for colon diverticulosis in swine model

Current leading managements for diverticular disease cannot prevent the recurrence of diverticulitis, bleeding and/or other complications. There is an immediate need for developing new minimal invasive therapeutic strategies to prevent and treat this disease. Through a biomechanical analysis of porcine colon with diverticular lesions, we proposed a novel adhesive patch concept aiming at mechanical reconstruction of the diseased colon wall. This study aims to evaluate the surgical feasibility (safety and efficacy) of pulmonary visceral pleura (PVP) patch therapy using a pig model of diverticulosis. Six female Yucatan miniature pigs underwent collagenase injection (CI) for the development of diverticular lesions. The lesions in each animal either received patch implantation (treated group, n = 40 for 6 pigs) or left intact (untreated group, n = 44 for 6 pigs). The normal colonic wall in each animal received patch implantation at two spots to serve as control (n = 12 for 6 pigs). After 3 months of observation, the performance and safety of the patch treatment were evaluated through macroscopic and histological examination. We found that 95% of pouch-like herniation of the mucosa was prevented from the colon wall with the treatment. The pouch diameter was significantly reduced in the treated group as compared to the untreated group (p < 0.001). The patch application caused a significant increase in the levels of collagen of the colon tissue as compared to the untreated and control groups (p < 0.001). No difference was found in the lymphocyte and macrophage inflammatory infiltrate between the groups. Our results suggest that patch treatment efficiently inhibits the diverticular pouch deformation and promotes the healing of the colon wall with a normal inflammatory response, which may minimize the risk of diverticulosis reoccurrence and complications over time

Comparison of Skeletal Effects of Ovariectomy Versus Chemically Induced Ovarian Failure in Mice

Bone loss associated with menopause leads to an increase in skeletal fragility and fracture risk. Relevant animal models can be useful for evaluating the impact of ovarian failure on bone loss. A chemically induced model of menopause in which mice gradually undergo ovarian failure yet retain residual ovarian tissue has been developed using the chemical 4-vinylcyclohexene diepoxide (VCD). This study was designed to compare skeletal effects of VCD-induced ovarian failure to those associated with ovariectomy (OVX). Young (28 day) C57Bl/6Hsd female mice were dosed daily with vehicle or VCD (160 mg/kg/d, IP) for 15 days (n = 6–7/group) and monitored by vaginal cytology for ovarian failure. At the mean age of VCD-induced ovarian failure (∼6 wk after onset of dosing), a different group of mice was ovariectomized (OVX, n = 8). Spine BMD (SpBMD) was measured by DXA for 3 mo after ovarian failure and OVX. Mice were killed ∼5 mo after ovarian failure or OVX, and bone architecture was evaluated by μCT ex vivo. In OVX mice, SpBMD was lower than controls 1 mo after OVX, whereas in VCD-treated mice, SpBMD was not lower than controls until 2.9 mo after ovarian failure (p < 0.05). Both VCD-induced ovarian failure and OVX led to pronounced deterioration of trabecular bone architecture, with slightly greater effects in OVX mice. At the femoral diaphysis, cortical bone area and thickness did not differ between VCD mice and controls but were decreased in OVX compared with both groups (p < 0.05). Circulating androstenedione levels were preserved in VCD-treated mice but reduced in OVX mice relative to controls (p < 0.001). These findings support that (1) VCD-induced ovarian failure leads to trabecular bone deterioration, (2) bone loss is attenuated by residual ovarian tissue, particularly in diaphyseal cortical bone, and (3) the VCD mouse model can be a relevant model for natural menopause in the study of associated bone disorders

Effect of chitosan treatment (all in 2 mg/mL) on the levels of proinflammatory cytokines released from (A) naïve mouse peritoneal macrophages and (B) LPS-challenged macrophages.

<p>Cytokine levels are determined by Milliplex Multi-Analyte Profiling cytokine/chemokine panel. Media of the LPS-challenged macrophages were 10 times diluted prior to analysis. Graphs on the right are displayed in narrow y-scales. ZWC (An/Am = 0.7); C-Gt: chitosan glutamate; Gly-C: glycol chitosan. Data are expressed as averages with standard deviations of three repeated measurements. *: p<0.05; **: p<0.01; ***: p<0.001 vs PBS.</p

Viability of mouse peritoneal macrophages in the presence of ZWC (An/Am ratio = 0.7), chitosan glutamate (C-Gt) and glycol chitosan (Gly-C).

<p>Data are expressed as averages with standard deviations of three repeated measurements. *: p<0.05; **: p<0.01 vs PBS.</p

MIP-2 production from macrophages incubated with ZWC (2 mg/mL), LPS (1 µg/mL), a mixture of LPS (1 µg/mL) and ZWC (2 mg/mL) (LPS co-inc w ZWC), or LPS pre-incubated with ZWC (equivalent to 1 µg/mL LPS under an assumption that all the LPS remained in the supernatant; LPS pre-inc w ZWC).

<p>ELISA was performed on 10 times-diluted culture media. Data are expressed as averages with standard deviations of three repeated measurements. ***: p<0.001.</p

Cytology of the peritoneal fluid from different treatment groups using hematoxylin and eosin staining.

<p>(A) PBS; (B) Glutamate buffer; (C) ZWC. (A-C) Peritoneal fluid composed of small macrophages (box) and lymphocytes (arrows). No chitosan precipitates were identified. (D) Glycol chitosan: peritoneal fluid is composed of large macrophages (box) containing chitosan. (E) Chitosan glutamate: peritoneal fluid composed of large macrophages with intracellular eosinophilic chitosan. Extracellular chitosan (Ch) is surrounded by numerous macrophages. All images are of 400× magnification.</p