The localisation of inflammatory cells and expression of associated proteoglycans in response to implanted chitosan

Implantation of a foreign material almost certainly results in the formation of a fibrous capsule around the implant however, mechanistic events leading to its formation are largely unexplored. Mast cells are an inflammatory cell type known to play a role in the response to material implants, through the release of pro-inflammatory proteases and cytokines from their α-granules following activation. This study examined the in vivo and in vitro response of mast cells to chitosan, through detection of markers known to be produced by mast cells or involved with the inflammatory response. Mast cells, identified as Leder stained positive cells, were shown to be present in response to material implants. Additionally, the mast cell receptor, c-kit, along with collagen, serglycin, perlecan and chondroitin sulphate were detected within the fibrous capsules, where distribution varied between material implants. In conjunction, rat mast cells (RBL-2H3) were shown to be activated following exposure to chitosan as indicated by the release of β-hexosaminidase. Proteoglycan and glycosaminoglycans produced by the cells showed similar expression and localisation when in contact with chitosan to when chemically activated. These data support the role that mast cells play in the inflammatory host response to chitosan implants, where mediators released from their α-granules impact on the formation of a fibrous capsule by supporting the production and organisation of collagen fibres.     

Promotion of cardiac differentiation of brown adipose derived stem cells by chitosan hydrogel for repair after myocardial infarction

The ability to restore heart function by replacement of diseased myocardium is one of the great challenges in biomaterials and regenerative medicine. Brown adipose derived stem cells (BADSCs) present a new source of cardiomyocytes to regenerate the myocardium after infarction. In this study, we explored an injectable tissue engineering strategy to repair damaged myocardium, in which chitosan hydrogels were investigated as a carrier for BADSCs. In vitro, the effect and mechanism of chitosan components on the cardiac differentiation of BADSCs were investigated. In vivo, BADSCs carrying double-fusion reporter gene (firefly luciferase and monomeric red fluorescent protein (fluc-mRFP)) were transplanted into infarcted rat hearts with or without chitosan hydrogel. Multi-techniques were used to assess the effects of treatments. We observed that chitosan components significantly enhanced cardiac differentiation of BADSCs, which was assessed by percentages of cTnT⁺ cells and expression of cardiac-specific markers, including GATA-4, Nkx2.5, Myl7, Myh6, cTnI, and Cacna1a. Treatment with collagen synthesis inhibitors, cis-4-hydroxy-d-proline (CIS), significantly inhibited the chitosan-enhanced cardiac differentiation, indicating that the enhanced collagen synthesis by chitosan accounts for its promotive role in cardiac differentiation of BADSCs. Longitudinal in vivo bioluminescence imaging and histological staining revealed that chitosan enhanced the survival of engrafted BADSCs and significantly increased the differentiation rate of BADSCs into cardiomyocytes in vivo. Furthermore, BADSCs delivered by chitosan hydrogel prevented adverse matrix remodeling, increased angiogenesis, and preserved heart function. These results suggested that the injectable cardiac tissue engineering based on chitosan hydrogel and BADSCs is a useful strategy for myocardium regeneration.     

The promotion of osteointegration under diabetic conditions using chitosan/hydroxyapatite composite coating on porous titanium surfaces

Composited Chitosan/Hydroxyapatite (CS/HA) material coated on titanium surface (cTi) is a promising approach to produce biomaterials with better osseointegration capacity, but its bio-performance under diabetic conditions and the mechanisms involved remain elusive. We propose that the alterations in the Wnt/β-catenin pathway may play a role in mediating the improvement effect of cTi on diabetes-induced impaired implant osteointegration. To confirm the hypothesis, primary rat osteoblasts incubated on Ti and cTi were subjected to normal serum (NS), diabetic serum (DS), DS + Wnt3a (a specific Wnt agonist) and DS + Dkk1 (a specific Wnt antagonist) treatment. In vivo study was performed on diabetic sheep implanted with Ti or cTi into the bone defect on crista iliaca. Results showed that diabetes depressed osteoblast function evidenced by impaired cell adhesion and morphology, decreased cell proliferation and ALP activity, and higher apoptotic rate on Ti. Importantly, both cTi and Wnt3a treatment ameliorated osteoblastic dysfunction and apoptosis under diabetic condition. Implantation with cTi significantly improved osteointegration evidenced by Micro-CT and histological examinations compared with Ti. Moreover, the aforementioned promotive effects afforded by cTi were abolished by blocking Wnt pathway with Dkk1. Our study explicitly demonstrates that CS/HA composite material improves diabetes-induced impaired osteointegration of Ti via the reactivation of Wnt/β-catenin pathway and provides a target point for biomaterial modification to attain better clinical performance in diabetic patients.     

透明质酸/壳聚糖复合支架的制备及其力学性能评价

目的　模拟天然骨组织的结构和成分,寻找适合骨组织工程的新型支架材料。 方法 以透明质酸、壳聚糖为基质材料,在微酸性环境中以一定配比与氯化钙和磷酸二氢钠混合,冷冻干燥得到多孔复合支架材料。然后在乙醇/水/尿素环境中分别陈化0、2、4、8、12和24 h,以生成产物钙磷盐前驱体转变为羟基磷灰石,最终制备出一种深度矿化的透明质酸/壳聚糖复合支架。并通过SEM、EDS等对支架进行表征,研究支架的形貌、成分及力学强度等性能。 结果 SEM观察显示,支架材料具有比较均匀的多孔结构,孔径大小为100~200 μm。EDS结果表明,复合支架在一次冻干之后形成的是磷酸氢钙（DCPD）,随着陈化时间的延长,DCPD逐渐向羟基磷灰石（HAP）转化。而压缩强度则表明经过原位矿化的支架力学性能显著提高。 结论 通过该法得到的透明质酸/壳聚糖复合支架可作为骨组织工程的新型支架材料。     

Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone

Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were assessed. Finally, the cultivation of osteoblastic SaOS-2 cells on the surface-functionalized CHS-g-PCL was followed by analyses of cell viability, mineral deposition and alkaline phosphatase activity. These characterizations revealed a composite that combines the versatile properties of CHS-g-PCL with the osteogenic activity of the silicatein/biosilica coating and, hence, represents an innovative alternative to conventionally used CHS/PCL composites for biomedical applications, where stable bone–material interfaces are required.     

Effectiveness of tissue engineered based platelet gel embedded chitosan scaffold on experimentally induced critical sized segmental bone defect model in rat

BackgroundHealing and regeneration of large bone defects are a challenging problem for reconstructive orthopedic surgeons.PurposeThis study investigated the effectiveness of chitosan scaffold (CS), platelet gel (PG) and their combination (CS-PG) on healing process of an experimentally induced critical sized segmental bone defect model in rat.MethodsFifty bilateral defects were created in the mid diaphysis of the radial bones of 25 Sprague-Dawley rats. The animals were randomly divided into five equal groups. The bone defects were either left untreated or treated with corticomedullary autograft, CS, PG or CS-PG. Plain radiographs were provided from the radial bones on weeks 2, 5, and 8 after injury. In addition, clinical examinations were done for the healing radial bones. The animals were euthanized after 8 weeks of injury, and their harvested samples were evaluated by gross morphology, histopathology, scanning electron microscopy, CT-scan, and biomechanical testing.ResultsCompared with the defect group, the PG and autograft treated bone defects had significantly superior radiological scored values, bone volume and biomechanical performance which had positive correlation with their superior gross pathological, histopathological and ultra-structural features. Compared with the untreated defects, the PG and CS-PG treated defects showed significantly superior structural and functional properties so that PG had the highest value. In addition, CS had low value in bone regeneration. Although combination of CS and PG improved the healing efficacy of the CS, this strategy reduced the ability of PG to increase osteoconduction and osteoinduction during bone regeneration.ConclusionApplication of PG alone enhanced bone healing and can be regarded as a promising option for bone tissue engineering in clinical settings. Chitosan was not effective in bone reconstruction surgery and further investigations should be conducted to find a suitable carrier for PG.     

Feasibility study of chitosan as intravitreous tamponade material

BACKGROUND: Chitosan can inhibit fibroblastic proliferation by suppressing fibroblast cells, and has the similar physiological characteristics as normal vitreous body, so it might have the potential to become vitreous filling material and might possibly inhibit proliferative vitreous retinopathy. To investigate the possibility of chitosan as vitreous filling material, this study was designed to investigate retina, ciliary body, lens and cornea morphology changes, intraocular pressure and intraocular inflammatory factors fluctuating after chitosan intravitreous tamponade. METHODS: Fifteen healthy chinchilla rabbits were chosen; three of them were a blank (negative) control group without any surgical procedure. The remaining 12 rabbits received vitrectomy on both eyes; all the right eyes (experimental group) were given 1.2-1.8 ml (average 1.5 ml) of chitosan intravitreously, while sodium hyaluronate were given in the left eyes (control group). All eyes underwent slit-lamp biomicroscope and indirect ophthalmoscope examination and intraocular pressure measurement pre- and post-op. The concentration of IL-6, IL-8 (radioimmunoassay), NO (nitrate reductase method) in aqueous humor and vitreous body were tested at day 15 and day 30 post-op. At day 30 post-op, the cornea, ciliary body, and lens were dissected for light microscopy examination, and the retinal tissues 2PD away from the optic disc on the vertical orientation of posterior pole were dissected for light- and electro-microscope examination. RESULTS: The conjunctival congestion and slight inflammatory response in the anterior chamber disappeared within 7 days post-op. During the 30-day experiment, cornea, lens and the filling material in vitreous cavity were transparent in all animals. The retina was attached without proliferation. The intraocular pressure in the experimental group post-op fluctuated between 4.55 +/- 2.94 and 6.25 +/- 2.37 mmHg, which was not significantly different from the situation pre-op (6.18 +/- 1.19 mmHg) (P > 0.05). The intraocular pressure in the control group post-op fluctuated between 5.10 +/- 2.51 and 5.90 +/- 2.49 mmHg, which was not significantly different from the situation pre-op (6.50 +/- 0.94 mmHg) (P > 0.05). There was also no significant difference in the intraocular pressure post-op at different time points between the experimental group and control group (all P > 0.05). At day 15 post-op, IL-6 concentration in aqueous humor were 37.31 +/- 8.59 ng/ml and 39.52 +/- 9.69 ng/ml in experimental group and control group respectively, both higher than those in the blank control group (26.55 +/- 9.34 ng/ml) (P < 0.05). IL-8 concentration were 7.00 +/- 3.79 ng/ml and 6.32 +/- 3.68 ng/ml respectively, no significant difference to the blank control group (4.72 +/- 1.71 ng/ml) (P > 0.05): the concentrations of NO were 63.94 +/- 26.80 micromol/ml and 51.81 +/- 13.19 micromol/ml respectively, no significant difference to the blank control group (50.36 +/- 15.67 micromol/ml) (P > 0.05). At day 30 post-op, the concentrations of IL-6, IL-8 and NO in aqueous humor showed no significant difference among all three groups (P > 0.05). In vitreous body at day 30 post-op, the concentrations of IL-8 in experimental group and control group were 10.17 +/- 3.63 ng/ml and 10.69 +/- 3.52 ng/ml, and those of NO were 50.23 +/- 19.69 micromol/mL and 50.60 +/- 12.72 micromol/mL respectively, all higher than in the blank control group (30.37 +/- 14.63 micromol/ml) (P < 0.05); the concentrations of IL-6 were 24.51 +/- 10.71 ng/ml and 26.36 +/- 13.00 ng/ml, no significant difference to the blank control group (24.06 +/- 5.98 ng/ml) (P > 0.05). At various time points, there was no significant difference in the concentrations of IL-6, IL-8 and NO in aqueous humor and vitreous body in the experimental group and the control group (P > 0.05). There was no morphological change found under light microscopy in cornea, ciliary body and lens. The outer plexiform layer of retina was thinner, but no significant degeneration, necrosis, karyopyknosis or lysis were found under the ultrastructural microscopy. CONCLUSION: Chitosan intravitreous tamponade has no significant effect on the histology of the eye, doesn't cause intraocular pressure to fluctuate, and slightly increases inflammatory factors (IL-6, IL-8, NO) in comparison to the normal levels, but with no significant difference from the effects caused by sodium hyaluronate, which indicated chitosan might not lead to a clinically significant inflammatory response. It suggests that chitosan could be used as intravitreous tamponade material.     

壳聚糖对甲硝唑凝胶体外促透作用研究

目的 考察壳聚糖(CS)对甲硝唑凝胶体外透皮速率的影响。方法 将1.0% CS作为吸收促进剂用于甲硝唑凝胶中,以泊洛沙姆p407为凝胶基质,以含2%氮酮的甲硝唑凝胶作为阳性对照,不含任何促渗剂的甲硝唑凝胶作为阴性对照,采用改良Franz扩散池进行大鼠体外皮肤渗透实验,反相高效液相色谱法（RP HPLC）法测定接受液中甲硝唑含量,计算累积透过量Q,得出Q t回归方程及稳态渗透速率J。结果 1.0%CS组和氮酮组J分别为2.841, 2.874 μg&#8226;（cm2） 1&#8226;h 1,两者差异无显著性(P＞0.05),而与阴性组相比均差异有显著性(均P＜0.05),其透皮吸收行为符合一级方程。结论 CS对甲硝唑凝胶的体外透皮吸收有较好的促进作用,值得进一步研究。     

几丁糖顺铂缓释药膜的制备及体外抑制人胃腺癌SGC--7901细胞生长的研究

目的观察几丁糖-顺铂缓释药膜体外缓释性和对人胃腺癌SGC-7901细胞生长的抑制作用。方法将几丁糖与顺铂混合,加入交联剂戊二醛,真空干燥,制成几丁糖-顺铂缓释药膜,检测药膜的自然降解性、药膜顺铂含量和药物包封率,同时观察药膜体外缓释顺铂及其对胃癌细胞生长的抑制作用。结果几丁糖-顺铂缓释药膜在含溶菌酶的生理盐水中于8天后开始自然降解,60天后降解率达83.33%;药膜载药率为(7.30±0.20)%,药物包封率为(51.78±2.10)%;药膜体外释放顺铂量于第1天达75.40μg/ml,第2天释放量明显降低,为21.35μg/ml,以后呈较低水平缓慢释放,第7天达3.94μg/ml,并且,其浸出液体外抑制胃癌细胞生长的抑制率第1天为84.24%,以后渐降低,第7天达29.81%。结论几丁糖-顺铂缓释药膜在体外可较好地缓释顺铂,对人胃腺癌SGC--7901细胞的生长有较持久地抑制作用。     

壳聚糖纳米粒制备的研究进展

载药纳米粒作为药物、基因传递和控释的载体,是近年来出现的药物控释和缓释的新剂型。壳聚糖具有较好的生物黏附性、促吸收效应和酶抑制载体作用等特性。壳聚糖纳米粒作为一种新型药物载体,已成为目前国内外研究开发的热点。本文就壳聚糖纳米粒制备的研究进展情况进行了综述。