Controlled release of fibroblast growth factors and heparin from photocrosslinked chitosan hydrogels and subsequent effect on in vivo vascularization

Application of ultraviolet (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted within 10 s in an insoluble, flexible hydrogel. A low molecular weight acidic molecule like trypan blue and various high molecular weight molecules such as bovine serum albumin (BSA), heparin and protamine were all retained within the hydrogel, while a low molecular weight basic molecule like toluidine blue was rapidly released from the hydrogel. In the present work, we examined the retaining capability of the chitosan hydrogel for growth factors and controlled release of growth factors from the chitosan hydrogel in vitro and in vivo. Fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor(165) (VEGF(165)), heparin-binding epidermal growth factor (HB-EGF) in phosphate buffered saline (PBS) were mixed with Az-CH-LA aqueous solution to form growth factor-incorporated chitosan hydrogels. About 10-25% of the growth factor was released from a growth factor-incorporated chitosan hydrogel into PBS within the first day, after which no further substantial release took place. The growth factors interacted with Az-CH-LA molecules poly-ion complexation, and probably were unable to be released after the first day under the in vitro nondegradation conditions of the hydrogel. Although the FGF-1, FGF-2, and VEGF(165)-incorporated chitosan hydrogels on a culture plate significantly stimulated HUVEC growth, the stimulating activity of the growth factor-incorporated chitosan hydrogel was completely cancelled out by washing the hydrogel with PBS solution for 3 days or more. The stimulating activity on the HUVEC growth were however highly recovered by treating the washed growth factor-incorporated chitosan hydrogel during 7 days with chitinase and chitosanase to partly degrade the hydrogel, strongly suggesting that the growth factors within the hydrogel retained their biologically active forms. The chitosan hydrogel (100 microl) when implanted into the back of a mouse was biodegraded in about 10-14 days. When FGF-1- and FGF-2-incorporated chitosan hydrogels were subcutaneously implanted into the back of a mouse, significant neovascularization was induced near the implanted site of the FGF-1- and FGF-2-incorporated chitosan hydrogels. Furthermore, addition of heparin with either FGF-1 or FGF-2 into the hydrogel resulted in a significantly enhanced and prolonged vascularization effect. These results indicate that the controlled release of biologically active FGF-1 and FGF-2 with heparin is caused by biodegradation of the chitosan hydrogel, and subsequent induction of vascularization.     

Chitosan hydrogel as a drug delivery carrier to control angiogenesis

An aqueous solution of photocrosslinkable chitosan containing azide groups and lactose moieties (Az-CH-LA) incorporating paclitaxel formed an insoluble hydrogel within 30 s of ultraviolet light (UV) irradiation. The chitosan hydrogel showed strong potential for use as a new tissue adhesive in surgical applications and wound dressing. The fibroblast growth factor (FGF)-2 molecules retained in the chitosan hydrogel and in an injectable chitosan/IO₄-heparin hydrogel remain biologically active, and were gradually released from the hydrogels as they biodegraded in vivo. The controlled release of biologically active FGF-2 molecules from the hydrogels caused induction of angiogenesis and collateral circulation occurred in healing-impaired diabetic (db/db) mice and in the ischemic limbs of rats. Paclitaxel, which is an antitumor reagent, was also retained in the chitosan hydrogel and remained biologically active as it was released on degradation of the hydrogel in vivo. The chitosan hydrogels incorporating paclitaxel effectively inhibited tumor growth and angiogenesis in mice. The purpose of this review is to describe the effectiveness of chitosan hydrogel as a local drug delivery carrier for agents (e.g., FGF-2 and paclitaxel) to control angiogenesis. It is thus proposed that chitosan hydrogel may be a promising new local carrier for drugs such as FGF-2 and paclitaxel to control vascularization.     

Vascularization in vivo caused by the controlled release of fibroblast growth factor-2 from an injectable chitosan/non-anticoagulant heparin hydrogel

Addition of various heparinoids to the lactose-introduced, water-soluble chitosan (CH-LA) aqueous solution produces an injectable chitosan/heparinoid hydrogel. In the present work, we examined the capability of the chitosan/non-anticoagulant heparin (periodate-oxidized (IO(4)-) heparin) hydrogel to immobilize fibroblast growth factor (FGF)-2, as well as the controlled release of FGF-2 molecules from the hydrogel in vitro and in vivo. The hydrogel was biodegraded in about 20 days after subcutaneous injection into the back of a mouse. When the FGF-2-incorporated hydrogel was subcutaneously injected into the back of both mice and rats, a significant neovascularization and fibrous tissue formation were induced near the injected site. These results indicate that the controlled release of biologically active FGF-2 molecules is caused by biodegradation of the hydrogel, and that subsequent induction of the vascularization occurs.     

Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process.

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations.     

The interaction of chitosan with fibroblast growth factor-2 and its protection from inactivation

Application of ultraviolet light (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) results within 30s in an insoluble, flexible hydrogel. The retained FGF-2 molecules in the chitosan hydrogel remain biologically active, and are released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In view of these findings, we here tested the interaction of chitosan with FGF-2, thereby modifying and stabilizing the FGF-2 activity from inactivations. The photocrosslinkable chitosan hydrogel has a low affinity for FGF-2 (Kd = 6.12 x 10(-7) M). Soluble chitosan (CH-LA; Az-CH-LA without photocrosslinkable azide group) substantially prolonged the biological half-life time of FGF-2. Furthermore, CH-LA could protect the FGF-2 activity from inactivation, such as heat, proteolysis, and acid. The effect of chitosan on the FGF-2 activity is of a protective nature, since it had no effect of modifying the FGF-2 activity directly on growth of human umbilical vein endothelial cells (data not shown). Thus, one of the ways by which the chitosan potentiated the FGF-2 activity could be through protecting it from inactivations by the interaction between FGF-2 and chitosan molecules.     

Impaired liver regeneration after partial hepatectomy in db/db mice

Fatty liver is the most common hepatic disorder in humans and supposed to be a cause of poor prognosis after liver transplantation and hepatic resection which could be resulted from impaired liver regeneration. This study was carried out to analyze the process of liver regeneration in db/db mice which show severe steatosis because of abnormal leptin receptor. We performed 70% partial hepatectomy (PH) on db/db mice and normal +m/+m mice, and then sacrificed the animals 1, 2, 3, 5, 7 and 10 days later. The liver samples were weighed and examined histologically or immunohistochemically. As a result, the liver mass restitution was significantly inhibited in db/db mice compared with +m/+m mice. The BrdU labelling index peaked at 2 days after PH in both strains, although the value was lower in db/db mice. After that, interestingly, it decreased to the control level at 5 days in +m/+m mice while the recovery was delayed in db/db mice. Similar sequence was also observed in the PCNA labelling index. In addition, the peak time of the mitosis index was 2 days and 5 days after PH in +m/+m mice and in db/db mice, respectively. Thus, although not significant, the proliferative response of hepatocytes to PH occurred somewhat more transient and sharply in +m/+m mice while it lasted somewhat longer in db/db mice. This suggests that db/db mice may be valuable as one of the animal models for the investigation of the effects of steatosis on the liver regeneration.     

Effect of controlled release of fibroblast growth factor-2 from chitosan/fucoidan micro complex-hydrogel on in vitro and in vivo vascularization

We produced a chitosan/fucoidan micro complex-hydrogel as a carrier for controlled release of heparin binding growth factors such as fibroblast growth factor (FGF)-2. Material consisting of a soluble chitosan (CH-LA) mixed with fucoidan yielded a water-insoluble and injectable hydrogel with filamentous particles. In this study, we examined the ability of the chitosan/fucoidan complex-hydrogel to immobilize FGF-2 and to protect its activity, as well as the controlled release of FGF-2 molecules. The chitosan/fucoidan complex-hydrogel has high affinity for FGF-2 (K(d) = 5.4 x 10(-) (9)M). The interaction of FGF-2 with chitosan/fucoidan complex-hydrogel substantially prolonged the biological half-life time of FGF-2. It also protected FGF-2 from inactivation, for example by heat and proteolysis, and enhance FGF-2 activity. When FGF-2-containing complex-hydrogel was subcutaneously injected into the back of mice, significant neovascularization and fibrous tissue formation were induced near the site of injection at 1 week, and the complex-hydrogel was biodegraded and disappeared by 4 weeks. These findings indicate that controlled release of biologically active FGF-2 molecules is caused by both slow diffusion and biodegradation of the complex-hydrogel, and that subsequent induction of vascularization occurs. FGF-2-containing chitosan/fucoidan micro complex-hydrogel is thus useful and convenient for treatment of ischemic disease.     

Comparison of mitochondrial and macrophage content between subcutaneous and visceral fat in db/db mice

Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than peripheral (subcutaneous) obesity, however the underlying differences in morphology and pathophysiology between subcutaneous and visceral adipose are largely unknown. To evaluate the effects of diabetes and rosiglitazone (RSG) treatment, the expression of mitochondrial Hsp60, UCP-1 and F4/80 in inguinal subcutaneous (SC) fat, composed of white and brown adipose tissues, and epididymal (EP) fat, mainly white adipose tissue, were evaluated. In diabetic db/db mice, there was significant increased number of aggregated macrophage foci compared to db/+ mice, especially in EP fat. On the other hand, the expression of mitochondrial Hsp60 protein was suppressed in both SC and EP fat of db/db mice compared to db/+ mice, and the expression level of mitochondrial Hsp60 in db/+ mice was lower in EP fat compared with SC. In db/db mice, RSG suppressed the number of aggregated macrophage foci in EP fat, but not in SC fat. RSG ameliorated the mitochondrial Hsp60 expression and induced the expression of UCP-1 in both SC and EP fat. Taken together, these data suggest that differences exist in mitochondrial and macrophage content, and in the response to RSG between visceral and subcutaneous adipose tissue, and adipose type and distribution may be important for obesity-linked insulin resistance.     

Controlled release of fibroblast growth factor-2 from an injectable 6-O-desulfated heparin hydrogel and subsequent effect on in vivo vascularization

We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding growth factors, such as fibroblast growth factor (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo. Only 10% of FGF-2 was gradually released from the FGF-2-containing 6-O-DS-Hep hydrogel (photocrosslinked 6-O-DS-Hep (4%; w/w) hydrogel containing 50 microg/mL FGF-2) into PBS (phosphate-buffered saline) within first 7 days. The 6-O-DS-Hep hydrogel in vitro maintained the original form through 1 weeks incubation in PBS, but it was gradually fragmented and could not maintain the original form by 2-3 week-washing. When the FGF-2-containing 6-O-DS-Hep hydrogel was subcutaneously injected into the back of rats, significant neovascularization and fibrous tissue formation were induced near the injected site from day 3 after the injection. And, the hydrogel had been biodegraded and completely disappeared from the injected sites in vivo within about 15-20 days after the injection. These findings indicate a controlled release of biologically active FGF-2 molecules together with fragmentation and biodegradation of 6-O-DS-Hep hydrogel and the subsequent induction of neovascularization in vivo.     

Gender-dependent attenuation of cardiac potassium currents in type 2 diabetic db/db mice

Single ventricular myocytes were prepared from control db /+ and insulin-resistant diabetic db/db male mice at 6 and 12 weeks of age. Peak and sustained outward potassium currents were measured using whole-cell voltage clamp methods. At 6 weeks currents were fully developed in control and diabetic mice, with no differences in the density of either current. By 12 weeks both currents were significantly attenuated in the diabetic mice, but could be augmented by in vitro incubation with the angiotensin-converting enzyme (ACE) inhibitor quinapril (1 μ m , 5–9 h). In cells from female db/db mice (12 weeks of age), K⁺ currents were not attenuated and no effects of quinapril were observed. To investigate whether lack of insulin action accounts for these gender differences, cells were also isolated from cardiomyocte-specific insulin receptor knockout (CIRKO) mice. Both K⁺ currents were significantly attenuated in cells from male and female CIRKO mice, and action potentials were significantly prolonged. Incubation with quinapril did not augment K⁺ currents. Our results demonstrate that type 2 diabetes is associated with gender-selective attenuation of K⁺ currents in cardiomyocytes, which may underlie gender differences in the development of some cardiac arrhythmias. The mechanism for attenuation of K⁺ currents in cells from male mice is due, at least in part, to an autocrine effect resulting from activation of a cardiac renin–angiotensin system. Insulin is not involved in these gender differences, since the absence of insulin action in CIRKO mice diminishes K⁺ currents in cells from both males and females.     

Fibrin-based scaffold incorporating VEGF- and bFGF-loaded nanoparticles stimulates wound healing in diabetic mice

Diabetic skin ulcers are difficult to heal spontaneously due to the reduced levels and activity of endogenous growth factors. Recombinant human vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are known to stimulate cell proliferation and accelerate wound healing. Direct delivery of VEGF and bFGF at the wound site in a sustained and controllable way without loss of bioactivity would enhance their biological effects. The aim of this study was to develop a poly(ether)urethane–polydimethylsiloxane/fibrin-based scaffold containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with VEGF and bFGF (scaffold/GF-loaded NPs) and to evaluate its wound healing properties in genetically diabetic mice (db/db). The scaffold application on full-thickness dorsal skin wounds significantly accelerated wound closure at day 15 compared to scaffolds without growth factors (control scaffold) or containing unloaded PLGA nanoparticles (scaffold/unloaded NPs). However, the closure rate was similar to that observed in mice treated with scaffolds containing free VEGF and bFGF (scaffold/GFs). Both scaffolds containing growth factors induced complete re-epithelialization, with enhanced granulation tissue formation/maturity and collagen deposition compared to the other groups, as revealed by histological analysis. The ability of the scaffold/GF-loaded NPs to promote wound healing in a diabetic mouse model suggests its potential use as a dressing in patients with diabetic foot ulcers.     

绿原酸改善db/db小鼠血管内皮功能障碍的作用初探

目的:探讨绿原酸(CGA)对肥胖型2型糖尿病小鼠血管内皮功能障碍的作用并初步分析相关的机制。方法:雄性db/db小鼠12只,随机分对照组和CGA组,每组6只。CGA组以含0.02%CGA的饲料喂养,对照组给予普通饲料喂养,干预时间为12周。每周测空腹血糖、体重和鼠尾血压。实验结束后取血分离血浆,采用ELISA法测血红素氧合酶1(HO-1)、过氧化氢酶(CAT)、醌NAD(P)H脱氢酶1(NQO1)和谷胱甘肽过氧化物酶1(GPx-1)的水平。取胸主动脉以DHE和DAF-2 DA染色观察血管内膜超氧阴离子和一氧化氮(NO)水平,以Wire Myograph System观察胸主动脉张力,Western blot观察血管组织中核因子E2相关因子2(Nrf2)、过氧化物酶体增殖物激活受体α(PPARα)、磷酸化AMP活化蛋白激酶(p-AMPK)、磷酸化内皮型NO合酶(p-eNOS)、P22~(phox )和P47~(phox)的蛋白水平。结果:膳食CGA可显著降低小鼠的空腹血糖和体重,增加血浆中HO-1、CAT、NQO1和GPx-1水平,减少血管内皮超氧阴离子的水平,增加NO水平,改善小鼠血管内皮依赖性舒张功能(P0.01)。Western blot结果发现CGA可显著上调血管组织中PPARα、Nrf2、p-AMPK和p-eNOS的蛋白水平,降低P22~(phox )和P47~(phox)的水平(P0.01)。结论:膳食CGA显著改善db/db小鼠血管内皮依赖性舒张功能。这可能与其上调PPARα、Nrf2和AMPK等抗氧化应激分子,降低氧化应激水平,促进eNOS磷酸化有关,但确切机制尚需进一步研究。     

Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice

Receptor for advanced glycation end-products (RAGE), and two of its ligands, AGE and EN-RAGEs (members of the S100/calgranulin family of pro-inflammatory cytokines), display enhanced expression in slowly resolving full-thickness excisional wounds developed in genetically diabetic db+/db+ mice. We tested the concept that blockade of RAGE, using soluble(s) RAGE, the extracellular ligand-binding domain of the receptor, would enhance wound closure in these animals. Administration of sRAGE accelerated the development of appropriately limited inflammatory cell infiltration and activation in wound foci. In parallel with accelerated wound closure at later times, blockade of RAGE suppressed levels of cytokines; tumor necrosis factor-alpha; interleukin-6; and matrix metalloproteinases-2, -3, and -9. In addition, generation of thick, well-vascularized granulation tissue was enhanced, in parallel with increased levels of platelet-derived growth factor-B and vascular endothelial growth factor. These findings identify a central role for RAGE in disordered wound healing associated with diabetes, and suggest that blockade of this receptor might represent a targeted strategy to restore effective wound repair in this disorder.     

Tissue regeneration using macrophage migration inhibitory factor-impregnated gelatin microbeads in cutaneous wounds

Migration inhibitory factor (MIF) responds to tissue damage and regulates inflammatory and immunological processes. To elucidate the function of MIF in cutaneous wound healing, we analyzed MIF knockout (KO) mice. After the excision of wounds from the dorsal skin of MIF KO and wild-type (WT) mice, healing was significantly delayed in MIF KO mice compared to WT mice. Lipopolysaccharide treatment significantly increased [(3)H]thymidine uptake in WT mouse fibroblasts compared to MIF KO mouse fibroblasts. Furthermore, there was a significant reduction in fibroblast and keratinocyte migration observed in MIF KO mice after 1-oleoyl-2-lysophosphatidic acid treatment. We subsequently examined whether MIF-impregnated gelatin slow-release microbeads could accelerate skin wound healing. Injection of more than 1.5 microg/500 microl of MIF-impregnated gelatin microbeads around a wound edge accelerated wound healing compared to a single MIF injection without the use of microbeads. MIF-impregnated gelatin microbeads also accelerated skin wound healing in C57BL/6 mice and diabetic db/db mice. Furthermore, incorporating MIF-impregnated gelatin microbeads into an artificial dermis implanted into MIF KO mice accelerated procollagen production and capillary formation. These findings suggest that MIF is crucial in accelerating cutaneous wound healing and that MIF-impregnated gelatin microbeads represent a promising treatment to facilitate skin wound healing.     

A bilayer composite composed of TiO2-incorporated electrospun chitosan membrane and human extracellular matrix sheet as a wound dressing

We designed bilayer composites composed of an upper layer of titanium dioxide (TiO₂)-incorporated chitosan membrane and a sub-layer of human adipose-derived extracellular matrix (ECM) sheet as a wound dressing for full-thickness wound healing. The dense and fibrous top layer, which aims to protect the wound from bacterial infection, was prepared by electrospinning of chitosan solution followed by immersion in TiO₂ solution. The sponge-like sub-layer, which aims to promote new tissue regeneration, was prepared with acellular ECM derived from human adipose tissue. Using a modified drop plate method, there was a 33.9 and 69.6% reduction in viable Escherichia coli and Staphylococcus aureus on the bilayer composite, respectively. In an in vivo experiment using rats, the bilayer composites exhibited good biocompatibility and provided proper physicochemical and compositional cues at the wound site. Changes in wound size and histological examination of full-thickness wounds showed that the bilayer composites induced faster regeneration of granulation tissue and epidermis with less scar formation, than control wounds. Overall results suggest that the TiO₂-incorporated chitosan/ECM bilayer composite can be a suitable candidate as a wound dressing, with an excellent inhibition of bacterial penetration and wound healing acceleration effects.     

In vivo collagen metabolism in spontaneously diabetic (db/db) mice

To further define the pathogenesis of diabetic connective tissue lesions, collagen synthesis and degradation were measured in vivo in spontaneously diabetic db/db mice. A double isotopic labeling technique, in which 14C-labeled and 3H-labeled proline were injected into the same mouse 7 days apart, was applied. Collagen synthesis and degradation were assessed in skins, intestines, hearts, and kidneys. There were no changes in collagen metabolism in the intestines of the diabetic mice. In all other tissues, collagen degradation was accelerated. Collagen synthesis was decreased in skins, but increased in the hearts and kidneys of the diabetic mice. These tissue-specific changes in collagen metabolism resulted in a net loss of collagen in all tissues examined except intestines. The results of this study provide insight into the mechanisms leading to connective tissue defects occurring in diabetes mellitus.     

Ramipril improves oxidative stress-related vascular endothelial dysfunction in db/db mice

Endothelial dysfunction often precedes Type 2 diabetes-associated cardiovascular complications. One important cause of endothelial dysfunction is oxidative stress, which can lead to reduced nitric oxide (NO) bioavailability. In this study, we examined the effects of ramipril (an angiotensin-converting enzyme inhibitor, ACEI) on reactive oxygen species (ROS) production and endothelium-dependent vasodilation using a Type 2 diabetic (db/db) murine model. Plasma concentration of 8-isoprostane ([8-isoP]) was measured and used as an indication of the amount of ROS production. Six weeks of ramipril (10 mg/kg/day) treatment significantly reduced [8-isoP] and improved acetylcholine(ACh)-induced vasodilation in db/db mice without altering responses in wild-type (WT) mice. Responsiveness of smooth muscle cells to NO, assessed by sodium nitroprusside-induced vasodilation, was not different between db/db and WT mice regardless of ramipril or vehicle treatment. Our results suggest that ramipril specifically improved endothelium-dependent vasodilation in Type 2 diabetic mice, possibly by reducing ROS levels.     

甜菊糖甙对db/db小鼠胰岛素抵抗和胰岛炎症的影响

目的：大量证据显示,胰岛的炎症反应和胰岛素抵抗是db/db小鼠发生2型糖尿病（DM2）的两个主要因素。甜菊糖甙是一种从菊科植物中提取的天然化合物对于糖尿病病人有许多益处。本实验试图观察甜菊糖甙是否能够改善db/db小鼠的胰岛素抵抗和胰岛炎症反应,从而改善糖代谢。方法：给予db/db小鼠甜菊糖甙或者空白溶剂处理2个月。在处理结束时观察空腹血糖和胰岛素耐量,同时分离胰岛行葡萄糖刺激的胰岛素分泌试验,并行胰腺组织免疫荧光观察胰岛内巨噬细胞浸润情况。结果：甜菊糖甙干预2个月不影响db/db小鼠体重,但显著降低小鼠的空腹血糖,明显改善胰岛素抵抗。同时葡萄糖刺激的胰岛素分泌试验结果提示,甜菊糖甙可显著提高db/db小鼠胰岛在体外分泌胰岛素的能力。此外,免疫荧光提示,甜菊糖甙能显著减轻db/db小鼠胰岛内巨噬细胞浸润程度明显减轻和改善胰岛炎症反应。结论：甜菊糖甙能通过改善胰岛素抵抗和胰岛炎症反应来改善db/db小鼠的糖代谢和胰岛素分泌能力。