胶原/PLLA纳米粒子复合海绵的制备及其生物相容性评价

通过紫外和自由基聚合在PLLA上接枝聚丙烯酸（PAA）,制备了平均粒径316nm,zeta电位-39.88mV的表面亲水性PLLA纳米粒子,与胶原溶液混合通过冷冻干燥法制备了胶原/PLLA纳米粒子复合多孔支架。复合支架材料的孔隙率与纯胶原支架相近,而密度、湿态压缩模量有明显提高,降解率降低显著。随着复合支架中PLLA纳米粒子的增加,L929细胞在材料中的播种效率和增殖有明显提高,表明胶原/PLLA纳米粒子复合多孔支架是一种理想的组织工程支架材料。     

人肌腱胶原蛋白的提取及凝胶制备

目的探索人肌腱胶原蛋白在组织工程皮肤及整形外科中的应用。方法从人肌腱提取酸溶性胶原,制备人肌腱胶原蛋白。结果所提取的胶原在pH5.8左右迅速形成凝胶。光镜下可见酸溶性胶原纤维呈长细丝状,交织成网状。纯度以羟脯氨酸质量分数为基准,分光光度法测定为95%。氨基酸成分主要为甘氨酸占35%,脯氨酸占10%。结论氨基酸分析表明,所提取的胶原为典型的Ⅰ型胶原蛋白,热稳定性测定Td为39～41℃。     

The effect of hyaluronic acid on interleukin-1-induced deregulation of collagen metabolism in cultured human skin fibroblasts.

Although hyaluronic acid (HA) has been used in the treatment of osteoarthritis for 30 years, the mechanism of its protective action on collagen metabolism disturbances in tissues during inflammation is not known. The present study was undertaken to evaluate the mechanism of Interleukin-1 (IL-1)-induced deregulation of collagen metabolism in cultured human skin fibroblast and the effect of HA on the process. In normal fibroblasts IL-1 strongly induced inhibition of collagen biosynthesis, while HA counteracted the process. The mechanism of this phenomenon was independent of prolidase activity, an enzyme that plays an important role in collagen biosynthesis at the post-translational level. Instead, IL-1 was found to inhibit the expression of insulin-like growth factor-I receptor (IGF-IR) and MAP kinases-ERK1 and ERK2, while HA was shown to counteract this process. Since insulin-like growth factor-I (IGF-I) is a most potent stimulator of collagen biosynthesis in fibroblasts the mechanism of IL-1-dependent inhibition of collagen biosynthesis may be related to inhibition of IGF-IR expression and signaling. The data suggest that hyaluronic acid protects collagen against IL-1-induced inhibition of biosynthesis of this protein in cultured human skin fibroblasts at the level of IGF-IR signaling.     

Electrophoretic analysis of type I collagen from bovine Achilles tendon: comparison between the extracted raw material and the freeze-dried product

The present study was aimed at verifying if the freeze-drying process has any effect on the polypeptide composition of the collagen extracted from bovine Achilles tendon in an acetic acid gelified form. Data from our laboratories showed that the freeze-drying process renders the collagen gel essentially insoluble; under these conditions the collagen sample can no longer be analyzed by gel electrphoresis. We found that treatment of the sample with pepsin in acid environment, followed by precipitation with ammonium sulphate yields an insoluble fraction that is susceptible of being analyzed by polyacrylamide gel electrophoresis. The The electrophoresis, run under standard conditions, shows that six major subunits, corresponding to alpha, beta and gamma polypeptides, can be revealed in the sample treated in this way. So the freeze-dried collagen exhibits a polypeptidic composition that is basically identical to that shown by the collagen gel, with regard to the fraction precipitated with ammonium sulphate. Otherwise the pattern of the enzymatic hydrolysis was investigated by measuring the hydroxyproline content, and so the collagen content using the 7.46 conversion factor from hydroxyproline to the scleroprotein collagen, in the various steps of the hydrolysis itself: the analytical results showed no differences between the freeze-dried collagen and the gelified form; this confirms that the lyophilization process does not alter the polypeptidic composition of the collagen in any way.     

Marine sponge collagen: isolation, characterization and effects on the skin parameters surface-pH, moisture and sebum.

A previously described isolation procedure for collagen of the marine sponge Chondrosia reniformis Nardo was modified for scaling-up reasons yielding 30% of collagen (freeze-dried collagen in relation to freeze-dried sponge). Light microscope observations showed fibrous structures. Transmission electron microscopy studies proved the collagenous nature of this material: high magnifications showed the typical periodic banding-pattern of collagen fibres. However, the results of the amino acid analysis differed from most publications, presumably due to impurities that still were present. In agreement with earlier studies, sponge collagen was insoluble in dilute acid mediums and all solvents investigated. Dispersion of collagen was facilitated when dilute basic mediums were employed. The acid-base properties of the material were investigated by titration. Furthermore, a sponge extract was incorporated in two different formulations and compared with their extract-free analogues and a commercially available collagen containing product with respect to their effects on biophysical skin parameters. None of the preparations had a noticeable influence on the physiological skin surface pH. Skin hydration increased only slightly. However, all tested formulations showed a significant increase of lipids measured by sebumetry.     

胶原蛋白凝胶的制备及其中药物体外扩散性考察

目的:提取鱼鳞胶原蛋白,制成凝胶并考察其中药物的体外扩散性。方法:鱼鳞经胃蛋白酶提取、盐析、纯化、冷冻干燥得到胶原蛋白。以水杨酸为模型药物,加入柠檬酸溶液、甘油和乙醇混合液制备胶原蛋白凝胶,并采用琼脂扩散法以扩散系数k为指标评价其体外扩散性,同时与水杨酸水溶性和油脂性软膏进行比较。结果:所得胶原蛋白含量为91.04%;所制水杨酸胶原蛋白凝胶及水溶性和油脂性软膏扩散系数k值分别为68.11、58.04、17.67mm2·h-1。结论:所制凝胶中药物体外扩散速率较快,胶原蛋白可作为载体用于需较快释放药物的外用制剂。     

ARFI技术与血清铁、铁蛋白和肝纤维化四项检测在肝纤维化及肝硬化诊断中的相关性

目的：探讨声脉冲辐射力成像技术（ acoustic radiation force impulse imaging ,ARFI）、血清铁、铁蛋白及肝纤维化四项指标（Ⅲ型前胶原、Ⅳ型胶原、层黏蛋白、透明质酸）在诊断肝纤维化及肝硬化中的相关性。方法在2013年6-10月期间,选取20例正常者作为对照组和53例肝纤维化、肝硬化作为观察组。所有研究对象均进行上述7项指标的检测,比较对照组和观察组组间测量指标的差异;将观察组ARFI测量值与血清铁、铁蛋白、肝纤维化四项指标进行相关性分析。结果观察组的ARFI、血清铁、铁蛋白、Ⅲ型前胶原、Ⅳ型胶原、层黏蛋白和透明质酸的测值均高于对照组（ P均＜0.01）;观察组的ARFI分别与透明质酸（r＝0．48,P＜0．01）、层黏蛋白（r＝0．44,P＜0．01）、Ⅳ型胶原（r＝0．41,P＜0．01）和血清铁（r＝0.33,P＜0.05）均存在正相关;但与Ⅲ型前胶原（r＝0．07,P＞0．05）和铁蛋白（r＝0．12,P＞0．05）不存在相关性。结论 ARFI技术可无创反映肝组织的弹性硬度,且与透明质酸、层黏蛋白、Ⅳ型胶原、血清铁相关性良好,对肝纤维化及肝硬化的诊断具有一定临床价值。     

Acetylsalicylic acid prevents nickel-induced collagen biosynthesis in human fibroblasts

Exposure to nickel compounds that occurs mainly via inhalation can have adverse effects on human health. One of them is pulmonary fibrosis that results from accumulation of collagen in lung tissues. The mechanism of this process as well as effective treatment of the disease is not known. To evaluate the effect of nickel on collagen biosynthesis human dermal fibroblasts were treated with various concentrations of nickel chloride(II) for 72 h. The compound was found to stimulate collagen biosynthesis in dose-dependent manner. We considered prolidase as a potential target for nickel-dependent collagen biosynthesis regulation. Prolidase [E.C.3.4.13.9] is a cytosolic metalloproteinase, which specifically splits imidodipeptides with C-terminal proline that is recycled for collagen biosynthesis. However, it was found that 72 h treatment of confluent cells with Ni(II) did not affect significantly prolidase activity. An addition of acetylsalicylic acid, known, non-specific inhibitor of prolidase to the cells treated with 100 μM NiCl(II), significantly reduced both collagen biosynthesis and prolidase activity. It suggests that acetylsalicylic acid prevents nickel-induced increase in collagen biosynthesis through inhibition of prolidase activity in human fibroblasts. The results indicate that tissue fibrosis may be considered as a possible target for prolidase inhibitory therapy and acetylsalicylic acid may represent such an agent for potential application in tissue fibrosis prevention or early stages of tissue fibrosis.     

Influence of retinoids on skin fibroblasts metabolism in vitro

The most dangerous environmental factor for our skin condition is ultraviolet light radiation. Chronic exposition to ultraviolet light can induce epidermal atrophy, keratosis, depigmentation and dysplasia. In the dermis, UV light causes dramatic up-regulation of extracellular matrix-degrading enzymes. Matrix metalloproteinases (MMPs) are engaged in collagen, elastin and other extracellular matrix components degradation. In addition, to increase level of destructive enzymes, UV light has been shown to decrease collagen production. As a consequence of UV impact on skin, it shows signs of aging including loss of tone and elasticity, increased skin fragility, blood vessels weakness and wrinkles. The most dangerous effect of UV on skin is an increased risk of melanoma and other skin cancers. Retinoids are well known antiaging agents. For many years this vitamin has been used for the prevention and treatment of photoaging. Retinoids abolish cellular atypia, increase compacting of the stratum corneum and reduce skin hyperpigmentation caused by sun light. Recent evidence suggests that retinoids also play a role in the prevention of aging, because of its inhibitory effects on metalloproteinases expression. The aim of this study was to examine if all-trans-retinoic acid (ATRA) effects MMP-1, MMP-2, MMP-3 and MMP-14 gene expression in fibroblasts cultured in vitro.     

Acetylsalicylic acid prevents nickel-induced collagen biosynthesis in human fibroblasts

Exposure to nickel compounds that occurs mainly via inhalation can have adverse effects on human health. One of them is pulmonary fibrosis that results from accumulation of collagen in lung tissues. The mechanism of this process as well as effective treatment of the disease is not known. To evaluate the effect of nickel on collagen biosynthesis human dermal fibroblasts were treated with various concentrations of nickel chloride(II) for 72 h. The compound was found to stimulate collagen biosynthesis in dose-dependent manner. We considered prolidase as a potential target for nickel-dependent collagen biosynthesis regulation. Prolidase [E.C.3.4.13.9] is a cytosolic metalloproteinase, which specifically splits imidodipeptides with C-terminal proline that is recycled for collagen biosynthesis. However, it was found that 72 h treatment of confluent cells with Ni(II) did not affect significantly prolidase activity. An addition of acetylsalicylic acid, known, non-specific inhibitor of prolidase to the cells treated with 100 μM NiCl(II), significantly reduced both collagen biosynthesis and prolidase activity. It suggests that acetylsalicylic acid prevents nickel-induced increase in collagen biosynthesis through inhibition of prolidase activity in human fibroblasts. The results indicate that tissue fibrosis may be considered as a possible target for prolidase inhibitory therapy and acetylsalicylic acid may represent such an agent for potential application in tissue fibrosis prevention or early stages of tissue fibrosis.     

Role of betaine in liver injury induced by the exposure to ionizing radiation

Oxidative stress, apoptosis, and fibrosis may play a major role in the development of radiation‐induced liver damage. Betaine, a native compound widely present in beetroot, was reported to possess hepato‐protective properties. The objective of this study was to investigate the influence of betaine on radiation‐induced liver damage. Animals were exposed to 9 Gy applied in 3 doses of 3 Gy/wk. Betaine (400 mg/kg/d), was orally supplemented to rats after the first radiation dose, and daily during the irradiation period. Animals were sacrificed 1 day after the last dose of radiation. The results showed that irradiation has induced oxidative stress in the liver denoted by a significant elevation in malondialdehyde, protein carbonyl, and 8‐hydroxy‐2‐deoxyguanosine with a significant reduction in catalase activity and glutathione (GSH) content. The activity of the detoxification enzyme cytochrome P450 (CYP450) increased while GSH transferase (GSH‐T) decreased. The activity of the apoptotic marker caspase‐3 increased concomitant with increased hyaluronic acid, hydroxyproline, laminin (LN), and collagen IV. These alterations were associated with a significant increase of gamma‐glutamyl transferase, alkaline phosphatase and alanine and aspartate aminotransferase markers of liver dysfunction. Betaine treatment has significantly attenuated oxidative stress, decreased the activity of CYP450, enhanced GSH‐T, reduced the activity of caspase‐3, and the level of fibrotic markers concomitant with a significant improvement of liver function. In conclusion, betaine through its antioxidant activity and by enhancing liver detoxification and reducing apoptosis may alleviate the progression of liver fibrosis and exert a beneficial impact on radiation‐induced liver damage.     

Changes in rat lung collagen after life-time treatment with vanadium

Male Wistar rats were treated in the period from conception up to the 105th day of age with 40 and 60 ppm of vanadium, given as sodium metavanadate in the drinking water. It was found that total collagen and soluble collagen within the lungs of treated animals were significantly lower than in non-treated controls. The decrease in total collagen content observed was not dependent on the dose of vanadium administered. While the content of soluble collagen decreased significantly with increasing dose, the content of insoluble collagen in the lungs of the same rats was significantly higher than in controls and increased with an increase in the dose of vanadium administered. The increase in insoluble collagen was not accompanied by an increased content of hexoses and free aldehydes but by an increased content of collagen-bound vanadium. The results seem to indicate that this metal participates in an increased formation of insoluble collagen by forming additional cross-links between vanadium ions and the ligand groups in the amino acid side-chains.     

Effect of proline analogs on oxygen toxicity-induced pulmonary fibrosis in the rat

Proline analogs inhibit collagen biosynthesis and prevent accumulation of collagen in tissues. The antifibrotic effects of three proline analogs, cis-hydroxyproline, L-azetidine-2-carboxylic acid, and L-3,4-dehydroproline, were compared in a rat oxygen toxicity model. The specificity of these agents for collagen was examined by measuring their effects on noncollagen protein and elastin accumulation in the lung. Increased lung collagen was produced by exposing rats to 95% O2 for 60 hr followed by a 2-week recovery period. Animals were treated with the proline analogs for the 2-week period. Oxygen exposure in untreated animals increased lung collagen 26% above air-breathing controls, and this increase was prevented by all three analogs. Increased noncollagen protein was also prevented by these agents, suggesting they were not entirely specific for collagen. Elastin accumulation, however, was not inhibited by cis-hydroxyproline. It was concluded that proline analogs were antifibrotic, but affected the metabolism of noncollagen protein.     

Reversibility of d-penicillamine induced collagen alterations in rat skin and granulation tissue

Granulation tissue was produced in rats by subcutaneous implantation of Visella^® sponges. d-penicillamine (d-pen) 100 or 500 mg/kg was administered daily for 42 days by gastric tubing. Pairfed, placebo treated animals were included as controls. Half of the groups were kept for additionally 28 days without medication. The inhibitory effect of d-pen on cross-link formation in newly synthesized collagen was readily reversible. By contrast, cross-link deficiency lasting beyond the observation period was observed in the higher polymeric collagen variants released by dilute acid, heat exposure or limited pepsin proteolysis as estimated by solubility, α/β chain ratio and/or aldehyde content. By SDS-polyacrylamide gel electrophoresis on gels containing 3.6 M urea it was shown that purified dermal acid soluble collagen from treated animals consisted of a mixture of type I and III collagen, whereas only type I collagen was detected in controls. The band pattern was identical in reduced and unreduced collagen samples. Four weeks after d-pen discontinuance type III collagen had disappeared from the acid extract. Moreover, the ratio of type III to type I collagen in the pepsin digest from both granulation tissue and skin showed a persistent rise with d-pen. These observations indicate that d-pen destabilized type III collagen in particular by interference with its disulfide linkages. The amount of granulation tissue remained unaffected throughout the experiment, whereas the skin collagen content decreased at the higher dose level. The regeneration was not completed by the end of the observation period. Modulation of the molecular stability of granuloma collagens may be of relevance for the antirheumatoid effect of d-pen, but the sustained effect on normal tissues may imply a long standing impairment of their supportive capacity.     

Collagen Profile in Rat Granulation Tissue after Cyclophosphamide Treatment

Abstract: The collagen profile was studied in 2–3 week old granulation tissue, induced in rats by subcutaneous implantation of viscose cellulose sponges. Cyclophosphamide was given daily intraperitoneally, in a dose of 10 mg per kg during the entire experiment, beginning either 7 days before or on the day of the sponge implantation. Cyclophosphamide caused an increase in the water percentage, and decreased the dry weight and the total amount of collagen in the granulomas, reflecting a suppression of collagen synthesis. The catabolism of collagen was also inhibited in the cyclophosphamide treated rats, as indicated by a fall in the content of free OH-proline and a lowering of the alpha/beta ratio in acid extracted collagen. Cyclophosphamide treatment increased the collagen solubility in acetic acid and increased the aldehyde content in relation to OH-proline in purified collagen. This indicates that cyclophosphamide may inhibit the cross-linking of collagen by blocking the aldehydes or decrease the stability of the collagen molecule by decreasing the hydroxylation of proline. Pretreatment with cyclophosphamide did not influence the effect of cyclophosphamide given during the development of granulation tissue.     

Granuloma maturation in the rat is advanced by the oral administration of Eucommia ulmoides OLIVER leaf

We have reported that collagen metabolism was improved by the administration of Eucommia ulmoides OLIVER leaf. In this paper, we examine the granuloma maturation and deposition of collagen in the granuloma of rats due to the oral administration of this leaf. After 3 weeks of the oral administration, granuloma formation was induced by the formalin soaked filter paper-pellet method. A week later, the developing granuloma was dissected. Granuloma formation was significantly increased due to ingestion of the dried leaf at a dose of 1.8 g/kg of body weight/d. The collagen content in the granuloma was also significantly increased. In the case of the collagen profile, the pepsin-solubilized collagen content and its relative percentage to the total collagen were significantly higher than in the control. Histochemical examination showed that the granuloma tissues were well developed, and displayed many newly synthesized capillary vessels and a greater quantity of fibroblasts and monocytes in the 1.8 g leaf group. High density lipoprotein (HDL)-cholesterol and triglyceride content in the blood plasma were significantly higher than in the control. These results show that granuloma maturation was accelerated and the energy was supplied from fatty acid metabolism. The administration of Eucommia ulmoides OLIVER leaf may be effective at speeding up the wound healing process.     

Effect of acetylosalicylic acid and naproxen on collagen solubility and heterogeneity in the rat liver

Rats were treated acetylosalicylic acid (150 mg/kg b.w.) or naproxen (125 mg/kg b.w.) during 6 weeks. An increase of collagen content, elevated solubility of collagen and changes in the ratio of type I to type III collagen were observed in animals receiving acetylosalicylic acid, and only slight alterations were found in these treated with naproxen. A rise of indices of the liver injury in blood serum was shown after treatment with acetylosalicylic acid. It is suggested that changes of collagen metabolism in the liver of rats receiving anti-rheumatic drugs are caused by direct action of the drugs on collagen biosynthesis and catabolism as well as resulted from the toxic action of the drugs on the liver.     

The synthesis and evaluation of polymer prodrug/collagen hybrid gels for delivery into metastatic cancer cells

Metastatic cancer cells degrade extracellular matrix containing collagen. In this study, a variety of different polymer prodrugs have been synthesized and embedded in collagen gels for application in a metastasis-associated drug delivery system (DDS). Dendrimer-doxorubicin (Dox) prodrugs were prepared with different surfaces, including collagen peptides and polyethylene glycol. Furthermore, Dox was conjugated to linear poly(glutamic acid) (poly-Glu) instead of the dendrimer. The cytotoxicities of each of these polymer prodrug systems against the poorly invasive MCF-7 and highly invasive MDA-MB-231 cells were similar. The highly invasive MDA-MB-231 cells, however, were more sensitive than the MCF-7 cells to the polymer prodrugs-embedded collagen gels, suggesting that these polymer prodrugs/collagen hybrid gels would be useful for the development of metastasis-associated DDSs. The cytotoxicities of the polymer prodrugs were dependent on their chemical compositions. The collagen peptide-conjugated dendrimer prodrug/collagen hybrid gel demonstrated in vivo anticancer effects in an orthotopic metastatic mouse model.From the Clinical EditorIn this study, a variety of polymer prodrugs have been synthesized and embedded in collagen gels to be used in a metastasis-associated drug delivery system, demonstrating in vivo anticancer effects in an orthotopic metastatic mouse model.     

Inhibitory effect of acetylsalicylic acid on metalloproteinase activity in human lung adenocarcinoma at different stages of differentiation

The mechanism underlying the anticancer effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is not clear. We addressed the question whether the alterations in collagen content in lung adenocarcinomas reported in previous studies result from dysregulation of gelatinolytic activity and whether the activity is altered by acetylsalicylic acid in vitro. Human lung adenocarcinomas were divided into three groups: well-differentiated (G1), moderately differentiated (G2) and poorly differentiated (G3) tumors. Each group was compared with normal lung tissue with respect to tissue collagen and collagen degradation product content (hydroxyproline assay), gelatinolytic activity (zymography) and the expression of matrix metalloproteinases, MMP-2 and MMP-9 (Western immunoblot). Moreover, in the studied tissues, the effect of acetylsalicylic acid on gelatinolytic activity was measured. The lung adenocarcinoma G1 had a similar collagen content as normal lung tissue but increased amounts of collagen degradation products and free hydroxyproline. These phenomena were accompanied by a marked increase in gelatinolytic activity (MMP-2 and MMP-9) in the G1 tumor. In adenocarcinoma G2, the free hydroxyproline content and gelatinolytic activity were increased, while the collagen and collagen degradation product contents were not markedly altered, compared to control. In contrast, adenocarcinoma G3 had an increased tissue collagen content (by about 60%), decreased percentage of collagen degradation products and similar gelatinolytic activity, compared to normal lung. Acetylsalicylic acid was found to inhibit gelatinolytic activity both in control and adenocarcinoma tissues, preferentially the active forms of gelatinases MMP-2 and MMP-9. The results suggest that human lung adenocarcinoma G1, through an elevated expression of the activated forms of both MMP-2 and MMP-9, may represent a more invasive phenotype than less differentiated tumors G2 or G3. It indicates that lung adenocarcinoma G1 should be considered as a possible target for metalloproteinase inhibitory therapy. Acetylsalicylic acid may be such a therapeutical agent in cancer prevention or early stages of tumor growth.     

Salicylate-induced inhibition of collagen and mucopolysaccharide biosynthesis by a chick embryo cell-free system

The ability of a chick embryo cell-free system to synthesize collagen, mucopolysaccharide and non-collagen protein in the presence of sodium salicylate was studied. Added creatine phosphate together with endogenous creatine kinase was the ATP generating system. The incorporation of labelled proline or labelled glucose into collagen or mucopolysaccharide respectively depended on ATP level in the cell-free system used. Salicylate inhibited collagen and mucopolysaccharide synthesis to a greater extent than non-collagen protein synthesis. The ability of the cell-free system to hydroxylate labelled protocollagen was inhibited 50% by storage at ?18°. Ferrous iron reversed this inhibition. Salicylate prevented the restoration of the enzyme activity by ferrous iron. Incorporation of radioactivity into hyaluronic acid, when labelled UDP-glucuronic acid and UDP-N-acetylglucosamine were supplied, was inhibited 17% by salicylate. Under the same conditions 47% inhibition of incorporation into chondroitin sulphate was seen. This suggests that UDP-N-acetyl-glucosamine-UDP-N-acetylgalactosamine epimerase is inhibited by salicylate.