Evaluation of the physical and biological properties of hyaluronan and hyaluronan fragments

Hyaluronan (HA) has been extensively used for various medical applications, including osteoarthritis, tissue augmentation and ocular surgery. More recently, it has been investigated for use in polymer therapeutics as a carrier for drugs and biologically active proteins, thanks to its biodegradability, biocompatibility and inherent biological properties. Such biological functions are strongly dependent on HA's chain length, yet the molecular weight of HAs used in polymer conjugates varies widely and is inconsistent with its intended application. Therefore, this study aimed to determine the ideal chain length of HA to be used in polymer conjugates for enhanced tissue repair.HA fragments (M_w 45,000-900,000 g/mol) were prepared by acid hydrolysis of rooster comb HA and their physicochemical and biological properties were characterized. Such HA fragments had a highly extended, almost rod-like solution conformation and demonstrated chain length- and concentration-dependent viscosity, while exposure to HAase caused a rapid reduction in HA viscosity, which was most significant for the native HA. Initial HA hydrolysis rate by HAase varied strongly with HA chain length and was dependent on the formation of a stable enzyme-substrate complex. When normal human dermal fibroblasts were exposed to the different HA fragments for 72 h, only native (900,000 g/mol) HA reduced proliferation at 1000 μg/mL. Conversely, only the smallest HA fragment (70,000 g/mol) reduced the proliferation of chronic wound fibroblasts, at 1000 μg/mL. The 70,000 g/mol HA fragment also promoted the greatest cell attachment.These observations demonstrate that low molecular weight (70,000-120,000 g/mol) HA fragments would be best suited for the delivery of proteins and peptides with applications in chronic wound healing and paves the way for the rationalized development of novel HA conjugates.     

纳豆提取物的保湿性、抗过敏性和亚硝酸盐清除率的初步研究

近年来在外用美容药物和化妆品的透皮吸收研究中,高度生物靶向性和严格选择的生物导向制剂的研究开发十分引人注目。以γ-聚谷氨酸作为研究对象,验证它是否具有保湿、抗过敏以及对于亚硝酸盐的清除作用,对于开发透皮制剂具有重要意义。本实验通过3方面研究纳豆提取物的性质。在实验条件下,低浓度的纳豆提取物溶液保湿率均较低,具有明显的抗过敏性和对NaNO2的清除的作用。     

Percutaneous epididymal sperm aspiration and short time insemination in the treatment of men with obstructive azoospermia

Objective

To study the efficacy of percutaneous epididymal sperm aspiration (PESA) in combination with short time insemination to treat infertile men with obstructive azoospermia (OA).

Design

Paired randomized controlled trial in which each couple’s cohort of oocytes was divided into two equal groups.

Setting

Center for reproductive care.

Patients

Twenty men with OA.

Interventions

Motile spermatozoa were collected using PESA. Half of the oocytes were used for intracytoplasmic sperm injection (ICSI). The rest were inseminated briefly with PESA sperm in vitro fertilization (IVF). After 4–5 h, the remaining cumulus cells were removed mechanically for second polar body observation to decide whether to apply “rescue” ICSI (RE-ICSI).

Main outcome measures

Rates of oocyte maturation, fertilization, cleavage, and good quality embryos. Numbers of available embryos and good quality embryos were compared between PESA-IVF (using a short incubation protocol + rescue ICSI) group and PESA-ICSI group.

Results

In the short time insemination group, cumulus cells were dispersed by PESA spermatozoa. No second polar bodies were found, so RE-ICSI was done. PESA-IVF + RE-ICSI and PESA-ICSI outcomes were comparable in terms of fertilization rates, 2PN cleavage rate and good quality embryo rates with no statistically significant differences.

Conclusions

PESA sperm without centrifugation could disperse the cumulus cells but were infertile and therefore could substitute for synthetic hyaluronidase. The outcomes of PESA-IVF with rescue ICSI were equivalent to PESA-ICSI. Using spermatozoa obtained by PESA and IVF before RE-ICIS is a viable treatment for men with OA.     

精子透明质酸酶活性与精液动态参数的相关性

目的探讨不育男性精子顶体内透明质酸酶活性与精液有关参数的关系,以期为临床男性不孕不育的病因分析、诊断、治疗及精子的功能检查提供理论及实验依据。方法收集81例精液标本,其中正常生育对照组为20例,不育男性61例,以精子质量分析仪对每份标本进行计算机辅助精液分析（CASA）。并利用改良明胶底物膜法检测精子顶体内透明质酸酶活性。结果①不育组与对照组相关动态参数的分析结果：VCL、VSL、VAP、LIN、WOB的数据不育组与对照组具有统计学意义,表明不育组精子综合质量与对照组间存在差异;②精子运动活力的参数即精子的VCL、VSL、VAP以及ALH等精子参数与精子顶体内透明质酸酶活性有显著相关性,与LIN、STR、WOB、BCF及STR无统计学意义。结论①不育组与对照组相关动态参数VCL、VSL、VAP、LIN、WOB数据具有统计学意义,精子综合质量与对照组间存在差异;②精子参数VCL、VSL、VAP及ALH与精子顶体内透明质酸酶活性有显著相关性。     

Effects of cell damage and glycosaminoglycan degradation on available extravascular space of different dextrans in a rat fibrosarcoma

Drug delivery to solid tumors may be enhanced through increasing the available volume fraction ( K AV ) of drugs. Therefore, two approaches were investigated that may increase K AV of dextrans in a rat fibrosarcoma: (a) damaging cells in tumours via ex vivo incubation of tumour tissues, and (b) degrading tumour glycosaminoglycans (GAGs) with exogenous hyaluronidase. The molecular weights of dextrans used in this study were ~ 10 000 (D10), 70 000 (D70) and 2 000 000 (D2000), respectively. It was found that GAG degradation had minimal effects on K AV of dextrans. Ex vivo incubation at 37°C for up to 3 h caused only minor cell damage and had minimal effects on K AV of D10 and D70. However, the ex vivo incubation reduced K AV of D2000 ( p < 0.05). When the incubation at 37°C was maintained for 20 h, the amount of viable cells in tumours was reduced by 56% and K AV of all dextrans were significantly increased ( p < 0.05). Ex vivo incubation at 41°C for 3 h caused similar cell damage to that at 37°C for 20 h, but only K AV of D10 and D70 were increased significantly ( p < 0.05). There was no significant change in K AV of D2000, although it was higher than that in tumours incubated at 37°C for 3 h ( p < 0.05). These data suggest that cell damage is a more effective approach than GAG degradation for increasing K AV of macromolecules and that the amount of increase depends on the degree of cell damage and the size of molecules.     

Cartilage degradation independent of MMP/aggrecanases

OBJECTIVE: To identify and characterize a cartilage degradation mechanism that is independent of the proteolytic cleavages by matrix metalloproteinases (MMPs) and aggrecanases. METHODS: The sensitivity of glycosaminoglycan (GAG) release and collagen release to an MMP/aggrecanase inhibitor, AG3340, was compared using a bovine nasal cartilage explant culture. The release of matrix proteins and hyaluronan (HA) from the culture was analyzed by immunoblotting and radioimmunoassay, respectively. Induction of HA-degrading activity by retinoic acid was examined using the cartilage explant culture and a primary culture of chondrocytes. Degradation of the matrix components of cartilage was also characterized in vivo using an acute arthritis model induced by an intra-articular injection of interleukin 1alpha (IL-1alpha). RESULTS: AG3340 did not effectively inhibit GAG release at a concentration of more than 10muM, while 10nM of the inhibitor completely suppressed collagen degradation. Retinoic acid induced the release of the aggrecan G1 domain, link protein and HA into the culture medium, and the release of these molecules was not completely inhibited by 10muM of AG3340. The molecules were released as ternary complexes. Retinoic acid induced HA degradation in the explant culture and hyaluronidase activity in the primary culture of chondrocytes. The release of the G1 domain of aggrecan and link protein into the synovial fluid was also observed in the IL-1alpha-induced acute arthritis model. CONCLUSION: A novel mechanism by chondrocyte-derived hyaluronidase(s) is involved in the release of the matrix components from cartilage, and the hyaluronidase(s) and MMPs/aggrecanases act in a coordinated manner in cartilage degradation.     

Comparative Evaluation of the Efficacy of 2% Lidocaine Containing 1:200,000 Epinephrine with and without Hyaluronidase (75 IU) in Patients with Irreversible Pulpitis

Introductions

The purpose of this study was to determine the anesthetic efficacy of lidocaine containing epinephrine compared with lidocaine containing epinephrine plus hyaluronidase (75 IU) when performing an inferior alveolar nerve block.

Methods

Patients complaining of pain in the mandibular posterior teeth were selected. Based on their chief complaint, proper clinical and radiographic examinations were performed. Among them, 40 subjects diagnosed with irreversible pulpitis were selected. The inferior alveolar nerve block was induced using 3 mL 2% lidocaine with epinephrine. Hyaluronidase (75 IU) or a placebo was injected 30 minutes after the beginning of pulpal anesthesia (randomized and double-blind trial). The duration of the effect in the pulpal and gingival tissues was evaluated by the response to painful electrical stimuli applied to the adjacent premolar and by mechanical stimuli (pinprick) to the buccal gingiva, respectively.

Results

In both pulpal and gingival tissues, the duration of the anesthetic effects with hyaluronidase was longer than with placebo.

Conclusions

Hyaluronidase increased the duration of the effects of lidocaine in inferior alveolar nerve blocks.     

透明质酸酶与血管内皮生长因子在人乳腺癌侵袭与转移中的作用

目的研究透明质酸酶(H yase)与血管内皮生长因子(VEG F)在人乳腺癌侵袭与转移中的意义。方法应用组织匀浆、ELSA及免疫组化等方法测定H yase和VEG F在人乳腺癌中的表达并对其病理特征进行比较。结果乳腺癌组织学分级Ⅰ级、Ⅱ级、Ⅲ级组H yase的均值分别为(3.99±1.91)m U/g、(8.01±2.59)m U/g和(12.00±3.96)m U/g,Ⅲ级和Ⅱ级组H yase表达明显高于Ⅰ级组,Ⅲ级组明显高于Ⅱ级组,其差异具有显著的统计学意义(P<0.05)。乳腺癌无淋巴结转移组和淋巴结转移组H yase的均值分别为(5.25±2.84)m U/g和(8.21±3.26)m U/g,乳腺癌淋巴结转移组H yase表达明显高于无淋巴结转移组。VEG F阳性率在淋巴结转移组为82.6%,明显高于无淋巴结转移组33.3%(P<0.05)。VEG F阳性组和阴性组H yase为(8.39±3.26)m U/g和(5.16±2.58)m U/g,二者间具有显著性统计学差异(P<0.05)。结论H yase和VEG F一样,与人乳腺癌侵袭与转移相关。     

人精子膜蛋白P34H表达与透明质酸酶活性的关系

目的探讨人精子膜蛋白P34H表达和顶体内透明质酸酶活性的关系。方法收集88例精液标本,其中正常生育组20例,不育男性68例。参照世界卫生组织(WHO)标准对标本进行精液常规分析,根据精液参数的不同,将68例不育标本分为正常精液参数不育组和精液参数异常不育组。Western blotting检测P34H蛋白在人精子中的表达,免疫荧光观察P34H蛋白在精子表面的阳性表达率,采用改良透明质酸钠-明胶底物膜法检测精子顶体内透明质酸酶(HYD)活性(HYD阳性反应率、HYD活性强度)。结果正常精液参数不育组和精液参数异常不育组的精子P34H/β-actin平均吸光度、精子P34H标记阳性率均明显低于正常生育组,经统计学分析差异均有显著性(P0.05);正常精液参数不育组和精液参数异常不育组的HYD活性(HYD阳性反应率、HYD活性强度)与正常生育组比较均有显著下降(P0.05)。相关性分析提示,精子P34H蛋白表达量与HYD活性(HYD阳性反应率、HYD活性强度)存在正相关性,相关系数r=0.449、0.431,P0.01;精子P34H阳性率与HYD活性(HYD阳性反应率、HYD活性强度)存在正相关性,相关系数r=0.727、0.691,P0.01。结论男性不育患者精子P34H蛋白表达减少,精子P34H阳性率降低以及HYD活性(HYD阳性反应率、HYD活性强度)减弱。