变色硅胶用作菌种冷冻干燥的脱水剂

冷冻干燥法保存细菌的菌种和病毒的毒种，不仅保存时间长，而且可以避免变异，可保持原细菌和病毒的生物特性。冷冻干燥技术在生物学领域中占有重要的地位，是保存生物制剂及其标准品的优良方法。以往制备冷冻干燥菌、毒种和生物制剂时常以氯化钙或五氧化二磷为脱水剂，一般只能使用一次，不能重复使用。我们最近在制备冷冻干燥菌种时，采用新材料无水变色硅胶做脱水剂，获得了良好效果。无水变任．砗膝不仅可以脱水每原日能名次反复使用，既经济又方便。     

脱蛋白联合冷冻干燥法制备异种骨的实验研究

目的通过脱蛋白联合冷冻干燥的方法制备异种骨,探讨其能否在保持其生物力学特性的同时良好地消除异种骨的抗原性,以满足骨移植的需求。
　　方法取牛股骨上端去除表面筋膜、结缔组织和皮质骨部分,制备成骨粒和圆柱形骨棒。将十二烷基苯磺酸钠（ABS）和脂肪醇聚氧乙烯醚硫酸钠（AES）以及蒸馏水以重量比13：7：80的比例配制复合表面活性剂。将牛骨粒或者骨棒与复合表面活性剂以重量比1：10的比例置于烧瓶中,超声振荡清洗,去除其抗原性,电镜下观察其结构。骨粒和骨棒经乙醇和乙醚脱水、脱脂,冷冻干燥。骨粒用作溶血试验和细胞毒性检测,骨棒进行新西兰大白兔的长期骨植入实验（4、12、26、52周）检测其生物相容性。乙醇抽吸法检测脱蛋白-冻干骨与未脱蛋白的单纯冻干骨的孔隙率。并且比较牛松质骨骨粒与脱蛋白-冻干松质骨骨粒的生物力学特性差异。
　　结果电镜下观察,制备的异种骨材料为天然多孔结构,保留了骨组织的三维结构,骨小梁间有200~650μm 骨髓腔。溶血率检测未超过5%,判定溶血率合格。细胞毒性检测为1级,极低细胞毒性,材料合格。长期骨植入实验表明,植入4周即有植入的骨棒与兔自身骨出现融合,未见严重炎症反应;52周时,植入骨已完全吸收。孔隙率检测表明,脱蛋白-冻干骨与未脱蛋白的单纯冻干骨相比无显著性差异,均可达到60%以上,符合骨移植材料的要求。脱蛋白-冻干骨以及未脱蛋白的单纯冻干骨的力学特性与新鲜松质骨块相比均有显著降低,但两者之间无显著性差异。结论通过脱蛋白联合冷冻干燥的方法制备的异种骨,能够在保持其生物力学特性的同时良好地消除抗原性;满足骨移植的需求。     

真空冷冻干燥过程中猪主动脉血管的力学性能

背景:以往的冷冻干燥研究着重探讨冷冻保护剂、降温速率、保存温度等因素,目前尚缺乏冷冻干燥工艺对冻干后血管力学性能的研究。目的:使用质构仪对冻干后复水的猪主动脉与新鲜猪主动脉进行力学性能对比分析,揭示冷冻干燥过程对猪动脉血管力学性能的影响,从而选择一个适合血管冷冻干燥过程的控制参数。方法:采用真空冷冻干燥技术,对新鲜的猪主动脉血管进行冻干处理,猪主动脉经过微机控制程序降温仪进行预冻处理,然后由真空冷冻干燥机完成一次和二次干燥过程。冷冻干燥结束后,将冻干血管进行复水,然后使用质构仪对其穿刺应力、轴向拉伸应力和周向拉伸应力进行测量。结果与结论:血管冷冻干燥合适的预冻速率为1K/min,一次干燥温度为-20℃,二次干燥温度为10℃。血管冻干后复水,其力学性能同新鲜血管相比,穿刺和周向拉伸应力分别增大20%和30%左右,轴向拉伸应力减小约20%。结果表明冻干复水后的血管基本保持了新鲜血管的弹性,耐压性和顺应性,可望成为一种有效的血管保存手段。     

冷冻干燥法对人精子DNA的影响

目的探讨冷冻干燥法用于人类精子保存的安全性。方法取健康志愿者合格精液40份，平均分为4组，其中3组分别加入不同的冻干保护剂（ETBS；ETBS＋海藻糖：ETBS＋海藻糖＋蛋黄）后给予冷冻干燥处理，在4℃冰箱中保存3周；1组作为新鲜精液对照组。对4组标本分别以原位缺口末端标记（TUNEL）法和彗星试验进行DNA断裂精子百分率检测。结果ETBS、ETBS＋海藻糖、ETBS＋海藻糖＋蛋黄组和新鲜精液组DNA断裂精子百分率以TUNEL法检测，分别为（6．39±1．46）％、（5．75±1．29）％、（5．20±1．38）％、（4．94±1．86）％；以彗星试验检测，分别为（6．48±1．58）％、（5．83±1．48）％、（5．28±1．42）％、（5．12±1．65）％。冷冻干燥保存后的各组与新鲜精液相比较，DNA断裂精子百分率差异均无统计学意义（P〉0．05）。结论以ETBS或ETBS加海藻糖、蛋黄为保护剂的冷冻干燥法对人精子DNA无明显损伤，可有效地保护人精子的DNA。     

几种胶原型创伤敷料制作的实验研究

介绍了几各胶原型创伤敷料研制的方法。冷冻牛腱经０．０５Ｍ乙酸处理（ＰＨ３．２）４８－７２ｈ后，捣碎过滤、脱泡、加太酸软骨素（８％），制成１．５－２．５％胶原溶液。该溶液在预冷或不预冷的模具内冷冻干燥，冻干的胶的海绵在０．２５％戊二醛溶液中交联２４ｈ。并以类似的方法研制成聚氨酯膜－胶原海绵复合膜，涂聚氨酯胶原膜，和纱布胶原膜三种创伤敷料。结果表明冷冻牛腱胶原性能稳定，冻干的胶原海绵具有良好的孔洞结构     

冷冻干燥法保藏放线菌菌株32～39年存活性试验

<正>冷冻干燥保藏法,是目前被广泛采用的一种较理想的保藏方法。文献报道一般保藏时间达5～15年,所以传代转种的间隔是5～10年。国内尚未有保藏时间超过30年的文献报道。本文将1972-1979年间采用冷冻干燥法保存的120株菌种复壮,发现此法保藏的菌株经过32～39年的保存,总存活率高达83.3%。     

生物羊膜冷冻干燥工艺优化研究

为探索羊膜冷冻干燥保存的最佳工艺条件,取健康剖宫产产妇胎盘,钝性剥离羊膜。以保存羊膜组织形态变化、胶原蛋白酶降解速度、生物力学特征、细胞因子含量为考察指标,对羊膜冷冻干燥工艺中的关键因素进行优化。结果显示,改良冷冻干燥羊膜上皮细胞、纤维基质层形态结构与新鲜羊膜基本相同,但纤维基质厚度略有增加,上皮细胞表面微绒毛略有减少;Ⅳ型胶原酶在溶液中降解速度有所加快;生物力学特征与新鲜羊膜无明显差别;6种细胞因子含量明显低于新鲜羊膜。结合前期工作,与常规冷冻干燥法比较,改良冷冻干燥工艺对保存羊膜组织结构和生物学活性因子影响较小,并能使保存羊膜具有更好的生物力学特性。     

载氧药物的冷冻干燥和冻干制品的保存稳定性

对以聚合胎盘血红蛋白为有效成分的载氧药物进行冷冻干燥.采用蔗糖作保护剂,减少冷冻干燥过程中高铁血红蛋白(Methemoglobin,MetHb)的增加.测定冷冻干燥前后的MetHb含量、UV光谱、十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、高效液相色谱法(HPLC)等指标.结果表明:冷冻干燥中,当蔗糖/蛋白质的重量比≥0.5时,MetHb增加得到有效控制, UV光谱中血红蛋白(Hb)的特征吸收峰、SDS-PAG、HPLC无显著改变.冻干制品分别在室温或冰箱中保存3个月,考察其稳定性.结果表明:保存过程中,MetHb的增加与蔗糖/蛋白质的重量比、保存温度有关,试验组C(蔗糖/蛋白质的重量比为1.0)在室温或冰箱中保存3个月,MetHb含量、UV特征吸收峰、SDS-PAGE、 HPLC等指标均无显著改变.     

胶原海绵及其止血性能的研究

利用酸碱溶解法，从牛腱中提取了可溶性胶原材料。冻干法制得了胶原海绵止血材料。对胶原材料的氨基酸分析和紫外吸收光谱分析，证实了可溶性胶原的结构和氨基酸组成。物理性能测试和止血效果实验结果表明，胶原海绵亲水性强，而且具有优良的止血和粘附创而的能力。     

不同比例壳聚糖胶原复合骨、软骨缺损支架材料的制备及性状比较

背景:骨组织工程支架材料由最初的自体骨,软骨材料到生物活性陶瓷,乃至后来的有机材料胶原蛋白等细胞外基质材料,其生物相容性及性能越来越优越,越来越接近体内的真实情况。但是这些材料在抗压性及强度方面还有待进一步提高。目的:应用胶原与壳聚糖制备生物支架并对其检测其生物学性质,为骨、软骨缺损提供移植替代物。方法:将不同比例壳聚糖-胶原蛋白溶解,经冷冻冻干后紫外线交联后冷冻干燥,二次冻干制备好支架。检测不同比例支架的孔隙率,降解率,溶胀率。扫描电镜观察孔径的大小及形态。结果与结论:制备的支架外观呈海绵多孔状。支架的孔径大小随着胶原比例增加而减小。胶原比例的增加对支架孔隙率的影响较轻微。支架的溶胀率可达到80%左右,支架的溶胀程度随胶原比例增加而减少。胶原含量越大支架柔韧度增加明显。支架的降解率随着胶原比例增加而增加,而壳聚糖含量越高,降解速度越慢。结果提示,通过调整壳聚糖-胶原蛋白比例使支架具有作为骨、软骨缺损移植材料的替代物可能。     

Collagen-based wound dressings: control of the pore structure and morphology

Collagen-based sponges have been used as both temporary and permanent coverings for dermal defects in animals and humans. Cellular ingrowth within such a sponge has been shown to depend on the porosity and the presence of fibrous structure. Collagen sponges were made by freezing and freeze-drying dispersions under acidic conditions. These studies involved the effects of dispersion pH and viscosity as well as freezing temperature on the surface and bulk morphology of collagen-based sponges. Using scanning electron and light microscopy, the results of these studies indicated that large surface pores that form connections (channels) with the interior of the sponge were formed using low-viscosity collagen dispersions. At high dispersion pH (3.2) and at a moderate freezing temperature (-30 degrees C), fibrous structure and a large number of channels were present. When a lower dispersion pH (2.0) and freezing temperature (-80 degrees C) were used, pores sizes were smaller with channels and fibrous structure, whereas a higher freezing temperature (-20 degrees C) resulted in a sheet-like structure and increased pore sizes. Differences in pore size and surface morphology were explained on the basis of ice crystal growth. In the case of abundant free water (high pH) and high freezing temperature, the pore size was greatest because of enhanced ice crystal growth.     

Characterization of UV-irradiated dense/porous collagen membranes: morphology, enzymatic degradation, and mechanical properties

Collagen-based membranous materials of various shapes (gel, film, sponge) are known to be the most promising materials in terms of facilitating the regeneration of dermal defects. In this study, dense and porous collagen membranes were fabricated using air-drying and freeze-drying processes, respectively, and the effect of ultraviolet (UV) radiation on the degree of membrane crosslinking was evaluated by in vitro biodegradation and mechanical testing. A non-irradiated membrane group was used as the negative control and a glutaraldehyde (GA) treated group as the positive control. Scanning electron microscopy showed that, as the freezing temperature decreased to -196 degrees C, the resultant mean pore sizes also decreased; optimal pore size was obtained at a freezing temperature of -70 degrees C. In vitro biodegradation and mechanical testing demonstrated that GA treatment or 4 hours of exposure to UV radiation significantly increased both resistance to collagenase and mechanical strength versus the untreated controls, regardless of the collagen membrane type (dense or porous). Our results suggest that UV treatment is a useful tool for the fabrication of collagen membranes designed to be used as dermal dressings.     

Osteoblastic response to collagen scaffolds varied in freezing temperature and glutaraldehyde crosslinking

Collagen sponges are widely used scaffolds in bone engineering. To form bone, the osteoblastic cells undergo proliferation, differentiation, and mineralization stages in the scaffold. Crosslinking and freezing temperature are two important variables in fabricating collagen sponges. The purpose of this study was to examine the osteoblastic responses to collagen sponges prepared with or without glutaraldehyde crosslinking at different freezing temperatures (-20 degrees C or -80 degrees C). MC3T3-E1 osteoblastic cells were cultured in differently prepared sponges. Osteoblastic responses examined included cell numbers, osteocalcin expression, and calcium deposition. Cell numbers were measured by DNA content. Osteocalcin expression was determined by RT-PCR and real-time RT-PCR. Calcium deposition was assayed by ortho-cresophthalein complexone method and von Kossa stain. The osteoblastic cells grown in all collagen sponges did not show apparent signs of cytotoxicity. Collagen sponges differed in freezing temperatures resulted in similar osteoblastic responses. Glutaraldehyde-crosslinked sponges demonstrated less cell-mediated contraction and more cell numbers at day 7 (p < 0.005). However, they showed lower osteocalcin expression at day 7 (p < 0.05) and less calcium deposition at day 21 (p < 0.001). In summary, different freezing temperatures played a minor role in osteoblastic responses. Glutaraldehyde crosslinking process, though improved the dimensional stability of collagen sponges, might compromise the osteoblastic differentiation and mineralization.     

Structure and properties of bilayer chitosan-gelatin scaffolds

Chitosan-gelatin hybrid polymer network scaffolds were prepared via the freeze-drying technique by using the ice microparticle as a porogen. Monolayer and bilayer scaffolds were obtained by using different pre-freezing methods. The novel bilayer scaffolds were prepared via contact with -56 degrees C lyophilizing plate directly, then lyophilized. The properties of chitosan-gelatin scaffolds, such as microstructure, physical and mechanical and degradable properties, were studied. These results suggested that the porosity and pore size of the scaffolds could be modulated with thermodynamic and kinetic parameters of ice formation. The scaffolds prepared from chitosan and gelatin can be utilized as a promising matrix for tissue engineering.     

Cryopreservation of collagen-based tissue equivalents. I. Effect of freezing in the absence of cryoprotective agents

The effect of freezing on the viability and mechanical properties of tissue-equivalents (TEs) was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. TEs (a class of bioartificial tissues) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. TEs were detached from the substrate and frozen in phosphate-buffered saline using a controlled rate freezer (CRF) at various cooling rates (0.5, 2, 5, 20, and 40 degrees C/min to -80 or -160 degrees C) or in a directional solidification stage (DSS) (5 degrees C/min to -80 degrees C) or slam frozen (>1000 degrees C/min). Viability of the fibroblasts in the TEs was assessed by ethidium homodimer and Hoechst assays immediately after thawing. Uniaxial tension experiments were also performed on an MTS (Eden Prairie, MN) Micro Bionix system to assess the postthaw mechanical properties of the frozen-thawed TEs. Cooling rates of either 2 or 5 degrees C/min using the CRF were optimal for preserving both immediate cell viability and mechanical properties of the TEs, postthaw. By 72 h postthaw, TEs frozen in the CRF at 5 degrees C/min to -80 degrees C showed a slight decrease in cell viability, with a significant increase in tangent modulus and ultimate tensile stress suggesting a cell-mediated recovery mechanism. Both the postthaw mechanical properties and cell viability are adversely affected by freezing to the lower end temperature of -160 degrees C. Mechanical properties are adversely affected by freezing in the DSS.     

Studies of novel hyaluronic acid-collagen sponge materials composed of two different species of type I collagen

The authors have developed novel hyaluronic acid (HA)-collagen sponge materials (HACSMs) composed of various ratios of bird feet (BF) and pig skin (PS) collagen that are fabricated employing a combination of freezing, lyophilizing, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) crosslinking methods. Morphology, swelling ratio, resistance to collagenase, thermal stability, tensile strength, and free amine index are determined to evaluate the physical-chemical properties of various HACSMs. Different BF: PS ratios directly vary with the physical-chemical properties of HACSMs and control their biodegradability for multiple uses. Resistance to collagenase, thermal stability, and tensile strength of HACSMs increases as the ratio of BF collagen increases. On the contrary, the higher swelling ratio, free amine index, and pore size occur in materials composed of higher ratios of PS collagen. A linear relationship between the decreased ratio of PS collagen and the increase in tensile strength and biostability are observed. The materials of B4P1HA (BF: PS: HA=4: 1: 0.2) exhibit the highest value of tensile strength, but no significant difference exists between B4P1HA and B5P0HA (BF: PS: HA=5: 0: 0.2). These phenomena should be closely related to the BF collagen which contains a higher amount of carboxyl groups of glutamic or aspartic acid residues and forms more amine bonds under EDC cross-linking when compared to PS collagen. However, these results suggest that the B4P1HA and B5P0HA materials should be produced according to highest bio-stability and mechanical strength and, furthermore they may be suitable for artificial skin or drug delivery applications.     

Characterization of CO3Ap-collagen sponges using X-ray high-resolution microtomography

For reconstruction and regeneration of hard tissues, scaffold biomaterials with large size pores and high porosity are important, in addition to their roles as supporting frames. To develop a new biodegradable scaffold biomaterial, CO3Ap, which has crystallinity and a chemical composition similar to bone, was synthesized at pH 7.4 and 60 degrees C. Then, the CO3Ap was mixed with a neutralized collagen gel and the CO3Ap-collagen mixtures with different kinds of CO3Ap contents and porosity were lyophilized into sponges. Scanning electron micrography (SEM) observation of CO3Ap-collagen sponges showed favorable pores for cell invasion. Approximately 50-300 microm size pores appeared to continue through the bulk. Higher magnification of the sponge showed a better adhesion between CO3Ap crystals and collagen. X-ray high-resolution microtomography revealed a clear image of the 3D structure of the sponges. The porosity of 0, 70 and 90%(w/w) CO3Ap-collagen sponges was 79.2 +/- 2.8%, 72.6 +/- 2.4% and 48.9 +/- 6.1%, respectively. The 70%(w/w) CO3Ap-collagen sponge appeared to be the most favorable biomaterial from the viewpoint of natural bone properties. Mouse osteoblast MC3T3-E1 cells were cultured in alphaMEM with 10% FCS for 2 weeks. Hematoxylin-eosin staining confirmed osteoblast cells invaded well into the CO3Ap-collagen sponge. These sponges are expected to be used as hard tissue scaffold biomaterials for therapeutic uses.     

Stabilizing cold-adapted influenza virus vaccine under various storage conditions

Various diluents, stabilizers, buffers, and storage conditions were assessed for their efficacy in stabilizing cold-adapted influenza virus vaccine. Frozen liquid vaccine formulations, comprised of a normal uninfected allantoic fluid diluent and an SPG (sucrose-phosphate-glutamate) stabilizer, generated complete stability of H1N1, H3N2, and Type B strains for at least 1 year of storage at -20 degrees C. The ability to store live influenza virus frozen liquid vaccines, at the moderate temperature of -20 degrees C, has not been demonstrated previously. This significant advance could facilitate influenza vaccine storage and administration in the clinic, and subsequently increase marketability. The stability of lyophilized formulations was also augmented by the addition of 2% Casitone and the control of pH with 0.066 M phosphate in the SPG stabilizer. This alternative formulation may be useful in markets where freezing is not feasible or short-term room temperature storage is necessary.     

Factors affecting viability and growth in HeLa 229 cells of Chlamydia sp. strain TWAR.

Two prototype isolates (TW-183 and AR-39) of Chlamydia sp. strain TWAR were used to study factors affecting growth of this organism in HeLa 229 cells. The results showed that an incubation temperature of 35 degrees C was better than one of 37 degrees C for growth. The burst size after 3 days of incubation at 35 degrees C was found to be small (13 to 52), which partially explains the difficulty of serial passage in cell culture. Application of a higher centrifugal force (1,700 X g versus 900 X g) at the time of inoculation enhanced growth 2.2 to 3.6 times. Infectivity was enhanced by treatment of cells with DEAE-dextran (2.4 times) or poly-L-lysine (1.6 times), but not with Polybrene or polyethylene glycol. The viability of the TWAR organism in chlamydia transport medium SPG was also studied. It was shown that the organism was rapidly inactivated at room temperature (22 degrees C); only 1% remained viable after storage for 24 h. The viability was preserved at 4 degrees C, and 70% remained viable after storage for 24 h. Freezing at -75 degrees C inactivated 23% of the organisms when the organisms were frozen within 4 h after harvesting and stored at 4 degrees C before freezing.