原位生长纳米羟基磷灰石晶须增强β-磷酸三钙多孔支架的生物安全性

背景：通过纳米羟基磷灰石原位生长明显提高了磷酸钙支架的强度与韧性。 目的：体外评价纳米羟基磷灰石晶须/β-磷酸三钙(nHAW/β-TCP)作为人工骨支架材料的生物相容性。 方法：急性全身毒性试验：30只小白鼠随机分为静脉实验组,腹腔实验组和对照组,分别注射浸提液及生理盐水,24,48,72 h观察动物的一般状态。溶血试验：材料浸提液与稀释人鲜血混合观察红细胞溶解情况,545 nm下检测A值计算溶血率;致敏试验：16只豚鼠随机分为实验组、阴性对照组和阳性对照组,每只豚鼠脊柱两侧皮内注射等体积nHAW/β-TCP支架材料浸提液、生理盐水及二硝基氟苯。于注射后即刻和24,48,72 h观察局部皮肤反应。细胞毒性试验：材料浸提液培养细胞进行细胞形态大体观察,采用CCK-8法观察细胞活性。 结果与结论：急性全身毒性试验：人工骨浸提液静脉及腹腔注射后不引起小鼠呼吸、进食改变或死亡,体质量稳定。溶血试验：nHAW/β-TCP的溶血率小于ISO规定的5%,可认为这种材料无溶血作用。致敏试验：豚鼠皮内注射后未出现过敏反应。细胞毒性试验：CCK-8细胞毒性试验显示不同浓度人工骨浸提液的细胞毒性为0级。提示nHAW/β-TCP复合支架不引起全身毒性反应、溶血反应和过敏反应,且无细胞毒性,生物相容性良好,符合组织工程人工骨支架材料的应用要求。     

灌注法制备全肾脏脱细胞基质支架的体内生物相容性

背景：应用灌注法制备的大鼠全肾脏脱细胞基质支架具有良好的体外细胞相容性,但其体内生物相容性尚不明确。 目的：应用灌注法制备大鼠全肾脏脱细胞基质支架,检测其体内生物相容性。 方法：应用灌注法制备Wistar大鼠全肾脏脱细胞基质支架,进行以下实验：①急性毒性实验：在小鼠腹腔分别注射全肾脏脱细胞基质支架浸提液、生理盐水及苯酚。②溶血实验：将抗凝新西兰兔血分别与全肾脏脱细胞基质支架浸提液、生理盐水及蒸馏水混合。③热源实验：向新西兰兔耳缘静脉注射全肾脏脱细胞基质支架浸提液。④内皮刺激实验：在新西兰兔皮下注射全肾脏脱细胞基质支架浸提液,观察有无皮肤刺激反应。⑤皮下植入实验：将全肾脏脱细胞基质支架埋入新西兰兔背部皮下。 结果与结论：全灌注法制备的Wistar大鼠全肾脏脱细胞基质支架无细胞残留,未引起全身毒性反应、急性溶血反应、热源反应及皮肤刺激反应,植入兔体内具有良好的组织相容性。说明大鼠全肾脏脱细胞基质材料在动物体内具有很好的生物相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

冻干去抗原羊松质骨支架的生物相容性评价

背景:异种骨具有与人类骨类似的天然多孔结构,在治疗骨缺损时可引导骨组织再生,但植入过程中也会引起不同程度的免疫反应。目的:采用冻干法制备去抗原羊脊椎松质骨支架材料,并评价其生物相容性。方法:取羊脊椎松质骨,制备两组去抗原异种骨支架,化学组经H2O2、甲醇/氯仿混合液等化学试剂处理;冻干组将经化学处理的羊松质骨在-80℃冰箱中低温冷冻4周,真空干燥,60Co照射消毒。①细胞毒性实验:分别采用化学组材料浸提液、冻干组材料浸提液与DMEM/F12培养基培养羊骨髓间充质干细胞。②热源实验、急性毒性实验:从兔耳缘静脉分别注射化学组材料浸提液、冻干组材料浸提液与生理盐水。③皮内刺激实验:分别在兔脊柱背部皮下注射化学组材料浸提液、冻干组材料浸提液、生理盐水及乙醇。结果与结论:冻干组支架无细胞毒性、急性毒性及热源反应,皮内刺激实验阴性;化学组支架有细胞毒性及轻微急性毒性反应,有致热源作用及轻度皮肤刺激性。结果表明经过化学处理羊脊椎松质骨支架的生物相容性相对较差,而化学处理配合低温冷冻、真空干燥及60Co照射的羊脊椎松质骨生物相容性较好,基本能达到骨组织工程支架材料的要求。     

猪来源关节软骨脱细胞支架的生物安全性研究

目的评价猪来源关节软骨脱细胞支架的生物安全性。方法取猪四肢关节软骨,分别制备脱细胞和未脱细胞软骨支架。在人软骨细胞中分别加入浓度为100%、50%、25%的脱细胞软骨支架浸提液[无血清DMEM培养液与支架表面积按1ml:(3～6)cm2比例混合,37℃静置72h],计算相对增殖率,判定其体外细胞毒性。0.9%NaCl与支架表面积按1ml:(3～6)cm2比例混合,37℃静置72h制备脱细胞软骨支架浸提液,分别用于全身急性毒性实验(小白鼠)、溶血实验、热原检测实验、皮内刺激实验(大白兔),分别观察其体内毒性、溶血程度、热原和刺激情况。在大白兔体内分别埋植脱细胞软骨支架与未脱细胞软骨支架,观察炎症反应和细胞免疫情况。结果猪脱细胞软骨支架浸提液对细胞生长无抑制作用,无细胞毒性。注射支架浸提液后小白鼠一般情况良好,无全身急性毒性反应。支架浸提液溶血程度为1.3%,无溶血作用。支架浸提液无热原存在且原发刺激指数为0分,皮内刺激实验阴性。脱细胞软骨支架与未脱细胞软骨支架相比免疫原性小,无炎症反应和淋巴细胞浸润。结论猪来源关节软骨脱细胞支架具有良好的生物相容性,将有可能成为理想的天然生物材料,具有良好的应用前景。     

聚四氟乙烯联合Ⅰ型胶原作为隆鼻高分子材料的生物相容性

背景：高分子材料聚四氟乙烯膨体作为隆鼻填充材料具有耐腐蚀、化学性质稳定等优点,但其线膨胀系数较大,易引发感染及排异反应,故应用有一定局限性。目的：对比聚四氟乙烯和聚四氟乙烯联合Ⅰ型胶原作为隆鼻填充材料的细胞毒性、埋植后的炎性浸润及体内生物相容性。方法：采用MTT法检测聚四氟乙烯浸提液和聚四氟乙烯联合Ⅰ型胶原浸提液培养L929细胞的细胞增殖。采用电子显微镜观察聚四氟乙烯浸提液和聚四氟乙烯联合Ⅰ型胶原浸提液培养L929细胞后的细胞生长情况。将聚四氟乙烯和聚四氟乙烯联合Ⅰ型胶原材料分别埋置于新西兰白兔鼻背筋膜下7 d,苏木精-伊红染色观察鼻黏膜上皮组织炎性浸润情况。兔耳缘静脉分别注射聚四氟乙烯浸提液和聚四氟乙烯联合Ⅰ型胶原浸提液后,观察兔的全身毒性、过敏、热源反应及死亡情况。结果与结论：作为隆鼻填充材料,聚四氟乙烯联合Ⅰ型胶原材料在细胞毒性、埋植后的炎性浸润方面均优于单纯聚四氟乙烯材料(P < 0.05);兔耳缘静脉注射聚四氟乙烯联合Ⅰ型胶原材料后发生的过敏反应、热源反应少于注射单纯聚四氟乙烯材料(P < 0.05)。表明聚四氟乙烯联合Ⅰ型胶原作为隆鼻填充材料具有良好的生物相容性。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

脱细胞软骨生物支架材料的生物学特性

目的 探讨脱细胞软骨生物支架材料（ACM）在细胞毒性、溶血试验、急性全身毒性等方面的生物学特性：方法①ACM的制备：猪膝关节软骨冻干加工为粉末，胰酶消化，曲拉通洗脱，蒸馏水洗净冻干，紫外线照射（UVI）后成型；③细胞毒性测定：材料浸提液培养细胞进行细胞形态人体观察，MTT法观察细胞活性；③急性全身毒性反应：材料浸提液注射入SD大鼠腹腔观察材料对动物表现及体重变化的影响；④溶血试验：材料浸提液与稀释动物鲜血混合观察红细胞溶解情况，492nm下检测OD值计算相对溶血率；结果 ①细胞毒性试验：24h、48h、72h各时间段内三组细胞OD值两两比较（P〉0．05），无显著差异，ACM细胞毒性为0级；②动物急性毒性实验：Ⅰ生物材料处理组和Ⅱ生理盐水处理组对动物体重影响没有差异（P〉0．05），Ⅰ生物材料处珊组和Ⅲ苯酚处理组对动物体重有显著差异（P〈0．05）；③溶血试验：材料相对溶血率为2.92％，低于5％的标准，尢明显溶血现象；结论ACM存细胞毒性、溶血实验、动物急性毒件反应方面符合软骨组织工程中对于支架材料的要求，提示支架材料何良好的牛物相容性和安全性。     

纳米羟基磷灰石/硫酸钙复合人工骨的生物安全性研究

目的研制纳米羟基磷灰石/半水硫酸钙(n-HA/CSH)复合型人工骨,并对其进行体内、外生物安全性测试。方法对n-HA/CSH人工骨进行急性全身毒性试验、皮内刺激试验、致敏试验、MTT细胞毒性试验和遗传毒性实验(Ames试验)并与对照组比较。结果人工骨浸取液静脉及腹腔注射后不引起小鼠呼吸、进食改变或死亡,体重稳定。家兔皮内注射72小时后仅出现红斑或微弱水肿,豚鼠皮内注射后未出现过敏反应。MTT细胞毒性试验显示含HA10%、20%、40%人工骨及纯n-HA、CSH的细胞增殖率均在77%以上,细胞毒性均为0～1级,Ames试验表明含HA40%人工骨的不同浓度生理盐水浸取液引起鼠伤寒沙门氏菌回复突变数均不超过阴性对照组的2倍。结论n-HA/CSH复合材料不引起全身毒性反应、皮内刺激反应和急性过敏反应。且无MTT细胞毒性,细胞相容性良好。同时,复合材料的生理盐水浸取液不引起鼠伤寒沙门氏菌回复突变数增加。     

D,L-聚乳酸基形状记忆聚合物的生物安全性和细胞相容性

背景：课题组前期实验研制了输卵管避孕器材料D, L-聚乳酸基形状记忆聚合物,依据国内《生物材料和医疗器材生物学评价技术要求》规定,植入体内的组织工程材料必须进行生物安全评价和细胞相容性实验。 目的：观察D, L-聚乳酸基形状记忆聚合物的生物安全性。 方法：①内毒素实验：在鲎试剂中分别加入聚合物浸提液、内毒素工作标准品溶液和细菌内毒素检查用水。②致敏实验：在昆明小鼠肩胛骨内侧分别注射聚合物浸提液+弗氏完全佐剂+生理盐水、弗氏完全佐剂+生理盐水,通过皮内诱导、局部诱导和激发阶段,观察动物激发部位皮肤红斑和水肿反应程度。③急性毒性实验：分别在昆明小鼠腹腔注射100%,50%,25%聚合物浸提液及生理盐水。④细胞增殖MTT实验：直接法为将人脐静脉内皮细胞分别接种于聚合物膜、聚乳酸与玻璃片上;间接法为将人脐静脉内皮细胞分别接种于聚合物浸提液、丙烯酰胺溶液及1640培养液。 结果与结论：D, L-聚乳酸基形状记忆聚合物材料无细菌污染状况,符合生物安全标准,无致敏性及毒性,并且具有较好的细胞相容性。     

不同方法制备异种骨支架材料的生物相容性评价

背景：异种骨形态、结构与组成类似于人体骨组织,经理化处理后的异种骨抗原性降低,具有天然的多孔结构,被认为是解决自体骨与同种异体骨来源不足的有效方法。 目的：检测3种不同方法制备羊椎骨异种骨材料的生物相容性。 方法：将经过物理、化学和物理结合化学3种处理方法制备的羊椎骨异种骨材料,制成材料浸提液,植入新西兰大白兔体内,通过急性毒性实验、热源实验、皮内刺激实验和细胞毒性实验,初步评价不同处理组羊椎骨异种骨材料的生物相容性。 结果与结论：物理组和物理结合化学组羊椎骨异种骨材料无急性毒性、无热源反应、无刺激性与细胞毒性;化学组羊椎骨异种骨材料有急性毒性反应、有致热源作用、有轻度刺激性及存在细胞毒性。结果提示经过物理、物理结合化学处理的异种骨材料具有良好的生物相容性;单纯经过化学处理的异种骨材料生物相容性较差,不符合生物材料安全性标准。     

温敏性壳聚糖水凝胶对大鼠成骨细胞的毒性

背景：温敏性壳聚糖与多种细胞相容性良好,是组织工程中不可多得的优良载体,但其对成骨细胞毒性研究相对缺乏。 目的：验证温敏性壳聚糖水凝胶对成骨细胞的毒性。 方法：成骨细胞在温敏性壳聚糖水凝胶中进行培养,显微镜下观察细胞形态及扩增情况,同时,SD大鼠成骨细胞在不同浓度的温敏性壳聚糖水凝胶浸提液中体外培养24,48,72,96 h,MTT法测定细胞相对增殖率,判断细胞毒性的级别。 结果与结论：SD大鼠成骨细胞在温敏性壳聚糖水凝胶中培养24 h内镜下观察呈圆形,48 h后开始伸出触角并扩增;温敏性壳聚糖水凝胶浸提液中培养的各组细胞在不同时间点相对增殖率在92%~112%之间,各浓度的温敏性壳聚糖水凝胶材料浸提液的细胞毒性均为0级或1级,完全符合生物材料的安全评价标准。     

Effects of calcium on afferent responses of the ampullae of Lorenzini

Summary Afferent responses in single-fibre and multi-fibre preparations from isolated ampullae of Lorenzini to various concentrations of ionic calcium (from 0.2 g/l to 5.0 g/l) were investigated in the dogfish (Scylliorhinus canicula).At constant temperature (12.3° C), calcium concentrations between 0.2 and 1.6 g/l were practically ineffective. Concentrations between 1.8 and 5.0 g/l inhibited the static impulse discharge for time periods up to several minutes depending on the calcium charge. Concentration and diffusion parameters were decisive for the amount as well as the temporal course of this inhibition. Additional thermal or mechanical stimulation led to an answer superimposed on the effects of calcium. Calcium-free fluids produced a slight increase in average impulse rate.This investigation was supported by grant from the Deutsche Forschungsgemeinschaft.     

Sensory transmission of spinal heat and cold sensitivity in ascending spinal neurons

Summary In anesthetized cats, mostly spinalized at C₁/C₂, the thoracic and lumbar sections of the vertebral canal were selectively heated or cooled. Single unit activity was recorded with steel microelectrodes from the spinal cord at the level of C₂ to C₄. The positions of the electrode tips were determined by micromarking.The existence of two groups of temperature dependent ascending spinal units was confirmed. One group of units was activated by spinal cord cooling below normal body temperature. The other group was activated by spinal cord heating. No temperature dependent neurons were found, so far, exhibiting maximum discharge rates at normal body temperature. A roughly proportional relation between discharge rate and vertebral canal temperature seemed to exist in both heat sensitive and cold sensitive units within a limited range of spinal hyperthermia or hypothermia respectively. Part of the units exhibited dynamic responses to changes of vertebral canal temperature in addition to their static responses.As determined by micromarking, heat sensitive and cold sensitive ascending spinal units were conducted in the anterolateral tracts. Both types of units were observed also under neuromuscular blockage. It is concluded that the temperature dependent ascending spinal neurons are transmitting signals from basically afferent spinal thermosensitive structures existing as two functionally different sets, one heat sensitive and one cold sensitive.     

Behavioural thermosensitivity after bilateral lesions of the lateral funiculi in the cervical spinal cord of the cat

Summary The behavioural thermosensitivity of six cats was measured before and after single stage, symmetrical, bilateral, surgical lesions of the cervical spinal cord. The lesions were aimed at an area in the most ventral parts of the dorsal halves of the lateral funiculi. Unilateral lesions of that area have previously been found to cause reproducible, although subtotal, contralateral thermosensory defects, which were attributed to interruption of the thermosensory spinothalamic pathway. The lesions of three of the present cats were found to be incomplete, and those animals showed no postoperative thermosensory deficiency. Two of the cats with complete lesions showed marked post-operative defects, especially immediately after the operations, but the third cat with a complete lesion showed no postoperative thermosensory defect at all. The differences between the last three animals have been compared to the irregularity of previous reports about thermosensitivity after spinal cord lesions in man and animals, and may depend on the testing technique, rather than differences of thermosensitivity per se.     

Thermosensory defects after cervical spinal cord lesions in the cat

Summary The behavioural thermosensitivity of cat paws was examined before and/or after restricted uni- and/or bilateral lesions had been made in the spinal cord between the first and fifth cervical segments. Unilateral lesions of the lateral funiculus, which involved at least its whole width at the level of the central canal, reproducibly were found to interfere with the contralateral sensitivity for temperature increases and/or decreases. No corresponding thermosensory deficiencies were found after unilateral lesions involving the ventral spinal quadrant or the dorsal funiculus. Various bilateral and combined lesions were made, but no cat ever developed thermoanaesthesia. The bilateral lesions included bilateral transections of: the middle parts of the lateral funiculi, the dorsal halves of the lateral funiculi, the dorsal funiculi, and the ventral spinal half.Most of our knowledge about peripheral behavioural thermosensitivity after spinal cord injury is based on observations of human patients, especially after anterolateral chordotomies. The present finding of contralateral thermosensory deficiencies after lesions of the middle part of the lateral funiculus fits with some of the clinical reports. The present failure to cause thermoanaesthesia, on the other hand, is inconsistent with the theory of a single ascending spinal pathway for behavioural thermo-sensitivity, which has emanated mainly from the clinical observations.     

Behavioural thermosensitivity after unilateral,partial lesions of the lateral funiculus in the cervical spinal cord of the cat

Summary Adult cats were tested for unilateral, behavioural thermosensitivity, before and after unilateral, surgical lesions involving part of the lateral funiculus of the cervical spinal cord. Contralateral thermosensory deficiencies were found after lesions, which involved the middle parts of the lateral funiculus. No such deficiences were observed after lesions which were restricted to either the dorsal third, or the ventral half of the lateral funiculus. It was concluded that behaviourally important, thermosensory information is transmitted via an ascending pathway which is located ventrally in the dorsal half of the lateral funiculus.     

Special aspects of firing rates and thermosensitivity of preoptic neurons

Summary Mean firing rates (per 10 sec) of preoptic neurons of the rat were recorded at normal temperature and after central warming and cooling. Thermosensitivity was determined according to the reactions of the mean firing rate. In compatibility with results from other animals we obtained the following percentages (52 neurons): 23% warm-sensitive, 13% cold-sensitive, 64% non-thermosensitive neurons.However, it turned out that a lot of interesting special effects are not taken into account, using exclusively the criterion increasing/decreasing/constant mean firing rate. Particularly we observed long lasting adaption processes and stationary non-linear effects as well as striking oscillations which were dependent on the thermal stimuli, while the mean interval value remained constant.     

Differential thermosensitivity and electric prepolarization of the ampullae of Lorenzini

Summary In single-fibre preparations the afferent discharges from prepolarized ampullae of Lorenzini responding to graded temperature steps were investigated in the dogfish (Scyliorhinus canicula).The transformation characteristics of the ampullary receptors, especially their differential thermosensitivity interfering with electrosensitivity, were analyzed. Prepolarization significantly influenced the dynamic component of differential thermosensitivity, while the static component remained practically unchanged. Hyperpolarization reduced positive and negative dynamic thermal responses: depolarization amplified them.Biological consequences of this bimodal interference for receptor transformation of superimposed thermal and electric stimuli and for decoding afferent ampullary impulse patterns are discussed.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Analysis of periodic components of hypothalamic spike-trains after central thermal stimulation

Summary Stationary spike-trains of preoptic neurons of the rat were submitted to a correlation- and spectral-analysis at normal temperature and after central warming and cooling. Correlograms and periodograms were computed which enable the elimination of stochastic signal components and the identification of the relevant periodic components.About one half of the analysed neurons showed periodic correlograms. The following three classes may be distinguished: Neurons which showed correlograms with periodicities at each applied thermal state, at only one or two thermal states and those with no recognizable periodicities at all. Relations between periodicity and the thermosensitivity of the neurons are discussed.     

Effect of ouabain and potassium-free solution on mammalian thermosensitive afferents in vitro

Summary Studies have been performed on the afferent fibers innervating the scrotal skin of the rat to test the hypothesis that the generator potential mechanism underlying cold thermosensitivity of these afferent fibers is an electrogenic sodium pump. In these experiments a pudendal nerve-scrotal skin preparation was isolated from the animal and, maintained in oxygenated mammalian Ringer's where composition could be varied and drugs added. Application of ouabain resulted in an increase in the discharge of cold sensitive afferent fibers which was more pronounced at 38–41° C than at temperatures below 30° C. In most of the cases transient accelerations on cooling were reduced, but often a transient response to warming appeared. The effects of ouabain administration were reversible. Removal of extracellular K at 35° C resulted in an increased discharge of cold sensitive afferents. This observation is consistent with the effects of ouabain, since removal of extracellular K also blocks Na transport. The generator potential mechanism underlying cold sensitivity of these afferent fibers appears to be an electrogenic Na pump.