Sustained release of bioactive protein from a lyophilized tissue‐engineered construct promotes the osteogenic potential of mesenchymal stem cells

Tissue‐engineered constructs (TECs) seeded with mesenchymal stem cells (MSCs) represent a therapy for large bone defects. However, massive cell death in TECs in the early postimplantation period prompted us to investigate the osteoinductive mechanism of TECs. Previous studies demonstrated that stem cell extracts retained equivalent levels of bioactive proteins and exhibited an osteoinductive nature similar to that of intact cells. These data led us to hypothesize that despite the massive cell death in TECs, devitalized MSC‐derived proteins remain on the scaffolds and are released to improve cell function. Here, TECs were prepared using demineralized bone matrix seeded with human umbilical cord Wharton's jelly‐derived MSCs (hWJMSCs), and the cells seeded in TECs were devitalized by lyophilizing the TECs. Scanning electron microscopy, BCA protein assays, quantitative cytokine array analysis and immunofluorescent staining indicated that approximately 3 mg/cm³ of total protein and 49 types of cytokines derived from hWJMSCs were preserved in the lyophilized TECs (LTECs). The sustainable release of total protein and cytokines from LTECs lasted for more than 2 weeks. The released protein improved the osteogenic behavior of and gene expression in MSCs. Furthermore, the lyophilized hWJMSC‐derived proteins had immunoregulatory properties similar to those of live MSCs in mixed lymphocyte reactions. Collectively, we present a novel perspective on the osteoinductive mechanism of TECs and introduce LTECs as new systems for delivering multiple cytokines to enhance MSC behavior. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:386–394, 2016.     

Development of novel bioadhesive granisetron hydrochloride spanlastic gel and insert for brain targeting and study their effects on rats

The aim of this study was to formulate granisetron hydrochloride (GH) spanlastic in mucoadhesive gels and lyophilized inserts for intranasal administration to improve GH bioavailability and brain targeting. Carpapol 934 and HPMC were incorporated in GH spanlastic in nasal gels (GHSpNGs). Gelatin and HPMC as matrix former, glycine as a collapse protecting and mannitol as an insert filler and sweeting agent were used to prepare GH spanlastic loaded in lyophilized inserts (GHSpNIs). The prepared GHSpNGs were characterized for pH measurement, drug content, rheology, and in vitro drug release. The prepared GHSpNIs were characterized for drug content, surface pH, GH release, and mucoadhesion. Biological investigations including pharmacokinetics studies and brain drug targeting efficiency dimensions were performed on rats (LC–MS/MS). The results showed thixotropic pseudoplastic gels and white insert with pH values in a physiological range, drug content (89.9–98.6%), (82.4–98.38%) for gel and insert, respectively and rapid release rate of GH. Biological studies showed that C_max and AUC_0–6?h in brain and plasma after intranasal administration of gel and insert were higher compared to IV administration of GH solution. A high brain targeting efficiency (199.3%, 230%) for gel and insert, respectively and a direct nose to brain transport (49.8%, 56.95%) for gel and insert, respectively confirmed that there is a direct nose to brain transport of GH following nasal administration of GH spanlastic loaded in nasal gel and insert. GHSpNIs can be considered as potential novel drug delivery system intended for brain targeting via the nasal rout of administration than GHSpNGs.     

Improving the topical ocular pharmacokinetics of lyophilized cyclosporine A-loaded micelles: formulation,in vitro and in vivo studies

Dry eye syndrome (DES) is one of the most common disorders of the eye for which combined treatment includes modification of the ocular environment and pathogenic therapies. Cyclosporine A (CsA), a immunosuppressive agent, has been demonstrated to be effective for the treatment of DES but is limited clinically by its low ocular bioavailability due to poor water solubility. In this paper, methoxy poly (ethylene glycol)-poly (lactide) polymer (mPEG-PLA) micelles were investigated as alternative vehicles for the solubilization and delivery of CsA to the eye. The in vitro stability indicated that CsA-loaded micellar lyophilized powder was stable for at least 3?months and the release profile showed a sustained release manner of CsA from micelles physically. In vivo ocular distribution studies demonstrated that the micellar formulations exhibited a 4.5-fold increase in retention effect at eyes compared with 0.05% CsA emulsion. In addition, the in vivo pharmacokinetics profile showed that the CsA-loaded micelles could enhance the retention time, achieving longer effect toward the DES. These studies proposed an effective micelle formulation as a novel ocular drug delivery system to improve solubility and bioavailability of ophthalmic CsA-controlled delivery.     

蛹虫草液体发酵产物冻干粉的抗氧化活性及保肝作用

目的 研究蛹虫草液体发酵产物冻干粉的抗氧化活性及保肝作用。方法 采用真空冷冻干燥的方法将蛹虫草液体发酵产物制成干粉并对冻干粉主要成分进行分析测定,通过DPPH自由基清除实验和衰老小鼠模型验证蛹虫草发酵产物冻干粉的抗氧化活性,通过小鼠急性肝损伤实验研究冻干粉的保肝作用,采用急性毒性试验验证冻干粉的安全性。结果 蛹虫草发酵产物冻干粉含丰富的粗纤维、粗多糖、粗三萜及黄酮类物质。蛹虫草发酵产物冻干粉对DPPH自由基的半数有效浓度（EC₅₀）为8.12 mg/mL,在16 mg/mL时对DPPH自由基的清除率为61.56%,与0.02 mg/mL的维生素C（Vc）清除能力相当;蛹虫草发酵产物能显著提高衰老模型小鼠血清和肝脏中的总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-px）活力水平,降低丙二醛（MDA）含量,高剂量组的抗衰老效果与阳性药Vc组的抗氧化能力相当。小鼠急性肝损伤试验结果显示,冻干粉能有效降低模型组小鼠血液中门冬氨酸氨基转移酶（AST）与丙氨酸氨基转移酶（ALT）水平（P＜0.05）。冻干粉的半数致死量（LD₅₀）远大于国家无毒标准15 g/kg,属于无毒级别。结论 蛹虫草发酵产物具有良好的抗氧化活性和保肝作用,且安全无毒。     

新活素对急性心肌梗死患者心率变异性的影响

目的 观察急性心肌梗死(AMI)患者心率变异性(HRV)的改变及新活素——冻干重组人脑利钠肽(Lrh-BNP)对AMI患者HRV的影响.方法 选择AMI患者(AMI组,n=56)及同期健康体检者(正常对照组,n=30),将AMI组患者分为新活素治疗组(n=35,在基础AMI的治疗上静脉注射Lrh-BNP 24 h)和对照组(n=21,进行基础AMI的治疗).所有入选者均进行24h Hoher检查,对新活素治疗组、对照组和正常对照组进行心率、功率谱时域和频域的分析.结果 AMI组HRV各项时域指标较正常对照组显著降低(P＜ 0.01);Lrh-BNP治疗后,患者HRV值较对照组显著改善,时域指标(SDNN、SDANN、RMSSD、PNN50、心率)均显著改善(P＜0.01),频域指标(LF和HF)亦明显改善(P＜0.05);Lrh-BNP治疗1年后,患者心功能不全的发生率及病死率较对照组明显降低(P＜ 0.05);Lrh-BNP对不同部位的AMI患者HRV各项时域指标的影响无显著差异(P＞0.05).结论 应用Lrh-BNP可显著改善AMI患者降低的HRV,减少心功能不全的发生率,降低病死率.     

不同中药佐治小儿支气管肺炎的疗效观察

目的探讨痰热清注射液、注射用双黄连冻干粉及炎琥宁注射液佐治小儿支气管肺炎的疗效。方法 120例支气管肺炎患儿随机分为A、B、C三组,在常规抗感染、退热基础上A组佐用痰热清注射液,B组佐用注射用双黄连冻干粉,C组佐用炎琥宁注射液,每组40例。观察三组患儿临床疗效、病情恢复时间及不良反应发生情况。结果 A、B、C三组的治疗显效率分别为95.00%、92.50%和85.00%,A、B两组显著高于C组（P〈0.05）,A、B组间无统计学差异,（P〉0.05）。A、B两组患儿退热时间、咳嗽、肺部啰音消失时间及平均住院天数均明显短于C组（P〈0.05）。三组在治疗过程中未发现不良反应。结论痰热清注射液、注射用双黄连冻干粉佐治小儿支气管肺炎疗效优于炎琥宁注射液。     

注射用地龙冻干粉针对大鼠颈动脉血栓形成的影响

目的：评价注射用地龙冻干粉针的抗血栓作用。方法：SD雄性大鼠40只，利用血栓生成仪复制颈动脉血栓模型，模型成功后72h，将动物随机分成空白组、地龙冻干粉针1．8，0．9mg·kg^-1剂量组及阳性药疏血通注射液对照组，每组lO只，各组大鼠分别尾静脉注射（iv）生理盐水、地龙冻干粉针1．8，0．9mg·kg^-1剂量溶液和疏血通注射液（O．1mL·kg^-1）12d后，剥离颈动脉血栓，测定血栓重量，并对各组大鼠腹主动脉取血，利用ELISA法测定血浆组织型纤溶酶原激活剂（tissue type plasminogen activator，t-PA）含量，采用观察法测定各组大鼠的凝血时间（clotting time，CT）、血浆复钙时间（recalcification time，RT），利用血小板聚集仪测定血小板最大聚集率（maximal platelet aggregation rate，PAgR-max）。结果：与空白组相比较，地龙冻干粉针1．8mg·kg^-1剂量组大鼠血栓重量显著减少（P〈0．05）；阳性药疏血通组与地龙冻干粉针0．9mg·kg^-1剂量组大鼠血浆t-PA含量显著升高（P〈0．05）；阳性药疏血通组、地龙冻干粉针1．8mg·kg^-1剂量组大鼠CT、RT显著延长（P〈0．05），0．9mg·kg^-1剂量组RT显著延长（P〈0．05），且地龙冻干粉针对RT延长呈剂量依赖性。结论：注射用地龙冻干粉针具有显著的溶解血栓的作用，并能够抑制内源性凝血途径，溶血栓作用可能与提高血浆t-PA含量有关。     

常规加用血栓通(冻干粉)治疗不稳定性心绞痛临床观察

目的:观察血栓通(冻干粉)治疗不稳定型心绞痛的临床疗效。方法:选择104例不稳定型心绞痛患者,随机分为研究组(52例)和对照组(52例)。对照组给予抗血小板、抗凝、调脂、抗心肌缺血等常规治疗。研究组在对照组常规治疗基础上加用血栓通(冻干粉),治疗14 d后比较两组心绞痛症状及心电图变化情况。结果:研究组心绞痛临床症状及心电图改善的总有效率为90.38%和84.62%,均高于对照组71.15%和65.38%,差异有统计学意义(P<0.05)。结论:在常规药物基础上加用血栓通(冻干粉)治疗不稳定性心绞痛,可进一步改善心绞痛症状及心电图缺血情况。     

近红外漫反射光谱法快速判别注射用炎琥宁的冻干骨架结构

目的:建立快速判别注射用炎琥宁冻干骨架结构的对向传播人工神经网络(counter-propagation artificial neural net-work,CP-ANN)近红外漫反射光谱法。方法:采用配有积分球附件的Antaris II傅里叶变换近红外光谱仪测定注射用炎琥宁的近红外漫反射光谱;用TQ Analyst 8.0进行光谱处理及数据预处理;用注射用炎琥宁校正样品的近红外漫反射光谱数据、以Matlab 6.5建立未损与已损冻干骨架结构的CP-ANN判别模型,并对模型进行交叉验证;用所建CP-ANN模型预测注射用炎琥宁验证样品冻干骨架结构的完整性。结果:所建近红外漫反射光谱CP-ANN判别模型预测注射用炎琥宁冻干骨架结构的准确率为100.0%,且具有良好的可视化功能。结论:所建方法判断客观,无损、无污染,简便快速,可望用于冻干注射用制剂的生产过程质量控制或临床使用质量控制。     

四逆散冻干粉化学成分变化的研究

目的 采用RP-HPLC法分析四逆散冻干粉的化学成分变化,为其工艺控制和配伍规律提供可靠依据。方法 采用高效液相色谱法,色谱条件：Kromasil C18柱(250 mm×4.6 mm,5 μm),体积流量1.0 mL/min,柱温28 ℃,流动相A为乙腈;B为磷酸水(pH 3),在检测波长为210 nm和240 nm条件下,构建四逆散冻干粉、缺味四逆散及单味药冻干粉的HPLC色谱图,分析其化学成分的变化。结果 四逆散冻干粉240 nm波长下高效液相色谱图21个成分峰中,确认了有2个组分来自柴胡,有3个组分来自白芍,有10个组分来自枳实,有5个组分来自甘草,1个组分为新物质。210 nm波长下高效液相色谱图24个成分峰中,确认了有1个组分来自柴胡,有4个组分来自白芍,有14个组分来自枳实,有5个组分来自甘草。结论 本方法稳定可控,可用于四逆散冻干粉指纹图谱测定,可全面地反映其化学成分变化。