大鼠皮质骨上机械性显微损伤的修复机制研究

目的研究大鼠皮质骨上植入物造成的机械性显微损伤的修复机制。方法 30只SD大鼠随机分为两组,一组行卵巢切除术,另一组行假手术。3个月后在右侧胫骨行钻孔术,分别于钻孔后1、2、4周后处死,处死前注射四环素和钙黄绿素进行标记。将含有钻孔的骨段(1 cm)应用大块碱性品红染色,甲基丙烯酸甲酯包埋后切成约50μm厚的切片。应用Bioquant图象分析系统对切片进行骨形态计量学分析。结果去卵巢大鼠和假手术大鼠都出现了与显微损伤有关的骨吸收腔,其中去卵巢组的孔隙率和骨吸收腔明显高于假手术组(P<0.05);随着术后时间的延长,吸收腔的数目逐渐增加,各时间点之间的差异有明显的统计学意义(P<0.05)。结论去卵巢大鼠的孔隙率和骨吸收腔数明显高于假手术大鼠,反映了在雌激素缺乏和较多裂纹形成两种因素刺激下,去卵巢大鼠皮质骨内的重建更为活跃,这会降低骨的强度从而增加骨折的危险性。     

Highly Permeable Genipin‐Cross‐linked Gelatin Conduits Enhance Peripheral Nerve Regeneration

Here we have evaluated peripheral nerve regeneration with a porous biodegradable nerve conduit (PGGC), which was made from genipin‐cross‐linked gelatin. To examine the effect of pores, nonporous genipin‐cross‐linked gelatin conduit (GGC) was considered as the control. Both the PGGC and the GGC were dark blue in appearance with a concentric and round lumina. The PGGC featured an outer surface with pores of variable size homogeneously traversing, and a partially fenestrated inner surface connected by an open trabecular meshwork. The GGC had a rough outer surface whereas its inner lumen was smooth. Both PGGCs and GGCs had similar hydrophilicity on condition of the same material and cross‐linking degree. The porosity of PGGCs and GGCs was 90.8 ± 0.9% and 24.3 ± 2.9%, respectively. The maximum tensile force of the GGCs (0.12 ± 0.06 kN) exceeded that of the PGGCs (0.03 ± 0.01 kN), but the PGGCs had a higher swelling ratio than GGCs at 0.5, 1, 3, 6, 12, 24, 48, 60, 72, and 84 h after soaking in deionized water. Cytotoxic testing revealed the soaking solutions of both of the tube composites would not produce cytotoxicity to cocultured Schwann cells. After subcutaneous implantation on the dorsal side of the rat, the PGGC was degraded completely after 12 weeks of implantation whereas a thin tissue capsule was formed encapsulating the partially degraded GGC. Biodegradability of both of the tube groups and their effectiveness as a guidance channel were examined as they were used to repair a 10 mm gap in the rat sciatic nerve. As a result, fragmentation of the GGC was still seen after 12 weeks of implantation, yet the PGGC had been completely degraded. Histological observation showed that numerous myelinated axons had crossed over the gap region in the PGGCs after 8 weeks of implantation despite only few myelinated axons and unmyelinated axons mostly surrounded by Schwann cells seen in the GGCs. In addition, the regenerated nerves in the PGGCs presented a significantly higher nerve conductive velocity than those in the GGCs (P < 0.05). Thus, the PGGCs can not only offer effective aids for regenerating nerves but also accelerate favorable nerve functional recovery compared with the GGCs.     

Long-term culture of HL-1 cardiomyocytes in modular poly(ethylene glycol) microsphere-based scaffolds crosslinked in the phase-separated state

Poly(ethylene glycol) (PEG) microspheres were assembled around HL-1 cardiomyocytes to produce highly porous modular scaffolds. In this study we took advantage of the immiscibility of PEG and dextran to improve upon our previously described modular scaffold fabrication methods. Phase separating the PEG microspheres in dextran solutions caused them to rapidly deswell and crosslink together, eliminating the need for serum protein-based crosslinking. This also led to a dramatic increase in the stiffness of the scaffolds and greatly improved the handling characteristics. HL-1 cardiomyocytes were present during microsphere crosslinking in the cytocompatible dextran solution, exhibiting high cell viability following scaffold formation. Over the course of 2 weeks a 9-fold expansion in cell number was observed. The cardiac functional markers sarcomeric α-actinin and connexin 43 were expressed at 13 and 24 days after scaffold formation. HL-1 cells were spontaneously depolarizing 38 days after scaffold formation, which was visualized by confocal microscopy using a calcium-sensitive dye. Electrical stimulation resulted in synchronization of activation peaks throughout the scaffolds. These findings demonstrate that PEG microsphere scaffolds fabricated in the presence of dextran can support the long-term three-dimensional culture of cells, suggesting applications in cardiovascular tissue engineering.     

Prediction of Tablet Hardness and Porosity Using Near‐Infrared Diffuse Reflectance Spectroscopy as a Nondestructive Method

The main objective of this research is to use the near‐infrared diffuse reflectance method to evaluate and quantify the effects of hardness and porosity on the near‐infrared spectras of tablets. To develop a model equation, validate the model and test the model predictive ability. Seven theophylline tablet formulations of the same composition but with seven different hardness values (3 kp, 6, 8, 10, 12, 15, and 17 kp) were prepared. Another seven theophylline tablets formulation with seven different porosity values (57.4, 50.3, 41.9, 40.3, 39.9, 37.3, and 35.1%) were prepared. Five placebo tablets formulation with different hardness and porosity values were also prepared. Laboratory hardness and porosity values were compared to near‐infrared diffuse reflectance data. Linear regression, quadratic, cubic and partial least square techniques were used to determine the relationship between hardness, porosity and the near‐infrared spectras. The results demonstrated that an increase in tablet hardness and a decrease in tablets porosity produced an increase in near‐infrared absorbance. Series of model equations depending on the mathematical technique used for regression were developed from the calibration of hardness and porosity data using laboratory equipment vs. the near‐infrared diffuse reflectance for each formulations. The results of near‐infrared hardness and porosity predictions were very similar to laboratory hardness and porosity tests. The near‐infrared diffuse reflectance spectroscopy method is an alternative nondestructive method for measurement of hardness and porosity of tablets.     

Insights into the roles of carrier microstructure in adhesive/carrier-based dry powder inhalation mixtures: Carrier porosity and fine particle content

To gain insights into complex interactions in carrier-based dry powder inhalation mixtures, we studied the relationships between the carrier microstructural characteristics and performance. We used mercury intrusion porosimetry to measure the microstructural characteristics and to also derive the air permeability of eight carriers. We evaluated the performances of inhalation mixtures of each of these carriers and fluticasone propionate after aerosolization from an Aerolizer®. We did not observe a simple relationship between the carrier total porosity and the performance. Classification of the porosity according to pore size, however, provided interesting insights. The carrier nanoporosity, which refers to pores smaller than micronized drug particles, has a positive influence on the performance. Nanopores reduce the carrier effective contact area and the magnitude of interparticulate adhesion forces in inhalation mixtures. The carrier microporosity, which refers to pores similar in size to drug particles, also has a positive influence on the performance. During mixing, micropores increase the effectiveness of frictional and press-on forces, which are responsible for breaking up of cohesive drug agglomerates and for distribution of drug particles over the carrier surface. On the other hand, the carrier macroporosity, which refers to pores larger than drug particles, apparently has a negative influence on the performance. This influence is likely mediated via the effects of macropores on the powder bed tensile strength and fluidization behavior. The air permeability better represents these effects. The inhalation mixture performance improved as the carrier air permeability decreased. Interestingly, as the carrier fine particle content increased, the carrier microporosity increased and the carrier air permeability decreased. This proposes a new mechanism for the positive effect of fine excipient materials on the performance of carrier-based inhalation mixtures. Fine excipient materials apparently adhere to the surface of coarse carrier particles creating projections and micropores, which increase the effectiveness of mixing. The data also support the mechanism of powder fluidization enforcement by fine excipient materials. The current study clearly demonstrates that the microporosity and the air permeability are key dry powder inhalation carrier performance determinants. Mercury intrusion porosimetry is a useful tool in the dry powder inhalation field; it successfully allowed resolution of carrier pores which contribute differently to the performance.     

Enhanced angiogenesis of growth factor-free porous biodegradable adhesive made with hexanoyl group-modified gelatin

The bonding behavior of hexanoyl (Hx: C₆) group-modified alkaline-treated gelatin (HxAlGltn) porous films ((P)HxAlGltn) on the porcine intestine was evaluated. (P)HxAlGltns with various porosities were prepared by the salt-leaching method for various solid–liquid ratios. (P)HxAlGltns bonded more strongly to porcine intestine surfaces than did porous AlGltn films ((P)AlGltns). L929 cells cultured on (P)HxAlGltns showed adhesivity than cells cultured on (P)AlGltns. Faster tissue infiltration and a shorter degradation time of highly porous (P)HxAlGltns were observed after implantation in rat subcutaneous tissues. The angiogenic markers CD34 and α-SMA were highly expressed around (P)HxAlGltns that had high porosity. These results indicated that highly porous (P)HxAlGltns have advantages with respect to not only bonding strength on wet soft tissues, but also angiogenesis.     

Non-connected versus interconnected macroporosity in poly(2-hydroxyethyl methacrylate) polymers. An X-ray microtomographic and histomorphometric study

Poly(2-hydroxyethyl methacrylate) (pHEMA) has potentially broad biomedical applications: it is biocompatible and has a hardness comparable to bone when bulk polymerized. Porous biomaterials allow bone integration to be increased, especially when the pores are interconnected. In this study, three types of porogens (sugar fibers, sucrose crystals, and urea beads) have been used to prepare macroporous pHEMA. The pore volume and interconnectivity parameters of the porosity were measured by X-ray microtomography and image analysis. Sucrose crystals, having a high volumetric mass, gave large pores that were located on the block sides. Urea beads and sugar fibers provided pores with the same star volume (2.65 ± 0.46 mm³ and 2.48 ± 0.52 mm³, respectively) but which differed in interconnectivity index, fractal dimension, and Euler–Poincaré's number. Urea beads caused non-connected porosity, while sugar fibers created a dense labyrinth within the polymer. Interconnectivity was proved by carrying out surface treatment of the pHEMA (carboxymethylation in water), followed by von Kossà staining, which detected the carboxylic groups. Carboxymethylated surfaces were observed on the sides of the blocks and on the opened or interconnected pores. The disconnected pores were unstained. Macroporous polymers can be prepared with water-soluble porogens. X-ray microtomography appears a useful tool to measure porosity and interconnectedness.     

The Preparation of Rapidly Disintegrating Tablets in the Mouth

Elderly people, children and patients sometimes have difficulties swallowing tablets. To solve this problem, a novel method of preparing tablets that disintegrate rapidly in the mouth was developed. A tablet with high porosity is required for rapid disintegration, but such a tablet is generally fragile. To make a tablet having both high porosity and practical strength, amorphous sucrose, which has good compactability, was used. Mannitol powder with freeze-dried amorphous sucrose was tabletted at low compression and stored under certain conditions. The tablet disintegrated rapidly in the mouth, because of its high porosity. The tensile strength of the tablet increased remarkably during storage, while the porosity of the tablet seemed almost unchanged. The results of thermal analysis and powder x-ray diffraction measurement showed that the amorphous sucrose in tablet crystallized during storage. The increase in the tensile strength of the tablet was due to crystallization of the amorphous sucrose and formation of new internal contact points in the tablet. It was concluded that this crystalline transition method is a very useful method to prepare a rapidly disintegrating tablet.     

Modeling of Femoral Neck Cortical Bone for the Numerical Simulation of Ultrasound Propagation

Quantitative ultrasound assessment of the cortical compartment of the femur neck (FN) is investigated with the goal of achieving enhanced fracture risk prediction. Measurements at the FN are influenced by bone size, shape and material properties. The work described here was aimed at determining which FN material properties have a significant impact on ultrasound propagation around 0.5 MHz and assessing the relevancy of different models. A methodology for the modeling of ultrasound propagation in the FN, with a focus on the modeling of bone elastic properties based on scanning acoustic microscopy data, is introduced. It is found that the first-arriving ultrasound signal measured in through-transmission at the FN is not influenced by trabecular bone properties or by the heterogeneities of the cortical bone mineralized matrix. In contrast, the signal is sensitive to variations in cortical porosity, which can, to a certain extent, be accounted for by effective properties calculated with the Mori-Tanaka method.     

多孔纯钛孔隙率及孔隙尺寸对蛋白吸附及成骨细胞分化的影响

目的从分子生物学角度研究筛选利于蛋白质吸附、成骨细胞分化的合适孔隙结构,为多孔钛材料作为牙种植体提供研究基础及制备参数。 方法筛选不同粒径（Ⅰ组：0~200 μm、Ⅱ组：200~400 μm、Ⅲ组：400~600 μm）的NH₄HCO₃造孔剂颗粒,按照不同的质量分数（A组：20%、B组：30%）与钛粉均匀混合,粉末冶金法制备6组多孔纯钛试件,不添加造孔剂制备致密纯钛试件作为对照组;每组10个试件。蛋白定量试剂盒测定蛋白质吸附量以评价多孔纯钛试件的蛋白吸附能力,实时荧光定量聚合酶链反应（PCR）检测Runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）及骨钙素（OCN）基因表达量以评价成骨细胞分化水平。 结果多孔纯钛的蛋白质吸附量高于致密纯钛（P < 0.05）,其中小平均孔隙尺寸及高平均孔隙率组较其余多孔纯钛组吸附更多蛋白质（P < 0.05）。多孔纯钛的孔隙率和孔隙尺寸对成骨细胞分化的影响具有交互效应。成骨细胞培养第7天,仅AI组Runx2基因表达量高于对照组（P < 0.05）;培养第14天,多孔纯钛各组Runx2基因的表达量均低于对照组（P < 0.05）。成骨细胞培养7、14 d后,多孔纯钛各组ALP基因的表达量高于对照组（培养第7天,BⅢ组除外;培养第14天,AⅠ、AⅢ组除外）（P < 0.05）。成骨细胞培养第7天,AⅠ、BⅠ组OCN基因的表达量较对照组及其他平均孔隙尺寸组高（P < 0.05）;成骨培养第14天,仅BⅠ组OCN基因的表达量高于对照组（P < 0.05）。 结论本实验条件下,孔隙结构提高了纯钛试件的蛋白质吸附量,且平均孔隙率为53.3%、平均孔径为191.6 μm的多孔钛试件利于小鼠前成骨细胞MC3T3-E1的成骨向分化。