Preparation of fiber-microsphere scaffolds for loading bioactive substances in gradient amounts

Gradient scaffolds are needed for interface tissue regeneration. In this study, a technique combining electrospinning and electrospraying was developed for preparing poly(L-lactide-co-glycolide) (PLGA) fiber-microsphere scaffolds for loading bioactive substances in gradient amounts. The gradient fiber-microsphere scaffolds contain two sheets of electrospun membranes and a sheet of microspheres loaded with bioactive substances in gradient amounts between the electrospun membranes. The morphologies of the gradient scaffolds were characterized and bovine serum albumin (BSA) was loaded as a model bioactive substance. The amount of BSA-loaded microspheres decreased gradually along the length of the gradient scaffold. The addition of poly (ethylene glycol) significantly improved the hydrophilicity of the gradient scaffold and the release behavior of BSA with respect to the gradient became apparent, with differences in the release amounts along the length of the gradient scaffold being observed. The biocompatibility of the gradient scaffold was verified using MC3T3-E1 pre-osteoblastic cells. The study demonstrated that the combination of electrospinning and electrospraying was a feasible method for the preparation of gradient scaffolds for potential applications in interface tissue engineering.     

Immobilization of biomacromolecules on poly-L-lactide surface via a layer-by-layer method for the improving of its cytocompatibility to bone marrow stromal cells

Hyaluronic acid （HA） and chitosan （CS） were immobilized on the surface of poly-L-lactide （PLLA） by the following procedure： Firstly, PLLA was aminolyzed with 1, 6-hexanediamine, and part of the PLLA surface ester groups were converted to free amino groups. Then negatively charged hyaluronic acid and positively charged chitosan were deposited onto the surface of aminolyzed PLLA film in a layer-by-layer assembly manner. The effect of the layer-by- layer deposition was evaluated by ATRoFTIR spectroscopy, Raman spectroscopy and static contact angle measurements. The cytocompatibility of PLLA sample to bone marrow stromal cells （BMSCs） was improved after modification with chitosan and HA. The cell attachment, activity, and proliferation on CS/HA modified PLLA films were enhanced comparing with the control. The cells cultured on the modified PLLA samples excreted abundant cytoplasm and can differentiate to vascular smooth muscle （SM）-like （SM-like） cells. A macroporous three-dimensional PLLA scaffold was prepared by integrating both the technique of freeze-drying and particle leaching. Layer-by-layer modification by HA/CS and cell culture was also applied on this scaffold. The scaffold cultured with BMSCs for 2 weeks has been tested successfully in vivo as a patch for repairing the artificial incision on canine pulmonary artery.     

Advances in tissue engineering

Tissue engineering is a newly developed specialty involved in the construction of tissues and organs either in vitro or in vivo. Tremendous progress has been achieved over the past decade in tisse construction as well as in other related areas, such as bone marrow stromal cells, embryonic stem cells and tissue progenitor cells. In our laboratory, tissues of full-thickness skin, bone, cartilage and tendon have been successfully engineered, and the engineered tissues have repaired full-thickness skin wound, cranial bone defects, articular cartilage defects and tendon defects in animals. In basic research areas, bone marrow stromal cells have been induced and transformed into osteoblasts and chondrocytes in vitro. Mouse embryo stem cell lines we established have differentiated into neuron precursor, cardiac muscle cells and epithelial cells. Genetic modifications of seed cells for promoting cell proliferation, delaying cell aging and inducing immune tolerance have also been investigated.     

Distribution of bone marrow stem cells in large porous polyester scaffolds

This work examined the optimal syringing depth during in vitro cell loading in order to even cell distribution after syringing a drop of cell suspension in cylinder poly（lactide-co-glycolide） （PLGA） porous scaffolds. The scaffolds of 10 mm height and 10 mm diameter were fabricated via room-temperature compression molding ＆ particulate leaching technique based on spherical porogens. In vitro tests were employed for such examinations： a global observation of a cell-loaded scaffold stained by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） technique and a quantitative measurement of spatial distribution of cells after slicing the cell-loaded scaffolds into layers. It was found that an even distribution of cells was soon achieved only if the initial cell suspension was seeded on the layer that was below the top surface but above the middle of scaffolds. The availability of in vitro osteoblastic differentiation of rat bone marrow stem cells in such a kind of spherical-pore PLGA scaffolds was meanwhile confirmed.     

Preparation and bioactivity of sol-gel macroporous bioactive glass

Bioactive glass is well known for its ability of bone regeneration, and sol-gel bioactive glass has many advantages compared with melt-derived bioactive glass. 3-D scaffold prepared by the sol-gel method is a promising substrate material for bone tissue engineering and large-scale bone repair. Porous sol-gel glass in the CaO-SiO₂-P₂0₅ system with macropores larger than 100 μm was prepared by the addition of stearic acid as a pore former. The diameter of the pore created by the pore former varied from 100 to 300 μm. The formation of a hydroxyapatite layer on the glass was analyzed by studying the surface of the porous glass by scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and Raman spectra after they had been immersed in simulated body fluid (SBF) for some time, and the porous glass shows good bioactivity.     

Engineered polycaprolactone – magnesium hybrid biodegradable porous scaffold for bone tissue engineering

In this paper, we describe the fabrication of a new biodegradable porous scaffold composed of polycaprolactone(PCL) and magnesium(Mg)micro-particles. The compressive modulus of PCL porous scaffold was increased to at least 150% by incorporating 29% Mg particles with the porosity of 74% using Micro-CT analysis. Surprisingly, the compressive modulus of this scaffold was further increased to at least 236% when the silane-coupled Mg particles were added. In terms of cell viability, the scaffold modified with Mg particles significantly convinced the attachment and growth of osteoblasts as compared with the pure PCL scaffold. In addition, the hybrid scaffold was able to attract the formation of apatite layer over its surface after 7 days of immersion in normal culture medium, whereas it was not observed on the pure PCL scaffold. This in vitro result indicated the enhanced bioactivity of the modified scaffold. Moreover, enhanced bone forming ability was also observed in the rat model after 3 months of implantation. Though bony in-growth was found in all the implanted scaffolds. High volume of new bone formation could be found in the Mg/PCL hybrid scaffolds when compared to the pure PCL scaffold. Both pure PCL and Mg/PCL hybrid scaffolds were degraded after 3 months. However, no tissue inflammation was observed. In conclusion, these promising results suggested that the incorporation of Mg micro-particles into PCL porous scaffold could significantly enhance its mechanical and biological properties. This modified porous bio-scaffold may potentially apply in the surgical management of large bone defect fixation.     

A new bone repair scaffold combined with chitosan/ hydroxyapatite and sustained releasing icariin

Icariin, a plant-derived flavonol glycoside, has been proved as an osteoinductive agent for bone tissue engineering. A new bone repair scaffold was generated by thorough mixing of icariin and chitosan/ hydroxyapatite （icariin-CS/HA） using freeze-drying technigue. Characteristics of morphology, mechanical properties, biocompatibility, drug release behavior and bone repair abilities in vivo were evaluated. The results show that drug loading process of icariin did not affect physical structure of CS/HA composite significantly but decreased mechanical properies of CS/HA composite, which happened with a high dosage; icariin-CS/HA had favorable cell compatibility and promoted osteogenic differentiation of hBMSCs; the controlled release of icariin was satisfactory and the release retained after 90 d in vitro. In addition, icariin-CS/HA scaffolds had favorable osteoconduction and osteoinduction in vivo, and could fill bone defect sites and stimulate newborn bone tissues formation at early stage. On the basis of these data, icariin-CS/HA is believed to be an optical bone repair scaffold for tissue engineering.     

Processing, microstructure and mechanical properties of biomedical magnesium with a specific two-layer structure

A novel magnesium based scaffold with a two-layer structure was synthesized by powder metallurgical process using salt particles as space holder. The outer layer of the scaffold shows an interconnected porous structure and the inner layer presents a compact structure reinforced by the salt particles. Such a specific structure is introduced primarily for the purpose of a better combination of biocompatibility and mechanical compatibility. Experimental results demonstrate that the structural features and mechanical properties of the magnesium based scaffold with a salt content of 30 wt% prepared by the current method are quite compatible with the cancellous bone. Such a novel Mg-based scaffold has the potential to act as degradable implants for bone substitute application.     

Comparative study of microstructural remodification to porous β-TCP and HA in rabbits

To assess the remolding ability of repaired bone in hydroxyapatite （HA） and β-calcium phosphate （β-TCP） scaffold, two 75% porosity bioceramics with the same three-dimensional geometry were implanted into femoral condyles of rabbits. Histological and micro-computed tomography （micro-CT） results demonstrated abundant new bone formation in the porous HA scaffold along with indistinctive scaffold degradation. Results also indicated that scaffold resorption in the β-TCP group, which was followed by a replacement with newly formed bone, was significantly higher than that in the HA group. The crosslinking trabeculae remodeled from the mixtures of the newly formed bone and β-TCP scaffold remnants might be helpful to promoting even loading and reducing stress. The bone remodeling pattern resulted from bone formation and scaffold resorption was significantly different for the two bioceramics. The results demonstrated that the 75% porous β-TCP was more suitable for new bone remodification than HA scaffold.     

Fiber-Based Chitosan Tubular Scaffolds for Soft Tissue Engineering： Fabrication and in Vitro Evaluation

Porous, two-ply tubular chitosan conduits for guided tissue regeneration were fabricated by combining the textile technique （inner layer） with the thermally induced phase separation process （outer layer）. A hollow chitosan tube was prepared using an industrial warp knitting process with chitosan yarns. Then, an appropriate diameter mandrel was inserted into the pre-fabricated tube. The tube and the mandrel were dipped into the chitosan solution together, taken out, and freeze-dried. After being neutralized in alkaline solution and dried at room temperature, the mandrel was removed to create the chitosan tubular scaffold. Scanning electron micrographs show that the resulting tubes have a biphasic wall structure, with a fibrous inner layer and a semipermeable outer layer. The swelling properties and the mechanical strength before and after in vitro degradation were investigated. The biocompatibility of the scaffolds was also investigated by co-culturing neuroblastoma cells （N2A, mouse） with the scaffolds. The results suggest that these chitosan tubular scaffolds are useful for the regeneration of tissues requiring a tubular scaffold.     

Dexamethasone-Loaded PLGA Microspheres Incorporated PLLA/PLGA/PCL Composite Scaffold for Bone Tissue Engineering

The combination of micro-carriers and polymer scaffolds as promising bone grafts have attracted considerable interest in recent decades.The poly(L-lactic acid)/poly(lactic-co-glycolic acid)/polycaprolactone(PLLA/PLGA/PCL)composite scaffold with porous structure was fabricated by thermally induced phase separation(TIPS).Dexamethasone(DEX)was incorporated into PLGA microspheres and then loaded on the PLLA/PLGA/PCL scaffoldtopreparethedesiredcompositescaffold.The physicochemical properties of the prepared composite scaffold were characterized.The morphology of rat bone marrow mesenchymal stem cells(BMSCs)grown on scaffolds was observed using scanning electron microscope(SEM)and fluorescence microscope.The resultsshowedthatthePLLA/PLGA/PCLscaffoldhad interconnected macropores and biomimetic nanofibrous structure.In addition,DEX can be released from scaffold in a sustained manner.More importantly,DEX loaded composite scaffold can effectively support the proliferation of BMSCs as indicated by fluorescence observation and cell proliferation assay.The results suggested that the prepared PLLA/PLGA/PCL composite scaffold incorporating drug-loaded PLGA microspheres could hold great potential for bone tissue engineering applications.     

珊瑚／胶原／重组人骨形成蛋白-2复合人工骨修复兔颅骨缺损

为评价珊瑚／ 胶原／ 重组人骨形成蛋白２ 复合人工骨（ 简称复合骨） 的骨修复能力,将复合骨植入兔颅骨直径１５ｍ ｍ 的圆形缺损,以单纯珊瑚植入作对照;术后６ 周、１２ 周取材,通过组织学观察和计算机图像分析,评价其骨修复能力．发现复合骨植入区形成的骨组织量明显多于同期的珊瑚植入区（ Ｐ＜ ０ ．０１） ;术后１２ 周,复合骨完全被成熟的骨组织取代,其骨修复效果明显优于单纯的珊瑚．说明复合骨具有骨传导和骨诱导活性,骨修复能力较强,是一种较为理想的新型生物性植骨材料     

磷酸化壳聚糖-季铵化壳聚糖载体提高体内外基因转染功效

本研究通过磷酸化壳聚糖(PC)包被季铵化壳聚糖(TMC)/质粒(pDNA)纳米复合物,制备PC/TMC/pDNA三元复合物(PTC),用于提高体内外基因转染功效.PTC粒径为100~200nm,Zeta电势为-16~-34mV,可有效缩合pDNA,保护pDNA免遭核酶降解.PC降低PTC中pDNA结合力,增加体外释放量.表面荷负电的PTC抗非特异性蛋白吸附能力强,经小窝蛋白介导的细胞内吞途径入胞后逃避溶酶体降解,细胞核内分布比例高.HEK293细胞体外转染试验结果显示,PTC体外转染效率是TMC/pDNA纳米复合物(TC)的1.5~3.1倍;小鼠胫前肌注射给药试验结果表明,PTC可显著提高基因体内转染效率.因此,合适质量比的PTC有望作为功能性基因药物的递送载体,用于基因治疗.     

多孔CPC材料复合转染VEGF的骨髓间充质干细胞修复大鼠颅骨缺损的实验研究

为研究转染血管内皮生长因子(VEGF)基因的骨髓间充质干细胞(BMSCs)复合多孔CPC构建的组织工程化骨对骨缺损的修复作用,用脂质体介导质粒pIRES2-EGFP/VEGF165转染大鼠BMSCs,与多孔CPC复合体外构建组织工程化骨,植入大鼠颅骨缺损模型,未转染VEGF165的组织工程化骨作对照。分别在4周、12周取材,以组织学组织形态学分析骨缺损修复情况.比较两组的骨形成差异。结果转染VEGF组骨缺损的修复效果明显好于未转染组。组织形态计量学分析显示,4周时,转染组材料内新生骨面积为31.9%,未转染组为19.7%。12周时,转染组材料内新生骨面积为75.9%,未转染组仅为49.7%。差异具有统计学意义(P<0.000 1)。转染组的骨形成速率明显快于未转染组,两者分别为分别为6.18±1.62μm/d和3.56±0.93μm/d。说明VEGF转染细胞组较对照组血供加强,骨缺损修复加速。     

不同浓度掺镁透钙磷石骨水泥修复骨质疏松家兔骨缺损及BMP-2表达的研究

健康家兔36只,进行骨质疏松造模后随机分为空白对照组、透钙磷石组(DCPD组)、掺镁6.67%组、掺镁26.67%透钙磷石组,在家兔双侧前腿桡骨中段制作骨缺损,术后每组动物分别于4、8、12周各处死3只,观察组织切片中BMP-2表达情况与X线片显示骨缺损修复状况。免疫组化结果显示同组不同时期比较,4周时BMP-2表达最高,8周时较4周时减弱,12周时阳性表达最弱。同一时期组间比较掺镁6.67%组阳性表达最强。在骨质疏松家兔桡骨骨缺损修复中,掺镁6.67%透钙磷石组成骨效果较其他组显著,同时期组间比较BMP-2阳性表达最强。     

可降解Hr-BMP复合胶原膜修复腭部骨缺损的实验研究

目的：观察腭部扁平骨引导性骨再生现象（GBR），探讨利用GBR修复腭裂骨缺损的可能性。方法：建立幼犬腭部骨缺损实验动物模型，应用常规组织学检查术、免疫荧光显微法、X线及扫描电子显微术在实验过程的不同阶段进行观察研究。结果：1）借助复合人重组骨形成蛋白（Hr-BMP）胶原膜GBR，腭部骨缺损可以完全修复；2）胶原膜可提供成骨所需的密闭空间；3）复合Hr-BMP胶原膜具有良好的组织相容性和安全性；4）复合Hr-BMP胶原膜GBR所致骨形成的量及速度均高于其它对照。结论：1）复合Hr-BMP胶原GBR具有确实有效的骨引导和骨诱导性，在骨缺损修复的早期阶段即可产生大量的新骨；2）复合Hr-BMP胶原膜植入后4周内的成骨活动最活跃，胶原膜和复合Hr-BMP胶原膜在其降解吸收过程中，不干扰后续的成骨活动；3）仍有必要构建具有适合的吸收降 解性和一定力学强度的隔膜材料。     

长期递增负荷运动中雌性大鼠骨超微结构的变化

目的:长期递增负荷运动中雌性大鼠骨超微结构的变化的影响。方法将200只3月龄经过17周递增负荷训练后,利用透射电镜(TEM)和酶联免疫吸附法检测和观察长期递增负荷跑台运动对雌性大鼠股骨的超微结构的影响、测试分析骨形成和骨吸收生化标志物的变化。结果:(1)根据大鼠动情周期抑制率BUN、体重、BMD等指标的检测结果可判断出本实验动物模型建造成功。(2)透射电镜(TEM)实验结果表明,4、9周时的运动训练对大鼠骨超微结构无明显影响,13、15、17周大强度递增负荷运动对大鼠骨超微结构影响显著,定性比较,超微结构损伤较同期对照组差异显著;(3)生化指标变化趋势表明随着运动强度的加大骨处于高转换状态,结合BMD结果判断骨吸收大于骨形成。结论:实验中运动性动情周期抑制检查BUN、BMD变化结果证实,随着运动强度的加大,大鼠动情周期显著紊乱,明显抑制了下丘脑垂体性腺轴的正常生理功能。9周前的运动组大鼠骨超微结构较同期对照组无显著差异,证实了适宜的运动强度对大鼠骨组织有较好的促进作用,9周后大强度递增负荷的运动对大鼠股骨超微结构的负面作用显著;大鼠骨超微结构的异常具有位点特异性。17周大强度递增负荷的运动导致大鼠骨处于高转换状态,骨吸收占据优势。