TCP复合BMP-2诱导hMSCs成骨分化的协调机制研究

目的探讨TCP复合BMP-2诱导h MSCs成骨分化的协调机制。方法分别制备TCP支架及TCP复合BMP-2支架材料,自3名志愿者身上分离h MSCs进行传代扩增,取第3代细胞接种备好的支架材料,在复合后的第1、3、7天测量ALP的活性并进行比较。结果在复合后第1天,两组间ALP活性值比较无明显差异(P0.05);在复合后第3、7天两组间ALP活性值比较差异显著(P0.05);TCP与细胞培养组1、3、7天的ALP活性值差异不显著,无统计学意义(P0.05);TCP复合BMP-2与细胞培养组的1、3、7天的ALP活性值随着培养时间的延长,逐渐升高。结论 BMP-2能协调TCP促进h MSCs的分化效应,基因治疗可能为骨骼疾病提供一种新的治疗途径。     

载药脂质体复合缺钙磷灰石骨水泥支架的研究

采用食盐颗粒浸出法制备了缺钙磷灰石水泥(CPC)多孔支架;用脂质体包裹盐酸万古霉素制备了载药脂质体。将它们两者结合,制备了脂质体载药复合缺钙磷灰石水泥(dl-CPC)支架。结果表明:缺钙CPC多孔支架能够将载药脂质体吸附在其大孔表面或微孔里;dl-CPC支架对MC3T3-E1细胞的生长没有负面影响,显示出良好的细胞相容性。此外,dl-CPC支架具有很好的抗菌性能,能够抑制大肠杆菌生长,抗菌率达99%(12 h)。dl-CPC支架浸泡在磷酸缓冲溶液中,释放药物的速度比较缓慢(前4周);而直接吸附药物的CPC支架,在1周内大部分药物释放出来,出现暴释现象。另结果表明:dl-CPC支架具有缓释药物和骨再生的双重功能,可用于骨缺损的修复及治疗慢性骨髓炎。     

去卵巢大鼠骨基质明胶修复骨缺损骨痂的矿物质元素分析

应用等离子体发射光谱仪测定了去卵巢水平下骨基质明胶修复大鼠颅骨骨缺损新生骨痂的矿物质元素含量。将28只雌性SD大鼠随机分为2组。去卵巢组切除双侧卵巢，伪手术对照组保留卵巢。术后4周在颅骨上制备骨缺损并同期植入骨基质明胶。8周后，测定新生骨痂的矿物质元素含量。结果显示，去卵巢大鼠新生骨痂的S、Ca、P、Zn、Fe和Cu含量较伪手术组显著降低。实验表明雌激素水平影响骨基质明胶修复骨缺损骨痂的矿物质元素含量。     

肝素化丝素支架作为骨形态发生蛋白-2缓释载体的研究

骨形态发生蛋白-2(BMP-2)的缓释载体一直是骨组织工程中的研究热点.本研究通过化学改性制备了两种肝素化丝素支架,并浸渍吸附BMP-2,研究了BMP-2在不同丝素支架样品上的吸附能力、体外释放性能及其对人骨肉瘤细胞MG-63碱性磷酸酶活性(ALP)的影响.结果表明,肝素化丝素支架对BMP-2具有较强的吸附能力,并能保持其体外缓慢释放性能;MG-63细胞在肝素化支架上生长状态良好,并具有显著的增殖能力,负载BMP-2后的肝素化支架能显著促进MG-63细胞的分化.因此,肝素化丝素支架是一种较理想的BMP-2缓释载体.     

3D打印nHA/PEEK-AgNPs复合多孔支架的制备与性能

通过熔融沉积成型3D(FDM 3D)打印技术进行打印，制备成nHA/PEEK复合多孔支架。再采用多巴胺氧化聚合和硝酸银化学还原法，在支架表面形成银纳米颗粒涂层，从而制备出nHA/PEEK-AgNPs复合多孔支架。nHA/PEEK-AgNPs具有独特的三维多孔结构，表面接触角约为(33.2±3.65)(°)，展示出较好的亲水性。力学测试结果显示，nHA/PEEK-AgNPs多孔支架的最大压缩强度为(47.4±3.9) MPa，明显高于PEEK组的(36.3±7.3) MPa。同时，最大弹性模量与PEEK组无明显差异，表明复合材料的力学强度与PEEK多孔支架相比有所增强。抑菌实验结果显示，对于大肠杆菌和金黄色葡萄球菌具有明显的抑菌性，抑菌率分别达到(89.4±2.4)%和(85.8±4.4)%。细胞增殖检测结果显示，7 d内nHA/PEEK-AgNPs组在各个时间点的细胞增殖情况均明显优于PEEK组(P<0.05)。此外，nHA/PEEK-AgNPs具有较好细胞相容性和成骨活性。RT-PCR结果显示，14 d内，和PEEK相比，nHA/PEEK-AgNPs组细胞的Runx2基因表达水...     

Effect of Soybean Isoflavone on Bone Mass and Ultrastructural in Ovariectomized Rat Model of Osteoporosis

为研究大豆异黄酮(SI)对骨质疏松模型大鼠骨量及微观结构的影响,将46只雌性SD大鼠双侧摘除卵巢,术后一周按血清总胆固醇水平随机分为5组,分别给予雌激素,低、中、高剂量SI干预并设正常对照组,实验周期为12周,灌胃给药,每周称质量一次,分别在去卵巢、给药4、8周处死时抽取尾静脉血,实验结束后分别测定血清碱性磷酸酶(AKP)活性及骨密度等指标.结果表明,高剂量SI干预对维持大鼠骨密度的作用与雌激素相似,去卵巢后干预SI可提高碱性磷酸酶(AKP)活性并能缓解因去卵巢造成的骨丢失,全视野下骨形态学以雌激素组、高剂量SI干预组与正常组结构最为接近.提示大豆异黄酮对骨质疏松模型大鼠的骨量及微观结构存在一定影响,中、高剂量干预可使骨质疏松模型大鼠血清AKP酶活性增加、逆转因去势造成的骨密度下降,考虑到植物雌激素与哺乳动物的雌激素受体(ER)结合能力低下,采用SI在临床开展干预的剂量与远期效果尚待进一步论证.     

与胶原特异性结合的BMP2模拟肽/PLGA 3D打印复合支架的制备及成骨诱导活性

将胶原绑定结构域(CBD)多肽序列与骨形态发生蛋白2模拟肽(BMP2-MP)序列连接制备具有胶原绑定能力的CBD-BMP2-MP, 再将CBD-BMP2-MP与聚丙交酯-乙交酯/胶原(PLGA/COL)3D打印支架相结合, 以支架表面的胶原成分为媒介, 将CBD-BMP2-MP更有效地固定于骨修复材料上, 达到对其进行改性的目的. 利用扫描电子显微镜(SEM)、 电子万能试验机和接触角测量仪对复合支架表面形貌、 力学强度和亲水性等材料学性能进行评价. 用荧光成像法评测 CBD-BMP2-MP及BMP2-MP与支架材料的结合能力. 在各组支架材料表面接种MC3T3-E1细胞进行体外培养, 采用CCK-8、 鬼笔环肽荧光染色、 茜素红染色及qPCR综合评价细胞在材料表面的黏附、 增殖和成骨分化等细胞行为, 研究CBD-BMP2-MP修饰的3D多孔PLGA/COL复合支架的生物学性能. 研究结果表明, 利用3D打印技术制备的多孔支架具有形貌可控的孔隙结构, 为细胞生长创造更有利的细胞微环境, 支架表面胶原成分的加入提高了支架材料的亲水性, 同时对支架材料本身的力学性能无任何影响, 提高了复合支架本身的生物相容性. 与普通BMP2-MP相比, CBD-BMP2-MP具有更好的胶原绑定能力, 与复合支架的结合更稳定, 提高了PLGA/COL复合支架对BMP2-MP的负载能力. 支架表面负载CBD-BMP2-MP后具有极强的促细胞成骨分化能力. MC3T3-E1细胞表现出更高的钙沉积能力, 并且成骨分化相关基因Runx2, ALP, COL-I及OPN等水平也有了明显提升. 表明CBD-BMP2-MP多孔复合支架具有良好的生物相容性和成骨诱导活性, 在骨组织修复领域具有良好的应用前景.     

三维打印双固化POXC/CPC可降解骨修复支架

从成分设计和结构控制着手,在木糖醇部分取代1,8-辛二醇与柠檬酸聚合反应制备聚(柠檬酸-辛二醇-木糖醇)酯(POXC)的基础上,采用POXC预聚体与磷酸钙骨水泥(CPC)悬浮体三维打印了孔道贯通的POXC/CPC多孔复合预支架,并进一步采用固化反应制备得到该复合支架。探索了材料的可打印参数,评价了复合支架的降解性、润湿性以及生物相容性。结果表明,POXC的降解速率随着木糖醇取代度的增加而增大。56d后,POXC/CPC降解率高达43%,对照组聚(1,8-辛二醇-柠檬酸)酯/CPC(POC/CPC)降解率近10%,这是由于POXC与复合支架的贯通孔结构的协同作用所致。木糖醇的引入及其与CPC的复合大大提高了支架的亲水性,有利于细胞的黏附和增殖。POXC/CPC支架具有贯通的大孔结构、良好的生物相容性和降解性,可促进骨缺损的修复。     

大豆异黄酮对骨质疏松模型大鼠骨量及微观结构的影响

为研究大豆异黄酮（SI）对骨质疏松模型大鼠骨量及微观结构的影响,将46只雌性SD大鼠双侧摘除卵巢,术后一周按血清总胆固醇水平随机分为5组,分别给予雌激素,低、中、高剂量SI干预并设正常对照组,实验周期为12周,灌胃给药,每周称质量一次,分别在去卵巢、给药4、8周处死时抽取尾静脉血,实验结束后分别测定血清碱性磷酸酶（AKP）活性及骨密度等指标。结果表明,高剂量SI干预对维持大鼠骨密度的作用与雌激素相似,去卵巢后干预SI可提高碱性磷酸酶（AKP）活性并能缓解因去卵巢造成的骨丢失,全视野下骨形态学以雌激素组、高剂量SI干预组与正常组结构最为接近。提示大豆异黄酮对骨质疏松模型大鼠的骨量及微观结构存在一定影响,中、高剂量干预可使骨质疏松模型大鼠血清AKP酶活性增加、逆转因去势造成的骨密度下降,考虑到植物雌激素与哺乳动物的雌激素受体（ER）结合能力低下,采用SI在临床开展干预的剂量与远期效果尚待进一步论证。     

HA/GCPU复合多孔支架的可控制备及细胞相容性

以脂肪族异佛尔酮二异氰酸酯(IPDI)作为硬段、蓖麻油甘油酯(GCO)作为软段,通过原位聚合法制备了羟基磷灰石/蓖麻油甘油酯基聚氨酯(HA/GCPU)复合多孔支架.利用红外光谱和扫描电子显微镜对复合支架进行表征,并测试其力学性能及孔隙率.研究结果表明,HA/GCPU复合多孔支架的孔隙率和抗压强度依赖羟基磷灰石的含量,并具有明显的可控性.HA质量分数分别为0,20%和40%时,HA/GCPU复合多孔支架孔隙率分别为(61±3)%,(68±2)%和(57±3)%,抗压强度分别为(605±61),(2125±58)和(4588±260)k Pa,可见HA质量分数为20%时,HA/GCPU复合多孔支架具有与松质骨较为匹配的孔隙率和抗压强度.将MG63细胞与多孔支架共培养,通过倒置显微镜和扫描电子显微镜观察,用噻唑蓝(MTT)法表征HA/GCPU复合多孔支架的细胞相容性,结果表明,HA/GCPU复合多孔支架表面细胞黏附,生长良好,无细胞毒性,在骨组织工程领域具有一定的应用前景.     

Comparison between heparin-conjugated fibrin and collagen sponge as bone morphogenetic protein-2 carriers for bone regeneration

Bone morphogenetic protein-2 (BMP-2) is used to promote bone regeneration. However, the bone regeneration ability of BMP-2 relies heavily on the delivery vehicle. Previously, we have developed heparin- conjugated fibrin (HCF), a vehicle for long-term delivery of BMP-2 and demonstrated that long-term delivery of BMP-2 enhanced its osteogenic efficacy as compared to short-term delivery at an equivalent dose. The aim of this study was to compare the bone-forming ability of the BMP-2 delivered by HCF to that delivered by clinically utilized BMP-2 delivery vehicle collagen sponge. An in vitro release profile of BMP-2 showed that HCF released 80% of the loaded BMP-2 within 20 days, whereas collagen sponge released the same amount within the first 6 days. Moreover, the BMP-2 released from the HCF showed significantly higher alkaline phosphatase activity than the BMP-2 released from collagen sponge at 2 weeks in vitro. Various doses of BMP-2 were delivered with HCF or collagen sponge to mouse calvarial defects. Eight weeks after the treatment, bone regeneration was evaluated by computed tomography, histology, and histomorphometric analysis. The dose of BMP-2 delivered by HCF to achieve 100% bone formation in the defects was less than half of the BMP-2 dose delivered by collagen sponge to achieve a similar level of bone formation. Additionally, bone regenerated by the HCF-BMP-2 had higher bone density than bone regenerated by the collagen sponge-BMP-2. These data demonstrate that HCF as a BMP-2 delivery vehicle exerts better osteogenic ability of BMP-2 than collagen sponge, a clinically utilized delivery vehicle.     

Influence of the Degree of Deacetylation of Chitosan and BMP-2 Concentration on Biocompatibility and Osteogenic Properties of BMP-2/PLA Granule-Loaded Chitosan/β-Glycerophosphate Hydrogels

Compositions based on chitosan/β-glycerophosphate hydrogels with highly porous polylactide granules can be used to obtain moldable bone graft materials that have osteoinductive and osteoconductive properties. To eliminate the influence of such characteristics as chain length, degree of purification, and molecular weight on a designed material, the one-stock chitosan sample was reacetylated to degrees of deacetylation (DD%) of 19.5, 39, 49, 55, and 56. A study of the chitosan/β-glycerophosphate hydrogel with chitosan of a reduced DD% showed that a low degree of deacetylation increased the MSCs (multipotent stromal cells) viability rate in vitro and reduced the leukocyte infiltration in subcutaneous implantation to Wistar rats in vivo. The addition of 12 wt% polylactide granules resulted in optimal composite mechanical and moldable properties, and increased the modulus of elasticity of the hydrogel-based material by approximately 100 times. Excessive filling of the material with PLA (polylactide) granules (more than 20%) led to material destruction at a ~10% strain. Osteoinductive and osteoconductive properties of the chitosan hydrogel-based material with reacetylated chitosan (39 DD%) and highly porous polylactide granules impregnated with BMP-2 (bone morphogenetic protein-2) have been demonstrated in models of orthotopic and ectopic bone formation. When implanted into a critical-size calvarial defect in rats, the optimal concentration of BMP-2 was 10 μg/mL: bone tissue areas filled the entire material’s thickness. Implantation of the material with 50 μg/mL BMP-2 was accompanied with excessive growth of bone tissue and material displacement beyond the defect. Significant osteoinductive and osteoconductive properties of the material with 10 μg/mL of BMP-2 were also shown in subcutaneous implantation.     

Bioactive gelatin cryogels with BMP-2 biomimetic peptide and VEGF:A potential scaffold for synergistically induced osteogenesis

A poor biocompatibility and bioactivity of invasive materials remains major problems for biomaterialbased therapy. In this study, we introduced gelatin scaffolds carrying both bone morphogenetic protein-2(BMP-2) biomimetic peptide and vascular endothelial growth factor-165(VEGF) that achieved controlled release, cell attachment, proliferation and differentiation. To promote osteogenesis with VEGF, we designed the BMP-2 biomimetic peptide that comprised BMP-2 core sequence oligopeptide(SSVPT), ph...     

Effects of Dimephosphone on Mineral Metabolism of High Dose Prednisolone Treated Rats Compared to Etidronic Acid

Abstract

The aim of the study was to evaluate the effects of dimephosphone dimethyl ether of 1,1 -dimethyl-3-oxobutylphosphonic acid (monophosphonate) on mineral metabolism and to compare it to the effects of classical bisphosphonate - etidronic acid (Xydifonum as a medicinal preparation produced in Russia). Dimephosphone was synthesised according to Wiesel (Wiesel A.O. et all, 1998). The rat model of high dose prednisolone-induced mineral disturbance (50 mg/kg orally for 14 days) was used. Prednisolone increased urine calcium and phosphate level. The urine excretion of oxyproline - a marker of bone resorbtion was also significantly increased by prednisolone treatment. Prednisolon increased bone calcium level (atom-absorbtion spectrophotometry) and produced hyperplasia of adrenal glands. Dimephosphone effectively reduced urine phosphate and oxyproline excretion while xydiphonum had no effect on oxyproline excretion. Dimephosphone increased hrther bone and blood calcium level, while xydiphonum reduced it. Dimephosphone protected adrenal glands from hyperplasia, the effect of xydiphonum was less marked. The results suggest the possible use of dimephosphone in treatment of steroid-induced mineral metabolism disturbances including steroid osteoporosis.     

Highly porous PEEK and PEEK/HA scaffolds with Escherichia coli-derived recombinant BMP-2 and erythropoietin for enhanced osteogenesis and angiogenesis

The present study reports the results of structural and mechanical analysis, as well as proteins release kinetics and osteointegration in mice craniotomy model of highly porous PEEK (PolyEther Ether Ketone) and PEEK/HA (PolyEther Ether Ketone/HydroxyApatite) biomimetic scaffolds loaded with Escherichia coli-derived recombinant Bone Morphogenetic Protein-2 (BMP-2) and ErythroPOietin (EPO). Porous scaffolds were obtained by thermopressing with NaCl as a pore-forming filler. Two fractions of pore-forming filler were used to imitate natural trabecular bone tissue by making a preferential porosity using large fraction and creating an extended surface and special microrelief using small fraction. Hydroxyapatite (HA) was added up to 20% to activate bioinert PEEK providing loading of recombinant growth factors and osteointegration as well as sufficient level of mechanical properties imitating human trabecular bone. Unexpectedly, the non-activated PEEK produced by our technology was also able to spontaneously bind both BMP-2 and EPO. Loading of both BMP-2 and EPO to both types of implants resulted in enhanced neoosteogenesis and angiogenesis in a critical-size cranial defect model in mice in 3–6 weeks. Considering good mechanical characteristics and excellent osteoinductive and angiogenic properties, both materials in combination with BMP-2 and EPO can find their application in regenerative medicine.     

Longitudinal Bone Growth Stimulating Effect of Allium macrostemon in Adolescent Female Rats

Allium macrostemon (AM) may affect bone growth by regulating bone formation and resorption. To examine the effect of AM on bone growth, 48 rats were divided into four administration groups in which either distilled water, AM (100 and 300 mg/kg), or recombinant human growth hormone (rhGH; 20 μg/kg) was administered for 10 days. On day 9, all animals were intraperitoneally injected with tetracycline hydrochloride (20 mg/kg), and 48 h after the injection, the rats were sacrificed. Their tibial sections were photographed to measure bone growth. Antigen-specific immunohistochemistry was performed to detect insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2). The food intake of the AM 100 mg/kg group was higher; however, the food intake of the AM 300 mg/kg group was less than that of the control group. The rhGH and AM 100 mg/kg groups showed greater rates of bone growth (359.0 ± 23.7 and 373.1 ± 28.0 μm/day, respectively) compared with the control group. IGF-1 and BMP-2 in the AM and rhGH groups were highly expressed. Indigestion at higher doses of AM led to nonsignificant bone growth in spite of increased IGF-1 and BMP-2 expression. Therefore, a suitable amount of AM could increase bone growth.     

Homogeneous organic/inorganic hybrid scaffolds with high osteoinductive activity for bone tissue engineering

Composite scaffolds of polymers/β-tricalcium phosphate (TCP) have been widely used for bone regeneration due to the combination of osteoinductivity of TCP and mechanical properties of the polymers. However, the difference in surface properties of the two material causes composite has poor uniformity and weak two-phase interaction, resulting in poor TCP release and weak new bone-forming ability. In this research, a TCP sol was developed to replace traditional TCP nanoparticles for the preparation of homogeneous polycaprolactone (PCL)/TCP sol nanofibrous scaffolds. It was found that compared with TCP nanoparticles, TCP sol homogeneously distributed in PCL nanofibers, and greatly improved the hydrophilicity, biodegradability, and mechanical properties of the scaffolds. It is also confirmed that loading TCP sol promoted the formation of bone-like apatite on the surface of the scaffolds. Biological experiments showed that all scaffolds supported rat bone marrow mesenchymal stem cells (rBMSCs) proliferation, especially scaffolds loaded with TCP sol. The increase in alkaline phosphatase activity and collagen production, enhanced calcium deposition, and up-regulation of osteocalcin expression demonstrated that the loading TCP sol expanded an advantage of scaffolds in promoting rBMSCs osteogenic differentiation, suggesting it dramatically improved the osteoinductive activity of PCL/TCP hybrid system and had a great potential application in bone regeneration.     

Purification of squirt cellulose membrane from the cystic tunic of Styela clava and identification of its osteoconductive effect

The sea squirt has a tunic composed of cellulose fibers that are originated from animal cells and form a cellulose membrane that functions as a protective barrier and a skeleton of body. We purified the squirt cellulose membrane (SCM), about 100 μm thickness, from the cystic tunic of Styela clava. The SCM was turned out to be composed of pure native cellulose I in the analyses of X-ray diffraction, attenuated total reflectance Fourier transform-infrared spectroscopy. The inner surface of SCM showed numerous micropores in the meshworks of thin cellulose microfibrils, while the outer surface of SCM showed a few micropores. In order to know the osteoconductive effect of the SCM, it was applied to the perforated rat frontal bone, 8 mm diameter, for 4, 6, 8, 10, 12, and 16 weeks. The mesenchymal cells were tightly adhered to the inner surface of SCM and produced new bone there. The perforations to which the SCM was applied, were completely healed in 10 weeks, while the positive control group applied with the guided tissue regeneration (GTR) material, Bio-Gide^® showed incomplete healing until 16 weeks. Immunohistochemistry performed on the experimental group showed that BMP-2 and osteonectin were strongly positive in the early stage of bone healing at 4 weeks, while receptor activator of nuclear factor kappa B ligand (RANKL) became positive at 16 weeks. Particularly, the inner surface of SCM itself was consistently positive for the osteogenic proteins, indicating that the implanted SCM can absorb the osteogenic proteins from the stromal matrix of the host tissue. On the other hands, the positive control group implanted with Bio-Gide^® showed relatively weak reaction of the osteogenic proteins in the perforated bone area during the healing period, and the negative control group implanted none showed poor bony regeneration in the perforated wounds. Taken together, the SCM showed an excellent osteoconductive effect in the perforated rat frontal bone, which was quite comparable to the results of Bio-Gide^® implantation. Therefore, it is suggested that the SCM be a novel GTR material of a natural source with a strong osteoconductive effect on the injured bone.     

Lycopene effects on serum mineral elements and bone strength in rats

This study investigated the beneficial effect of lycopene on bone biomarkers in ovariectomized (OVX) rats. Female Wistar rats were either sham operated or surgically ovariectomized and then fed with lycopene for 8 weeks. Serum Ca, P, alkaline phosphatase (ALP), interleukin 6 (IL-6) and bone gla protein (BGP) concentration was significantly higher in the untreated OVX group compared with that of the sham group, whereas serum estrogen levels were lower. Bone mineral density (BMD), BMD/wt, bone mineral content (BMC), BMC/wt values, maximum load, stiffness, energy and maximum stress were significantly lower in the untreated OVX group compared with that of the sham group. Administration of lycopene (20, 30 and 40 mg/kg b.w.) for 8 weeks significantly decreased serum Ca, P, ALP, and IL-6 concentration, and enhanced serum estrogen level, BMD, BMD/wt, BMC, BMC/wt values, maximum load, stiffness, energy and maximum stress in lycopene-treated OVX groups. In conclusion, the consumption of lycopene may have the most protective effect on bone in OVX rats.