BIOACTIVITY OF BIORESORBABLE OSTEOSYNTHETICDEVICES MADE OF HYDROXYAPATITE／ POLY-DL- LAC- TIDE COMPOSITES： AN EXPERIMENTAL STUDY

Aim. To investigate the bioactivity of the serf-designed biodegradable osteosynthetic devices made of resorbable hydroxyapatite microparticles/ poly-DL-lactide (HA/PDLLA) composites. Method. Forty-three rabbits with a transverse transcondylar osteotomy of the distal femur were fLxed intramedullary by a HA/PDLLA rod, the duration of follow-up were 3,6,12, 24 and 36 weeks. Histological, scanning electron microscopic (SEM), energy dispersive X-ray (EDX) and biomechanical analyses were done. Resudts. Active new bone formation and direct bone-bonding were seen at the bone-implant interface. Generous apatite crystals deposited and grew on the surface of the composites at 3 - 6 weeks postoperation. The interracial shear strength increased significantly. Conclusion. Through the incorporating of resorbable HA microparticles, specific bone-bonding and active osteogenic capacity is introduced. This kind of bioactivity, together with other properties such as sufficient mechanical strength, enhanced biocompatibi]ity and mdiopacity, which are intrinsically unobtainable in totally resorbable polymer/polymer systems, make the HA/PDLLA composites become a desirable material for the internal fixation of cancellous bone.     

Bone Formation and Healing Study of hBMP-2 Transfected rMSCs Attached to DBM

Objective: To detect the effect of hBMP-2 transfected rMSCs on bone repair and the capability of the new biomaterial in enhancing bone repair. Methods: Auto-rMSCs were cultured and transfected with hBMP-2 by liposome. All the transfected and un-transfected riSCs were attached to Allo-DBMs. These new biomaterials were implanted in muscle bags and segmental radius defects of the New Zealand white rabbits, and some controlled material groups were established for comparison. All the biomaterials and the controlled materials were assessed by gross observation, radiographical and histological methods. Results: The osteoblasts could be seen in the biomaterials with transfected rMSCs, which have been implanted in muscle bags. There was no sign of bone formation in the biomaterials with untransfected rMSCs and the single DBM groups. With the segmental bone defects, all the transfected, untransfected and single DBM biomaterials could work, but the healing of the biomaterial with transfected hBMP-2 was the fastest and most effective. Conclusion: Delivery of rMSCs with transfected hBMP-2 genemay generate osteo.morphogenesis and promote skeletal repair in vivo.     

Modulation of transforming growth factor β to platelet-derived growth factor receptor-α of human osteoblasts

Objective To investigate the mechanism and the significance of TGFβ in modulating the expression of Platelet-Derived Growth Factor Receptor-α (PDGFR-α) in human osteoblasts. Methods The osteoblasts were isolated from human fetal calvaria. The percentage of cell increase (PCI) in every 4 hours was calculated to demonstrate the proliferation of osteoblasts affected by PDGF-AA and TGFβ. The osteoblasts were cultured with TGFβ for 24 hours and with PDGF-AA for another 24 hours, and the cells proliferation was shown by PCI too. The osteoblasts were cultured with TGFβ for 24 h, and the PDGFR-α of the cells were measured by immunofluorescent analysis.Results PCI was increased by 48.2% and 22.4% after PDGF-AA and TGFβ were added into the medium for 24 hours respectively (P<0.05), and PCI decreased after the removal of the two cytokines. Preincubated with TGFβ for 24 hours and then stimulated with PDGF-AA, PCI grew slowly. TGFβ downregulated the expression of the PDGFR-α.Conclusion TGFβ can downregulate the mitogenesis of PDGF-AA by lowering the number of PDG     

Role of p38MAPK in mediating TNF-α-induced apoptosis of rat glioma cell line C6

Objective: To study the role of p38MAPK in mediating TNF-α-induced apoptosis of rat glioma cell line C6. Methods: Effect of TNF-α on the proliferation of C6 cells was determined by MTT assay. The TNF-α induced apoptosis was detected by transmission electron microscopy and flow cytometry. The expression of p38MAPK was detected by SABC method and Western-blot. The effect of SB202190, a specific inhibitor of p38MAPK, on TNF-α-induced apoptosis was observed by flow cytometry and SABC method. Results: Inhibitory rate of TNF-α(2×105 U/L) on C6 cells was 43. 75% . In the TNF-α treated group, apoptotic cells were observed by transmission electron microscopy and the apoptotic rate was 37. 5% by flow cytometry. p38MAPK positive signals were detected by SABC method and Western-blot. In the SB202190 treated group, the apoptotic rate was 7. 0% and no p38MAPK signals were found. Conclusion: Apoptosis of C6 cells and expression of p38MAPK can be induced by TNF-α. The activation of p38MAPK promotes the apoptosi     

Polylactic Acid Nanoparticles Targeted to Brain Microvascular Endothelial Cells

In this work, blank polylactic acid （PLA） nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular endothelial cells （BMECs） targeting was examined by in vivo experiments and fluorescence microscopy. The results showed that PLA nanoparticles are less toxic than PACA nanoparticles but their BMECs targeting is similar to PACA nanoparticles. The experiments suggest that drugs can he loaded onto the particles and become more stable through adsorption on the surface of PLA nanoparticles with high surface activity. The surface of PLA nanoparticles was obviously modified and the hydrophilicity was increased as well in the presence of non-ionic surfactants on PLA nanoparticles. As a targeting moiety, polysobate 80 （T-80） can facilitate BMECs targeting of PLA nanoparticles.     

Mineralized and osteoid tissue from dental pulp stem cells on micro-arc oxidation titanium in vitro

The presence of insufficient bone volume affects the implant healing and success.The aim of this study was to evaluate osteogenic capacity of dental pulp stem cells(DPSCs) on micro-arc oxidation(MAO) titanium surface.DPSCs were challenged at MAO and smooth titanium surface separately for different durations,and the bone marrow mesenchymal stem cells(BMSCs) served as the positive controls.The osteogenic capacity of DPSCs on MAO titanium surface was assessed by using scanning electron microscopy,energy dispersive spectroscopy,biochemical tests and real-time quantitative PCR.Data showed that DPSCs differentiated into osteoblasts and expressed bone morphogenetic genes on MAO titanium surface.The results of this study revealed that DPSCs had good potential to generate mineralized tissue on MAO titanium plates.The differential potential of DPSCs may be regulated by MAO titanium surface.The osteogenesis potential of DPSCs on the MAO titanium was similar with BMSCs.     

In vitro and in vivo effects of puerarin on promotion of osteoblast bone formation

Objective:To assess the effect of puerarin,a natural flavonoid found in Chinese Pueraria Lobata (Wild.) Ohwi,on promotion of new bone formation.Methods:Osteoblasts isolated from calvarial of newborn rats were cultured in vitro in the presence of puerarin at various concentrations.The viability of osteoblasts and alkaline phosphotase activity and mineral node formation were determined.In addition,osteoblasts seeded in theβ-tricaclium phosphate scalfolds as bone substitute were implanted in rat dorsal muscles.Half of the recipient rats received intramuscular injection of puerarin at 10 mg/(kg·d) for 7 days.Osteogenesis was analyzed by examining the histology after 4 weeks of implantation.Results:The viability of osteoblasts treated with puerarin at either 40 or 80μmol/L was significantly higher than that of the control(P<0.05 and P<0.01, respectively).Alkaline phosphatase and mineral modules were significantly increased in osteoblasts cultured with puerarin at 40 or 80 mol/L when compared with that of the untreated cells.The puerarin-treated rats had a higher rate of bone formation in the osteoblast implants than the control rats(6.35%vs.1.32%,respectively, P<0.05).Conclusion:Puerarin was able to affect osteoblast proliferation and differentiation,and promote the new bone formation in osteoblast implants.     

Experimental Study on Allogenic Decalcified Bone Matrix as Carrier for Bone Tissue Engineering

The biocompatibility and osteogenic activity of allogenic decalcified bone matrix (DBM) used as a carrier for bone tissue engineering were studied. Following the method described by Urist, allogenic DBM was made. In vitro, DBM and bone marrow stromal cell (BMSC) from rab-bits were co-cultured for 3-7 days and subjected to HE staining, and a series of histomorphological observations were performed under phase-contrast microscopy and scanning electron microscopy (SEM). In vivo the mixture of DBM/BMSC co-cultured for 3 days was planted into one side of muscules sacrospinalis of rabbits, and the DBM without BMSC was planted into other side as con-trol. Specimens were collected at postoperative week 1, 2 and 4, and subjected to HE staining, and observed under SEM. The results showed during culture in vitro, the BMSCs adherent to the wall of DBM grew, proliferated and had secretive activity. The in vivo experiment revealed that BMSCs and undifferentiated mesenchymal cells in the perivascular region invaded gradually and proliferated together in DBM/BMSC group, and colony-forming units of chondrocytes were found. Osteoblasts,trabecular bone and medullary cavity appeared. The inflammatory reaction around muscles almost disappeared at the second weeks. In pure DBM group, the similar changes appeared from the sur-face of the DBM to center, and the volume of total regenerate bones was less than the DBM/BMSC group at the same time. The results indicated that the mixture of DBM and BMSC had good bio-compatibility and ectopic induced osteogentic activty.     

Clinicopathologic and ultrastructural observation of primary pulmonary cryptococcosis:a study of 37 cases

Objective To investigate the pathologic diagnosis and ultrastructure features of primary pulmonary cryptococcosis (PC). MethodsRetrospective analysis was made on 37 cases of PC with histochemical stain and light microscopy observations. Transmission electron microscopy was performed on 19 cases. ResultsThree of 37 cases were confirmed by fine needle aspiration biopsy while the other 34 patients by open-chest exploratory operation.Pathologic findings: cryptococcosis neoformans (CN) were detected in all cases,2 with gelatinous lesions and 35 non-casating granuloma. The revealing rate of CN with the mucicarmine stain(MC),periodic acid-Schiff stain(PAS), alcian blue (AB) and Grocott methenamine-silver (GMS) was 85.2% (23/27), 97.3% (36/37), 64.9% (24/37) and 96.3% (26/27), respectively. All CNs detected by transmission electron microscopy had capsules and most of them possessed simple structure with undeveloped cellular organelles and cell cataplasia. Some CNs had cellular nucleus, nucleoli, mitochondria and vacuoles. The detective rate of CNs by transmission electron microscope was 89.5 % (17/19). ConclusionAbsent of specific clinical manifestations and radiographic features, fine needle aspiration biopsy and open biopsy remain the chief approaches in the diagnosis of PC. It could be further helpful by using light microscopy and transmission electron microscopy to make definitive diagnosis and differential diagnosis as well as MS, GMC and PAS histochemical staining.     

消旋聚乳酸/羟基磷灰石/脱钙骨基质材料支架的体外细胞相容性实验研究

目的研究一种新的骨替代材料——消旋聚乳酸／羟基磷灰石／脱钙骨基质(PDLLA/HA／DBM)骨细胞界面形态，评价其生物相容性。方法采用体外细胞培养技术，选用培养的人成骨细胞，通过光镜和扫描电镜等观察，了解细胞在材料中的生长情况和相互关系，并与消旋聚乳酸(PDLLA)及脱钙骨(DBM)相比较。结果随着培养时间的延长，细胞在PDLLA／DBM材料表面的密度增加，未见细胞死亡。细胞以锚状结构牢固地附着于材料表面，并分泌细胞外基质，覆盖并完全包裹材料颗粒。结论PDLLA／HA／DBM与人成骨细胞能早期形成骨性结合界面，具有良好的牛物相容性，可望成为一种新的具有前景的骨替代材料。     

消旋聚乳酸/羟基磷灰石/脱钙骨基质人工骨修复兔桡骨大段骨缺损的实验研究

目的研制理想的,能够修复大段骨缺损的人工骨材料。方法采用乳液共混法将消旋聚乳酸(PDLLA)、羟基磷灰石(HA)、脱钙骨基质(DBM)结合,制成PDLLA/HA/DBM人工骨,并将PDLLA/HA/DBM和PDLLA进行兔桡骨大段骨缺损修复的对比研究。术后2、4、8和12周时摄X片及病理形态学观察及新骨形成定量分析。结果PDLLA/HA/DBM人工骨内新骨形成量明显多于PDLLA及空白对照组(P<0.01),且能够有效修复骨缺损。结论PDLLA/HA/DBM人工骨能促进长骨大段骨缺损的修复,是一种较理想的骨修复材料。     

兔骨髓基质细胞与聚DL-乳酸/羟基磷灰石生物相容性的实验研究

目的检测聚DL-乳酸/羟基磷灰石（PDLLA/HA）复合材料的特性,探讨骨髓基质细胞（BMSCs）与PDLLA/HA的生物相容性,为筛选骨组织工程的支架材料提供依据.方法将BMSCs与PDLLA/HA复合体外培养,通过形态学的观察、细胞增殖率及ALP活性的测定,观察PDLLA/HA对培养细胞的影响.结果复合培养时BMSCs能在PDLLA/HA上贴附、繁殖,其生长及功能不受影响.结论 PDLLA/HA具有良好的细胞相容性,能作为骨组织工程种子细胞的支架材料.     

Effect of Absorbable Hydroxyapatite/Poly-DL-Lactide Rods on Experimental Fracture Healing

Development of absorbable fracture fixationdevices that are degradable and biocompatiblewould obviate the need for a hardware removalprocedure after completion of union of a fracture.Most absorbable implants in clinical use today consist of a biodegradable alphahydroxy polyester, either self--reinforced polyglycolide (SRuPGA ) orself--reinforced poly--L--lactide (SR--PLLA ). However, there are some problems, which are yet to beresolved in search for better absorbable devices.The biodegrad…     

丝素蛋白与丝素蛋白/羟基磷灰石复合材料体外细胞相容性的比较

目的探讨丝素蛋白（SF）与丝素蛋白/羟基磷灰石复合材料（SF/HA）的体外细胞相容性,为组织工程提供理想的生物支架材料。方法将小鼠的骨髓间充质干细胞（C3H10T1/2）接种于上述两种生物材料上,运用四唑盐比色试验（MTT法）检测细胞的增殖能力,利用倒置显微镜和扫描电镜观察细胞在材料上的生长形态。结果 C3H10T1/2在SF与SF/HA上都能很好地黏附、生长和增殖。SF、SF/HA和C3H10T1/2都有良好的细胞相容性。结论 3%SF、6%SF、600%SF/HA3组多孔材料的体外细胞相容性最好。     

杜仲叶提取物/CPC复合载体对大鼠成骨细胞作用的实验研究

目的:观察不同浓度的杜仲叶提取物及其与CPC复合载体对MC3T3E1大鼠成骨细胞的影响。方法:将培养的MC3T3E1大鼠成骨细胞分为5组:空白组和0、0.152、0.304、0.608 mg/ml不同浓度杜仲叶提取物/CPC复合载体培养组。应用细胞增殖、MTT、扫描电镜法观察不同浓度杜仲叶提取物/CPC复合载体对体外培养成骨细胞48 h的增殖活性及生长、附着的影响。结果:杜仲叶提取物/CPC复合载体可促进成骨细胞的增殖率,尤其以浓度为0.152 mg/ml、0.304 mg/ml时作用较为明显(P<0.05)。结论:杜仲叶提取物/CPC复合载体具有促进成骨细胞的增殖和生长,以及附着的作用。     

复合BMSCs的异种骨移植材料异位成骨研究

目的:制备兔脱钙骨基质(DBM),研究复合骨髓间充质干细胞(BMSCs)的DBM的异位成骨能力。方法:制备DBM材料,将体外培养的ICR小鼠BMSCs与DBM复合培养后构建的组织工程骨植入ICR小鼠股部肌袋中,以单纯DBM植入组为对照,术后7d及28d行X线检测,并于术后7,14,28d取植入材料作组织学检测、钙含量测定。结果:DBM与BMSCs复合体植入后7d即有成骨细胞出现,随着时间增加成骨细胞增多,术后28d即有较成熟骨质形成,移植物钙含量随时间推移逐渐增高。单纯植入DBM术后14d出现少量成骨细胞,术后28d出现少量类骨质,钙含量持续处于较低水平。结论:DBM与BMSCs复合后具有很好的异位成骨能力,DBM是组织工程骨研究较理想的支架材料。     

飞秒激光在钛合金表面改性中的应用及其对成骨细胞黏附和增殖的影响

目的：采用不同能量密度飞秒激光处理钛合金表面,比较其对成骨细胞黏附和增殖的影响,探讨飞秒激光在种植材料表面改性中的作用。方法：钛合金Ti-6Al-4V圆片进行打磨抛光并采用不同能量密度的飞秒激光处理,分为低能量组（0.07 J•cm^-2）和高能量组（1.40 J•cm^-2）,同时以单纯打磨抛光样本为对照组,扫描电镜观察各组材料表面形貌;人成骨样细胞素MG63与各组材料进行共培养,扫描电镜观察材料表面黏附细胞的形态并采用丫啶橙染色和荧光显微镜观察黏附细胞的数量,用噻唑蓝（MTT）比色分析法测定MG63细胞的吸光度（A）值,分析不同时间点细胞增殖率。结果：飞秒激光处理钛合金表面形成2种不同的微形貌结构,即纳米级平行排列的条纹状结构和微米级的凸起与纳米级的条纹相结合的微纳复合结构;24 h各组黏附的细胞数量相近;48 h各组材料表面单个细胞形态均不明显,但微纳复合结构表面细胞覆盖面积大于纳米条纹表面和对照组的光滑表面,高能量组细胞增殖率明显高于对照组和低能量组（P＜0.05）。结论：飞秒激光处理钛合金表面产生的微纳复合结构有利于MG-63成骨细胞黏附和增殖。     

新型聚乳酸/骨基质多孔复合支架材料制备及初步研究

目的采用CO2超临界法(SC-CO2)制备一种新型的聚乳酸(PLA)/骨基质(BMG)多孔复合型生物活性骨支架材料,并评价其理化性能及细胞相容性,确定材料最佳复合比例,为进一步的实验提供实验依据。方法采用SC-CO2法制备不同比例的PLA/BMG多孔复合型骨植入材料,通过大体观察、孔隙率测定,力学检测、SEM观察评价其理化性能,并结合体外细胞相容性检测选择最佳复合比例。结果采用SC-CO2法制备的PLA/BMG多孔复合支架材料中BMG的比例与材料的孔隙率及细胞相容性成正相关,与力学性能成负相关;含30%BMG的复合材料具有良好的综合性能。结论采用SC-CO2法制备的PLA/BMG(7/3)多孔复合支架材料具有良好的理化性能及细胞相容性,可作为骨植入材料及骨组织工程支架进一步研究。