致密磷酸钙陶瓷在动态SBF中类骨磷灰石层形成研究

磷酸钙陶瓷植入体内后其表面类骨磷灰石层的形成是诱导成骨的先决条件．本实验在模拟体液(ｓｉｍｕｌａｔｅｄ ｂｏｄｙ ｆｌｕｉｄ,ＳＢＦ)以人体骨骼肌组织内体液的正常生理流率(２ｍＬ/１００ｍＬ·ｍｉｎ)和偏离正常生理流率流动的动态条件下,研究在动态ＳＢＦ中影响致密磷酸钙陶瓷表面类骨磷灰石层形成的因素．结果表明:在生理流率条件下,材料的粗糙表面有利于类骨磷灰石的形成,加大ＳＢＦ中Ｃａ^２＋、ＨＰＯ_４^２－离子浓度,类骨磷灰石层的形成速度加快．比起通常使用的静态浸泡试验,ＳＢＦ以生理流率流动的动态试验能够更好地模拟类骨磷灰石生长的体内环境．动态ＳＢＦ对了解类骨磷灰石形成,进而了解磷酸钙陶瓷在体内诱导成骨机理是十分有用的．     

多孔β-TCP生物陶瓷成骨过程的研究

主要对多孔β-TCP生物陶瓷在体内对新骨生成的诱导性进行研究.首先,人为造成兔股骨髁部骨腔洞,建立骨缺损模型,将自制β-TCP多孔陶瓷植入,然后逐月取一系列处死、取材,并综合运用扫描电镜观察(SEM)、电子探针X显微分析(EPMA)、甲苯胺蓝染色(toluidine blue staining,TBS)和四环素双标记法(tetracycline double labeling,TDL)等测试手段,对材料植入区进行研究,试图探明β-TCP多孔陶瓷修复骨缺损时的成骨过程.     

大孔孔径对含CO3^2-的双相HA／β-TCP多孔陶瓷表面类骨磷灰石形成的影响

钙磷陶瓷植入生物体内后其表面首先形成一层含CO3^2-的类骨磷灰石层。它对钙磷陶瓷诱导新骨的生成起非常重要的作用。本文以模拟体液SBF9^#为介质。利用体外模拟装置首次研究了以新工艺制备的含CO3^2-的双相HA／β-TCP多孔陶瓷其大孔孔径对表面类骨磷灰石形成的影响。结果表明该陶瓷因CO3^2-的掺入导致类骨磷灰石晶体的形成时间大大缩短(从14d缩短至6d)。且以300～400μm的大孔孔径最有利于类骨磷灰石晶体的形成。此外还有缺钙羟基磷灰石晶体的生成。而最不利于类骨磷灰石晶体形成的大孔孔径为400～500μm。大孔孔径的优化有利于该陶瓷材料骨诱导性的提高，进而有利于骨缺损的快速修复。     

大鼠骨髓间充质干细胞与β-磷酸三钙/聚L-乳酸支架材料的复合

将成骨诱导前后的大鼠骨髓间充质干细胞与不同比例、不同孔径和不同孔隙率的βTCP／PLLA材料复合,通过体内体外实验研究β-磷酸三钙／聚L-乳酸（β-TCP／PLLA）的成分、孔径和孔隙率等结构参数对复合材料在体内异位成骨能力的影响．结果表明,β-TCP／PLLA成分比为2：1,致孔剂含量为70％;孔径为200-450μm的支架材料在体外更有利于细胞的生长、增殖以及分化．这种支架材料在裸鼠体内具有异位成骨的能力,成骨诱导后的大鼠骨髓间充质干细胞比没有诱导的细胞更适合作为种子细胞．     

不同孔隙率锶磷灰石陶瓷体内异位成骨能力的比较

采用健康兔的骨髓组织,体外培养,诱导分化为成骨细胞,分别与孔径约为450～700μm、孔隙率分别为50%、60%、70%的Sr-HA以及孔隙率为70%的纯羟磷灰石陶瓷体复合,自体回植到兔背脊肌内.同时以未复合细胞的相同孔隙率的陶瓷材料作为对照,植入后4周、12周和24周取材,行四环素荧光染色观察,定量计算各组陶瓷材料新骨的形成量和速度,比较不同孔隙率锶磷灰石陶瓷材料的异位成骨能力.结果表明,复合成骨细胞的各组陶瓷在植入兔背脊肌内4周后均有新骨形成,随着植入时间的延长,骨组织的数量不断增加;孔隙率为70%的Sr-HA陶瓷和HA陶瓷的新骨形成数量和速度明显优于低孔隙率的Sr-HA陶瓷.四环素荧光标记还显示未复合成骨细胞的Sr-HA陶瓷和HA陶瓷孔隙内也有荧光沉积.Sr-HA多孔陶瓷是较理想的骨组织工程支架材料,成骨细胞复合Sr-HA陶瓷用于骨缺损的修复,具有广阔的临床应用前景.     

大孔孔径对含CO2-3的双相HA/β-TCP多孔陶瓷表面类骨磷灰石形成的影响

钙磷陶瓷植入生物体内后其表面首先形成一层含CO2-3的类骨磷灰石层.它对钙磷陶瓷诱导新骨的生成起非常重要的作用.本文以模拟体液SBF9#为介质,利用体外模拟装置首次研究了以新工艺制备的含CO2-3的双相HA/β-TCP多孔陶瓷其大孔孔径对表面类骨磷灰石形成的影响.结果表明该陶瓷因CO2-3的掺入导致类骨磷灰石晶体的形成时间大大缩短(从14d缩短至6d),且以300～400μm的大孔孔径最有利于类骨磷灰石晶体的形成.此外还有缺钙羟基磷灰石晶体的生成.而最不利于类骨磷灰石晶体形成的大孔孔径为400～500μm.大孔孔径的优化有利于该陶瓷材料骨诱导性的提高,进而有利于骨缺损的快速修复.     

等离子体表面改性的双相磷酸钙陶瓷在SBF中类骨磷灰石形成的研究

由于材料表面类骨磷灰石的形成是材料是否具有生物活性的关键因素。因此,本文研究了等离子体活化改性的双相钙磷陶瓷(HA／TCP)在模拟体液(SBF)中形成类骨磷灰石的表面形貌、组成和结构,并探讨活化和沉积的机理。结果发现等离子体处理的HA／TCP更容易形成类骨磷灰石。其机理是等离子体中的高能、高活性的粒子轰击HA／TCP,使其表面刻蚀和粗化,也使HA／TCP晶体产生畸变活化,从而增加了钙磷陶瓷的溶解性,易使局部钙、磷离子浓度达到过饱和,有利于类骨磷灰石的成核和生长。表明等离子体表面改性提高了材料的活性。有利于促进骨的形成和生长。     

磷酸钙陶瓷在不同动物的模拟体液中类骨磷灰石的形成研究

根据人、狗、猪、猴和兔五种动物体液的钙离子浓度和pH值的差异,配制了不同组分的模拟体液,将孔壁致密和有微孔的多孔磷酸钙陶瓷分别浸泡在这些模拟体液中,研究陶瓷孔隙表面类骨磷灰石的形成情况.结果表明:在模拟体液中浸泡14天后,孔壁致密的材料未见有类骨磷灰石层形成;有微孔的多孔磷酸钙陶瓷,材料孔壁表面(包括陶瓷表面较深孔隙)有类骨磷灰石层的形成,这与体内植入实验观察到的类骨磷灰石层形成和诱导成骨情况相似,可以推论类骨磷灰石层的形成的确是骨诱导的先决条件.随着钙离子浓度的增加,其孔壁表面类骨磷灰石层的形成也更为均匀,但类骨磷灰石生长快慢顺序与动物组织学观察到的骨诱导性高低的次序不完全一致.     

掺锶羟磷灰石固溶体陶瓷不同孔径对成骨能力的影响

通过控制发泡剂粒径的方法制得两组孔隙率相同孔径尺寸不同的掺锶羟磷灰石多孔体陶瓷:S组平均孔径200μm;L组平均孔径750μm.植入兔桡骨缺损部位,植入后3、6和12周取材,行X线观察、大体标本观察、组织学图像观察和四环素荧光染色观察,来对比不同孔径的锶磷灰石陶瓷多孔体的骨融合能力.结果表明锶磷灰石陶瓷多孔体具有良好的生物相容性及骨传导性;两种孔径材料都有良好的成骨性能,大孔径组锶磷灰石陶瓷多孔体材料比小孔径组材料有更佳的骨修复效果;且大孔径组在12周就能观察到陶瓷材料的降解以及新生骨包绕降解陶瓷颗粒生长的现象.     

不同表面特征的多孔钛与皮下软组织间生物力学性质的研究

为解决经皮器械长期生物密封问题, 选择具有三维支架结构和合适的表面微孔多孔钛作为其皮下固定材料, 表面预沉积一层类骨磷灰石, 空白样品作为对照, 皮下植入兔子的背部. 术后40天和70天对样品与周围软组织间的结合强度进行测定, 并对断口处组织与材料间的结合形貌和区域成分进行分析. 结果表明, 随着时间的延长, 不同表面特征的材料与组织的结合强度都不断增大; 表面具有类骨磷灰石层的样品无论是结合强度还是结合形态都优于空白样品, 涂层样品40天时与组织的结合强度几乎与对照样品70天时与组织的结合强度相当. 说明类骨磷灰石层在体内有利于较早形成活跃的细胞材料表面, 有助于皮下结缔组织的长入以及与材料表面的牢固结合.     

Ultrastructural Analysis on the Osteogenesis and Transformation of Calcium Phosphate Ceramics in Vivo

To study the osteogenesis and transformation process of calcium phosphate bioceramic in vivo, biodegradable porous β-tricalcium phosphate ceramics (β-TCP, φ5×8 mm) were implanted in the tibia of rabbits. β-TCP ceramics with surrounding bone tissue were retrieved and observed by SEM, TEM and EPMA every month after implantation.The results showed that osteogenesis was active and β-TCP ceramics bonded to bones directly. The new bones were forming and maturing as materials were continuously degrading, and materials were finally replaced by new bone. Parts of the materials were degraded, absorbed and recrystallized, while the rest were dispersed to the spongy bone and the Haversian lamella in an irregular arrangement, becoming incorporated into bone formation directly by remodeling the structure. Some β-TCP crystals cleaved along its (001) rhombohedral plane and formed lath-like crystals in vivo.     

Effect of silicate incorporation on in vivo responses of α-tricalcium phosphate ceramics

In addition to calcium phosphate-based ceramics, glass-based materials have been utilized as bone substitutes, and silicate in these materials has been suggested to contribute to their ability to stimulate bone repair. In this study, a silicate-containing α-tricalcium phosphate (α-TCP) ceramic was prepared using a wet chemical process. Porous granules composed of silicate-containing α-TCP, for which the starting composition had a molar ratio of 0.05 for Si/(P + Si), and silicate-free α-TCP were prepared and evaluated in vivo. When implanted into bone defects that were created in rat femurs, α-TCP ceramics either with or without silicate were biodegraded, generating a hybrid tissue composed of residual ceramic granules and newly formed bone, which had a tissue architecture similar to physiological trabecular structures, and aided regeneration of the bone defects. Supplementation with silicate significantly promoted osteogenesis and delayed biodegradation of α-TCP. These results suggest that silicate-containing α-TCP is advantageous for initial skeletal fixation and wound regeneration in bone repair.     

In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/     

A long-term evaluation of osteoinductive HA/β-TCP ceramics in vivo: 4.5 years study in pigs

It has been proved that some material-dependent calcium phosphate ceramics have intrinsic potentials to induce osteogenesis. But there is little literature concerning about the tissue response in long-term. The aim of this study is to evaluate the safety of the osteoinductive biocreamics and the stability of the newly formed bone after long-term tissue response. Porous calcium phosphate ceramics rods which contain hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) were implanted in the dorsal muscles of Banna Minipig Inbreding Line. After 4.5 years, all the implanted rods with surrounding tissues were harvested and stained with hematoxylin and eosin for histological observation. The 7 months’ rods were also harvested as short-term comparison. The histological results showed that compared with the short-term rods, amount of bone tissue formed after 4.5 years. And the newly formed bone in this bioceramics neither disappeared nor gave rise to uncontrolled growth. The bone growth in this bioceramics seemed to be self-confined. The surrounding soft tissues were normal and no tumor cell was found. We conclude that instead of disappearing or giving rise to out of control, the induced bone tissue trends to be further matured. And this bioceramics thus might have potentials in future clinical use.     

Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

Abstract

Because calcium phosphate (Ca–P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca–P ceramics with different Ca–P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca–P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca–P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca–P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca–P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.     

β-磷酸三钙/聚L-乳酸与大鼠骨膜成骨细胞复合骨修复材料的研究

采用溶液浇铸-模压成型-沥滤方法制备了β-TCP/PLLA多孔支架材料, 将支架材料与大鼠骨膜成骨细胞复合获得新型组织工程骨修复材料. 通过抗压强度及压缩模量的表征研究了支架材料的力学性能; 采用SEM观测、MTT法、碱性磷酸酶活性及骨钙素分泌量检测细胞复合材料的体外成骨特性; 通过裸鼠肌袋种植, 以组织学方法评价细胞复合材料的异位成骨能力. 结果表明: β-TCP/PLLA多孔支架材料孔隙率可调, 孔径为100～00μm, 孔道相互贯通; 材料抗压强度和压缩模量随孔隙率的增大而降低, β-TCP复合PLLA后材料的力学性能高于同孔隙率的纯PLLA多孔材料; 复合支架材料适宜骨膜成骨细胞粘附和生长, 无细胞毒性; 骨膜成骨细胞复合β-TCP/PLLA支架材料的体外成骨特性良好, 且具有体内异位成骨能力.     

Processing, physico-chemical characterisation and in vitro evaluation of silicon containing β-tricalcium phosphate ceramics

For bone grafting applications, the elaboration of silicon containing beta-tricalcium phosphate (β-TCP) was studied. The synthesis was performed using a wet precipitation method according to the hypothetical theoretical formula Ca_3 − x(PO₄)_2 − 2x(SiO₄)_x. Two silicon loaded materials (0.46 wt.% and 0.95 wt.%) were investigated and compared to a pure β-TCP. The maturation time of the synthesis required in order to obtain β-TCP decreased with the amount of silicon. Only restrictive synthesis conditions allow preparing silicon containing β-TCP with controlled composition. To obtain dense ceramics, the sintering behaviour of the powders was evaluated. The addition of silicon slowed the densification process and decreased the grain size of the dense ceramics. Rietveld refinement may indicate a partial incorporation of silicon in the β-TCP lattice. X-ray photoelectron spectroscopy and transmission electron microscopy analyses revealed that the remaining silicon formed amorphous clusters of silicon rich phase. The in vitro biological behaviour was investigated with MC3T3-E1 osteoblast-like cells. After the addition of silicon, the ceramics remained cytocompatible, highlighting the high potential of silicon containing β-TCP as optimised bone graft material.     

Clinical evaluation of β-TCP in the treatment of lacunar bone defects: A prospective,randomized controlled study

The aim of this study was to investigate the potential wide application of beta tricalcium phosphate (β-TCP) only for bone defects as compared to allograft. 95 patients with a solitary bone cyst were randomly assigned to the treatment. A new radiographic scoring system was employed to calculate the biodegradation of bone graft and to evaluate the influence of multiple factors. At an average of 28.43 months after surgery, a radiographic semi-quantitative analysis revealed that the degradation rates of β-TCP and the allograft were comparable (p > 0.05). Age, complication, packing methods and granule diameters have a significant influence on β-TCP degradation. The loose packing method and 3–5 mm granule size should be employed in clinical practice. A histological analysis of biopsy showed that β-TCP supported the growth of fibrous tissue, vascular tissue, as well as bone tissue into the implants. The results proved that single β-TCP is an advantageous alternative to allografts for lacunar bone defect repair and would well guide the design and clinical application of the β-TCP.     

Effects of MgO addition on sintering of calcium phosphate ceramics and composites

Bioactive ceramics attracts much attention as materials for bone implants, because of their high biocompatibility. For example, hydroxyapatite (HA) has bone-bonding ability through a bone-like apatite layer in body environment and β-tricalcium phosphate (β-TCP) has a high bioresorbability in body environment. In addition, HA/β-TCP composites has the characteristics of both HA and TCP. However, it is difficult to sinter the composite, so that MgO has been used as a sintering agent. In the present study, effects of MgO addition on sintering calcium phosphate ceramics and composites were investigated. In order to evaluate the effect of MgO on the composites, HA, HA/β-TCP(30wt%), and HA/β-TCP(50wt%) with 1wt% MgO were prepared and characterized. To clarify the role of MgO on sintering of calcium phosphate ceramics, HA, β-TCP, and α-TCP with different TCP content (0, 1, 2, 3, 4, and 5 wt%) were also prepared. The results suggest that MgO addition densified HA/β-TCP composites and gave higher strength composites. The results of monolithic calcium phosphate ceramics indicated MgO addition was effective on β-TCP and α-TCP, not on HA. The maximum content of Ca atom in β-TCP displaced with Mg atoms in MgO might be 24 atm%.