磷酸钙骨水泥生物材料用磷酸四钙的制备

采用CaCO3和CaHPO4的混合粉分别在空气和真空2种气氛条件下制备磷酸四钙。结果表明:在空气中较难制得磷酸四钙,这主要是由于潮湿的空气中含有较多水分和制备工艺中采取随炉缓慢冷却,对反应产生了不利影响;在真空条件下,采用n(Ca)∶n(P)=2∶1,1.8∶1和1.5∶1的3种混合粉均容易制得磷酸四钙,但同时都含有其它杂质相,对其纯度有影响,其中以n(Ca)∶n(P)=2∶1的混合粉制备的磷酸四钙纯度最高,其产物中仅含少量CaO杂质相,这种磷酸四钙可用于磷酸钙骨水泥。     

Formation of calcium-deficient hydroxyapatite from alpha-tricalcium phosphate

This study investigated the factors influencing the kinetics of Ca9HPO4(PO4)5OH (calcium deficient hydroxyapatite or CDHAp) formation from alpha-Ca3(PO4)2 (alpha-TCP). The kinetics of CDHAp formation were investigated by isothermal calorimetry at constant temperatures ranging between 30 and 75 degrees C and by changes in pH at 37.4 and 70 degrees C. The calorimetric curves were characterized by two reaction peaks. Activation energies were calculated for the events resulting in these peaks. Values obtained were 48.4 and 67.7 kJ mol(-1), respectively, indicating nucleation and growth mechanisms for both events. Temperature had a significant effect on the growth rate as indicated by a decrease in surface area (26.5-15.0 m2 g(-1)) of the CDHAp with increasing temperature (30-75 degrees C). A linear relationship between hydrolysis temperature and CDHAp surface area was observed. The morphology of the CDHAp was plate-like and the crystallites became more regular as the reaction temperature was increased. A rapid elevation in pH upon mixing with water indicated the synthesis method initially used did not entirely eliminate slight compositional variations within the alpha-Ca3(PO4)2. Rapid elevation in pH retarded subsequent reaction. This effect was eliminated by increasing the duration of high-temperature firing during alpha-TCP synthesizing.     

成骨细胞复合原位成型磷酸钙骨水泥的细胞学研究

目的:通过体外细胞培养实验,观察分析壳聚糖微球/磷酸钙骨水泥、β-磷酸三钙(β-tricalcium phosphate,β-TCP)/磷酸钙骨水泥、含钾磷酸钙骨水泥浆料固化过程对成骨细胞的影响以及固化后10 d细胞的生物活性变化.方法:首先以兔的骨髓为组织来源,采用密度梯度分离法和贴壁分离法分离培养原代兔骨髓基质细胞(rabbit marrow stromal cell,rMSC),通过流式细胞分析和分选得到成分比较单一的兔骨髓基质细胞,经过体外诱导得到兔成骨细胞,用茜素红染色法验证其成骨功能.将rMSC分别与上述3种骨水泥同化块及其浆料复合培养,空白对照组是直接在24孔板上接种细胞,每组4个样本.在复合培养的第1、4、7、10天,通过酸性磷酸酶(acid phosphatase assay,APA)和碱性磷酸酶(alkaline phosphatase,ALP)活性测定,检测细胞的增殖和分化能力;吖啶橙(acridine or-ange,AO)染色后荧光显微镜观察,对细胞进行计数并分析.环境扫描电子显微镜观察细胞在材料表面的生长、黏附状况.各组结果进行双因素方差分析,用LSD法进行组问比较.结果:3种骨水泥浆料在同化过程中均对rMSC有较大影响,细胞的增殖活性(复合培养第10天APA活性浆料组吸光度平均值分别为0.049,0.050,0.049;固化块组分别为0.898,0.867,0.909;P<0.001)和分化能力(复合培养第10天ALP活性浆料组平均值分别为0.775,0.782.0.798 U/g protein;固化块组分别为49.288,49.631,49.744 U/g protein;P<0.001)均明显低于固化块组,细胞数目也明显减少(复合培养第10天细胞数目每视野平均为3.7,3.7,3.7个;固化块组分别为91.1,89.7,93.7个,P<0.001),且这种影响是不可恢复的;rMSC在3种骨水泥的同化块上能较好地黏附,细胞数目、增殖和分化能力基本不受影响.结论:可注射性磷酸钙骨水泥浆料固化过程对成骨细胞有较大的影响,用成骨细胞复合浆料前需对细胞采取保护措施.     

Growth and differentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements

Materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications for bone regeneration and for use in skeletal reconstruction and joint replacement. We have studied the growth and interactions of human bone marrow cells on a variety of new cement composites in vitro. These cement materials are composed of calcium-deficient hydroxyapatites, carbonated apatite and amorphous calcium phosphate. Cell proliferation was significantly reduced and cell differentiation increased in the presence of these cements compared with cells cultured on tissue culture plastic. Alkaline phosphatase, one of the markers of the osteoblast phenotype, was dramatically stimulated by 3 of the 4 cements examined between day 4 and day 10, above levels observed following culture of human osteoblasts on plastic alone. Photomicroscopic examination demonstrated growth and close integration of bone marrow cells and 3 of the composites. Longer term marrow cultures (15 day) on the cements confirmed the stimulation of cell differentiation over proliferation. From these studies, enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas, cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro culture system demonstrates the use of human bone marrow cells for the potential evaluation of new biomaterials and the development of a novel carbonated apatite that may be of potential use in orthopaedic implants.     

磷酸钙骨水泥/聚乳酸-聚羟基乙酸复合材料的制备及性能

为深入研究磷酸钙骨水泥(CPC)/聚乳酸-聚羟基乙酸(PLGA)生物复合材料的制备工艺及性能,也为临床应用提供理论依据,实验制备了CPC-PLGA生物复合材料;并通过测定该材料在模拟体液中浸泡后引起的浸泡液pH值的变化,以及采用JSM 5600LV型扫描电镜观察该材料在体外降解后的微观形貌,间接评估了该材料引起体液pH值的变化以及该材料降解性能对人体细胞和骨组织可能存在的影响。结果表明,所制备的CPCPLGA复合材料没有引起浸泡液pH值的较大波动,该值始终处于人体安全范围之内,对人体细胞的刺激影响将会较小;由于该材料复合了高分子聚合物PLGA,使其具有良好的降解性,可为骨组织细胞、血管等的粘附和生成创造良好的条件。     

Histological, chemical, and crystallographic analysis of four calcium phosphate cements in different rabbit osseous sites

Four calcium phosphate cement formulations were implanted in the rabbit distal femoral metaphysis and middiaphysis. Chemical, crystallographic, and histological analyses were made at 2, 4, and 8 weeks after implantation. When implanted into the metaphysis, part of the brushite cement was converted into carbonated apatite by 2 weeks. Some of the brushite cement was removed by mononuclear macrophages prior to its conversion into apatite. Osteoclastlike cell mediated remodeling was predominant at 8 weeks after brushite had converted to apatite. The same histological results were seen for brushite plus calcite aggregate cement, except with calcite aggregates still present at 8 weeks. However, when implanted in the diaphysis, brushite and brushite plus calcite aggregate did not convert to another calcium phosphate phase by 4 weeks. Carbonated apatite cement implanted in the metaphysis did not transform to another calcium phosphate phase. There was no evidence of adverse foreign body reaction. Osteoclastlike cell mediated remodeling was predominant at 8 weeks. The apatite plus calcite aggregate cement implanted in the metaphysis that was not remodeled remained as poorly crystalline apatite. Calcite aggregates were still present at 8 weeks. There was no evidence of foreign body reaction. Osteoclastlike cell remodeling was predominant at 8 weeks. Response to brushite cements prior to conversion to apatite was macrophage dominated, and response to apatite cements was osteoclast dominated. Mineralogy, chemical composition, and osseous implantation site of these calcium phosphates significantly affected their in vivo host response.     

In vitro evaluation of a new injectable calcium phosphate material

BACKGROUND: Verbal autopsies have been widely used to determine the levels and causes of maternal death but few studies have assessed the reliability of various methods. METHODS: We compared the levels and causes of maternal mortality in three data sources from Matlab, Bangladesh: (1) maternal deaths identified through a unique demographic surveillance system (DSS); (2) maternal deaths identified as a result of a previous detailed investigation into the levels and causes of maternal mortality; and (3) maternal deaths identified in the current special study. All studies used lay reporting, but differed in terms of the nature of the study, the sex of the interviewer, the format of the questionnaire and the procedure to derive the diagnosis. RESULTS: There were substantial disagreements between the routine reporting and the special studies. The DSS identified 67.2% of all deaths occurring during pregnancy or within 42 days postpartum (82.3% of direct obstetric deaths, 70.0% of deaths due to induced abortions and 42.4% of indirect obstetric deaths). Extending the definition of maternal deaths to 90 days postpartum increased the numbers of maternal deaths between 1987 and 1993 from 174 to 196. The two special studies also disagreed in the ascertainment of the causes of maternal deaths and yielded different cause of death distributions; the proportion of direct obstetric deaths (excluding abortion) was 50.4% in the current system compared to 44.5% previously (P = 0.001). CONCLUSIONS: This study confirms the known difficulties in the ascertainment of the levels and causes of maternal mortality. The large disparities in the levels and causes of maternal mortality using three different methods of lay reporting in a population with an almost complete vital registration system add to the growing concern about the inaccuracies in the measurement of maternal mortality.     

Mechanical and histological evaluation of amorphous calcium phosphate and poorly crystallized hydroxyapatite coatings on titanium implants

The effect of amorphous calcium phosphate (Ca/P) and poorly crystallized (60% crystalline) hydroxyapatite (HA) coatings on bone fixation to "smooth" and "rough" (Ti-6A1-4V powder sprayed) titanium-6Al-4V (Ti) implants was investigated. Implants were evaluated histologically, mechanically, and by scanning electron microscopy (SEM) after 4 and 12 weeks of implantation in a rabbit transcortical femoral model. Histological evaluation of amorphous vs. poorly crystallized HA coatings showed significant differences in bone apposition (for rough-coated implants only) and coating resorption (for smooth- and rough-coated implants) that were increased within cortical compared to cancellous bone. The poorly crystallized HA coatings showed most degradation and least bone apposition. Mechanical evaluation, however, showed no significant differences in push-out shear strengths between the two types of coatings evaluated. Differences between 4 and 12 weeks were significant for coating resorption and push-out shear strength but not for bone apposition. Significant enhancement in interfacial shear strengths for bioceramic coated as compared to uncoated implants were seen for smooth-surfaced implants (3.5-5 times greater) but not for rough-surfaced implants at 4 and 12 weeks. Rough implants showed greater mean interfacial strengths than uncoated smooth implants at 4 and 12 weeks (seven times greater) and to coated smooth implants at 12 weeks only (two times greater). Mechanical failure of the bone/coating/implant interface consistently occurred within the bone, even in the case of the poorly crystallized HA coatings, which had almost completely resorbed on rough implants. These results suggest that once early osteointegration is achieved biodegradation of a bioactive coating should not be detrimental to the bone/coating/implant fixation.     

Surface-induced mineralization: a new method for producing calcium phosphate coatings

Calcium phosphate coatings were nucleated and grown from aqueous solution onto titanium metal substrates via surface-induced mineralization (SIM) processing techniques. This process is based on the observation that in nature organisms use biopolymers to produce ceramic composites, such as teeth, bones, and shells. The SIM process involves modification of a surface to introduce surface functionalization followed by immersion in aqueous supersaturated calcium phosphate solutions. This low-temperature process (< 100 degrees C) has advantages over conventional methods of calcium phosphate deposition in that uniform coatings are produced onto complex-shaped and/or microporous samples. Additionally, because it is a low-temperature process, control of the phase and crystallinity of the deposited material can be maintained.     

Oral calcium phosphate: a new therapy for Crigler-Najjar disease?

BACKGROUND & AIMS: Calcium phosphate binds unconjugated bilirubin in vitro, and dietary calcium phosphate supplementation reduces the serum bilirubin level in rats with hereditary unconjugated hyperbilirubinemia (Gunn rats). The aim of this study was to evaluate the effect of oral calcium phosphate supplementation on plasma bilirubin levels in patients with Crigler-Najjar disease. METHODS: A placebo-controlled, double-blind, crossover design was used. Eleven patients, 2-42 years of age, participated. The group included 5 patients with type I disease who were all treated with phototherapy and 6 patients with type II disease who were primarily treated with phenobarbital. In addition to plasma bilirubin levels, dietary intake and urinary and fecal excretion of calcium and phosphate were evaluated. RESULTS: A modest but significant decrease in serum bilirubin was observed in patients with type I disease (18% +/- 6%, P = 0.03) but not in patients with type II disease during treatment with calcium phosphate. Urinary output of calcium and phosphate did not change during the treatment period. CONCLUSIONS: Oral calcium phosphate may be a useful adjuvant to photo-therapy in Crigler-Najjar type I disease.     

Reactivity of fluoride-containing calcium aluminosilicate glasses used in dental glass-ionomer cements

The glass component critically determines the properties of glass-ionomer cements (GIC). However, the exact relationship between the composition of the glass and these properties is not yet fully understood. To investigate this relationship, we studied the reactivity of glasses used in commercial GIC in acetic acid solutions, using a pH-stat method. Qualitative differences in the leaching behavior of these glasses can be explained by different pre-treatments. Acid-washing and silanization modify the surfaces of the glass particles, thus inducing a delay of the leaching process, whereas untreated glasses exhibit a fast initial leaching, but their acid reactivity slows very soon. Quantitative differences in acid reactivity can be correlated with the mean chemical composition of the glasses. In this respect, the leaching tends to increase with an increasing ratio of network-dwelling cations to Al3+ ions. These results provide a fundamental basis for the explanation, prediction, and control of cement properties as a function of glass characteristics.     

Resorbable calcium phosphate bone substitute

Bacteroides fragilis strains isolated from different sources, i.e. 1 strain (AA1) from an aquatic environment, 1 strain from normal flora (118310) and the type strain (ATCC 25285) originally isolated from clinical material, were analysed for both cell envelope proteins composition and surviving under oxidative stress starvation. All strains examined showed a similar survival response when cultured in drinking water with a ten-fold decrease in viable counts per day during the 7 days of analysis. The outer membrane protein (OMP) profiles of all strains were quite similar during the stress period as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the periplasmic proteins of the strain 118310 showed two protein bands at 48 and 58 kDa, respectively, that were absent in the strains AA1 and ATCC 25285 during the incubation period in potable water. Whole cells and periplasmic 35S-labelled proteins from bacteria cultured in drinking water showed a significant increase in proteins at 16, 18, 24, 26, 35, 48, and 58 kDa and 18, 22, 24, 48, 58, and 70 kDa, respectively, in all strains when compared to cells grown in BHI-PRAS media as detected by autoradiography following SDS-PAGE. These data suggest that B. fragilis may have a synthesis mechanism that allows them to adapt to adverse environments.     

Evaluation of cytotoxicity of calcium phosphate cement consisting of alpha-tricalcium phosphate and dicalcium phosphate dihydrate

Elimination of the data processing bottleneck in high-throughput sequencing will require both improved accuracy of data processing software and reliable measures of that accuracy. We have developed and implemented in our base-calling program phred the ability to estimate a probability of error for each base-call, as a function of certain parameters computed from the trace data. These error probabilities are shown here to be valid (correspond to actual error rates) and to have high power to discriminate correct base-calls from incorrect ones, for read data collected under several different chemistries and electrophoretic conditions. They play a critical role in our assembly program phrap and our finishing program consed.     

Interface potential of calcium phosphate in simulated body fluid

Calcium phosphate ceramics are used in the substitution of injured or damaged bones. Nevertheless, the behaviour of these materials, and in particular, the mechanisms guiding their interface response in physiological environment is still unknown. This work describes the construction of hydroxyapatite and tricalcium phosphate electrodes used to determine the interface potential behaviour of these materials in a simulated body fluid, in a pH range corresponding to the variation observed in human body injuries, at ambient and physiological temperatures. These measurements are associated with the adsorption/desorption of ions from the materials. The results show that hydroxyapatite and tricalcium phosphate have similar behaviour in that they reach an interface potential equilibrium state faster when the solution pH is decreased and the temperature increased. This behaviour may be attributed to their ability to form a calcium-rich layer and is relevant to their quality as implantable materials.     

Interaction of human gallbladder mucin with calcium hydroxyapatite: binding studies and the effect on hydroxyapatite formation

Calcium hydroxyapatite (HAP) crystals formed in vitro in the presence of polymeric human gallbladder mucin (1.0 mg/mL) were smaller (0.75 +/- 0.39 microns) than control crystals (7.86 +/- 2.76 microns), but the mucin did not affect the kinetics of crystal formation or alter the amount of mineral phase present at equilibrium. In contrast, glycopeptide subunits produced by proteolysis of the native mucin had no effect on HAP crystal size. Both native mucin and glycopeptides bound to mature HAP crystals, but the glycopeptides were much more readily displaced by phosphate ions. Therefore, in experiments where HAP was being formed, the phosphate ions inhibited the interaction of glycopeptides with the nascent HAP. These results indicate that gallbladder mucin may modulate HAP formation in vivo, and that this ability may be altered during pathological states, such as neutrophil infiltration or bacterial colonization, that may cause the release of proteinases capable of digesting mucin.     

Serum calcium, phosphate and alkaline phosphate levels in epileptic children treated with phenobarbitone

A longitudinal study to estimate the serum calcium, phosphate and alkaline phosphatase levels of 89 ambulatory epileptic children, aged between 3 years and 12 years, and having generalised tonic-clonic seizures, was carried out. None was on any form of medication for the treatment of seizures prior to presentation. Each patient received only phenobarbitone during the period of study. Serum levels of the biochemical parameters were determined at presentation, 6 months and 12 months, while serum phenobarbitone levels were estimated at 6 months and 12 months. Mean serum calcium, phosphate and alkaline phosphatase of the patients remained within the normal range. Using the paired 't' test, the differences in the levels of the parameters at the three measurements were not statistically significant (P > 0.05). Serum phenobarbitone levels remained within the therapeutic range during the period of study. Our results show that over a 12-month period, serum levels of calcium, phosphate, and alkaline phosphatase, remain normal in ambulant epileptic children treated with phenobarbitone.     

Liposomes-coated hydroxyapatite and tricalcium phosphate implanted in the mandibular bony defect of miniature swine

Mutations, expected to affect the intracellular routing, i.e. additional nuclear localization sequences (NLS; the natural 23 kDa isoform and a 17D27R mutant) and/or a deletion of amino acids 26-29 (23 delta 26-29 and 17 delta 26-29), were introduced in basic fibroblast growth factor (bFGF). The mutants were assayed for their mitotic activity and their capacity to induce a tissue-specific response in human umbilical vein endothelial cells [HUVECs; induction of urokinase plasminogen activator receptor (u-PAR)], or in rat lens epithelial cells (fibre cell differentiation). In HUVECs, the 17D27R mutant had wild type activity, the 23 kDa and the delta 26-29 proteins were impaired in the induction of both mitosis and u-PAR. The delta 26-29 proteins, but not the 23 kDa protein or 17D27R mutant, were also impaired in receptor binding in that they bound only to a subset of receptors. The concentration of 17 kDa bFGF required for half maximal u-PAR response was 30 fold higher than for the half maximal 3H-thymidine incorporation. Addition of an NLS to bFGF strongly inhibited the induction of fibre cell differentiation, though it had little effect on the stimulation of DNA synthesis. The 17 delta 26-29 kDa mutant had wild type differentiation activity but was a poor mitogen for lens epithelial cells.