磷酸钙骨水泥的生物学研究进展

本文综述了磷酸钙骨水泥作为一种新型人工骨替代材料近年来的生物学基础及提高其生物学性能的研究 ,阐明了磷酸钙骨水泥以其良好的生物学特性 ,使其在骨缺损修复领域具有广阔的应用前景。     

Development of a new calcium phosphate cement that contains sodium calcium phosphate

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.     

Tissue response to a newly developed calcium phosphate cement containing succinic acid and carboxymethyl-chitin

We developed a new calcium phosphate cement containing succinic acid and carboxymethyl-chitin in the liquid component. In this study, the biocompatibility and osteoconductivity of this new cement were investigated. After mixing, cement in putty form was implanted immediately between the periosteum and parietal bone and in the subcutaneous tissues of rats. In control cement, distilled water was used instead of the liquid component. In addition to histological evaluations, analyses with X-ray diffraction and Fourier transform infrared were performed for the subcutaneously implanted cements. Histological examination showed slight inflammation around the new cement on the bone and in the subcutaneous tissue at 1 week after surgery. At 2 weeks, the cement was partially bound to the parietal bone. The extent of the surface of the new cement directly in contact with the bone increased with time, and most of the undersurface of the new cement bound to the host parietal bone by 8 weeks. Analysis by X-ray diffraction showed that the new cement in the subcutaneous tissue was transformed into hydroxyapatite by 8 weeks. These results indicate that this new calcium phosphate cement is useful as a bone substitute material.     

Self-setting kinetics of new type calcium phosphate bioactive bone cement: a thermokinetics study

Thermokinetics method was used to study the self-setting kinetics of a new kind of calcium phosphate cement (CPC) in the present study. A calcium-deficient hydroxyapatite CPC was developed by using alpha-TCP and other calcium phosphate bioceramics. The mixing liquids used were deionized water and 0.25 M NaH2PO4/Na2HPO4, respectively. The calorimetric curves, heat evolution curves and total heat evolution in the setting and hardening process of CPC were determined. It has been found that mixing liquids, reaction temperature had influences on the calorimetric curves and heat evolution, and mixing liquids exhibited the greatest influence on the kinetics of CPC during the self-setting and hardening process. Based on the calorimetric curves obtained, the kinetic model equation was simulated, and the reaction control step was determined.     

Biocompatibility and resorption of a brushite calcium phosphate cement

A hydraulic calcium phosphate cement with beta-tricalcium phosphate (TCP) granules embedded in a matrix of dicalcium phosphate dihydrate (DCPD) was implanted in experimentally created defects in sheep. One type of defect consisted of a drill hole in the medial femoral condyle. The other, partial metaphyseal defect was located in the proximal aspect of the tibia plateau and was stabilized using a 3.5 mm T-plate. The bone samples of 2 animals each per group were harvested after 2, 4, 6 and 8 weeks. Samples were evaluated for cement resorption and signs of immediate reaction, such as inflammation, caused by the cement setting in situ. Differences regarding these aspects were assessed for both types of defects using macroscopical, radiological, histological and histomorphometrical evaluations. In both defects the brushite matrix was resorbed faster than the beta-TCP granules. The resorption front was followed directly by a front of new bone formation, in which residual beta-TCP granules were embedded. Cement resorption occurred through (i) extracellular liquid dissolution with cement disintegration and particle formation, and (ii) phagocytosis of the cement particles through macrophages. Signs of inflammation or immunologic response leading to delayed new bone formation were not noticed at any time. Cement degradation and new bone formation occurred slightly faster in the femur defects.     

A self-setting single-component calcium phosphate cement

Following an original synthesis route, we have prepared a single-component calcium phosphate apatite-like powder which settles and hardens when mixed with deionized water in an approximative 1.2 g:1 ml ratio. This paper describes the first physico-chemical studies and characterizations of the material. Observations of its in vitro behavior show a slight volume contraction and toxicity against fibroblasts bone marrow cells on disks of compacted powder. It is suggested that after an improvement of the powder characters such as grain size, and the choice of another hardening liquid, to name a few, this material should be a potential--or an ingredient of--bone cement.     

Fiber-enriched double-setting calcium phosphate bone cement

Calcium phosphate bone cements are useful in orthopedics and traumatology, their main advantages being their biocompatibility and bioactivity, which render bone tissue osteoconductive, providing in situ hardening and easy handling. However, their low mechanical strength, which, in the best of cases, is equal to the trabecular bone, and their very low toughness are disadvantages. Calcium phosphate cement compositions with mechanical properties more closely resembling those of human bone would broaden the range of applications, which is currently limited to sites subjected to low loads. This study investigated the influence of added polypropylene, nylon, and carbon fibers on the mechanical properties of double setting alpha-tricalcium phosphate-based cement, using calcium phosphate cement added to an in situ polymerizable acrylamide-based system recently developed by the authors. Although the addition of fibers was found to reduce the compression strength of the double-setting calcium phosphate cement because of increased porosity, it strongly increased the cement's toughness (J(IC)) and tensile strength. The composites developed in this work, therefore, have a potential application in shapes subjected to flexure.     

Cell seeding into calcium phosphate cement

To improve the effectiveness of calcium phosphate cement (CPC), we have developed a method to seed osteoblasts into the cement. CPC powder is mixed with water to form a paste that can be shaped to fit a bone defect in situ. The paste hardens in 30 min, reacts to form hydroxyapatite, and is replaced with new bone. Reacted CPC is biocompatible but unreacted CPC paste was found to have toxic effects when placed on cell monolayers (MC3T3-E1 cells). In contrast, when cells were indirectly exposed to CPC paste using a porous membrane or by placing a coverslip containing adherent cells onto a bed of CPC paste, the unreacted CPC was nontoxic. These results suggested that gel encapsulation of the cells might protect them from the CPC paste. Thus, cells were encapsulated in alginate beads (3.6-mm diameter), mixed with CPC paste, and incubated overnight. Both vital staining (calcein-AM and ethidium homodimer-1) and the Wst-1 assay (measures dehydrogenase activity) showed that cell survival in alginate beads that were mixed with CPC was similar to survival in untreated control beads. These results suggest that gel encapsulation could be used as a mechanism to protect cells for seeding into CPC.     

Factors influencing calcium phosphate cement shelf-life

Long-term stability during storage (shelf-life) is one major criterion for the use of a material as medical device. This study aimed to investigate the ageing process of beta-tricalcium phosphate/monocalcium phosphate cement powders when stored in sealed containers at ambient conditions. This kind of cement type is of interest because it is forming dicalcium phosphate dihydrate (brushite) when set, which is in contrast to hydroxyapatite resorbable in physiological conditions. The stability of cements was checked by either measuring the phase composition of powders as well as the setting time and compressive strength when mixed with sodium citrate as liquid. Critical factors influencing ageing were found to be temperature, humidity and the mixing regime of the powders. Mechanically mixed cement powders which were stored in normal laboratory atmosphere (22 degrees C, 60% rel. humidity) converted to dicalcium phosphate anhydrous (monetite) within a few days; this could be mechanistically related to a dissolution/precipitation process since humidity condensed on the particles' surfaces and acted as reaction medium. Various storage conditions were found to be effective in prolonging cement stability which were in order of effectiveness: adding solid citric acid retardant>dry argon atmosphere=gentle mixing (minimal mechanical energy input) low temperature.     

Transmission electron microscopic study on setting mechanism of tetracalcium phosphate/dicalcium phosphate anhydrous-based calcium phosphate cement

This work studied transmission electron microscopy on the setting mechanism of tetracalcium phosphate/dicalcium phosphate anhydrous (TTCP/DCPA)-based calcium phosphate cement. The results suggest the process for early-stage apatite formation as the follows: when TTCP and DCPA powders are mixed in the phosphate-containing solution, the TTCP powder is quickly dissolved because of its higher solubility in the acidic solution. The dissolved calcium and phosphate ions, along with those ions readily in the solution, are then precipitated predominantly on the surface of DCPA particles. Few apatite crystals were observed on the surface of TTCP powder. During the later stages of reaction, the extensive growth of apatite crystals/whiskers, with a calcium/phosphorous ratio very close to that of hydroxyapatite, effectively linked DCPA particles together and also bridged the larger TTCP particles. It is suggested that, when the large TTCP particles are locked in place by the bridging apatite crystals/whiskers, the CPC is set and would not dissolve when immersed in Hanks' solution after 20-40 min of reaction.     

Immersion behavior of gelatin-containing calcium phosphate cement

Calcium phosphate cements (CPCs) have many favorable properties that support their clinical use as bone defect repair. However, it is difficult to deliver to the required site and hard to compact adequately due to inherently low ductility of ceramics. The aim of this study focused on the effect of the gelatin content on properties of CPCs. The diametral tensile strength, morphology, and weight loss of gelatin cements were evaluated after immersion in physiological solution, in addition to setting time. The results indicated that the setting time significantly increased with increasing gelatin amount. The 2 wt.% gelatin could make CPCs attain the maximum strength value of 2.1 MPa at 15-day immersion, while 1.6 MPa for the cement without gelatin. It is concluded that the presence of gelatin improved mechanical properties of CPCs; in particular, 2 wt.% gelatin. CPCs containing 2 wt.% gelatin hardened in an acceptable time recommended for clinical applications.     

Self-setting properties of a beta-dicalcium silicate reinforced calcium phosphate cement

Beta-dicalcium silicate was used to reinforce the injectable calcium phosphate cement (iCPC) for the first time in this study. The influence of the content of beta-dicalcium silicate on the mechanical properties, setting time, rheological properties, injectability, phase evolution, microstructure, and biodegradability of iCPC was systematically investigated. The results demonstrated that the addition of 8 wt % beta-dicalcium silicate obviously enhanced the compressive strength of the CPC from 26.5 to 47.5 MPa, and did not significantly influence the biodegradability, setting time, injectability, phase evolution, and microstructure of the CPC. The beta-dicalcium silicate-reinforced iCPC with relatively high mechanical property should have potential prospects for the wider applications in surgery such as orthopedics, oral, and maxillofacial surgery.     

Characterization of a novel calcium phosphate/sulphate bone cement

Apatitic cements have shown excellent biocompatibility and adequate mechanical properties but have slow resorption in the human body. To assure that new bone tissue grows into the bone defect, a certain porosity is necessary although hard to achieve in injectable cements with suitable mechanical properties. An attempt was made by mixing alpha-tricalcium phosphate (alpha-TCP), calcium sulphate hemihydrate (CSH) and an aqueous solution containing 2.5 wt% of Na(2)HPO(4). The aim was to obtain a material containing two phases: a) one apatitic phase (calcium-deficient hydroxyapatite; CDHA) and b) one resorbable phase (calcium sulphate dihydrate; CSD). alpha-TCP and CSH mixtures were produced at relative intervals of 20 wt%. The liquid-to-powder (L/P) ratio to obtain a paste was 0.32 mLg(-1). The highest compressive strength (34 MPa) was obtained for the pure alpha-TCP sample. The strength was, in a first approximation, directly correlated to the weight proportions of the powders. X-ray diffraction analysis showed that the relative intensity for CDHA increased linearly, and the one for CSD decreased exponentially, when the amount of alpha-TCP increased. Thus, CSH ceased to transform to CSD when the amount of alpha-TCP increased. Observations in environmental scanning electron microscopy confirmed the X-ray diffraction results. CSH-crystals (100 microm) were embedded in the HA-matrix permitting gradual porosity in the material when resorbed.     

新型可注射可降解磷酸钙骨水泥的生物相容性

背景：体外实验已证实新型磷酸钙骨水泥有良好的可注射性、力学性能、抗溃散性及体外降解性能。 目的：验证新型可注射、可降解磷酸钙骨水泥的生物相容性。 方法：①急性毒性实验：分别向昆明小鼠尾静脉可注射新型磷酸钙骨水泥浸提液与生理盐水。②热源实验：在新西兰兔耳缘静脉注射新型磷酸钙骨水泥浸提液。③溶血实验：在兔抗凝血分别加入新型磷酸钙骨水泥浸提液、生理盐水及双蒸水。④迟发型超敏反应实验：在豚鼠肩胛骨内侧部位分别注射可注射新型磷酸钙骨水泥浸提液与生理盐水,并进行敷贴激发实验。⑤体外细胞毒性实验：在L929系小鼠成纤维细胞株培养液中分别加入可注射新型磷酸钙骨水泥浸提液、聚乙烯浸提液及苯酚溶液。⑥微核实验：分别在昆明小鼠腹腔注射可注射新型磷酸钙骨水泥浸提液、生理盐水与环磷酰胺。⑦肌肉植入实验：将新型磷酸钙骨水泥植入新西兰兔脊柱两侧肌肉内。 结果与结论：新型可注射磷酸钙骨水泥无毒,无刺激性及致敏性,无热源反应,具有良好的血液相容性,植入动物肌肉后为非组织刺激物,具有良好的生物相容性,因而具有较好的生物安全性。     

Biocompatibility and resorption of a radiopaque premixed calcium phosphate cement

Calcium phosphate cements (CPC) are used as bone void filler in various orthopedic indications; however, there are some major drawbacks regarding mixing, transfer, and injection of traditional CPC. By using glycerol as mixing liquid, a premixed calcium phosphate cement (pCPC), some of these difficulties can be overcome. In the treatment of vertebral fractures the handling characteristics need to be excellent including a high radio-opacity for optimal control during injection. The aim of this study is to evaluate a radiopaque pCPC regarding its resorption behavior and biocompatibility in vivo. pCPC and a water-based CPC were injected into a ? 4-mm drilled femur defect in rabbits. The rabbits were sacrificed after 2 and 12 weeks. Cross sections of the defects were evaluated using histology, electron microscopy, and immunohistochemical analysis. Signs of inflammation were evaluated both locally and systemically. The results showed a higher bone formation in the pCPC compared to the water-based CPC after 2 weeks by expression of RUNX-2. After 12 weeks most of the cement had been resorbed in both groups. Both materials were considered to have a high biocompatibility since no marked immunological response was induced and extensive bone ingrowth was observed. The conclusion from the study was that pCPC with ZrO(2) radiopacifier is a promising alternative regarding bone replacement material and may be suggested for treatment of, for example, vertebral fractures based on its high biocompatibility, fast bone ingrowth, and good handling properties.     

Reinforcement of a self-setting calcium phosphate cement with different fibers

A water-based calcium phosphate cement (CPC) has been used in a number of medical and dental procedures due to its excellent osteoconductivity and bone replacement capability. However, the low tensile strength of CPC prohibits its use in many unsupported defects and stress-bearing locations. Little investigation has been carried out on the fiber reinforcement of CPC. The aims of the present study, therefore, were to examine whether fibers would strengthen CPC, and to investigate the effects of fiber type, fiber length, and volume fraction. Four different fibers were used: aramid, carbon, E-glass, and polyglactin. Fiber length ranged from 3-200 mm, and fiber volume fraction ranged from 1.9-9.5%. The fibers were mixed with CPC paste and placed into molds of 3 x 4 x 25 mm. A flexural test was used to fracture the set specimens and to measure the ultimate strength, work-of-fracture, and elastic modulus. Scanning electron microscopy was used to examine specimen fracture surfaces. Fiber type had significant effects on composite properties. The composite ultimate strength in MPa (mean +/- SD; n = 6) was (62+/-16) for aramid, (59+/-11) for carbon, (29+/-8) for E-glass, and (24+/-4) for polyglactin, with 5.7% volume fraction and 75 mm fiber length. In comparison, the strength of unreinforced CPC was (13+/-3). Fiber length also played an important role. For composites containing 5.7% aramid fibers, the ultimate strength was (24+/-3) for 3 mm fibers, (36+/-13) for 8 mm fibers, (48 +/-14) for 25 mm fibers, and (62+/-16) for 75 mm fibers. At 25 mm fiber length, the ultimate strength of CPC composite was found to be linearly proportional to fiber strength. In conclusion, a self-setting calcium phosphate cement was substantially strengthened via fiber reinforcement. Fiber length, fiber volume fraction, and fiber strength were found to be key microstructural parameters that controlled the mechanical properties of CPC composites.     

Incorporation of bioactive glass in calcium phosphate cement: An evaluation

Bioactive glasses (BGs) are known for their unique ability to bond to living bone. Consequently, the incorporation of BGs into calcium phosphate cement (CPC) was hypothesized to be a feasible approach to improve the biological performance of CPC. Previously, it has been demonstrated that BGs can successfully be introduced into CPC, with or without poly(d,l-lactic-co-glycolic) acid (PLGA) microparticles. Although an in vitro physicochemical study on the introduction of BG into CPC was encouraging, the biocompatibility and in vivo bone response to these formulations are still unknown. Therefore, the present study aimed to evaluate the in vivo performance of BG supplemented CPC, either pure or supplemented with PLGA microparticles, via both ectopic and orthotopic implantation models in rats. Pre-set scaffolds in four different formulations (1: CPC; 2: CPC/BG; 3: CPC/PLGA; and 4: CPC/PLGA/BG) were implanted subcutaneously and into femoral condyle defects of rats for 2 and 6 weeks. Upon ectopic implantation, incorporation of BG into CPC improved the soft tissue response by improving capsule and interface quality. Additionally, the incorporation of BG into CPC and CPC/PLGA showed 1.8- and 4.7-fold higher degradation and 2.2- and 1.3-fold higher bone formation in a femoral condyle defect in rats compared to pure CPC and CPC/PLGA, respectively. Consequently, these results highlight the potential of BG to be used as an additive to CPC to improve the biological performance for bone regeneration applications. Nevertheless, further confirmation is necessary regarding long-term in vivo studies, which also have to be performed under compromised wound-healing conditions.     

Cephalexin-loaded injectable macroporous calcium phosphate bone cement

Different types of calcium phosphate cements (CPCs) have been studied as potential matrices for incorporating different types of antibiotics. All of these matrices were morphologically microporous whereas macroporosity is essential for rapid cement resorption and bone replacement. In this study, liberation of cephalexin monohydrate (CMH) from a macroporous CPC was investigated over 0.5-300 h in simulated body fluid and some mathematical models were fitted to the release profiles. Macroporosity was introduced into the cement matrix by using sodium dodecyl sulfate molecules as air-entraining agents and the effect of both surfactant and CMH on basic properties of the CPC was studied. Incorporation of CMH into the CPC composition increased the setting time, decreased the crystallinity of the formed apatite phase, and improved the injectability of the paste. The use of both CMH and sodium dodecyl sulfate did not affect the rate of conversion of the reactants into apatite phase while soaking the cements in simulated body fluid. Results showed that the liberation rate of the drug from porous CPC was higher than that of the nonporous CPC but same release patterns were experienced in both types of cements, that is, like to nonporous CPC, a time-dependent controlled release of the incorporated drug was obtained from macroporous CPC. The Weibull model was the best fitting-equation for release profiles of all cements. The liberated CMH was as active as fresh cephalexin. It is concluded that this macroporous CPC can be successfully used as drug carrier with controlled release profile for the treatment of bone infections.     

Injectable and macroporous calcium phosphate cement scaffold

Calcium phosphate cement (CPC) can be molded and self-hardens in vivo to form resorbable hydroxyapatite with excellent osteoconductivity. The objective of this study was to develop an injectable, macroporous and strong CPC, and to investigate the effects of porogen and absorbable fibers. Water-soluble mannitol was used as porogen and mixed with CPC at mass fractions from 0% to 50%. CPC with 0-40% mannitol was fully extruded under a syringe force of 10 N. The paste with 50% mannitol required a 100-N force which extruded only 66% of the paste. At fiber volume fraction of 0-5%, the paste was completely extruded. However, at 6% and 7.5% fibers, some fibers were left in the syringe after the paste was extruded. The injectable CPC scaffold had a flexural strength (mean+/-sd; n=5) of (3.2+/-1.0) MPa, which approached the reported strengths for sintered porous hydroxyapatite implants and cancellous bone. In summary, the injectability of a ceramic scaffold, a macroporous CPC, was studies for the first time. Processing parameters were tailored to achieve high injectability, macroporosity, and strength. The injectable and strong CPC scaffold may be useful in surgical sites that are not freely accessible by open surgery or when using minimally invasive techniques.     

Polymerizable nanoparticulate silica-reinforced calcium phosphate bone cement

Bone cements based on calcium phosphate powder and different concentrations of colloidal silica suspensions were developed. Setting time and washout behavior of the cements were recorded and compared with those of a control group prepared by the same powder phase and distilled water as liquid. The phase composition, compressive strength, and morphology of the cements were determined after incubation and soaking in simulated body fluid. Proliferation of osteoblasts seeded on samples was also determined as a function of time. The results showed that the long setting time, poor compressive strength, and undesirable washout behavior of the cement made with distilled water were considerably improved by adding colloidal silica in a dose-dependent manner. On the basis of XRD and SEM results, both control group and nanosilica-added cements composed of nanosized apatite flakes after 7 days soaking, in addition to tetracalcium phosphate residual for the latter. It was found that the rate of hydraulic reactions that are responsible for conversion of the cement reactants to nanostructured apatite was increased by the presence of colloidal silica. Furthermore, the osteoblasts exhibited better proliferation on nanosilica added cements compared to control one. This study suggests better applied properties for nanosilica-added calcium phosphate cement compared to traditional cements.