Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits

In this study, the osteoinductive properties of porous calcium phosphate (Ca-P) cement loaded with bone morphogenetic protein 2 (rhBMP-2) were evaluated and compared with rhBMP-2 loaded absorbable collagen sponge (ACS). Discs with a diameter of 8mm were loaded with a buffer solution with or without 10 microg rhBMP-2 and inserted in 8mm full thickness cranial defects in rabbits for 2 and 10 weeks of implantation. Histological analysis revealed excellent osteoconductive properties of the Ca-P material. It maintained its shape and stability during the implantation time better than the ACS but showed no degradation like the ACS. Quantification of the Ca-P cement implants showed that bone formation was increased significantly by administration of rhBMP-2 (10 weeks pore fill: 53.0+/-5.4%), and also reached a reasonable amount without rhBMP-2 (43.1+/-10.4%). Remarkably, callus-like bone formation outside the implant was observed frequently in the 2 weeks rhBMP-2 loaded Ca-P cement implants, suggesting a correlation with the presence of growth factor in the surrounding tissue. However, an additional in vitro assay revealed an accumulative release of no more than 9.7+/-0.9% after 4 weeks. We conclude that: (1). Porous Ca-P cement is an appropriate candidate scaffold material for bone engineering. (2). Bone formation can be enhanced by lyophilization of rhBMP-2 on the cement. (3). Degradation of porous Ca-P cement is species-, implantation site- and implant dimension-specific.     

The kinetic and biological activity of different loaded rhBMP-2 calcium phosphate cement implants in rats

The healing of large bone defects can be improved by osteogenic bone graft substitutes, due to growth factor inclusion. A sustained release of these growth factors provides more efficient bioactivity when compared with burst release and might reduce the dose required for bone regeneration, which is desirable for socioeconomical and safety reasons. In this study, we compared different rhBMP-2 loadings in a sustained release system of CaP cement and PLGA-microparticles and were able to couple kinetic to biological activity data. Fifty-two rats received a critical-size cranial defect, which was left open or filled with the cement composites. The implants consisted of plain, high, and five-fold lower dose rhBMP-2 groups. Implantation time was 4 and 12 weeks. Longitudinal in vivo release was monitored by scintigraphic imaging of (131)I-labeled rhBMP-2. Quantitative analysis of the scintigraphic images revealed a sustained release of (131)I-rhBMP-2 for both doses, with different release profiles between the two loadings. However, around 70% of the initial dose was retained in both implant formulations. Although low amounts of rhBMP-2 were released (2.4 +/- 0.8 mug in 5 weeks), histology showed defect bridging in the high-dose implants. Release out of the low-dose implants was not sufficient to enhance bone formation. Implant degradation was limited in all formulations, but was mainly seen in the high-dose group. Low amounts of sustained released rhBMP-2 were sufficient to bridge critically sized defects. A substantial amount of rhBMP-2 was retained in the implants because of the slow release rate and the limited degradation.     

Maxillary sinus floor elevation using a tissue-engineered bone with rhBMP-2-loaded porous calcium phosphate cement scaffold and bone marrow stromal cells in rabbits

The aim of this study was to evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex with recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded porous calcium phosphate cement (CPC) scaffold and bone marrow stromal cells (bMSCs) in rabbits. bMSCs were cultured and osteogenically induced. The osteoblastic differentiation of expanded bMSCs was detected by alkaline phosphatase activity, and calcium deposits in vitro. Thirty-six rabbits were randomly allocated into week 2, 4 and 8 observation groups. At each time point, 24 maxillary sinus floor elevation surgeries in 12 rabbits were performed bilaterally and randomly implanted by (1) CPC materials alone (group A, n = 6), (2) rhBMP-2/CPC composite materials alone (group B, n = 6), (3) CPC/bMSCs complex (group C, n = 6) and (4) rhBMP-2/CPC/bMSCs complex (group D, n = 6). As for maxillary sinus floor elevation, rhBMP-2-loaded CPC could promote new bone formation as compared to CPC, while addition of bMSCs could further enhance its new bone formation and maturity significantly, as detected by histological findings, and fluorochrome labeling. Our data suggested that rhBMP-2/CPC possessed excellent osteoinductive ability, while combining with bMSCs could further promote new bone formation and maturation in maxillary sinus elevation.     

rhBMP-2／无定形磷酸钙纳米缓释微粒成骨活性的实验研究

目的探讨无定形磷酸钙（ACP）对rhBMP-2的缓释作用，检测rhBMP-2／ACP纳米缓释微粒的成骨活性。方法用扫描电镜观察已制备rhBMP-2／ACP缓释微粒的大小、形态：测定rhBMP-2的体外释放情况并描绘曲线；用MTT法检测缓释微粒对兔骨髓间充质干细胞（MSC）增殖情况的影响：用碱性磷酸酶（ALP）试剂盒检测细胞ALP活性以反映缓释微粒对其分化的影响．并与ACP的作用进行比较；将缓释微粒植入大鼠股部肌袋．通过X线、组织形态学观察评价缓释微粒的异位成骨能力。结果缓释微粒大小为100nm左右．具有典型的无定形球形面貌。开始时为快速释放期．随后呈缓慢持续释放。缓释微粒能显著促进MSC的增殖和分化，植入大鼠股部肌袋12周．材料大部分降解．有明显的骨形成。结论ACP可作为rhBMP-2合适的缓释载体材料．生成的纳米缓释微粒具有良好的降解性能和成骨活性。     

Bone healing response to an injectable calcium phosphate cement with enhanced radiopacity

The aim of this study was to determine the impact of barium sulfate on remodeling and regeneration in standard tibial defects in rabbits treated with the Norian skeletal repair system (SRS). Two formulations of SRS (with and without barium sulfate) were injected into the medullary canal of the tibia of New Zealand white rabbits. Animals were sacrificed at 6 weeks, 6 months, 1 year, and 2 years. Over the 2-year duration of the study, standard SRS and SRS with barium sulfate appeared to be biocompatible and osteoconductive with no evidence of either inflammation or fibrous tissue around the implant materials or at the bone-material interfaces. This outcome underscores the osteophilic property of the SRS. A difference we observed between the standard SRS and the SRS with barium sulfate was the appearance of acellular material contiguous to the SRS with barium sulfate. Energy dispersive X-ray spectroscopy (EDX) analysis was conducted and confirmed that the acellular material was barium sulfate. Pathological examination of additional tissues including regional lymph nodes revealed neither dissemination of calcium phosphate nor barium sulfate. We concluded that the residual barium sulfate detected by EDX was localized to the intramedullary canal of the tibia.     

一种可注射体内成孔的磷酸钙骨水泥

背景：磷酸钙骨水泥存在脆性大、抗水溶性(血溶性)差、力学性能不足、降解缓慢等缺点,其临床应用受到一定限制,故需要对其进行改性研究。 目的：制备一种具有一定强度、孔隙率、适合骨生长的多孔磷酸钙骨水泥生物支架材料。 方法：以磷酸钙骨水泥为基本体系,液相采用壳聚糖的弱酸溶液,以提高磷酸钙骨水泥的可塑性和黏弹性,使骨水泥具有可注射性,显著提升骨水泥的应用范围及应用舒适度。固相为双相磷酸钙(磷酸四钙+磷酸氢钙)粉体,并在固相中添加一定量的甘露醇及聚乳酸-乙醇酸共聚物作为造孔剂,制备磷酸钙支架材料。 结果与结论：此材料孔径可达到10~300 μm。添加60%致孔剂时,磷酸钙骨水泥固化体孔隙率可达到(68.3±1.5)%。磷酸钙骨水泥孔隙率的增加使材料的力学性能下降,其抗压强度从最初不含致孔剂时的(53.0±1.4) MPa下降到含60%致孔剂的(2.5±0.2) MPa。实验制备的此种多孔磷酸钙骨水泥材料,是具有一定抗压强度、较好的孔隙率,并能体内降解的可注射生物支架材料。     

Morphological and phase characterizations of retrieved calcium phosphate cement implants

A self-hardening calcium phosphate cement (CPC), consisting of equimolar amounts of tetracalcium phosphate and dicalcium phosphate anhydrous, hardens when mixed with water and forms a resorbable hydroxyapatite (HA) as the end-product. The objective of this study was to investigate the changes of the phase and morphology of the CPC during hardening and aging under in vivo conditions. CPC samples retrieved 12 h after hardening in vivo had already contained carbonated HA (type B), even though the initial cement mixture did not contain carbonate as one of the solid components. The mass fraction of carbonate in the 12-h sample was about 1%. The results suggested that under in vivo conditions carbonate is readily available and this allows formation of carbonated HA in favor of carbonate-free HA. The carbonate content of the CPC samples retrieved 3 months after implantation was similar to that of the 12-h samples, and the exterior surfaces of the 3-month samples appeared less crystalline than that of the 12-h samples.     

双相钙磷生物陶瓷/硫酸钙骨水泥多孔三维支架的生物性能

背景：随着组织工程技术的发展,多孔生物陶瓷被越来越多的运用到骨缺损的修复中,当前的研究主要集中在这种生物陶瓷的合成及其各项性能的评价。 目的：研究一种新型骨水泥的制备方法并测定其理化性能及与成骨细胞的生物相容性。 方法：共沉淀法制备双相钙磷生物陶瓷粉体,利用胶体团聚成颗粒,烧结后得到颗粒状、多孔羟基磷灰石/磷酸三钙生物陶瓷,并按不同比例与高纯度医用半水硫酸钙混合制备钙磷陶瓷/硫酸钙骨水泥。 结果与结论：X射线衍射证实合成物质为双相钙磷陶瓷,颗粒状双相钙磷陶瓷具有多孔网状结构,骨水泥在   3 min内保持可塑状态,固化时间为15 min,固化温度为36.5 ℃,压缩强度最高为5.82 MPa,MTT毒性级为0级,成骨细胞在材料表面生长良好。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Bulk properties and bioactivity assessment of porous polymethylmethacrylate cement loaded with calcium phosphates under simulated physiological conditions

Polymethylmethacrylate (PMMA) cements are widely used in spinal surgery. Nevertheless, these types of cements present some documented drawbacks. Therefore, efforts have been made to improve the properties and biological performance of solid PMMA. A porous structure would seem to be advantageous for anchoring purposes. This work studied the bulk physicochemical, mechanical and interconnectivity properties of porous PMMA cements loaded with various amounts of calcium phosphate (CaP). As a measure of bioactivity, changes of PMMA cements under simulated physiological conditions were studied in a calcium phosphate solution for 0, 3, 7, 14, 21 and 28 days. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), micro-computed tomography (μ-CT) and mechanical compression tests were performed to characterize the morphology, crystallographic and chemical composition, interconnectivity and mechanical properties, respectively. SEM allowed observing the result of loading CaP into the porous PMMA, which was corroborated by XRD, FTIR and μ-CT. No interference of the CaP with the PMMA was detected. μ-CT described similar interconnectivity and pore distribution for all CaP percentages. Mechanical properties were not significantly altered by the CaP percentages or the immersion time. Hence, porous PMMA was effectively loaded with CaP, which provided the material with properties for potential osteoconductivity.     

Bone response to radio frequency sputtered calcium phosphate implants and titanium implants in vivo.

The objective of this study was to evaluate the effect of radio frequency sputtered calcium phosphate (CaP) coatings of titanium (Ti) implants on the bond strength at the bone-implant interface and percent bone contact length. Cylindrical coated or noncoated implants (4.0-mm diameter by 8-mm long) were implanted for 3 and 12 weeks. At 3 weeks after implant placement, the ultimate interfacial strengths for as-deposited CaP-coated and heat-treated CaP-coated implants were 2.29 +/- 0.14 MPa and 1.28 +/- 0.04 MPa, respectively. These ultimate interfacial strength values at 3 weeks were statistically greater than the mean ultimate interfacial strength for control Ti implants (0.67 +/- 0.13 MPa). At 12 weeks after implant placement, no statistical differences in the mean ultimate interfacial strengths were observed between the as-deposited CaP-coated, heat-treated CaP-coated, and control Ti implants. Histomorphometric evaluation indicated greater percent bone contact lengths for the as-deposited CaP-coated implants compared with the heat-treated CaP-coated and control Ti implants 3 and 12 weeks after implant placement.     

Injectable rhBMP-2-loaded chitosan hydrogel composite: osteoinduction at ectopic site and in segmental long bone defect

Carriers for bone morphogenetic protein-2 (BMP-2) used in clinical practice still suffer from limitations such as insufficient protein retention. In addition, there is a clinical need for injectable carriers. The main objective of this study was to assess bone forming ability of rhBMP-2 combined either with chitosan hydrogel (rhBMP-2/CH) or chitosan hydrogel containing β-tricalcium phosphate (β-TCP) (rhBMP-2/CH/TCP). Formulations were first compared in a rat ectopic intramuscular bone formation model, and the optimal formulation was further evaluated in healing of 15-mm critical size defect in the radius of a rabbit. Three weeks after injection ectopically formed bone was analyzed by microcomputerized tomography (micro-CT) and histology. Significantly higher (4.7-fold) mineralized bone formation was observed in the rhBMP-2/CH/TCP group compared to rhBMP-2/CH group. In a pilot study, defect in a rabbit radius treated with rhBMP-2/CH/TCP showed incomplete regeneration at 8 weeks with composite leakage from the defect, indicating the need for formulation refinement when segmental defect repair is foreseen.     

The fabrication and biochemical evaluation of alumina reinforced calcium phosphate porous implants

Alumina reinforced calcium phosphate porous implants were manufactured to improve the mechanical strength while maintaining the bioactivity of calcium phosphate ceramics. The alumina porous bodies, which provided the mechanical strength, were fabricated by a polyurethane sponge method and multiple coating techniques resulted in the porous bodies with a 90-75% porosity and a compressive strength of up to approximately 6MPa. The coating of hydroxyapatite (HAp) or tricalcium phosphate (beta-TCP) was performed by dipping the alumina porous bodies into calcium phosphate ceramic slurries and sintering the specimens. The fairly strong bonding between the HAp or TCP coating layer and the alumina substrate was obtained by repeating the coating and sintering processes. The biochemical evaluations of the porous implants were conducted by in vitro and in vivo tests. For in vitro test, the implants were immersed in Ringer's solution and the release of Ca and P ions were detected and compared with those of calcium phosphate powders. For in vivo test, the porous bodies were implanted into mixed breed dogs and bone mineral density measurements and histological studies were conducted. The alumina reinforced HAp porous implants had a higher strength than the HAp porous implants and exhibited a similar bioactivity and osteoconduction property to the HAp porous implants.     

Bioresorption behavior of tetracalcium phosphate-derived calcium phosphate cement implanted in femur of rabbits

One primary focus of the present study was to clarify the crucial resorption-location relationship of a recently developed single-phase TTCP-derived calcium phosphate cement (CPC) implanted in rabbit femur in a systematic and quantitative way. Gross examination of retrieved CPC/bone composite samples indicated that the CPC implant did not evoke inflammatory response, necrosis or fibrous encapsulation in surrounding bony tissues. Histological examination revealed excellent CPC-host bone bonding. At 4 weeks, the resorption-induced voids between terminals of bone defects and implants were largely filled with new bone. CPC resorption, new blood vessels, osteocytes, osteons and osteoblast-like cells lining up with active new bone were observed at remodeling sites. At 12 weeks, a new bone network was developed within femoral defect, while CPC became islands incorporated in the new bone. At this stage, crevices filled with lamellar new bone structure were frequently observed. At 24 weeks, bone ingrowth and remodeling activities became so extensive that the interface between residual cement and new bone became less identifiable. In general, at all implant locations the resorption ratio values increased with implantation time, while at all implantation times the resorption ratios decreased from the exterior (cortical site) to the interior (cancellous site) of implants. At the end of 24 weeks, CPC was almost completely resorbed and bone remodeling almost finished at the cortical site.     

Mechanical properties of porous, electrosprayed calcium phosphate coatings

Mechanical properties of calcium phosphate coatings (CaP), deposited using the electrostatic spray deposition (ESD) technique, have been characterized using a range of analytical techniques, including tensile testing (ASTM C633), fatigue testing (ASTM E855), and scratch testing using blunt and sharp scratch styli. Moreover, a simple explantation procedure was successfully introduced using ESD-coated, threaded dental implants to characterize the mechanical performance of CaP coatings qualitatively under conditions that mimic clinical situations as close as possible. Generally, all analysis techniques revealed that ESD coatings need to be crystallized in order to ensure interfacial adhesion to the substrate and sufficient mechanical strength of the superficial reticular structure. Crystalline carbonated hydroxyapatite coatings (CHA, heat-treated at 700 degrees C) were resistant to fatigue as well as to plastic ploughing deformation by means of various scratch styli, and the fragile surface structure of ESD coatings was maintained to a large extent after unscrewing CHA-coated dental implants from femoral condyles of goat cadavers. From these experiments, it was concluded that interfacial adhesion of crystalline CHA ESD coatings to the titanium substrate was sufficient, but that mechanical strength of the superficial architecture of ESD coatings need to be optimized for applications where high shear and compressive stresses are imposed onto the rather fragile coating surface of reticular ESD morphologies.     

Effect of the calcium to phosphate ratio of tetracalcium phosphate on the properties of calcium phosphate bone cement

Six different tetracalcium phosphate (TTCP) products were synthesized by solid state reaction at high temperature by varying the overall calcium to phosphate ratio of the synthesis mixture. The objective was to evaluate the effect of the calcium to phosphate ratio on a TTCP-dicalcium phosphate dihydrate (DCPD) cement. The resulting six TTCP-DCPD cement mixtures were characterized using X-ray diffraction analysis, scanning electron microscopy, and pH measurements. Setting times and compressive strength (CS) were also measured. Using the TTCP product with a Ca/P ratio of 2.0 resulted in low strength values (25.61 MPa) when distilled water was used as the setting liquid, even though conversion to hydroxyapatite was not prevented, as confirmed by X-ray diffraction. The suspected CaO presence in this TTCP may have affected the cohesiveness of the cement mixture but not the cement setting reaction, however no direct evidence of CaO presence was found. Lower Ca/P ratio products yielded cements with CS values ranging from 46.7 MPa for Ca/P ratio of 1.90 to 38.32 MPa for Ca/P ratio of 1.85. When a dilute sodium phosphate solution was used as the setting liquid, CS values were 15.3% lower than those obtained with water as the setting liquid. Setting times ranged from 18 to 22 min when water was the cement liquid and from 7 to 8 min when sodium phosphate solution was used, and the calcium to phosphate ratio did not have a marked effect on this property.     

Influence of a novel radiopacifier on the properties of an injectable calcium phosphate cement

An injectable calcium phosphate cement (CPC) with excellent radiopacity was proposed by introducing a novel radiopacifier, strontium carbonate, into the powder phase of CPC. The results showed that the cement showed improved radiopacity even when the content of strontium carbonate was only 8 or 12wt.%. The addition of 8 or 12wt.% strontium carbonate clearly improved the injectability and compressive strength of the cement. Furthermore, the addition of strontium carbonate influenced the pore distribution in the cement. An injectable CPC containing 8 or 12wt.% strontium carbonate has the potential for use in procedures such as vertebroplasty and kyphoplasty.     

The physicochemical properties of the solidification of calcium phosphate cement

In this article, the physicochemical properties of the solidification of calcium phosphate cement (CPC) were investigated, and the components of the solidified body and the characteristics involved in the application were measured. The setting process of CPC was moderate and slightly exothermic, the heat liberation rate was slow, and the temperature increased little. At the initial stage of the hydration, the compressive strength rose linearly with time, and reached the maximum value after 4 hours. The volume was slightly shrunken by 1% during the setting process, and the total heavy metal content was below 2 ppm. The porosity of the solidified body was about 30%, which was mainly the gel pore with size below 0.1 microm. The final product of hydration was low crystalline hydroxyapatite (HAP). Its real density was 2.859 g/cm(3), which was lower than the theoretical value. This may be due to the existence of lattice defects, which will lead to a slight rise in solubility when leached in physiological saline.     

载药型磷酸钙骨水泥的制备及体外释放性能

背景：磷酸钙骨水泥具有良好的生物相容性,可作为骨修复材料与药物载体。 目的：制备载药磷酸三钙骨水泥,并分析其体外释放性能。 方法：采用共沉淀法制备磷酸三钙前躯体,经高温煅烧研磨获得α-磷酸三钙粉体,测试含不同浓度(1.25%,2.5%,3.75%,5%)抗生素(头孢拉定或头孢氨苄或环丙沙星)骨水泥,浸泡不同时间后(6 h、12 h、24 h、2 d、3 d、4 d、 5 d、6 d、7 d、8 d)的药物体外释放浓度。 结果与结论：制备的磷酸三钙粉体粒度约2 μm,结晶度良好。载不同抗生素的骨水泥体外释放都受自身物理性质的影响。载药骨水泥中环丙沙星能够满足长时间缓释,并能达到一个比较理想的缓释浓度,头孢类药物由于自身稳定性等原因,缓释效果并不理想。头孢氨苄的水解速率较低,环丙沙星的光降解条件比较苛刻,因此两者释放未受太大影响,与Higuchi模型基本吻合;头孢拉定的水解速率相对较高,对体系的释放驱动力产生较大影响,使得释放不再遵循Higuchi模型。     

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.     

Rheological properties of concentrated aqueous injectable calcium phosphate cement slurry

In this paper, the steady and dynamic rheological properties of concentrated aqueous injectable calcium phosphate cement (CPC) slurry were investigated. The results indicate that the concentrated aqueous injectable CPC showed both plastic and thixotropic behavior. As the setting process progressed, the yield stress of CPC slurry was raised, the area of the thixotropic hysteresis loop was enlarged, indicating that the strength of the net structure of the slurry had increased. The results of dynamic rheological behavior indicate that the slurry presented the structure similar to viscoelastic body and the property of shear thinning at the beginning. During the setting process, the slurry was transformed from a flocculent structure to a net structure, and the strength increased. Different factors had diverse effects on the rheological properties of the CPC slurry in the setting process, a reflection of the flowing properties (or injection), and the microstructure development of this concentrated suspension. Raising the powder-to-liquid ratio decreased the distance among the particles, increased the initial strength, and shortened the setting time. In addition, raising the temperature improved the initial strength, increased the order of reaction, and shortened the setting time, which was favorable to the setting process. The particle size of the raw material had much to do with the strength of original structure and setting time. The storage module G' of CPC slurry during the setting process followed the rule of power law function G'=A exp(Bt), which could be applied to forecast the setting time, and the calculated results thereafter are in agreement with the experimental data.