壳聚糖对纳米碳管／磷酸钙骨水泥性能的影响

通过不同添加方式将壳聚糖与纳米碳管／磷酸钙骨水泥混合，研究壳聚糖对复合材料性能的影响。结果表明：壳聚糖与复合粉体充分混合后，再加入去离子水时，可以更好提高复合材料的弯曲强度，壳聚糖含量为0．5％时可以得到较短的凝固时间和较高的弯曲强度（12．99MPa）。     

A novel injectable,cohesive and toughened Si-HPMC (silanized-hydroxypropyl methylcellulose) composite calcium phosphate cement for bone substitution

This study reports on the incorporation of the self-setting polysaccharide derivative hydrogel (silanized-hydroxypropyl methylcellulose, Si-HPMC) into the formulation of calcium phosphate cements (CPCs) to develop a novel injectable material for bone substitution. The effects of Si-HPMC on the handling properties (injectability, cohesion and setting time) and mechanical properties (Young’s modulus, fracture toughness, flexural and compressive strength) of CPCs were systematically studied. It was found that Si-HPMC could endow composite CPC pastes with an appealing rheological behavior at the early stage of setting, promoting its application in open bone cavities. Moreover, Si-HPMC gave the composite CPC good injectability and cohesion, and reduced the setting time. Si-HPMC increased the porosity of CPCs after hardening, especially the macroporosity as a result of entrapped air bubbles; however, it improved, rather than compromised, the mechanical properties of composite CPCs, which demonstrates a strong toughening and strengthening effect. In view of the above, the Si-HPMC composite CPC may be particularly promising as bone substitute material for clinic application.     

Energy dispersive X-ray diffraction study of phase development during hardening of calcium phosphate bone cements with addition of chitosan

The aim of this work was to study the phase transformation during the setting reaction of two calcium phosphate bone cements based on either alpha tricalcium phosphate (alpha-TCP) or tetracalcium phosphate (TetCP) initial solid phase, and a magnesium carbonate-phosphoric acid solution as the hardening liquid. Low molecular weight (38.2 kDa) chitosan was used to retard the cement's setting reaction. To follow the kinetics of the phase development, an energy dispersive X-ray diffraction technique was applied. This technique allowed the collection of diffraction patterns from the cement pastes in situ starting from 1 min of the setting process. In the case of the TetCP-based cement, the appearance and evolution of an intermediate phase was detected.     

磷酸钙人工骨修复骨缺损的研究进展

继发于各类病因的大段骨缺损通常需要人工骨材料进行修复,目前常用的人工骨材料包括磷酸钙和硫酸钙基人工骨、生物活性玻璃等,以磷酸钙为主要成分的人工骨,复合其他一种或多种材料以期改善人工骨的性能是目前的研究热点。本文将总结以磷酸钙为基质的各类复合材料,包括与聚合物复合的磷酸钙材料、以磷酸钙为基质的合金材料、药物缓释材料以及骨组织工程材料在骨缺损修复中的研究进展,为以磷酸钙为基质新材料的研究奠定基础。     

Tendon-to-bone healing using an injectable calcium phosphate cement combined with bone xenograft/BMP composite

Injectable calcium phosphate cement (ICPC) has been applied to enhance the tendon-to-bone healing. However, its slow degradation delays the osteointegration of grafted tendon in bone tunnels. We therefore constructed a synthetic biomaterial of ICPC combined with recombined bone xenograft granules (RBX). In this study, the first stage study demonstrated that the ICPCB contained 3 mg BMPs (ICPCB-3) obtained a porous structure. More importantly, the values of ICPCB-3 were highest in cell proliferation, alkaline phosphatase (ALP) activity, expression of osteogenic genes, and newly ectopic bone-forming area (P < 0.05). Then, ICPCB-3 was used in an anterior cruciate ligament (ACL) reconstruction model. Ninety skeletal mature rabbits underwent bilateral ACL reconstructions and were assigned to 3 groups: control group, ICPC alone group, and ICPCB-3 group. Animals were sacrificed at 6, 12 and 24 weeks. The results showed compared with ICPC, ICPCB-3 composite markedly accelerated tendon-to-bone healing. In addition, little remnants were observed in ICPCB-3 group. Moreover, the maximum loads to failure of ICPCB-3 group was significantly higher than ICPC group at 24 weeks (P < 0.01). We conclude that the ICPCB composite, with a porous structure and better osteointegration effect, has direct clinical instruction to arthroscopic techniques of the ACL reconstruction.     

RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration

Calcium phosphate cement scaffold (CPC) has been widely used as bone graft substitutes, but undesirable osteoinductivity and slow degradability greatly hamper their clinic application. To address these problems, a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium silicate/calcium phosphate cement scaffold (CSPC) with hierarchical pores was developed in this study. The CSPC scaffold with both interconnected macropores on the order of 200–500 μm and micropores of 2–5 μm was synthesized from CPC and calcium silicate (CS) by a NaCl particulate-leaching method. In vitro cell culture with C2C12 model cells, in vivo ectopic bone formation and rabbit femur cavity defect repair were performed to evaluate the osteogeneic capacity of the CSPC/rhBMP-2 scaffold. CPC, CSPC and CPC/rhBMP-2 scaffolds were parallelly investigated for comparison. The results demonstrated that the hierarchical macro/microporous structure, whether in presence of CS or rhBMP-2, highly favored the adhesion of C2C12 cells and bone in-growth into the CPC-based scaffolds. But, in comparison to the CPC-based scaffolds with CS or rhBMP-2 alone, the CSPC/rhBMP-2 scaffold strongly promoted osteogenic differentiation in vitro and osteogenetic efficacy in vivo. Further studies demonstrated that Si ions derived from CSPC contributed mainly to maintain the conformation of rhBMP-2 and thus stimulate the synergistic action of CS and rhBMP-2 in osteogenic differentiation and osteoinductivity. Additionally, the incorporation of CS was also beneficial for the dissolution of the scaffold. Those results suggest that the CSPC has superior properties for incorporation of rhBMP-2 and our developed CSPC/rhBMP-2 scaffold have great potential for future use in bone tissue regeneration.     

Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials.

We developed a calcium phosphate cement that could be molded into any desired shape due to its chewing-gum-like consistency after mixing. The powder component of the cement consists of alpha-tricalcium phosphate and tetracalcium phosphate, which were made by decomposition of hydroxyapatite ceramic blocks. The liquid component consists of citric acid, chitosan and glucose solution. In this study, we used 20% citric acid (group 20) and 45% citric acid (group 45). The mechanical properties and biocompatibility of this new cement were investigated. The setting times of cements were 5.5 min, in group 20 and 6.4 min, in group 45. When incubated in physiological saline, the cements were transformed to hydroxyapatite at 3, and 6 weeks, the compressive strengths were 15.6 and 20.7 MPa, in group 45 and group 20, respectively. The inflammatory response around the cement implanted on the bone and in the subcutaneous tissue in rats was more prominent in group 45 than in group 20 at 1 week after surgery. After 4 weeks, the inflammation disappeared and the cement had bound to bone in both groups. These results indicate that this new calcium phosphate cement is a suitable bone substitute material and that the concentration of citric acid in the liquid component affects its mechanical properties and biocompatibility.     

纳米羟基磷灰石/壳聚糖/半水硫酸钙为可注射骨组织工程支架材料的可行性

背景：前期实验采用仿生学原理制备了可注射性纳米羟基磷灰石/壳聚糖/半水硫酸钙复合材料,但其与骨髓间充质干细胞的生物相容性还不十分清楚。目的：探讨纳米羟基磷灰石/壳聚糖/半水硫酸钙作为注射型骨组织工程支架材料的可行性。方法：将第3代兔骨髓间充质干细胞与可注射纳米羟基磷灰石/壳聚糖/半水硫酸钙支架复合培养,作为实验组;以单纯接种培养的骨髓间充质干细胞为对照组,倒置显微镜下观察细胞生长情况,MTT法检测细胞增殖,扫描电镜观察细胞在材料表面生长与增殖。将纳米羟基磷灰石/壳聚糖/半水硫酸钙支架埋植在家兔背部肌袋内,埋植后2,4,6,8周进行病理学观察。结果与结论：实验组细胞生长、增殖良好,与对照组无明显差异。支架埋植后2周,材料周围有中等量中性粒细胞、淋巴细胞和巨细胞浸润,可见小血管与纤维母细胞增生,材料已被炎性细胞分割、围绕散碎;埋植后4周,可见少量淋巴细胞、纤维母细胞聚集,炎症反应进一步消退,肌纤维排列、形态正常;埋植后6周,材料周围炎症反应轻微,组织水肿不明显;埋植后8周,炎症反应基本消退,材料基本降解完成,肌纤维形态基本正常。表明纳米羟基磷灰石/壳聚糖/半水硫酸钙复合物具有良好的细胞相容性和生物降解性,可作为注射型支架材料。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：     

3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration

Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1–2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.     

3D生物描绘孔结构可控钙磷硅基骨修复支架的生物力学性能

目的设计和制备新型钙磷硅基骨修复支架,研究其在不同外力作用下体外生物力学性能。方法以自固化磷酸钙骨水泥(calcium phosphate cement,CPC)、介孔硅酸钙(mesporous calcium silicate,MCS)为原料,通过3D生物描绘技术构建孔径分别为350、500μm的MCS/CPC复合支架。采用扫描电镜观察支架表面形貌;分别通过万能力学试验机和动态力学分析仪,考察具有不同孔道结构MCS/CPC支架的抗压力学性能和不同频率动态周期性载荷作用下的力学性能。结果通过3D生物描绘技术能够实现对钙磷硅基骨修复支架内部孔道结构的可控制备。孔径为350μm的MCS/CPC支架具有较高的抗压力学强度[(9.80±0.39)MPa]和抗压模量[(132.50±4.30)MPa];此外,载荷频率在1~100 Hz范围内,孔径为350μm的支架具有较高的储能模量。结论通过3D生物描绘技术制备的孔径为350μm的MCS/CPC复合支架不仅具有规则的连通孔道,还具有较高的抗压力学性能,能在动态载荷作用下保持结构稳定,适合作为一种新型的骨缺损修复材料。     

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.     

Designing optimal calcium phosphate scaffold-cell combinations using an integrative model-based approach

Bone formation is a very complex physiological process, involving the participation of many different cell types and regulated by countless biochemical, physical and mechanical factors, including naturally occurring or synthetic biomaterials. For the latter, calcium phosphate (CaP)-based scaffolds have proven to stimulate bone formation, but at present still result in a wide range of in vivo outcomes, which is partly related to the suboptimal use and combination with osteogenic cells. To optimize CaP scaffold selection and make their use in combination with cells more clinically relevant, this study uses an integrative approach in which mathematical modeling is combined with experimental research. This paper describes the development and implementation of an experimentally informed bioregulatory model of the effect of calcium ions released from CaP-based biomaterials on the activity of osteogenic cells and mesenchymal stem cell driven ectopic bone formation.The amount of bone formation predicted by the mathematical model corresponds to the amount measured experimentally under similar conditions. Moreover, the model is also able to qualitatively predict the experimentally observed impaired bone formation under conditions such as insufficient cell seeding and scaffold decalcification. A strategy was designed in silico to overcome the negative influence of a low initial cell density on the bone formation process. Finally, the model was applied to design optimal combinations of calcium-based biomaterials and cell culture conditions with the aim of maximizing the amount of bone formation. This work illustrates the potential of mathematical models as research tools to design more efficient and cell-customized CaP scaffolds for bone tissue engineering applications.     

Synergistic effects of bisphosphonate and calcium phosphate nanoparticles on peri-implant bone responses in osteoporotic rats

The prevalence of osteoporosis will increase within the next decades due to the aging world population, which can affect the bone healing response to dental and orthopedic implants. Consequently, local drug targeting of peri-implant bone has been proposed as a strategy for the enhancement of bone-implant integration in osteoporotic conditions. In the present study, an established in-vivo femoral condyle implantation model in osteoporotic and healthy bone is used to analyze the osteogenic capacity of titanium implants coated with bisphosphonate (BP)-loaded calcium phosphate nanoparticles (nCaP) under compromised medical conditions. After 4 weeks of implantation, peri-implant bone volume (%BV; by μCT) and bone area (%BA; by histomorphometry) were significantly increased within a distance of 500 μm from implant surfaces functionalized with BP compared to control implants in osteoporotic and healthy conditions. Interestingly, the deposition of nCaP/BP coatings onto implant surfaces increased both peri-implant bone contact (%BIC) and volume (%BV) compared to the deposition of nCaP or BP coatings individually, in osteoporotic and healthy conditions. The results of real-time PCR revealed similar osteogenic gene expression levels to all implant surfaces at 4-weeks post-implantation. In conclusion, simultaneous targeting of bone formation (by nCaP) and bone resorption (by BP) using nCaP/BP surface coatings represents an effective strategy for synergistically improvement of bone-implant integration, especially in osteoporotic conditions.     

可降解生物材料聚乳酸/磷酸钙陶瓷在骨组织工程中的应用

聚乳酸和磷酸钙陶瓷都是骨组织工程中常用的可降解生物材料。前者是人工合成的多聚物,在体内降解时间较长,可起到临时支架的作用,不同结构的聚乳酸又有不同的生物特性;后者生物活性好,亲和性高,但是脆性大,抗折强度低。两者的复合物在一定程度上弥补了各自的不足,能成为新型的骨组织工程支架材料。     

硫酸钙/羟基磷灰石可注射性骨替代物凝固时间的影响因素

目的分析可注射性硫酸钙/羟基磷灰石骨替代物凝固时间的影响因素。方法分别在改变凝固条件后,或改变羟基磷灰石含量,液固比,固化液成分后用 Gillmore 双针法测定硫酸钙/羟基磷灰石骨替代物凝固时间的变化。结果当减低液固比,提高羟基磷灰石含量或升高温度时可注射性硫酸钙／羟基磷灰石骨替代物凝固时间缩短,使用固化液欧乃派克或蒸馏水对其凝固时间的影响无显著性差异。结论硫酸钙/羟基磷灰石骨替代物的凝固时间通过改变影响因素可以达到临床操作的要求。     

新型大孔隙磷酸钙骨水泥作为骨组织工程支架的相关研究

目的 探讨新型大孔隙磷酸钙骨水泥(CPC)材料支架的细胞毒性和对细胞黏附、生长和增殖的影响.方法 通过添加甘露醇制孔剂和应用磷酸钠溶液作为CPC固化液的方法合成新型CPC材料.通过CCK8法检测细胞在新型CPC材料浸提液中的生长增殖情况;通过电子扫描电镜测试材料孔径和细胞在材料表面上黏附生长情况;应用力学三点弯曲实验测试新型CPC的生物力学性能.结果 新型CPC材料的孔径值达到(267.43±118.01)μm,孔隙率为(66.15±6.91)％.新型CPC材料的最大负荷、抗弯强度和坚韧度较传统CPC均增加了约1倍(P＜0.05).新型CPC材料浸提液与细胞共培养2、4、6、8d后CCK8法测试吸光度(OD)值与阴性对照组比较其差异无统计学意义(P＞0.05).结论 新型CPC材料具有强大的生物力学性能、大孔隙、高孔隙率和良好的生物相容性,有望成为理想的骨组织工程支架.     

PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy

Lack of safe and effective delivery vehicle is the main obstacle for siRNA mediated cancer therapy. In this study, we synthesized a pH-sensitive polymer of PEG grafted carboxymethyl chitosan (PEG-CMCS) and developed anionic-charged hybrid nanoparticles of PEG-CMCS and calcium phosphate (CaP) for siRNA delivery through a single-step self-assembly method in aqueous condition. The formed nanoparticles with charge of around −8.25 mv and average diameter of 102.1 nm exhibited efficient siRNA encapsulation and enhanced colloidal and serum stability. The test in vitro indicated that the nanoparticles entered into HepG2 cells by endocytosis, and achieved endosomal escape of siRNA effectively due to the pH-responsive disassembly of nanoparticles and dissolution of CaP in the endosome. Reporter gene silencing assay showed that luciferase siRNA delivered by the anionic nanoparticles could achieve gene silencing efficacy comparable to that of conventional Lipofectamine 2000. Additionally, dramatic hTERT knockdown mediated by the anionic nanoparticles transfection induced significant apoptosis of HepG2 cells in vitro. After intravenous injection in tumor-bearing BALB/c nude mice, the nanoparticles specifically accumulated into tumor regions by EPR effect, leading to efficient and specific gene silencing sequentially. Most importantly, the nanoparticles carrying hTERT siRNA inhibited tumor growth significantly via silencing hTERT expression and inducing cells apoptosis in HepG2 tumor xenograft. Moreover, comprehensive safety studies of the nanoparticles confirmed their superior safety both in vitro and in vivo. We concluded that the PEG-CMCS/CaP hybrid anionic nanoparticles possessed potential as a safe and effective siRNA delivery system for anticancer therapy.     

Synthesis of a novel b-tricalcium phosphate/hydroxyapatite biphasic calcium phosphate containing niobium ions and evaluation of its osteogenic properties

To promote the osteogenic properties of osteoblasts, we synthesized a hydroxyapatite (HAp) with β-tricalcium phosphate (β-TCP) biphasic calcium phosphate containing Nb ions (NbTCP/HAp). NbTCP/HAp was prepared by annealing precipitates obtained by coprecipitation of an aqueous solution of Ca(NO₃)₂ and a mixture of (NH₄)₂HPO₄ and aqueous Nb solution. The precipitates can be regarded as a calcium-deficient HAp, the PO₄ sites of which are partly occupied by Nb ions. NbTCP/HAp was successfully synthesized by thermal decomposition of the precipitates. NbTCP/HAp enhanced the calcification of normal human osteoblasts (NHOst), and the amount of calcified tissue increased in proportion to the Nb ion concentration in the NbTCP/HAp. The alkaline phosphatase (ALP) activity of NHOst was also enhanced by NbTCP/HAp. Because Nb ions significantly enhance the ALP activity of NHOst, calcification by NbTCP/HAp is considered to be due to enhancement of ALP activity induced by Nb ions dissolved from NbTCP/HAp. These results indicate that NbTCP/HAp can be an effective bone repair material.     

Developing novel Ca-zeolite/poly(amino acid) composites with hemostatic activity for bone substitute applications

Abstract

The novel Ca-zeolite/poly(amino acid) (CaY/PAA) composites for bone substitute applications with hemostatic activity were prepared using the in situ melting polymerization method. In this study, Ca-zeolite (CaY) loaded with Ca²⁺ was obtained from Y-type zeolite (NaY) by ion-exchange method. The properties of the CaY/PAA composites and PAA, including composition, structure, compressive strength, in vitro degradability in phosphate-buffered solution (PBS), bioactivity, cytocompatibility and in vitro coagulation tests were characterized and investigated. The results showed that compressive strength of the CaY/PAA composites ranged from 145 to 186?MPa, demonstrating sufficient mechanical strength for load-bearing bone substitute. After soaking in PBS for 16 weeks, the weight loss of 25CaY/PAA and 50CaY/PAA were 4.1 and 1.6?wt%, respectively, and the pH values for CaY/PAA composites increased to about 8.0 in 2 weeks and then gradually stabilized around 7.4, indicating good stability in PBS. Scanning electron microscope and energy dispersive spectrometer results showed that the composites were bioactive and new apatite layers attached on their surfaces. Mesenchymal stem cells (MSCs) exhibited high-proliferation in the extract solution of the CaY/PAA composites and were well spread on the surfaces of the composites. Cells on the CaY/PAA composite groups showed higher alkaline phosphatase (ALP) activity indicating the potential to promote cell differentiation. The in vitro coagulation tests showed that CaY/PAA composites have shorter clotting time and better performance of promoting blood coagulation than other samples, presenting improved hemostatic activity. In summary, the results demonstrated that the CaY/PAA composites had good mechanical strength, stability, bioactivity, cytocompatibility and hemostatic activity for bone substitute applications.