纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性与体内降解研究

对制备的纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性及体内降解情况进行研究,为临床提供实验依据。参照GB/T16886医疗器械生物学评价标准和要求,对纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥进行急性细胞毒性试验、溶血试验、热源试验、急性全身毒性试验及体内植入试验等系列体内外生物学试验研究,以进行有效的生物相容性和安全性评价。纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的溶血率小于国家规定的5%,在体外不引起溶血反应;浸提液注入动物体内后无死亡,活动进食正常;无细胞毒性反应;热原试验动物体温升高均在0.7℃以下,3只兔体温升高值的总数〈1.5℃,无致热作用;材料植入体内初期有轻度炎症反应,随植入时间延长逐渐减轻,材料也逐渐降解吸收。纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥具有良好的生物相容性和降解性能,具有临床开发应用前景。     

纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性与体内降解研究

对制备的纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性及体内降解情况进行研究,为临床提供实验依据.参照GB/T16886医疗器械生物学评价标准和要求,对纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥进行急性细胞毒性试验、溶血试验、热源试验、急性全身毒性试验及体内植入试验等系列体内外生物学试验研究,以进行有效的生物相容性和安全性评价.纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的溶血率小于国家规定的5％,在体外不引起溶血反应;浸提液注入动物体内后无死亡,活动进食正常;无细胞毒性反应;热原试验动物体温升高均在0.7℃以下,3只兔体温升高值的总数＜1.5℃,无致热作用;材料植入体内初期有轻度炎症反应,随植入时间延长逐渐减轻,材料也逐渐降解吸收.纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥具有良好的生物相容性和降解性能,具有临床开发应用前景.     

壳聚糖/纳米羟基磷灰石分层复合支架的生物相容性研究

制备壳聚糖/纳米羟基磷灰石(CS/nHA)分层复合支架,对其进行细胞毒性评价.分离培养大鼠软骨细胞接种于支架,相差显微镜和扫描电镜观察细胞的黏附及生长情况.动物皮下埋植试验观察其组织相容性.实验结果证实壳聚糖/纳米羟基磷灰石分层复合支架具有良好的生物相容性,有望成为较好的骨软骨组织工程支架.     

复合壳聚糖微球-丝素基载药α-磷酸三钙骨水泥的细胞相容性及毒性

背景:磷酸钙骨水泥具有良好的生物相容性和骨传导性,已被应用于临床,但其不良的力学性能和缺乏骨诱导性限制了其进一步发展和应用。目的:制备壳聚糖微球-丝素基载药α-磷酸三钙骨水泥,验证其细胞相容性及细胞毒性。方法:分别以含体积分数10%胎牛血清和1%双抗的α-MEM培养基、苯酚、100%及50%复合壳聚糖微球-丝素基载药α-磷酸三钙骨水泥材料的浸提液培养MC3T3-E1细胞,采用MTT法评估细胞生长增殖情况,采用乳酸脱氢酶活性检测法判断复合壳聚糖微球-丝素基载药α-磷酸三钙骨水泥材料的毒性。将MC3T3-E1细胞系与复合壳聚糖微球-丝素基载药α-磷酸三钙骨水泥材料共培养,扫描电镜观察细胞在材料表面的附着及生长。结果与结论:复合壳聚糖微球-丝素基载药α-磷酸三钙骨水泥材料浸提液对MC3T3-E1细胞的生长增殖无明显影响,无明显细胞毒性。MC3T3-E1细胞在复合壳聚糖微球-丝素基载药α-磷酸三钙骨水泥材料表面生长良好,伸展充分,在材料表面伸出伪足,与材料贴附紧密,表明壳聚糖微球-丝素基载药α-磷酸三钙骨水泥细胞相容性良好。     

纳米碳管处理磷酸钙骨水泥的生物相容性及其强度和韧性

背景:由于骨缺损和骨质疏松等治疗需要大量的骨修复材料。但常用的骨修复材料之——磷酸钙骨水泥存在脆性大等缺陷,所以临床工作者一直期盼研制一种生物相容性和生物力学更好的骨科修复材料。目的:观察以纳米碳管处理的磷酸钙骨水泥材料的生物相容性和体外生物力学性能。方法:根据国际标准化组织颁布的ISO10993系列,对新型骨水泥材料进行体外溶血试验、细胞毒性试验、急性毒性试验、皮肤过敏性试验;取6具老年尸体胸腰段脊柱标本(T12～L4)进行体外生物力学性能测试,建立压缩性骨折模型后采用球囊扩张后凸椎体成形术恢复高度,分别注射填充纳米碳管处理的磷酸钙骨水泥和普通磷酸钙骨水泥,再进行前屈压缩,测量极限载荷、抗压强度、刚度。结果与结论:①新型材料浸提原液对健康人血红细胞溶血率为1.81%,无溶血现象。材料浸提液对L929细胞毒性分级为0级,无细胞毒性。材料浸提原液未引起小鼠急性毒性反应、小鼠遗传毒性及豚鼠过敏反应。②填充纳米碳管处理的磷酸钙骨水泥组成形后的极限载荷、抗压强度、刚度均高于普通磷酸钙骨水泥组(P0.05)。结果表明以纳米碳管处理的磷酸钙骨水泥材料生物相容性符合国际规定的体内植入物的生物学评价标准,其强度和韧性较普通骨水泥有较大的提高。     

重组人骨形态发生蛋白2壳聚糖纳米微球的制备及体外细胞毒性

背景：通过各种微球负载骨生长因子使骨形态发生蛋白达到缓释效果逐渐成为研究热点,但关于载药壳聚糖纳米微球的生物相容性特别是细胞毒性的报道较少。 目的：对重组人骨形态发生蛋白2壳聚糖纳米微球进行细胞毒性检测,评估应用壳聚糖纳米微球作为重组人骨形态发生蛋白2缓释载体的生物安全性。 方法：通过离子交联法制备空白壳聚糖纳米微球,应用透视电镜观察微球的形态,激光粒径分析其粒径分布;通过重组人骨形态发生蛋白2壳聚糖纳米微球体外细胞毒性试验评估微球的生物安全性。 结果与结论：离子交联法制备的壳聚糖微球,球形规整,分散均匀,微球平均粒径为230 nm,分布较集中。载药及空白微球的反应分级为0或1级,均为合格。提示,离子交联法制备可成功制备出负载重组人骨形态发生蛋2的纳米微球,且微球细胞毒性检测合格,为进一步的骨组织工程研究提供理论实验基础。     

胶原/壳聚糖复合纳米纤维膜引导骨再生的细胞学研究

采用静电纺丝技术制备胶原/壳聚糖复合纳米纤维膜,研究其作为引导骨再生生物膜的细胞生物相容性及诱导成骨性。以乙酸为溶剂,聚环氧乙烯(PEO)为增塑剂,采用静电纺丝技术制备胶原纳米纤维膜及不同比例的胶原/壳聚糖复合纳米纤维膜(胶原、壳聚糖、PEO质量比5∶1∶4,5∶2∶3,5∶4∶1),电子显微镜观察4种纳米纤维膜的表面形态;将骨髓间充质干细胞种植于胶原纳米纤维膜及表面形态较好的胶原/壳聚糖纳米纤维膜上,通过MTT法、碱性磷酸酶检测、细胞内胶原检测、免疫荧光染色及茜素红染色法观察,研究其细胞生物相容性及诱导成骨性。扫描电子显微镜观察胶原纳米纤维膜及质量比为5∶1∶4的胶原/壳聚糖复合纳米纤维膜的纤维光滑,直径均一。MTT法检测显示,胶原纳米纤维膜和胶原/壳聚糖复合纳米纤维膜均可促进骨髓间充质干细胞的粘附和增殖。细胞培养14 d后,胶原/壳聚糖复合纳米纤维膜上细胞内胶原含量检测为2.02 mg/gport,高于胶原纳米纤维膜组的1.63 mg/gport胶原含量(P<0.05),且胶原/壳聚糖复合纳米纤维膜上细胞内碱性磷酸酶、骨钙素及钙化结节的形成均高于胶原纳米纤维膜组。胶原/壳聚糖复合纳米纤维膜可促进骨髓间充质干细胞的增殖和分化,有望应用于骨再生的研究。     

纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥与骨髓基质细胞的生物相容性*☆

背景：在前期的试验中,通过共沉淀法合成了纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合粉体,并与柠檬酸衍生物溶液调和制备出可生物降解、适当力学性能以及较好黏合强度的骨水泥。 目的：验证纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥材料对体外兔骨髓基质细胞黏附及增殖的影响,了解材料的生物相容性。 方法：应用共沉淀法制备纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合材料作为骨水泥的固相粉体,将柠檬酸衍生物配制成溶液作为液相调和制备黏合性骨水泥。培养兔骨髓基质细胞,传代扩增后接种到材料上,体外继续培养;以细胞加入无材料的培养皿培养为对照。 结果与结论：体外培养的兔骨髓基质细胞2 d后呈梭形成纤维细胞样,生长良好。有材料实验组细胞数显著多于对照组(P < 0.01)。扫描电镜下骨水泥材料具有良好的多孔网状结构,兔骨髓基质细胞伸出多个伪足样突起,紧密贴附在材料表面。两组细胞均保持持续增殖,2,4,6,和8 d实验组增殖均显著快于对照组(P < 0.01)。提示纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥材料具有良好的生物相容性。      

聚左旋乳酸/壳聚糖电纺丝纳米纤维支架与兔内皮祖细胞的生物相容性

背景：电纺丝技术能够使许多高分子材料制备出与细胞外基质相似的三维纳米纤维支架。聚乳酸/壳聚糖纳米纤维复合支架材料能够克服材料的不足,提高组织工程支架生物相容性。 目的：评价聚左旋乳酸/壳聚糖电纺丝纳米纤维支架与兔内皮祖细胞的生物相容性。 方法：电纺丝技术制备聚左旋乳酸,壳聚糖,聚左旋乳酸/壳聚糖的纳米纤维支架,扫描电镜观察其形貌结构。纳米纤维支架与内皮祖细胞进行复合培养后,观察细胞在不同材料上的黏附率、一氧化氮分泌,生长特征和在聚左旋乳酸/壳聚糖纳米纤维支架上的细胞表型特征。 结果与结论：聚左旋乳酸/壳聚糖纳米纤维支架比聚左旋乳酸、壳聚糖具有更合适的纤维直径,具有与细胞外基质相似的纳米纤维三维多孔结构。聚左旋乳酸/壳聚糖纳米纤维支架能够促进内皮祖细胞黏附率和细胞的一氧化氮分泌(P < 0.05,P < 0.01)。内皮祖细胞能够在聚左旋乳酸/壳聚糖复合材料膜上融合成片,保持了细胞的完整形态和分化功能,显示了内皮细胞特异性的vWF表型。提示聚左旋乳酸/壳聚糖电纺丝纳米纤维支架与兔内皮祖细胞具有良好的生物相容性。     

基于壳聚糖的纳米材料在骨组织工程与再生医学中的研究进展

壳聚糖是目前发现的唯一与细胞外基质糖胺聚糖的化学结构相似的天然阳离子多聚糖,具有极为优良的生物相容性、生物可降解性和生物学活性.近年来,基于壳聚糖的纳米材料在组织工程中的研究较为广泛.对壳聚糖的纳米材料、壳聚糖复合纳米材料、壳聚糖纳米纤维和壳聚糖纳米粒子等在骨组织工程与再生医学中的研究进展进行回顾和阐述.近年来的研究显示,壳聚糖复合纳米材料生物支架、壳聚糖纳米纤维支架及包载具有骨诱导性的生物活性因子,以及外源基因的壳聚糖纳米粒子及纳米纤维,在骨组织工程与再生医学中具有良好的应用前景.     

Mechanical and biocompatible influences of chitosan fiber and gelatin on calcium phosphate cement

Calcium phosphate cement (CPC) is a widely used bone substitute in the clinic; however, the low strength of CPC limits its utilization. In this study, we investigated mechanical influences of chitosan fiber combined with gelatin on CPC, and examined the biocompatibility of the new composite with rat bone marrow stromal cells. Compared to the fiber impregnated in phosphate buffered saline (80.5 MPa), our study showed that tensile strength of chitosan fiber increased 106 and 114% with the impregnation of gelatin at the mass fraction 5 and 10%, although this increase was not statistically significant. It was demonstrated by Fourier transform infrared spectroscopy that the characteristic absorption bands of chitosan were changed with the addition of gelatin. The optimal flexural strength enhancement was obtained when CPC was reinforced with fiber at volume fraction of 30% and gelatin at mass fraction of 5% (maximum: 12.31 MPa). The fiber morphology was more compact when the chitosan fibers impregnated with gelatin at mass fraction of 5 or 10% than chitosan alone. The fracture analysis showed that the new CPC-chitosan fiber-gelatin composite presented many remnants of CPC adhered to fibers. Short minimum essential medium extract test showed no cell growth inhibition after the addition of the new composite. Rat bone marrow stromal cells retain the ability to spread and grow on the composite. Our studies demonstrated that the flexural strength is greatly increased by using CPC incorporated with proper ratio of CF and gelatin. More over, the new composite demonstrated biocompatibility in vitro.     

Effects of synergistic reinforcement and absorbable fiber strength on hydroxyapatite bone cement

Approximately a million bone grafts are performed each year in the United States, and this number is expected to increase rapidly as the population ages. Calcium phosphate cement (CPC) can intimately adapt to the bone cavity and harden to form resorbable hydroxyapatite with excellent osteoconductivity and bone-replacement capability. The objective of this study was to develop a strong CPC using synergistic reinforcement via suture fibers and chitosan, and to determine the fiber strength-CPC composite strength relationship. Biopolymer chitosan and cut suture filaments were randomly mixed into CPC. Both suture filaments and composite were immersed in a physiological solution. After 1-day immersion, cement flexural strengths (mean +/- SD; n = 6) were: (2.7 +/- 0.8) MPa for CPC control; (11.2 +/- 1.0) MPa for CPC-chitosan; (17.7 +/- 4.4) MPa for CPC-fiber composite; and (40.5 +/- 5.8) MPa for CPC-chitosan-fiber composite. They are significantly different from each other (Tukey's at 0.95). The strength increase from chitosan and fiber together in CPC was much more than that from either fiber or chitosan alone. The composite strength became (9.8 +/- 0.6) MPa at 35-day immersion and (4.2 +/- 0.7) MPa at 119 days, comparable to reported strengths for sintered porous hydroxyapatite implants and cancellous bone. After suture fiber dissolution, long macropore channels were formed in CPC suitable for cell migration and tissue ingrowth. A semiempirical relationship between suture fiber strength S(F) and composite strength S(C) were obtained: S(C) = 14.1 + 0.047 S(F), with R = 0.92. In summary, this study achieved substantial synergistic effects by combining random suture filaments and chitosan in CPC. This may help extend the use of the moldable, in situ hardening hydroxyapatite to moderate stress-bearing orthopedic applications. The long macropore channels in CPC should be advantageous for cell infiltration and bone ingrowth than conventional random pores and spherical pores.     

Synergistic reinforcement of in situ hardening calcium phosphate composite scaffold for bone tissue engineering

Calcium phosphate cement (CPC) hardens in situ to form solid hydroxyapatite, can conform to complex cavity shapes without machining, has excellent osteoconductivity, and is able to be resorbed and replaced by new bone. Therefore, CPC is promising for use in craniofacial and orthopaedic repairs. However, the low strength and lack of macroporosity of CPC limit its use. The aim of the present study was to increase the strength and toughness of CPC while creating macropores suitable for cell infiltration and bone ingrowth, and to investigate the effects of chitosan and mesh reinforcement on the composite properties. Specimens were self-hardened in 3 mm x 4 mm x 25 mm molds, immersed in a physiological solution for 1-84 d, and tested in three-point flexure. After 1d, the unreinforced CPC control had a flexural strength (mean+/-s.d.; n=6) of (3.3+/-0.4)MPa. The incorporation of chitosan or mesh into CPC increased the strength to (11.9+/-0.8) and (21.3+/-2.7)MPa, respectively. The incorporation of both chitosan and mesh synergistically into CPC dramatically increased the strength to (43.2+/-4.1)MPa. The work-of-fracture (WOF) (toughness) was also increased by two orders of magnitude. After 84 d immersion in a simulated physiological solution, the meshes in CPC dissolved and formed interconnected cylindrical macropores. The novel CPC scaffold had a flexural strength 39% higher, and WOF 256% higher than the conventional CPC without macropores. The new composite had an elastic modulus within the range for cortical bone and cancellous bone, and a flexural strength higher than those for cancellous bone and sintered porous hydroxyapatite implants. In conclusion, combining two different reinforcing agents together in self-hardening CPC resulted in superior synergistic strengthening compared to the traditional use of a single reinforcing agent. The strong and macroprous CPC scaffold may be useful in stress-bearing craniofacial and orthopaedic repairs.     

Strong calcium phosphate cement-chitosan-mesh construct containing cell-encapsulating hydrogel beads for bone tissue engineering

Calcium phosphate cement (CPC) can conform to complex cavity shapes and set in situ to form bioresorbable hydroxyapatite. The aim of this study was to introduce cell-encapsulating alginate hydrogel beads into CPC and to improve the mechanical properties using chitosan and fiber mesh reinforcement. Because the CPC setting was harmful to the MC3T3-E1 osteoblast cells, alginate was used to encapsulate and protect the cells in CPC. Cells were encapsulated into alginate beads, which were then mixed into three pastes: conventional CPC, CPC-chitosan, and CPC-chitosan-mesh. After 1 day culture inside the setting cements, there were numerous live cells and very few dead cells, indicating that the alginate beads adequately protected the cells. Cell viability was assessed by measuring the mitochondrial dehydrogenase activity, using a Wst-1 colorimetric assay. Absorbance at 450 nm (arbitrary units) (mean +/- SD; n = 5) was 1.36 +/- 0.41 for cells inside conventional CPC, 1.29 +/- 0.24 for cells inside CPC-chitosan composite, and 0.73 +/- 0.22 for cells inside CPC-chitosan-mesh composite. All three values were similar to 1.00 +/- 0.14 for the control with cells in beads in the cell culture medium without any CPC (Tukey's at p = 0.05). Flexural strength for conventional CPC containing cell-encapsulating beads was 1.3 MPa. It increased to 2.3 MPa when chitosan was incorporated. It further increased to 4.3 MPa with chitosan and the reinforcement from one fiber mesh, and 9.5 MPa with chitosan and three sheets of fiber mesh. The latter two strengths matched reported strengths for sintered porous hydroxyapatite implants and cancellous bone. In summary, cell-encapsulated-alginate-CPC constructs showed favorable cell viability. The use of chitosan and mesh progressively improved the mechanical properties. These strong, in situ hardening, and cell-seeded hydroxyapatite cements may have potential for bone tissue engineering in moderate stress-bearing applications.     

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.     

Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC–chitosan and CPC–chitosan–fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC–chitosan–fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC–chitosan–fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.     

Fast setting calcium phosphate-chitosan scaffold: mechanical properties and biocompatibility

Calcium phosphate cement (CPC) sets in situ to form hydroxyapatite and is highly promising for a wide range of clinical applications. However, its low strength limits its use to only non-stress applications, and its lack of macroporosity hinders cell infiltration, bone ingrowth and implant fixation. The aim of this study was to develop strong and macroporous CPC scaffolds by incorporating chitosan and water-soluble mannitol, and to examine the biocompatibility of the new graft with an osteoblast cell line and an enzymatic assay. Two-way ANOVA identified significant effects on mechanical properties from chitosan reinforcement and powder:liquid ratio (p<0.001). The flexural strength of CPC-chitosan composite at a powder:liquid ratio of 2 was (13.6+/-1.2) MPa, which was significantly higher than (3.2+/-0.6) MPa for CPC control without chitosan (Tukey's at 0.95). At a powder:liquid ratio of 3.5, CPC-chitosan had a strength of (25.3+/-2.9) MPa, which was significantly higher than (10.4+/-1.7) MPa for CPC control. The scaffolds possessed total pore volume fractions ranging from 42.0% to 80.0%, and macroporosity up to 65.5%. At total porosities of 52.2-75.2%, the scaffold had strength and elastic modulus values similar to those of sintered porous hydroxyapatite and cancellous bone. Osteoblast mouse cells (MC3T3-E1) were able to adhere, spread and proliferate on CPC-chitosan specimens. The cells, which ranged from about 20 to 50 microm including the cytoplasmic extensions, infiltrated into the 165-271 microm macropores of the scaffold. In summary, substantial reinforcement and macroporosity were imparted to a moldable, fast-setting, biocompatible, and resorbable hydroxyapatite graft. The highly porous scaffold may facilitate bone ingrowth and implant fixation in vivo. In addition, the two to three times increase in strength may help extend the use of CPC to larger repairs in moderately stress-bearing locations.     

Calcium phosphate cement containing resorbable fibers for short-term reinforcement and macroporosity.

Calcium phosphate cement (CPC) sets to form hydroxyapatite and has been used in medical and dental procedures. However, the brittleness and low strength of CPC prohibit its use in many stress-bearing locations, unsupported defects, or reconstruction of thin bones. Recent studies incorporated fibers into CPC to improve its strength. In the present study, a novel methodology was used to combine the reinforcement with macroporosity: large-diameter resorbable fibers were incorporated into CPC to provide short-term strength, then dissolved to create macropores suitable for bone ingrowth. Two types of resorbable fibers with 322 microm diameters were mixed with CPC to a fiber volume fraction of 25%. The set specimens were immersed in saline at 37 degrees C for 1, 7, 14, 28 and 56d, and were then tested in three-point flexure. SEM was used to examine crack-fiber interactions. CPC composite achieved a flexural strength 3 times, and work-of-fracture (toughness) nearly 100 times, greater than unreinforced CPC. The strength and toughness were maintained for 2-4 weeks of immersion, depending on fiber dissolution rate. Macropores or channels were observed in CPC composite after fiber dissolution. In conclusion, incorporating large-diameter resorbable fibers can achieve the needed short-term strength and fracture resistance for CPC while tissue regeneration is occurring, then create macropores suitable for vascular ingrowth when the fibers are dissolved. The reinforcement mechanisms appeared to be crack bridging and fiber pullout, the mechanical properties of the CPC matrix also affected the composite properties.     

Strong and bioactive composites containing nano-silica-fused whiskers for bone repair

Self-hardening calcium phosphate cement (CPC) sets to form hydroxyapatite with high osteoconductivity, but its brittleness and low strength limit its use to only non-stress bearing locations. Previous studies developed bioactive composites containing hydroxyapatite fillers in Bis-GMA-based composites for bone repair applications, and they possessed higher strength values. However, these strengths were still lower than the strength of cortical bone. The aim of this study was to develop strong and bioactive composites by combining CPC fillers with nano-silica-fused whiskers in a resin matrix, and to characterize the mechanical properties and cell response. Silica particles were fused to silicon carbide whiskers to roughen the whisker surfaces for enhanced retention in the matrix. Mass ratios of whisker:CPC of 1:2, 1:1 and 2:1 were incorporated into a Bis-GMA-based resin and hardened by two-part chemical curing. Composite with only CPC fillers without whiskers served as a control. The specimens were tested using three-point flexure and nano-indentation. Composites with whisker:CPC ratios of 2:1 and 1:1 had flexural strengths (mean+/-SD; n=9) of (164+/-14) MPa and (139+/-22) MPa, respectively, nearly 3 times higher than (54+/-5) MPa of the control containing only CPC fillers (p<0.05). The strength of the new whisker-CPC composites was 3 times higher than the strength achieved in previous studies for conventional bioactive composites containing hydroxyapatite particles in Bis-GMA-based resins. The mechanical properties of the CPC-whisker composites nearly matched those of cortical bone and trabecular bone. Osteoblast-like cell adhesion, proliferation and viability were equivalent on the non-whisker control containing only CPC fillers, on the whisker composite at whisker:CPC of 1:1, and on the tissue culture polystyrene control, suggesting that the new CPC-whisker composite was non-cytotoxic.     

High early strength calcium phosphate bone cement: effects of dicalcium phosphate dihydrate and absorbable fibers

Calcium phosphate cement (CPC) sets in situ to form resorbable hydroxyapatite with chemical and crystallographic similarity to the apatite in human bones, hence it is highly promising for clinical applications. The objective of the present study was to develop a CPC that is fast setting and has high strength in the early stages of implantation. Two approaches were combined to impart high early strength to the cement: the use of dicalcium phosphate dihydrate with a high solubility (which formed the cement CPC(D)) instead of anhydrous dicalcium phosphate (which formed the conventional cement CPC(A)), and the incorporation of absorbable fibers. A 2 x 8 design was tested with two materials (CPC(A) and CPC(D)) and eight levels of cement reaction time: 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, and 24 h. An absorbable suture fiber was incorporated into cements at 25% volume fraction. The Gilmore needle method measured a hardening time of 15.8 min for CPC(D), five-fold faster than 81.5 min for CPC(A), at a powder:liquid ratio of 3:1. Scanning electron microscopy revealed the formation of nanosized rod-like hydroxyapatite crystals and platelet crystals in the cements. At 30 min, the flexural strength (mean +/- standard deviation; n = 5) was 0 MPa for CPC(A) (the paste did not set), (4.2 +/- 0.3) MPa for CPC(D), and (10.7 +/- 2.4) MPa for CPC(D)-fiber specimens, significantly different from each other (Tukey's at 0.95). The work of fracture (toughness) was increased by two orders of magnitude for the CPC(D)-fiber cement. The high early strength matched the reported strength for cancellous bone and sintered porous hydroxyapatite implants. The composite strength S(c) was correlated to the matrix strength S(m): S(c) = 2.16S(m). In summary, substantial early strength was imparted to a moldable, self-hardening and resorbable hydroxyapatite via two synergistic approaches: dicalcium phosphate dihydrate, and absorbable fibers. The new fast-setting and strong cement may help prevent catastrophic fracture or disintegration in moderate stress-bearing bone repairs.