负载rhBMP-2钙磷硅基活性骨修复材料的3D打印构建及生物学性能研究

目的 构建一种新型钙磷硅基活性骨修复材料,研究材料成分、孔道结构和是否负载生长因子对支架力学性能、细胞粘附性能和骨组织形成过程的影响。方法 以磷酸钙骨水泥(CPC)、介孔硅酸钙（MCS）为原料,采用3D打印技术构建不同孔道结构的MCS/CPC（MCS/CPC）复合支架,在扫描电子显微镜下观察支架内部孔道结构,通过万能力学试验机测试各组支架的最大抗压力学性能,用四甲基偶氮唑盐法测试各组支架的细胞粘附性能。建立大鼠颅骨原位缺损修复模型,在支架上负载重组人骨形态发生蛋白（rhBMP-2）制备得到钙磷硅基活性多孔支架（MCS/CPC/rhBMP-2）,考察CPC、MCS/CPC和MCS/CPC/rhBMP-2支架的组织相容性与成骨性能。结果 采用3D打印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。垂直孔设计大小为350 μm 的MCS/CPC支架具有适宜的孔隙率和抗压力学强度,分别为56.6%和9.8 MPa;细胞相容性良好,有利于细胞粘附。负载rhBMP-2可显著加快新生骨组织形成,植入MCS/CPC/rhBMP-2复合支架初期纤维组织即可在连通孔道中自由生长,在植入12周后,MCS/CPC/rhBMP-2支架新生骨面积明显高于CPC和MCS/CPC支架,且新生骨组织形成过程与颅骨膜内成骨过程相似,具有一定仿生效应。结论 采用3D打印法制备的MCS/CPC/rhBMP-2支架材料具有规则的连通孔道,力学性能良好,促进成骨效果优异,是理想的新型骨缺损修复材料。     

三种钙磷陶瓷材料复合重组人骨形成蛋白-2体内成骨的研究

目的 探讨和比较3种钙磷陶瓷材料HA、TCP、HA／TCP复合重组入骨形成蛋白。2(rhBMP—2)体内异位成骨效果，为临床应用提供依据。方法 取35只3月龄Wistar大鼠，将复合rhBMP—2的3种钙磷陶瓷材料(1：1)植于鼠背部肌袋内，未复合rhBMP-2的上述3种单纯陶瓷材料为对照组。术后2、4和8周取材，测定植入物碱性磷酸酶(ALP)活性，通过HE染色和计算机图像分析进行组织学和组织计量学观察，比较新生骨组织的形成。结果 术后2、4周复合植入物ALP活性测定从高到低依次为HA、HA／TCP、TCP，但在相同rhBMP—2剂量下，其差异无统计学意义(P＞0．05)，与相对应单纯支架材料比较有统计学意义(P＜0．05)；组织学和组织计量学检测结果显示各复合材料组均有新骨形成，成骨量随时间推移而增加，2周时以HA/rhBMP—2成骨量较多，但3组间差异无统计学意义(P＞0．05)；8周时新骨形成以双相陶瓷HA／TCP最佳，相关参数和图像分析有统计学意义(P＜0．05)，成骨量8周较2、4周多，有统计学意义(P＜0．01)；3种单纯支架材料各观察期均无骨样组织形成。结论 双相陶瓷材料HA／TCP是携带rhBMP—2的钙磷陶瓷良好支架材料。     

骨组织工程中硅磷酸钙材料研究进展

硅磷酸钙是一种具有良好的骨传导性能、成骨性能及可控降解性能的新型多孔生物陶瓷材料,主要包括含硅羟基磷灰石和含硅磷酸三钙.在骨组织工程中,硅磷酸钙有较高的生物学活性,对成骨细胞的黏附、增殖及分化等起着重要作用,并可减少环境改变引起的细胞损害,是较为理想的骨替代材料.硅磷酸钙自身不具有成骨性能,这是作为骨支架材料的一大缺憾,有待更深入研究.该文就硅在骨组织代谢中的作用、硅磷酸钙结构和理化性质以及硅磷酸钙实验研究和临床应用进展作一综述.     

胶原支架增强自固化磷酸钙骨水泥的力学及成骨性能研究

[目的]研究胶原支架（CS）对磷酸钙骨水泥（CPC）的力学及其在体内成骨的影响。[方法]试验分CPC／CS及CPC两组，三点弯曲试验测试材料的强度和弹性模量；组织学观察材料植入兔股骨22及54周的成骨状况。[结果]CPC／CS比CPC的弯曲强度、韧性强度分别提高了64．2％、3933．3％，弹性模量降低了45．7％；组织学显示22周CPC／CS内的胶原支架已完全被新骨替代，CPC只在边缘有少量成骨及材料降解而内部无成骨；54周CPC／CS已大部分降解孔化，孔内充满大量新骨及髓样组织，而CPC边缘区的成骨及材料降解虽比22周时明显，但其内部仍未见成骨。[结论]胶原支架既可改善CPC的力学性能，又能促进新骨长入CPC／CS复合材料内部，因此，CPC／胶原支架是较好的骨缺损修复材料。     

应用活性玻璃/rhBMP2构建生物活性材料支架的相容性研究

[目的]观察BG／rhBMP2构建的三维立体材料支架的细胞相容性、组织相容性，为进一步改善材料性能提供依据。[方法]体外培养的兔成骨细胞分别与BG和BG／rhBMP2材料支架联合培养，倒置显微镜、扫描电镜观察细胞黏附情况，MTT法分析细胞增殖情况；将复合培养的两种支架材料植入兔肌袋中，同期观察复合材料及空白对照组，分别于术后2、4、8、12周来评价其组织相容性。[结果]MTT法结果显示：随着培养时间的延长，各组细胞数量都明显增加，各时间点复合材料支架组细胞数高于空白对照组，差异有显著性意义，复合材料支架BG／rhBMP2在细胞贴壁时间和细胞活性方面均较单纯支架优良；两种支架材料在体内均无明显炎症反应。[结论]BG／rhBMP2是一种具有良好生物相容性的支架材料，有望在骨组织工程中得到广泛应用。     

无机活性元素骨组织工程支架材料的细胞亲和性

目的 评价作为骨修复材料的无机活性元素骨组织工程支架材料的细胞亲和性。方法 测试无机活性元素骨组织工程支架材料的比表面积、孔径分布、孔隙率、吸水率。将体外培养扩增并经过流式细胞仪鉴定的人骨髓间充质干细胞（MSCs）与无机活性元素骨组织工程支架材料复合培养,以单纯接种培养MSCs为对照组,噻唑蓝（MTT）法检测材料对细胞增殖的影响,扫描电镜观察材料表面细胞生长状况。结果 无机活性元素骨组织工程支架材料比表面积为210m^2／g,平均微孔直径为6nm,孔隙率90％,有34％的吸水率。MIT法显示培养至第7天时材料组细胞增殖与对照组有显著性差异（P〈0．05）,扫描电镜下见材料表面细胞生长状况良好。结论 无机活性元素骨组织工程支架材料具有良好的细胞亲和性,是一种较为合适的极具潜力的骨修复材料。     

3D打印PLA-HA复合材料构建组织工程骨的实验研究

目的探讨并观察3D打印聚乳酸(PLA)-羟基磷灰石(HA)复合支架材料在体内成骨的可行性。方法将骨髓基质细胞与3D打印PLA-HA复合材料进行体外复合培养,通过扫描电镜观察细胞在支架材料上的生长黏附情况。选择成年新西兰大白兔单侧胫骨内侧建立体内生物反应器模型,分离隐动、静脉血管束,并剥离胫骨内侧骨膜,将游离的动静脉血管束完全穿越支架材料内部并固定于胫骨骨膜囊内,此种方法构建的组织工程骨为实验组;单纯将复合骨髓基质细胞的3D打印PLA-HA复合材料回植于胫骨内侧骨膜囊内,此种方法构建的组织工程骨为对照组。术后6周取材,分别采用聚合酶链式反应(PCR)检测骨分化相关基因,并进行显微CT三维重建及骨形态计量分析、HE染色组织学观察。结果骨髓基质细胞能在3D打印PLA-HA复合材料上黏附生长。定量PCR检测成骨分化基因OPN及COLⅠ的表达,结果显示实验组OPN及COLⅠ相对表达量分别为8.0333±0.5820和7.5452±0.5608;对照组OPN及COLⅠ相对表达量分别为5.7248±0.9975和4.0173±0.2654,差异有统计学意义(P0.05)。显微CT扫描显示,实验组新生骨组织体积及骨小梁相对数目较对照组高,且差异有统计学意义(P0.05)。组织学观察可见实验组有新生骨组织形成,部分新生骨组织为编织骨,骨细胞体积大、数量多,呈编织状排列,骨细胞分化成熟;对照组可见部分新生骨样组织形成,骨细胞分化较成熟。结论以骨髓基质细胞为种子细胞,3D打印PLA-HA复合材料为支架,复合隐动、静脉血管束及自体骨膜,在体内能构建出功能相对完善的组织工程骨;3D打印PLA-HA复合材料可作为骨组织工程中支架材料。     

同种异体脂肪干细胞复合脱钙骨修复大鼠尺骨缺损

目的探讨同种异体脂肪干细胞修复管状骨缺损的可行性。方法获取SD大鼠的腹股沟处脂肪,分离培养脂肪干细胞（Adipose-Derived Stem Cells,ADSCs）;鼠第3代ADSCs与脱钙骨复合,24 h后进行成骨诱导培养。检测细胞在材料表面的生长及成骨分化能力。建立鼠两侧尺骨缺损模型,分别植入鼠ADSCs-脱钙骨复合物（实验侧）和单纯脱钙骨材料（对照侧）;8周、24周后取样,行DR和组织学检测,观察成骨情况。结果 ADSCs能在脱钙骨上很好地黏附和生长,并维持成骨分化能力。细胞-材料复合物植入24周后,DR显示实验侧有新生骨基质长成,对照侧未见骨组织生成。组织学检测显示,实验侧缺损区被典型的骨组织取代,可见新生骨小梁附着于脱钙骨表面;对照侧只有少量的骨组织和纤维组织充填。结论 ADSCs-脱钙骨材料复合物植入,能成功修复临界大小的管状骨缺损。     

多孔钽材料细胞毒性、生物相容性及体内成骨性研究

 目的 探讨国产多孔钽材料的细胞毒性和生物相容性,并通过兔骨内植入成骨示踪观察其成骨作用。方法 扫描电镜观察并测量多孔钽的形态学特征。来源于新西兰胎兔颅盖骨的成骨细胞以多孔钽浸提液（实验组）及完全培养基（对照组）培养,MTT法检测多孔钽的细胞毒性及其对增殖的影响。成骨细胞与多孔钽体外复合培养,观察成骨细胞的黏附、生长及增殖。雄性新西兰大白兔24只,制备股骨髁上多孔钽棒植入模型;4只动物于术后第5天和第19天分别肌肉注射荧光素钙黄绿素和茜素红,术后10 周取材,于488 nm（钙黄绿素激发光）和543 nm（茜素红激发光）波长处,激光扫描共聚焦显微镜观察多孔钽-骨界面成骨;20只动物于术后2、4、8和12 周取材行大体及硬组织切片观察。结果 多孔钽表面及断面可见均匀分布的三维立体连通孔隙结构。MTT法检测显示实验组与对照组细胞随培养时间的延长,其OD值的差异均无统计学意义。扫描电镜显示复合培养早期,细胞在多孔支架表面和孔壁上黏附,相互连接;晚期汇合成片并分泌细胞外基质覆盖材料表面。体内成骨实验显示植入的多孔钽棒与宿主骨结合紧密。硬组织切片显示术后2、4周时多孔钽-骨界面已出现新生骨及小血管,并向孔隙内生长;8、12周时多孔钽表面和孔隙内已长满新生骨组织,新生骨小梁已发育成熟并与材料直接接触。激光共聚焦扫描显微镜显示多孔钽-骨界面及孔隙内可见绿色荧光（钙黄绿素）和红色荧光（茜素红）标记的新生骨组织,红色荧光带位于绿色荧光带周围,早期均呈不连续性,晚期则融为一体。结论 国产多孔钽材料无细胞毒性,具有良好的生物相容性,多孔钽-骨界面为接触及传导性成骨并呈时间依赖性。     

壳聚糖/胡须/磷酸钙骨水泥复合生物材料的力学性能及对诱导多能干细胞成骨潜能的影响

目的观察新型强化型磷酸钙骨水泥(calcium phosphate cement,CPC)生物支架材料的力学性能以及对诱导多能干细胞(induced potential stem cells,i PS)生物活性及成骨潜能的影响。方法根据制备的支架材料不同,将实验分为4组,A组为单纯CPC支架组,B组为CPC∶10%wt壳聚糖为2∶1比例混合支架组,C组为CPC∶10%wt壳聚糖∶胡须为2∶1∶1比例混合支架组,D组为CPC∶10%wt壳聚糖∶胡须为2∶1∶2比例混合支架组。检测各组支架材料力学性能(抗弯强度、断裂功、硬度及弹性模量)。与第5代i PS-MSCs复合培养1、3、7、14 d采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测各组吸光度(A)值;1、7、14 d行ALP活性检测、Live/Dead荧光染色及定量检测、RT-PCR检测成骨基因ALP、Runx2、Ⅰ型胶原、骨钙蛋白(osteocalcin,OC)及BMP-2表达;1、7、14、21 d行茜素红染色观察。取3月龄雄性SD大鼠24只,建立8 mm颅骨骨缺损模型,随机分为4组(n=6),分别植入对应的4组支架材料,术后8周取材行HE染色观察骨缺损修复情况,并检测新生骨体积百分比和新生血管密度。结果 B、C、D组抗弯强度、断裂功、弹性模量及硬度均显著高于A组,C、D组高于B组,D组高于C组,差异均有统计学意义(P0.05)。CCK-8法检测示随培养时间增加,细胞活性逐渐增加,3、7、14 d时B、C、D组A值均显著高于A组,C、D组高于B组(P0.05),C、D组间比较差异无统计学意义(P0.05)。Live/Dead荧光染色示,7、14 d时B、C、D组活细胞比例均显著高于A组(P0.05),7 d时C、D组显著高于B组(P0.05);RT-PCR检测示,7、14 d时B、C、D组各基因相对表达量均显著高于A组,C、D组显著高于B组(P0.05),C、D组间比较差异无统计学意义(P0.05);茜素红染色示随培养时间延长,各组支架材料表面的红色钙盐沉积均逐渐加深变厚,14、21 d时B、C、D组A值均显著高于A组,C、D组高于B组(P0.05),C、D组间差异无统计学意义(P0.05)。动物体内修复实验显示,各组新生骨主要填充于支架材料间隙中,成骨细胞和新生血管被基质中的新生骨组织包围,成骨细胞排列在新生骨边界上,B、C、D组新生骨显著多于A组,C、D组显著多于B组。B、C、D组新生骨体积百分比和新生血管密度均显著高于A组,C、D组高于B组,差异有统计学意义(P0.05);C、D组间差异无统计学意义(P0.05)。结论复合壳聚糖、胡须和CPC制备的新型强化型复合生物支架材料的机械性能明显优于单纯CPC支架材料,可满足皮质骨及松质骨的机械性能要求。i PS-MSCs在新型强化型复合生物支架材料上黏附、增殖,骨组织修复效果良好,可满足骨植入材料的生物活性及成骨活性要求。     

Adult-type hypolactasia and calcium availability: decreased calcium intake or impaired calcium absorption?

Summary Adult-type hypolactasia, as mediated by a widespread genetic predisposition, not only reduces calcium intake but also calcium absorption in the presence of high amounts of lactose and may, therefore, promote osteoporosis. A lactose-reduced diet and lactose-free calcium supplements may reverse this imbalance. Introduction and hypothesis Adult-type hypolactasia (HL) defined by the LCT_(−13910) polymorphism may reduce calcium intake by reducing dairy consumption and, therefore, promote osteoporosis. This study aimed to evaluate whether lactose also decreases intestinal calcium absorption in subjects with HL and whether lactose-reduced diet and lactose-free calcium supplementation as recommended could maintain bone mineral density (BMD). Methods Based on LCT genotyping, 73 postmenopausal women with and without HL underwent a conventional H₂ breath test with a concomitant oral strontium absorption test lasting 150 minutes, which closely reflects intestinal calcium absorption. In addition, we compared bone-specific laboratory parameters, lumbar and femoral BMD, and spinal radiographs to a similar bone assessment 5 years earlier. Results LCT genotyping and functional lactose malabsorption tests were highly correlated. Dairy product consumption was reduced by 80% in HL individuals. During concomitant lactose application, mean strontium absorption was blunted by 54% in HL subjects after 150 minutes (1272 ± 629 μg/L vs. 2020 ± 1130 μg/L in lactose tolerant subjects, p = 0.001). Nevertheless, BMD in HL subjects remained stable with lactose-free calcium supplements during the observation period. Conclusion Both decreased calcium intake as well as lactose-associated impaired calcium absorption may predispose subjects with HL to osteoporosis. Lactose-free calcium supplementation may help to maintain BMD in HL subjects.     

Dose dependency of calcium absorption: a comparison of calcium carbonate and calcium citrate

Calcium supplementation is recommended as a prophylaxis against bone loss. This study was performed to determine the dose dependency of calcium absorption in an attempt to derive an optimum dose schedule. Using the well-described oral calcium load technique, we measured the calcium absorption from three different calcium doses (0.5, 1.0, and 2.0 g) of both calcium carbonate and calcium citrate administered to 21 normal subjects (4 men and 17 women, 22-60 years). Nine subjects underwent two additional loads with 0.2 g of elemental calcium as calcium carbonate and as calcium citrate. The intestinal calcium absorption from calcium carbonate and calcium citrate was estimated from the rise in urinary calcium following oral ingestion of the respective calcium salt. The increment in urinary calcium post-load, reflective of intestinal calcium absorption, rose rapidly from 0 to 0.5 g calcium loads with only slight subsequent increases from the 0.5 g to 2.0 g calcium doses. Thus, results indicate that 0.5 g of calcium is the optimum dose of either calcium salt. Moreover, the increment in urinary calcium post-load was higher from calcium citrate than from calcium carbonate at all four dosage levels. The increment in urinary calcium (during the second 2 hr) following calcium citrate load (0.5 g calcium) was 0.104 +/- 0.096 mg/dl glomerular filtrate (GF), which was higher than that of 0.091 +/- 0.068 mg/dl GF obtained from 2.0 g calcium as calcium carbonate. These results confirm the superior calcium bioavailability from calcium citrate as compared with calcium carbonate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extra-intestinal calcium handling contributes to normal serum calcium levels when intestinal calcium absorption is suboptimal

The active form of vitamin D, 1,25(OH)₂D, is a crucial regulator of calcium homeostasis, especially through stimulation of intestinal calcium transport. Lack of intestinal vitamin D receptor (VDR) signaling does however not result in hypocalcemia, because the increased 1,25(OH)₂D levels stimulate calcium handling in extra-intestinal tissues. Systemic VDR deficiency, on the other hand, results in hypocalcemia because calcium handling is impaired not only in the intestine, but also in kidney and bone. It remains however unclear whether low intestinal VDR activity, as observed during aging, is sufficient for intestinal calcium transport and for mineral and bone homeostasis. To this end, we generated mice that expressed the Vdr exclusively in the gut, but at reduced levels. We found that ~ 15% of intestinal VDR expression greatly prevented the Vdr null phenotype in young-adult mice, including the severe hypocalcemia. Serum calcium levels were, however, in the low-normal range, which may be due to the suboptimal intestinal calcium absorption, renal calcium loss, insufficient increase in bone resorption and normal calcium incorporation in the bone matrix. In conclusion, our results indicate that low intestinal VDR levels improve intestinal calcium absorption compared to Vdr null mice, but also show that 1,25(OH)₂D-mediated fine-tuning of renal calcium reabsorption and bone mineralization and resorption is required to maintain fully normal serum calcium levels.     

Intestinal absorption of calcium from calcium ascorbate in rats

The intestinal absorption of calcium (Ca) from Ca ascorbate (Ca-AsA) was investigated in normal rats. Each animal was perorally administered either 5 mg (low dose) or 10 mg (high dose) of Ca in 1 ml of distilled water as Ca-AsA, Ca carbonate (CaCO₃), or Ca chloride (CaCl₂), which were intrinsically labeled with ⁴⁵Ca using ⁴⁵CaCl₂. The amount of radioactivity in plasma was measured periodically up to 34 h after dosing, and pharmacokinetic parameters were calculated from the radioactivity in plasma. The time taken to reach the maximum ⁴⁵Ca level (T_max) did not differ among the three groups. The area under the plasma ⁴⁵Ca level/time curve (AUC∞) value for the Ca-AsA group was significantly higher than those for the CaCO₃ and the CaCl₂ groups. The radioactivity at T_max (C_max) for the Ca-AsA group was significantly higher than those for the CaCO₃ and the CaCl₂ groups for the low dose, and comparable with or significantly higher than those for the CaCl₂ and CaCO₃ groups for the high dose. Similar results were observed for whole-body ⁴⁵Ca retention. Radioactivity in the femur 34 h after dosing was the highest in the Ca-AsA group and the lowest in the CaCO₃ group. The rank order of solubility in water, the first fluid (pH 1.2, JP-1) of JPXIII disintegration medium, acetate buffer solution (pH 4.0), triethanolamine-malate buffer solution (pH 7.0) and ammonium chloride buffer solution (pH 10.0) at 37°C was CaCl₂ > Ca-AsA > CaCO₃. In contrast, the rank order of the solubility in the second fluid (pH 6.8, JP-2) of JPXIII disintegration medium at 37°C was Ca-AsA > CaCl₂ > CaCO₃. These results indicate that the absorbability of Ca from Ca-AsA is almost comparable with, or higher than, that from CaCl₂ and significantly higher than that from CaCO₃ because of its high degree of solubility in the intestine. Therefore, Ca-AsA would be useful as a Ca supplement with relatively high absorption from intestine. Received: March 31, 1998 / Accepted: June 16, 1998     

Fecal calcium density: a measure of calcium compliance

We evaluated fecal calcium density (mass of calcium per g dry weight of feces) as a measure of compliance with a prescribed calcium intake regimen using 4 day fecal pools collected on a metabolic research unit from subjects ingesting measured, constant intakes. Fecal calcium density was highly correlated with intake (r = 0.897, P less than 0.001). Intake estimates based on fecal calcium density exhibited a standard error of the mean equal to 3.76 mmol calcium. Since a typical calcium supplement table contains 12.5 mmol calcium, the measurement of fecal calcium density is sensitive enough to detect regular omission of one or more pills daily. Applicability of this approach to convenience samples of feces was evaluated in 15 individuals by testing homogeneity of fecal calcium density values on up to six different 3-9 g portions (wet weight) of each volunteer's fecal sample. The within-sample coefficient of variation was 9.5% for all subsamples and 7.3% for samples from individuals with intakes above 25 mmol calcium per day. Thus feces are reasonably homogeneous in regard to calcium density. Accordingly, reasonably small fecal collections should suffice for its measurement.     

Epithelial calcium channel: gate-keeper of active calcium reabsorption

The epithelial calcium channel present in the apical membrane of 1,25-dihydroxyvitamin D3-responsive nephron segments represents the first member of a new family of calcium channels. This review covers the distinctive properties of this highly calcium-selective channel and highlights the implications for our understanding of the process of calcium reabsorption.     

Intestinal mitochondrial calcium uptake during adaptation to dietary calcium restriction

In order to evaluate the role of mitochondrial calcium uptake in intestinal calcium absorption, the effect of adaptation to dietary calcium deficiency on the in vitro uptake of calcium by isolated duodenal mitochondria was studied. Experiments were performed utilizing 28-day-old cockerels which had received a diet adequate in vitamin D₃ and phosphate and containing either 0.08% or 1.20% calcium. Mitochondrial⁴⁵Ca uptake from chicks deprived of dietary calcium was not significantly different from controls. These results suggest that increased calcium uptake by intestinal mitochondria is not crucial for adaptation to a low calcium diet.