Cells responding to surface structure of calcium phosphate ceramics for bone regeneration

Surface structure largely affects the inductive bone‐forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical‐sized bone defects. Surface‐dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone‐forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone‐related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3‐E1 (osteogenic precursors), SV‐HFO (pre‐osteoblasts), MG63 (osteoblasts) and SAOS‐2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron‐scaled surface structure (TCP‐B) or submicron‐scaled surface structure (TCP‐S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis‐related gene expression (i.e. vascular endothelial growth factor) on TCP‐S. Without additional osteogenic inducing factors, submicron‐scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3‐E1, MG63 and SAOS‐2, and increased ALP activity of MC3T3‐E1 and SV‐HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron‐structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone regeneration. Copyright © 2017 John Wiley & Sons, Ltd.     

Bio-Gide胶原膜对兔骨髓间充质干细胞增殖和成骨分化的影响

背景：相关实验表明Bio-Gide胶原膜与细胞有良好的生物相容性,但有关与其复合培养干细胞成骨分化能力的报道少见。 目的：观察Bio-Gide胶原膜对骨髓间充质干细胞增殖及成骨分化的影响。 方法：全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,将第3代兔骨髓间充质干细胞分别接种于覆盖Bio—Gide胶原膜的培养板（实验组）与单纯培养板（对照组）培养。于培养1,4,7,14d利用CCK-8试剂盒检测细胞增殖;成骨分化诱导培养1,4,7,14d收集细胞培养液上清,检测细胞碱性磷酸酶活性。 结果与结论：两组细胞数量均随着培养时间的增加而不断增加,对照组培养7d细胞数量明显多于实验组（P〈0．05）,其他时间点组间比较差异无显著性意义。两组细胞碱性磷酸酶活性均随着培养时间的增加而不断增加,实验组成骨诱导14d细胞碱性磷酸酶活性高于对照组（P〈0．05）,其他时间点组间比较差异无显著性意义。表明Bio-Gide胶原膜可促进兔骨髓间充质干细胞的增殖及成骨分化。     

Octacalcium phosphate collagen composite facilitates bone regeneration of large mandibular bone defect in humans

Recently it was reported that the implantation of octacalcium phosphate (OCP) and collagen composite (OCP–collagen) was effective at promoting bone healing in small bone defects after cystectomy in humans. In addition, OCP–collagen promoted bone regeneration in a critical‐sized bone defect of a rodent or canine model. In this study, OCP–collagen was implanted into a human mandibular bone defect with a longer axis of approximately 40 mm, which was diagnosed as a residual cyst with apical periodontitis. The amount of OCP–collagen implanted was about five times greater than the amounts implanted in previous clinical cases. Postoperative wound healing was satisfactory and no infection or allergic reactions occurred. The OCP–collagen‐treated lesion was gradually filled with radio‐opaque figures, and the alveolar region occupied the whole of the bone defect 12 months after implantation. This study suggests that OCP–collagen could be a useful bone substitute material for repairing large bone defects in humans that might not heal spontaneously. Copyright © 2015 John Wiley & Sons, Ltd.     

Influence of electron beam irradiation doses on bone regeneration by octacalcium phosphate collagen composites

An octacalcium phosphate and collagen composite (OCP/Col) achieved efficient bone regeneration with excellent resorbability. After the confirmation of its safety and efficacy in preclinical animal studies, the present study investigated the influence of electron beam irradiation doses on bone regeneration by OCP/Col in order to secure its commercialization. OCP/Col was prepared as previously described and the packed OCP/Col was sterilized using different doses of electron beam irradiation. A standardized defect, 9 mm in diameter, was made in the rat calvarium. A disk of OCP/Col (diameter 9 mm, thickness 1.5 mm) sterilized using different doses of electron beam irradiation was then implanted into the defect. Five defects in each group were treated and fixed 4, 12 and 24 weeks after implantation. Specimens were decalcified and stained with haematoxylin and eosin. In a histomorphometrical analysis, the percentage of newly formed bone in the defect (n‐Bone%) was calculated. In OCP/Col 15 kGy, newly formed bone was enhanced and present throughout the defect with the resorption of OCP/Col. Furthermore, vigorous bone remodelling and bone maturation were observed. In OCP/Col 40 kGy, newly formed bone was not as prominent as that with OCP/Col 15 kGy. A histomorphometrical analysis using Student's t‐test at 24 weeks revealed that the n‐Bone% of OCP/Col 15 kGy (65.9 ± 7.14%) was significantly higher than that of OCP/Col 40 kGy (38.0 ± 10.2%). These results suggest that different electron beam irradiation doses influence bone regeneration by OCP/Col.     

Comparative study of NMP‐preloaded and dip‐loaded membranes for guided bone regeneration of rabbit cranial defects

Guided bone regeneration (GBR) has been utilized for several decades for the healing of cranio‐maxillofacial bone defects and, particularly in the dental field, by creating space with a barrier membrane to exclude soft tissue and encourage bone growth in the membrane‐protected volume. Although the first membranes were non‐resorbable, a new generation of GBR membranes aims to biodegrade and provide bioactivity for better overall results. The Inion GTR? poly(lactide‐co‐glycolide) (PLGA) membrane is not only resorbable but also bioactive, since it includes N‐methylpyrrolidone (NMP), which has been shown to promote bone regeneration. In this study, the effects of loading different amounts of NMP onto the membrane through chemical vapour deposition or dipping have been explored. In vitro release demonstrated that lower levels of NMP led to lower NMP concentrations and slower release, based on total NMP loaded in the membrane. The dipped membrane released almost all of the NMP within 15 min, leading to a high NMP concentration. For the in vivo studies in rabbits, 6 mm calvarial defects were created and left untreated or covered with an ePTFE membrane or PLGA membranes dipped in, or preloaded with, NMP. Evaluation of the bony regeneration revealed that the barrier membranes improved bony healing and that a decrease in NMP content improved the performance. Overall, we have demonstrated the potential of these PLGA membranes with a more favourable NMP release profile and the significance of exploring the effect of NMP on these PLGA membranes with regard to bone ingrowth. Copyright © 2014 John Wiley & Sons, Ltd.     

Dehydrothermal treatment of collagen influences on bone regeneration by octacalcium phosphate (OCP) collagen composites

We have engineered a scaffold constructed of synthetic octacalcium phosphate (OCP) and collagen composites (OCP-collagen) and report that OCP-collagen significantly enhanced bone regeneration more than the implantation of OCP. We hypothesized that the dehydrothermal treatment (DHT) during the fabrication of OCP-collagen might influence bone regeneration by OCP-collagen. To examine this hypothesis, bone regeneration by the implantation of OCP-collagen with DHT [OCP/Col(+)] was compared with that by OCP-collagen without DHT [OCP/Col(-)]. It was confirmed that both OCP/Col(+) and OCP/Col(-) contained the characteristics of OCP structure in X-ray diffraction. Before implantation, calcium deposition derived from OCP was observed within the collagen of both OCP/Col(+) and OCP/Col(-) by undecalcified histological sections. OCP/Col(+) or OCP/Col(-) was implanted into the critical-sized defects in rat crania. Radiographic and histological examination was performed and the percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. N-Bone% treated with OCP/Col(+) was significantly higher than that with OCP/Col(-) at 4 and 12 weeks after implantation, because fast degradation of the implanted collagen of OCP/Col(-) elicited disappearance of the scaffold for bone regeneration. The stiffness of the calcified collagen in OCP-collagen would be more important than the existence of calcified collagen to enhance the bone regeneration by OCP-collagen composites. The present study suggests that the dehydrothermal treatment would influence effective bone regeneration by OCP-collagen.     

Bone marrow mesenchymal stem cells,platelet‐rich plasma and nanohydroxyapatite–type I collagen beads were integral parts of biomimetic bone substitutes for bone regeneration

Platelet rich plasma (PRP), which includes many growth factors, can activate osteoid production, collagen synthesis and cell proliferation. Nanohydroxyapatite‐type I collagen beads (CIB), which mimetic natural bone components, are not only flexible fillers for bone defect but also encourage osteogenesis. Bone marrow mesenchymal stem cells (BMSCs) are often used as an abundant cell source for tissue engineering. We used a rabbit model to combine PRP, CIB and BMSCs (CIB+PRP+BMSC) into a bone‐like substitute to study its impact on bone regeneration, when compared to defect alone, PRP, CIB+PRP, and PRP+BMSC. CIB+PRP upregulated more alkaline phosphatase (ALP) activity in BMSCs than PRP alone at 4 weeks postoperation. CIB+PRP+BMSC and PRP+BMSC did not differ significantly in DNA content, total collagen content, and ALP activity at 8 weeks. In histological assay, both CIB+PRP+BMSC and PRP+BMSC showed more bone regeneration at 4 and 8 weeks. Higher trabecular bone volume in tissue volume (BV/TV) (31.15±2.67% and 36.93±1.01%), fractal dimension (FD) (2.30±0.18 and 2.65±0.02) and lower trabecular separation (Tb.Sp) (2.30±0.18 and 1.35±0.16) of CIB+PRP+BMSC than of other groups at 4 and 8 weeks, and approach to of bone tissue (BV/TV=24.35±2.13%; FD=2.65±0.06; Tb.Sp=4.19±0.95). CIB+PRP+BMSC significantly enhanced new bone formation at 4 week. Therefore, nanohydroxyapatite‐type I collagen beads combined with PRP and BMSCs produced a bone substitute with efficiently improved bone regeneration that shows promise to repair bone defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Cultivation of human bone marrow stromal cells on three-dimensional scaffolds of mineralized collagen: influence of seeding density on colonization, proliferation and osteogenic differentiation

In this study human bone marrow stromal cells (hBMSCs) were cultured on three-dimensional porous scaffolds of biomimetically mineralized collagen type I developed for bone engineering. Three different cell numbers were used for seeding of the nanocomposites, and the impact of the seeding density on proliferation and osteogenic differentiation of hBMSCs was investigated. In addition, the effect of the seeding cell number on seeding efficiency and distribution of the cells within the scaffolds was studied. Our data revealed that the open and interconnecting porosity of the mineralized collagen scaffolds allows a very efficient seeding for all seeding densities tested. Although penetration of the cells into the interior of the scaffolds was demonstrated for all seeding densities, the application of higher cell numbers resulted in a better colonization also of the deeper scaffold regions. A substantial influence of the seeding density was observed on proliferation and osteogenic differentiation of hBMSCs. Thus, the highest proliferation rate and specific alkaline phosphatase activity was found for the cell matrix constructs seeded with the lowest density. RT-PCR analyses revealed a higher expression of alkaline phosphatase and bone sialoprotein II at lower seeding densities; however, expression of osteopontin was unaffected by the seeding cell number. Our results demonstrated that the seeding density might be an important factor for the development of optimal cell matrix constructs for bone tissue engineering.     

种植体周围炎骨缺损重建的研究

目的：临床评价引导骨再生（GBR）技术进行种植体周围炎导致的骨缺损重建的效果。方法：10颗因种植体周围炎导致牙槽骨吸收大于5mm但稳固的种植体,其中TPS界面9颗,HA界面1颗,拆除上部修复体,取下基台接入覆盖螺帽,口服抗生素及0.2%氯已定漱口水含漱控制感染。7d后翻瓣,清除骨袋内的炎性组织,种植体表面用0.2%氯已定纱布和生理盐水纱布交替浸泡后再重复上述步骤一次。骨缺损区充填Bio-Oss,覆盖Bio-Gide。6个月后行2期手术,评估种植体周围骨再生情况。结果：9颗TPS界面种植体骨再生理想,完成烤瓷修复。1颗HA界面种植体感染失败拔除。结论：TPS界面种植体周围炎造成的环沟状骨缺损采用GBR技术进行骨再生效果理想,HA界面种植体周围炎造成的环沟状骨缺损采用GBR技术效果有待进一步研究。     

可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损

背景：自体髂骨移植一直被认为是骨缺损修复的“金标准”,但其来源有限。目的：验证应用可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损的效果。方法：80只雄性SD大鼠建立双侧颅骨缺损模型,随机分为3组：骨修复材料＋骨碎补总黄酮组采用可注射骨修复材料结合骨碎补总黄酮灌胃修复大鼠颅骨缺损：骨修复材料＋去离子水组采用可注射骨修复材料结合去离子水灌胃修复大鼠颅骨缺损;羟基磷灰石＋去离子水组采用羟基磷灰石结合去离子水灌胃修复大鼠颅骨缺损,1次／d,持续8周。于建模后2,4,8周取颅骨标本进行苏木精一伊红染色和Masson染色组织学观察。结果与结论：羟基磷灰石组新骨形成和材料降解速度较慢;可注射骨修复材料组新骨形成和材料降解较羟基磷灰石组快,利于血管及纤维组织长入;骨碎补总黄酮灌胃可以促进血管及纤维组织长入材料,促进成骨。与羟基磷灰石相比,可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损,可促进新骨形成,缩短骨缺损修复时间。     

亲水性钛表面微纳米形貌对大鼠骨髓间充质干细胞增殖与成骨分化的影响

目的探讨亲水性钛表面微纳米形貌对大鼠骨髓间充质干细胞（rBMSCs）增殖与成骨分化的影响。 方法采用阳极氧化法和喷砂碱热法分别构建微纳米形貌钛表面,检测其亲水性。钛片表面接种rBMSCs,采用细胞计数试剂盒-8（CCK8）法在培养1、3、5、7 d时检测rBMSCs细胞活性;第7天和14天时检测总蛋白浓度与碱性磷酸酶（ALP）活性,并在第7天时检测成骨标志物ALP、I型胶原（COL1）与成骨特异性转录因子2（RUNX2）的mRNA表达水平。采用t检验比较2组水静态接触角、CCK-8、总蛋白浓度以及成骨分化PCR检测指标。 结果阳极氧化组钛表面呈规则有序纳米管阵列,喷砂碱热组呈三维网状纳米多孔结构,二者具有相似的微纳米形貌,阳极氧化组水静态接触角大于喷砂碱热阻[（83.3±2.3）^° vs （47.7±2.0）^°],差异具有统计学意义（t=11.54,P<0.001）。相比阳极氧化组,喷砂碱热组rBMSCs细胞增殖能力均有所增强,接种3、5、7 d后喷砂碱热组rBMSCs细胞活性均明显高于阳极氧化组（0.66 ±0.03 vs 0.52 ±0.03;1.15 ±0.06 vs 0.85 ±0.05;1.58 ±0.07 vs 1.26 ±0.07）,且差异具有统计学意义（t=2.962、3.845、3.183,P=0.042、0.018、0.033）;培养7、14 d后,喷砂碱热组rBMSCs的总蛋白浓度高于阳极氧化组[（389±45）μg/ml vs（226±32）μg/ml;（1070±59）μg/ml vs （760±65）μg/ml],差异具有统计学意义（t=3.319、3.518,P=0.029、0.025）;第7、14天时喷砂碱热组钛表面rBMSCs的ALP活性高于阳极氧化组[（2.11±0.32）U/gprot vs （1.00±0.21）U/gprot;（6.13±0.57）U/gprot vs （3.92±0.51）U/gprot],差异具有统计学意义（t=2.912、2.976,P=0.043、0.041）;第7天时,喷砂碱热组rBMSCs中ALP、COL1与RUNX2的mRNA表达水平均高于阳极氧化组（1.86 ±0.24 vs 1.00 ±0.15;2.05 ±0.16 vs 1.00 ±0.14;2.28 ±0.18 vs 1.00 ±0.12）,差异具有统计学差异（t=3.383、5.012、5.710,P=0.028、0.007、0.005）。 结论相比阳极氧化组,喷砂碱热组钛表面微纳米形貌具有更强的促rBMSCs增殖与成骨分化能力,该过程可能与其良好的亲水性有关。