利用新型聚羟丁酸酯材料构建组织工程软骨

目的:研究新型聚羟丁酸酯作为组织工程软骨支架材料的可行性.方法:取幼兔软骨组织中软骨细胞体外培养扩增.实验组接种软骨细胞于支架材料上,体外培养两周后埋植于新西兰大白兔背部皮下;对照组埋入未接种细胞的支架材料.扫描电镜观察材料表面形态及细胞生长情况.分别于第4、8、12周取出标本,大体观察后进行HE和Masson染色,观察组织工程软骨形成情况.结果:扫描电镜观察可见裸材料孔隙分布均匀,形状不规则;细胞材料复合体体外培养两周后材料表面爬满细胞且生长状态良好.埋植材料取出后可见不同时间点实验组标本大小无明显变化,对照组标本逐渐变小.HE和Masson染色显示各组支架材料至12周时已被完全吸收;实验组12周时可见较成熟软骨组织;对照组支架材料被吸收后最终被纤维结缔组织取代.结论:此新型聚羟丁酸酯材料可作为组织工程软骨支架材料.     

纳米双相磷酸钙陶瓷支架材料肌内降解性能的实验研究

目的:纳米双相磷酸钙陶瓷(Biphasic calcium phosphate nanocomposite,NanoBCP)支架是一种新型支架材料,具有三维立体多孔结构,孔隙率可达60%～80%。本研究观察了纳米双相磷酸钙陶瓷肌内降解情况。方法:将NanoBCP制备为5mm×5mm×1.5mm大小各8块的支架植入SD大鼠腿部肌袋内,相同孔径、孔隙率的羟基磷灰石(Hydroxyapatite,HA)及普通双相磷酸钙陶瓷(Biphasic calciam phosphate,BCP)作为对照,于4、12、24周取材,测定材料降解率(失重率),从大体、组织学观察以了解材料降解情况。结果:材料肌内植入后降解率测定结果:NanoBCP降解率为32%,BCP的降解率为13%,HA的降解率为3%。组织学观察发现,NanoBCP肌内植入24周后,大部分NanoBCP支架已经将解,并且将解的碎片已埋入纤维结缔组织里。结论:NanoBCP与BCP、HA相比有良好的降解性能。     

干湿纺聚乳酸／壳聚糖纤维交织织物作为细胞体外培养载体的实验观察

在体外培养的条件下观察干湿纺聚乳酸/壳聚糖纤维交织织物与成骨细胞的相容性,探讨其作为人工胸壁支架材料和人工骨支架材料的可行性。将hFOB1.19人SV40转染的成骨细胞与干湿纺聚乳酸/壳聚糖纤维交织织物体外联合培养。用扫描电镜对体外联合培养早期细胞的形态学进行观察。结果表明,成骨细胞与干湿纺聚乳酸/壳聚糖交织织物间黏附良好,具有良好的相容性,干湿纺聚乳酸/壳聚糖纤维交织织物有可能成为一种理想的可用于修复胸壁缺损和骨缺损的成骨细胞载体。     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

可控多孔结构支架制备及灌注式体外培养

利用CAD和快速成形技术设计制造具有可控多孔结构的支架。构建灌注式生物反应器系统,实现氧气和营养物质的大量输送,同时产生一定流体剪应力,调节细胞功能的发挥。根据支架负型结构制造出相应的树脂原型,用磷酸钙骨水泥进行填充烧结,得到与设计相符的多孔支架。接种兔成骨细胞,分别采用静态和灌注式三维动态培养方法,观察不同培养条件下细胞在支架表面以及所构造微管道内的生长情况。试验结果表明,灌注式体外培养方法更有利于细胞在支架微管道内的存活和功能的发挥,此灌注式系统能够改善支架微管道内细胞生存的微环境,增强黏附在支架微管道内细胞的活性,促进细胞进一步的增殖和矿化基质的产生。     

应用3D适形打印制备网状复合体对兔颅骨缺损的修复作用及安全性研究

目的:探讨采用3D适形打印技术制备的羟基磷灰石/聚乳酸网状复合体在兔颅骨缺损中的修复作用及安全性。方法:以24只新西兰兔为研究对象,以羟基磷灰石/聚乳酸为材料,采用3D适形打印技术制备网状复合体,于兔颅骨顶部制成两个颅骨全层缺损,分别为孔A(左)和孔B(右),孔A(阳性对照组)以自体颅骨为修复材料,孔B(实验组)以复合体为修复材料,观察缺损修复区域的形态学、影像学(X线及CT扫描)及组织学检查结果。结果:植入后24周时,形态学显示:阳性对照组可见致密的骨组织修复,与缺损边缘界限不清,实验组中支架孔隙内纤维组织由新生骨质取代,且新生骨成熟度较提高,材料表面有部分吸收。CT扫描观察显示:冠状面上,阳性对照组缺损修复区域与周围正常骨组织融合为一体,实验组修复材料与缺损边缘融合紧密,与周围正常骨组织结合良好,部分边缘结合不连贯。组织学观察显示:实验组材料部分降解,材料间隔可见新生骨小梁。研究中无实验动物死亡,皮肤切口处缝合良好,无皮下积液,无移植物脱出、红肿感染等情况出现。结论:以3D适形打印技术制备的羟基磷灰石/聚乳酸复合体对兔颅骨缺损有较好的修复作用,能促进缺损区域新骨的形成和生长,且安全性较高。     

牛煅烧骨的表征及其对成骨细胞的作用

以天然牛骨为原料,经过加工处理,分别在1120和1250℃烧结得到两组牛煅烧骨样品。测定了材料的物理性能,讨论了烧结温度对材料孔隙率及其结构等的影响。将两组煅烧骨样品分别与成骨前体细胞在体外复合培养,通过对细胞生长和分化情况的观察和测定,对两组多孔材料与成骨细胞的亲和性作出了评价。结果表明牛煅烧骨的主要成分为羟基磷灰石,保留了天然骨的网状孔隙结构,成骨细胞在两组材料上均能正常生长和分化,而且在1120℃烧结的样品具有更好的成骨细胞亲和性,说明牛煅烧骨是一种有发展前途和应用前景的骨修复材料和骨组织工程支架材料。     

γ-PGA/HEMA/PEG聚合物晶胶支架修复兔眼眶骨折缺损的实验研究

目的 近年来,眶骨骨折发生率逐年增加,其治疗关键旨在修复缺损的眶骨,聚（γ-谷氨酸）/2-羟乙基甲基丙烯酸酯/聚（乙二醇）（γ-PGA/HEMA/PEG）聚合物晶胶是一种具有互连多孔结构的新型支架材料,研究旨在检验其在眼眶骨折缺损修复中的骨修复效果。方法 采用低温凝胶技术制备了γ-PGA/HEMA/PEG聚合物晶胶。制备了兔的眼眶骨缺损模型,根据植入支架材料的不同分为3组,空白对照组、聚合物晶胶组（Gel组）、矿化聚合物晶胶组（M-gel组）。植入后8周和16周标本取材进行大体观察,通过影像学和组织学检查观察其血管生成和成骨效果。结果 影像学检查结果表明,支架材料能有效促进眶骨缺损的修复,缺损区被骨组织完全替代。组织学结果表明,支架材料可以增加Runt相关转录因子2（Runx-2）、碱性磷酸酶（ALP）、骨桥蛋白（OPN）和血小板内皮细胞黏附分子1（CD31）的表达,这表明支架材料植入后血管生成和成骨能力增强。结论 矿化聚合物晶胶是一种良好的眼眶骨折缺损修复的支架材料。     

国产多孔钽材料修复兔胫骨缺损的实验研究

摘要 目的：研究国产多孔钽材料能否在兔胫骨缺损模型中顺利实现骨长入,用于修复胫骨缺损。方法：在36只新西兰大白兔双侧胫骨骨干处建立骨缺损模型,每只动物左右侧缺损随机分组,分别进入实验组(植入多孔坦材料)和对照组（不植入多孔坦材料）。植入后4周、8周和12周取材,通过X线检测以及硬组织切片苏木精伊红染色,检测多孔钽材料与骨界面的骨整合情况。采用推出实验检测多孔钽材料与骨界面的结合强度。结果：将术后不同时间点取得的胫骨标本作X射线拍片分析,4周时,骨缺损端与材料结合部位有骨质生成,在8周时材料表面有骨形成现象,逐渐完全覆盖材料表面,在12周时骨量继续增加,形成覆盖材料并桥接骨缺损断端的骨痂。样本行硬组织切片并行HE染色后检测,植入4周后实验组材料两端被新生骨所覆盖,材料深部的孔隙中也可见少量骨组织长入;植入8周后发现实验组材料与骨组织生长良好,多孔钽材料表面和两端材料孔隙内均有骨组织长入,材料孔隙与组织紧密连接,有骨小梁长入;植入12周时两端骨组织长入深度没有明显变化,但材料表面骨组织继续长入,并完全嵌入圆柱体材料内。材料植入后4周与8周比较差异无统计学意义（P＞0.05）,材料植入后8周与12周比较差异有统计学意义（P<0.05）。将植入4周、8周和12周后含材料样本置于动态疲劳试验机上进行推出实验,随时间延长所需推出力明显增加,植入后4周和8周相比,虽然后者所需推力较大,但两者比较差异无统计学意义（P＞0.05）,而8周和12周比较则差异有统计学意义（P<0.05）。结论：国产多孔坦材料能在胫骨缺损中实现与骨整合,能用于皮质骨缺损修复。     

不同来源成骨细胞构建组织工程骨之分子生物学评价

目的:探讨骨膜来源成骨细胞(POB)和盖骨来源成骨细胞(COB)在构建组织工程骨中的黏附、增殖和分化能力.方法:研究分别选用人胚POB和COB为种子细胞,接种于生物衍生骨上,经过10天的体外培养,提取总RNA,经过逆转录成cDNA,采用实时定量PCR对目的基因成骨细胞特异转录因子(Cbfa1)、成骨细胞特异基因(osterix)、Ⅰ型胶原(ColI)、骨钙素(osteocalcin,OC)以及整合素Integfina5β1读取扩增循环数(cycle threshold,Ct).结果:在组织工程骨中,POB和COB对ColI、OC、Cbfal以及osterix的表达没有统计学意义的差别,Integrinα5β1的表达可见POB明显高于COB.结论:POB与COB在与生物衍生材料接触后,仍可保持终末的分化性状,三维孔隙结构促进成骨细胞迅速而活跃的完成增殖期.POB对生物衍生骨的粘附能力优于COB.     

In vitro ligament-bone interface regeneration using a trilineage coculture system on a hybrid silk scaffold

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor β3 (TGF-β3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-β3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-β3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-β3 delayed the mineralization process of chondrocytes.     

A novel collagen scaffold supports human osteogenesis—applications for bone tissue engineering

Collagen glycosaminoglycan (CG) scaffolds have been clinically approved as an application for skin regeneration. The goal of this study has been to examine whether a CG scaffold is a suitable biomaterial for generating human bone tissue. Specifically, we have asked the following questions: (1) can the scaffold support human osteoblast growth and differentiation and (2) how might recombinant human transforming growth factor-beta (TGF-β₁) enhance long-term in vitro bone formation? We show human osteoblast attachment, infiltration and uniform distribution throughout the construct, reaching the centre within 14 days of seeding. We have identified the fully differentiated osteoblast phenotype categorised by the temporal expression of alkaline phosphatase, collagen type 1, osteonectin, bone sialo protein, biglycan and osteocalcin. Mineralised bone formation has been identified at 35 days post-seeding by using von Kossa and Alizarin S Red staining. Both gene expression and mineral staining suggest the benefit of introducing an initial high treatment of TGF-β₁ (10 ng/ml) followed by a low continuous treatment (0.2 ng/ml) to enhance human osteogenesis on the scaffold. Osteogenesis coincides with a reduction in scaffold size and shape (up to 70% that of original). A notable finding is core degradation at the centre of the tissue-engineered construct after 49 days of culture. This is not observed at earlier time points. Therefore, a maximum of 35 days in culture is appropriate for in vitro studies of these scaffolds. We conclude that the CG scaffold shows excellent potential as a biomaterial for human bone tissue engineering.     

多孔beta-磷酸三钙与磷灰石复合材料的制备及其细胞相容性研究

综合运用三维凝胶叠层法和发泡法制备了多孔β-磷酸三钙支架。将多孔支架在1．5倍模拟体液中浸泡14天,得到材料1;或者将其在氢氧化钠溶液中浸泡4天,再在1．5倍模拟体液中浸泡14天,得到材料2。测定了两种材料的物理性能,讨论了类骨磷灰石层对材料矿物组成及其显微结构等的影响。将两组材料分别与成骨前体细胞在体外复合培养,观察和测定了细胞的形态和增殖情况。结果表明复合材料的主要成分为β-磷酸三钙,表面具有结构不完整的含有碳酸磷灰石的类骨磷灰石,成骨细胞能在两组材料上正常粘附和增殖,而且材料2上的细胞粘附情况更好,说明多孔β-磷酸三钙与磷灰石的复合材料有望成为一种有应用前景的骨修复材料和骨组织工程支架材料。     

In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering

The selection of a suitable scaffold matrix is critical for cell-based bone tissue engineering. This study aimed to identify and characterize natural marine sponges as potential bioscaffolds for osteogenesis. Callyspongiidae marine sponge samples were collected from the Fremantle coast of Western Australia. The sponge structure was assessed using scanning electron microscopy (SEM) and Hematoxylin and eosin. Mouse primary osteoblasts were seeded onto the sponge scaffold and immunostained with F-actin to assess cell attachment and aggregation. Alkaline phosphatase expression, von Kossa staining and real-time PCR were performed to examine the osteogenic potential of sponge samples. SEM revealed that the sponge skeleton possessed a collagenous fibrous network consisting of interconnecting channels and a porous structure that support cellular adhesion, aggregation and growth. The average pore size of the sponge skeleton was measured 100 to 300 μm in diameter. F-actin staining demonstrated that osteoblasts were able to anchor onto the surface of collagen fibres. Alkaline phosphatase expression, a marker of early osteoblast differentiation, was evident at 7 days although expression decreased steadily with long term culture. Using von Kossa staining, mineralisation nodules were evident after 21 days. Gene expression of osteoblast markers, osteocalcin and osteopontin, was also observed at 7, 14 and 21 days of culture. Together, these results suggest that the natural marine sponge is promising as a new scaffold for use in bone tissue engineering.     

Preparation and chemical and biological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications

In this work, porous scaffolds obtained from the freeze-drying of pectin/chitosan polyelectrolyte complexes were prepared and characterized by FTIR, SEM and weight loss studies. Additionally, the cytotoxicity of the prepared scaffolds was evaluated in vitro, using human osteoblast cells. The results obtained showed that cells adhered to scaffolds and proliferated. The study also confirmed that the degradation by-products of pectin/chitosan scaffold are noncytotoxic.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.     

Sprouty2 is involved in the control of osteoblast proliferation and differentiation through the FGF and BMP signaling pathways

Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential roles in bone formation and osteoblast activity through the extracellular signal‐regulated kinase 1/2 (ERK1/2) and Smad pathways. Sprouty family members are intracellular inhibitors of the FGF signaling pathway, and four orthologs of Sprouty have been identified in mammals. In vivo analyses have revealed that Sprouty2 is associated with bone formation. However, the mechanism by which the Sprouty family controls bone formation has not been clarified. In this study, we investigated the involvement of Sprouty2 in osteoblast proliferation and differentiation. We examined Sprouty2 expression in MC3T3‐E1 cells, and found that high levels of Sprouty2 expression were induced by basic FGF stimulation. Overexpression of Sprouty2 in MC3T3‐E1 cells resulted in suppressed proliferation compared with control cells. Sprouty2 negatively regulated the phosphorylation of ERK1/2 after basic FGF stimulation, and of Smad1/5/8 after BMP stimulation. Furthermore, Sprouty2 suppressed the expression of osterix, alkaline phosphatase, and osteocalcin mRNA, which are markers of osteoblast differentiation. Additionally, Sprouty2 inhibited osteoblast matrix mineralization. These results suggest that Sprouty2 is involved in the control of osteoblast proliferation and differentiation by downregulating the FGF‐ERK1/2 and BMP‐Smad pathways, and suppresses the induction of markers of osteoblast differentiation.