Bio-oss骨粉对人胎盘来源间充质干细胞体外成骨活性影响的研究

目的: 研究人胎盘源间充质干细胞(HPMSCs)与Bio-oss骨粉结合后的增殖分化情况,探讨以人胎盘源干细胞(HPMSCs)作为种子细胞,Bio-oss骨粉作为支架材料联合构建组织工程化生物衍生骨的可能性。方法: 无菌条件下分离培养人胎盘源间充质干细胞(HPMSCs)并检测其分化能力。将细胞分为单独培养组与复合Bio-oss骨粉培养组。观察细胞与支架材料结合情况并在细胞形态特点、增殖分化能力方面将两组细胞进行比较。结果: 人胎盘源间充质干细胞与Bio-oss骨粉复合培养后,细胞形态、增殖分化能力与对照组人胎盘源间充质干细胞无明显差异。结论: 以人胎盘源干细胞(HPMSCs)为种子细胞、Bio-oss骨粉为支架复合培养构建组织工程化衍生骨是可行的。     

不同比例的聚乳酸-聚乙醇酸共聚物与羟基磷灰石复合支架对人牙髓干细胞增殖及矿化能力影响研究

目的　探讨不同比例的聚乳酸-聚乙醇酸共聚物与羟基磷灰石（PLGA/HA）复合支架的降解速率及其对人牙髓干细胞（human dental pulp stem cells,hDPSCs）增殖及矿化能力的影响。方法    采用溶液浇筑/颗粒沥析技术构建含质量分数分别为10%、20%及30% HA的PLGA/HA复合支架的3个实验组,单纯PLGA支架作为对照组。采用降解实验检测支架的降解速率。将hDPSCs接种于不同比例的PLGA/HA复合支架进行培养,扫描电镜观察细胞形态,应用MTT法检测细胞增殖能力。hDPSCs接种于各组支架培养72 h后,将复合支架植入裸鼠体内,分别于饲养1个月及3个月后处死裸鼠取出支架,应用HE染色观察组织学形态,应用牙本质基质蛋白1（DMP1）免疫组化染色观察细胞的矿化程度。结果    10%HA组和20%HA组具有较适宜的降解速率。各组均具有较高的促hDPSCs增殖能力,促进效果随HA含量的增加而加强。HE染色和免疫组化染色结果显示,HA可促进hDPSCs在体内的成牙本质向分化,分化程度与HA含量成正比,20% HA组及30% HA组矿化程度均较佳。结论   含20%HA的PLGA/HA复合支架具有较佳的降解速率及促细胞增殖和矿化能力,是比较理想的细胞支架材料。     

不同比例的聚乳酸-聚乙醇酸共聚物与羟基磷灰石复合支架对人牙髓干细胞增殖及矿化能力影响研究

 目的　探讨不同比例的聚乳酸-聚乙醇酸共聚物与羟基磷灰石（PLGA/HA）复合支架的降解速率及其对人牙髓干细胞（human dental pulp stem cells，hDPSCs）增殖及矿化能力的影响。方法    采用溶液浇筑/颗粒沥析技术构建含质量分数分别为10%、20%及30% HA的PLGA/HA复合支架的3个实验组，单纯PLGA支架作为对照组。采用降解实验检测支架的降解速率。将hDPSCs接种于不同比例的PLGA/HA复合支架进行培养，扫描电镜观察细胞形态，应用MTT法检测细胞增殖能力。hDPSCs接种于各组支架培养72 h后，将复合支架植入裸鼠体内，分别于饲养1个月及3个月后处死裸鼠取出支架，应用HE染色观察组织学形态，应用牙本质基质蛋白1（DMP1）免疫组化染色观察细胞的矿化程度。结果    10%HA组和20%HA组具有较适宜的降解速率。各组均具有较高的促hDPSCs增殖能力，促进效果随HA含量的增加而加强。HE染色和免疫组化染色结果显示，HA可促进hDPSCs在体内的成牙本质向分化，分化程度与HA含量成正比，20% HA组及30% HA组矿化程度均较佳。结论   含20%HA的PLGA/HA复合支架具有较佳的降解速率及促细胞增殖和矿化能力，是比较理想的细胞支架材料。     

凝胶样支架材料对三维培养的人牙髓干细胞增殖的影响

目的　评价牙髓组织工程中不同浓度的两种可注射性凝胶样支架材料对牙髓干细胞增殖的影响。方法：配制不同浓度的I型胶原、肽段水凝胶（Puramatrix）支架材料,将牙髓干细胞分别接种于各组支架材料上;CCK-8方法检测细胞增殖情况;活死细胞染色观察细胞数量及形态。 结果　接种在各浓度支架材料上的牙髓干细胞均生长良好,其中1%的胶原支架和0.25%的水凝胶支架对牙髓干细胞的增殖有明显的促进作用;活死细胞染色观察牙髓干细胞在各浓度的两种支架材料中均生长良好,细胞数量随培养时间的延长呈递增趋势;细胞充分伸展,呈纺锤型。     

Pulp ECM-derived macroporous scaffolds for stimulation of dental-pulp regeneration process

ObjectiveRecent studies suggest xenogeneic extracellular matrices as potential regenerative tools in dental pulp regeneration. This study aimed to fabricate and characterize a novel three-dimensional macroporous pulp-derived scaffold that enables the attachment, penetration, proliferation and differentiation of mesenchymal stem cells.MethodBovine pulp was decellularized and characterized with histological and DNA content methods. This scaffold was prepared using finely milled lyophilized decellularized pulp extracellular matrix (ECM) digested with pepsin. Three different concentrations of ECM (1.50, 2.25 and 3.00 mg/ml) were freeze-dried and were tested with/without chemical crosslinking. The specimens were subjected to physicochemical characterization, cell viability and quantitative real time polymerase chain reaction assessments with human bone marrow mesenchymal stem cells (hBMMSCs). All scaffolds were subcutaneously implanted in rats for two weeks and histological and immunostaining analyses were performed.ResultsHistological and DNA analysis confirmed complete decellularization. All samples demonstrated more than 97% porosity and 1.50 mg/ml scaffold demonstrated highest water absorption. The highest cell viability and proliferation of hBMMSCs was observed on the 3.00 mg/ml crosslinked scaffolds. The gene expression analysis showed a significant increase of dmp-1 and collagen-I on 3.00 mg/ml crosslinked scaffolds compared to the other scaffolds. Histological examination of subcutaneous implanted scaffolds revealed low immunological response, and enhanced angiogenesis in cross-linked samples compared to non-crosslinked samples.SignificanceThe three-dimensional macroporous pulp-derived injectable scaffold developed and characterized in this study displayed potential for regenerative therapy. While the scaffold biodegradability was decreased by crosslinking, the biocompatibility of post-crosslinked scaffold was significantly improved.     

模拟微重力环境对人牙髓干细胞体外增殖能力的影响

目的：研究模拟微重力环境对人牙髓干细胞（human dental pulp stem cells,hDPSCs）在PLGA支架上体外增殖能力的影响。方法：将分离、鉴定后的人牙髓干细胞接种在PLGA支架上,采用MTT法检测普通环境和模拟微重力环境中培养24、48、72h的人牙髓干细胞在PLGA支架上的细胞活性。DAPI荧光染色和流式细胞术比较牙髓干细胞在两种环境培养3d的细胞数量以及细胞周期分布情况。结果：MTT显示模拟微重力组中各时间点的A值均高于普通环境培养组;DAPI荧光染色显示在模拟微重力下培养3d的人牙髓干细胞数量明显多于普通环境培养组;细胞周期分析结果表明模拟微重力组中S期细胞比例明显高于普通环境培养组（P〈0.05）。结论：接种在PLGA支架上的人牙髓干细胞在模拟微重力环境下较普通环境具有更高的体外增殖能力。     

人牙髓干细胞在聚乳酸-聚乙醇酸共聚物支架上粘附与增殖的研究

目的：研究成人牙髓干细胞（HDPSCs）在聚乳酸-聚乙醇酸共聚物（PLGA）支架上粘附与增殖的情况。方法：采用酶消化法分离、培养人牙髓干细胞,免疫组化法及体外诱导分化对细胞进行鉴定。将人牙髓干细胞与PLGA支架材料进行复合培养,扫描电镜（SEM）观察支架材料形态,细胞粘附、增殖及基质分泌情况;细胞计数检测其增殖力。结果：细胞接种2、5、10d,扫描电镜及细胞计数均显示HDPSCs与PLGA支架材料粘附紧密,生长状态良好,细胞明显增殖（P〈0.05）,有丰富的细胞外基质形成。结论：PLGA是一种适宜人牙髓干细胞粘附与增殖的支架材料。     

Effects of 3-dimensional Bioprinting Alginate/Gelatin Hydrogel Scaffold Extract on Proliferation and Differentiation of Human Dental Pulp Stem Cells

IntroductionAlginate/gelatin hydrogel (Alg-Gel) scaffold has been applied in tissue engineering, but the research on its application in dental tissues regeneration is still lacking. We investigated the effect of this scaffold on human dental pulp stem cells (hDPSCs).MethodshDPSCs were cultured in both Alg-Gel and 3D-printed Alg-Gel scaffolds. Cell growth and adhesion were compared using fluorescein isothiocyanate–phalloidin staining and scanning electron microscopic micrographs. Changes in the proliferation in hDPSCs cultured in the complete culture medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds were examined using Cell Counting Kit-8 assay and flow cytometry analysis. Cells were cultured in the mineralization medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds for 7 or 14 days, and the differentiation of cells was shown by alizarin red S staining and alkaline phosphatase staining. The messenger RNA and protein expression of mineralization-related genes were detected with real-time polymerase chain reaction and Western blotting. Elemental analysis was used to test the material extract composition.ResultsMore cells were grown and adhered to the 3D-printed Alg-Gel scaffolds than the Alg-Gel scaffolds. The aqueous extracts of 3D-printed scaffolds can promote cell proliferation, and compared with Alg-Gel scaffolds, the extracts of 3D-printed scaffolds were more effective. Compared with the negative control group, 3D-printed Alg-Gel scaffold and Alg-Gel scaffold aqueous extracts promoted osteogenic/odontoblastic differentiation of hDPSCs with the enhanced formation of bone-like nodules and the alkaline phosphatase staining. The expression of mineralization-related genes was also up-regulated. 3D-printed scaffold aqueous extract contained more calcium and phosphorus ions than the Alg-Gel scaffold.ConclusionsThese findings suggest that compared with the Alg-Gel scaffold, 3D-printed Alg-Gel is more suitable for the growth of hDPSCs, and the scaffold extracts can better promote cell proliferation and differentiation.     

PLGA静电纺丝纳米纤维结合诱导培养基和BMP4诱导鼠胚胎干细胞向牙上皮分化

目的:检测聚羟基乙酸聚乳酸共聚物(PLGA)纳米支架材料结合诱导培养基和BMP4对胚胎鼠干细胞(mES)向牙上皮细胞分化的影响.方法:用PLGA作为基底材料,通过静电纺丝技术构建纳米纤维支架材料,结合不同时间点BMP4的干预,将鼠胚胎干细胞向牙上皮细胞诱导,构建mES向牙上皮分化的诱导体系,并进一步筛选出mES细胞向牙...     

细胞外基质支架及其在牙髓再生中应用的研究进展

实现全牙髓功能性再生是牙髓及根尖周疾病治疗的理想目标。以干细胞为基础的再生医学技术为牙髓再生带来了新的希望,而支架材料在其中发挥重要作用。通过化学、物理或生物学等方法去除组织、器官中的细胞及抗原,可获得天然脱细胞的细胞外基质支架。其具有良好的物理特性、生物相容性以及生物降解性,能够促进干细胞黏附、生长、增殖及分化,同时具有低免疫原性,避免过敏反应和疾病传播,被认为是再生医学一种新型的生物支架。文章就细胞外基质支架的制备、特性及其在牙髓再生中应用的研究进展做一综述。     

体外观察三种支架材料对人乳牙牙髓干细胞生物学行为的影响

目的：观察乳牙牙髓干细胞（SHEDs）在3种羟基磷灰石（HA）复合支架材料上黏附、增殖、分化情况。方法：利用正常替换的乳牙分离培养 SHEDs,免疫组织化学法鉴定细胞来源,体外诱导实验观察细胞分化能力。将 SHEDs 分别接种在羟基磷灰石／β-磷酸三钙（HA／TCP）,羟基磷灰石／胶原（HA／COL）和羟基磷灰石／聚乙丙交酯（HA／PLGA）支架材料上复合培养,于4、6、8、10 h 4个时间点检测各细胞黏附率,MTT 比色法检测 SHEDs 增殖情况;碱性磷酸酶（ALP）活性检测、能谱分析钙含量;Von Kossa 染色和灰度扫描细胞矿化能力。结果：SHEDs 在 HA／COL 上的黏附少于 HA／PLGA 和 HA／TCP（P ＜0．05）;HA／PLGA对 SHEDs 矿化诱导能力最强,其次是 HA／TCP,HA／COL 最弱。结论：SHEDs 在 HA／PLGA 上的黏附率、增殖率及矿化能力优于HA／TCP 和 HA／COL。     

Epithelial-mesenchymal interaction reduces inhibitory effects of fluoride on proliferation and enamel matrix expression in dental epithelial cells

AimFluoride, well known as a specific and effective caries prophylactic agent, also affects the differentiation and function of ameloblasts. High dose sodium fluoride (NaF) induces enamel hypoplasia, also called enamel fluorosis, whereas the size and form of teeth except the enamel are not changed with its treatment. We examined the effects of fluoride on dental epithelium proliferation and differentiation using co-cultures of dental epithelial and mesenchymal cells.MethodsCultures of the dental epithelial cell line SF2 and dental mesenchymal cell line mDP were performed, as well as co-cultures. Enamel matrix expression in SF2 cells treated with NaF was analyzed by RT-PCR, while cell proliferation was examined using a trypan blue dye exclusion method and BrdU incorporation findings. The effects of NaF on NT-4-induced ERK1/2 phosphorylation were analyzed by western immunoblotting.ResultsNeurotrophic factor NT-4 induced enamel matrix expression, which was inhibited in the presence of NaF. Similar results were observed in regard to SF2 cell proliferation, but not with mDP cells. The levels of proliferation and ameloblastin expression in SF2-GFP cells co-cultured with mDP in the presence of NaF were lower as compared to those in SF2 cells cultured alone.ConclusionOur results indicate that dental epithelial cells co-cultured with dental mesenchymal cells are resistant to the inhibitory effects of NaF on proliferation and ameloblastin expression. They also suggest that the dental fluorosis phenotype may affect enamel, but not tooth size or shape, because of rescue of the inhibitory effects of NaF by culturing with dental mesenchymal cells.     

PRP对纳米HA/PLGA复合支架上鼠骨骼肌卫星细胞增殖和成骨活性的影响

目的:研究富血小板血浆(platelet-rich plasma,PRP)对纳米羟基磷灰石(nano-hydroxyapatite,nHA)/聚乳酸乙醇酸(poly lactic acid-co-glycolic acid,PLGA)(nHA/PLGA)支架上SD大鼠骨骼肌卫星细胞(muscle satellite cells,MSCs)黏附、增殖和成骨活性的影响。方法:将体外培养、扩增的鼠MSCs与同一供体来源的PRP混合,滴加到nHA/PLGA支架上,形成MSCs/PRP/nHA/PLGA复合物(实验组),继续在体外培养,以MSCs/nHA/PLGA复合物作为对照组。通过扫描电镜观察MSCs在支架上黏附、生长和增殖情况;水溶性四氮唑法(WST-1)法测定MSCs增殖情况;碘化丙啶(PI)与钙黄绿素-AM(Calcein-AM)检测MSCs的荧光活性;组织化学方法检测培养液中碱性磷酸酶(alkaline phosphatase,ALP)活性和骨钙素(osteocalcin,OC)含量;RT-PCR检测骨桥蛋白(osteopontin,OPN)mRNA的表达。结果:扫描电镜观察显示,实验组大量MSCs黏附在支架表面及其洞壁上,并大量增殖和分泌骨基质,而对照组复合材料表面细胞附着较少;WST-1测定实验组吸光度值明显大于对照组(P<0.05);PI荧光染色二组的死细胞数量均较少;实验组ALP活性和OC含量均较对照组增高明显(P<0.05);RT-PCR结果显示OPN在实验组中表达明显增强。结论:PRP促进了nHA/PLGA支架上鼠MSCs黏附、增殖和成骨分化。     

3D打印多孔磷酸镁生物陶瓷支架的制备及其体外骨诱导能力的研究

目的:应用3D打印技术及致孔剂浸出法制备具有两种不同尺度孔隙的磷酸镁多孔支架,研究其物理化学性能及骨诱导能力.方法:通过间接打印法制备多孔磷酸镁(MgP)支架,利用NaCl(粒径25～50μm)作为致孔剂引入微孔,制备MgP-Na微孔支架;通过扫描电镜、压汞仪、通用材料试验机及X线衍射仪检测支架的物理及化学性能;应用C...     

Osseointegration of 3D-printed titanium implants with surface and structure modifications

BackgroundThe purpose of this study was to establish a mechanical and histological basis for the development of biocompatible maxillofacial reconstruction implants by combining 3D-printed porous titanium structures and surface treatment. Improved osseointegration of 3D-printed titanium implants for reconstruction of maxillofacial segmental bone defect could be advantageous in not only quick osseointegration into the bone tissue but also in stabilizing the reconstruction.MethodsVarious macro-mesh titanium scaffolds were fabricated by 3D-printing. Human mesenchymal stem cells were used for cell attachment and proliferation assays. Osteogenic differentiation was confirmed by quantitative polymerase chain reaction analysis. The osseointegration rate was measured using micro computed tomography imaging and histological analysis.ResultsIn three dimensional-printed scaffold, globular microparticle shape was observed regardless of structure or surface modification. Cell attachment and proliferation rates increased according to the internal mesh structure and surface modification. However, osteogenic differentiation in vitro and osseointegration in vivo revealed that non-mesh structure/non-surface modified scaffolds showed the most appropriate treatment effect.Conclusion3D-printed solid structure is the most suitable option for maxillofacial reconstruction. Various mesh structures reduced osteogenesis of the mesenchymal stem cells and osseointegration compared with that by the solid structure. Surface modification by microarc oxidation induced cell proliferation and increased the expression of some osteogenic genes partially; however, most of the markers revealed that the non-anodized solid scaffold was the most suitable for maxillofacial reconstruction.     

PLGA微囊支架材料的制备及对成骨细胞粘附和增殖的影响

[摘要]目的：制备一种新型结构的聚乳酸-羟基乙酸共聚物[poly（1actic—co—glycolicacid,PLGA]微囊支架,对其进行表征,并检测其对成骨细胞增殖与粘附的影响。方法：采用复乳法制备PLGA微囊,以激光粒度仪分析其粒径分布,筛选出合适的粒径大小,通过二氯甲烷蒸汽熏蒸使微囊结合,再经过烧结最终形成三维支架,通过扫描电子显微镜观察微囊及微囊支架形态。将MC3T3-E1成骨细胞接种到该支架上,通过扫描电镜观察细胞在支架上的粘附形态,通过CCK-8法检测细胞增殖的情况,并与无支架对照组进行比较。结果：制备的PLGA微囊支架具有一定孔径和孔隙率,具有三维连通的多孔结构,且孔隙结构均匀。细胞可在支架表面粘附生长。微囊支架上的细胞增殖活性高于对照组。结论：本方法制备的PLGA微囊支架具有一定的孔隙率和内部连通性,有利于细胞的粘附和增殖,在骨修复中有应用前景。     

GSK3beta inhibitor-induced dental mesenchymal stem cells regulate ameloblast differentiation

ObjectivesEpithelial-mesenchymal interactions are extremely important in tooth development and essential for ameloblast differentiation, especially during tooth formation. We aimed to identify the type of mesenchymal cells important in ameloblast differentiation.MethodsWe used two types of cell culture systems with chambers and found that a subset of debtal mesenchimal cells is important for the differentiatiuon of dental spithelial cells into ameloblasts. Further, we induced dental pulp stem cell-like cells from dental pulp stem cells using the small molecule compound BIO ( a GSK-3 inhibitor IX) to clarify the mechanism involved in ameloblast differentiation induced by dental pulp stem cells.ResultsThe BIO-induced dental pulp cells promoted the expression of mesenchymal stem cell markers Oct3/4 and Bcrp1. Furthermore, we used artificial dental pulp stem cells induced by BIO to identify the molecules expressed in dental pulp stem cells required for ameloblast differentiation. Panx3 expression was induced in the dental pulp stem cell through interaction with the dental epithelial cells. In addition, ATP release from cells increased in Panx3-expressing cells. We also confirmed that ATP stimulation is accepted in dental epithelial cells.ConclusionsThese results showed that the Panx3 expressed in dental pulp stem cells is important for ameloblast differentiation and that ATP release by Panx3 may play a role in epithelial–mesenchymal interaction.     

Enhancing cell seeding and osteogenesis of MSCs on 3D printed scaffolds through injectable BMP2 immobilized ECM-Mimetic gel

ObjectiveDesign of bioactive scaffolds with osteogenic capacity is a central challenge in cell-based patient-specific bone tissue engineering. Efficient and spatially uniform seeding of (stem) cells onto such constructs is vital to attain functional tissues. Herein we developed heparin functionalized collagen gels supported by 3D printed bioceramic scaffolds, as bone extracellular matrix (ECM)-mimetic matrices. These matrices were designed to enhance cell seeding efficiency of mesenchymal stem cells (MSCs) as well as improve their osteogenic differentiation through immobilized bone morphogenic protein 2 (BMP2) to be used for personalized bone regeneration.MethodsA 3D gel based on heparin-conjugated collagen matrix capable of immobilizing recombinant human bone morphogenic protein 2 (BMP2) was synthesized. Isolated dental pulp Mesenchymal stem cells (MSCs) were then encapsulated into the bone ECM microenvironment to efficiently and uniformly seed a bioactive ceramic-based scaffold fabricated using additive manufacturing technique. The designed 3D cell-laden constructs were comprehensively investigated trough in vitro assays and in vivo study.ResultsIn-depth rheological characterizations of heparin-conjugated collagen gel revealed that elasticity of the matrix is significantly improved compared with freely incorporated heparin. Investigation of the MSCs laden collagen-heparin hydrogels revealed their capability to provide spatiotemporal bioavailability of BMP2 while suppressing the matrix contraction over time. The in vivo histology and real-time polymerase chain reaction (qPCR) analysis showed that the designed construct supported the osteogenic differentiation of MSCs and induced the ectopic bone formation in rat model.SignificanceThe presented hybrid constructs combine bone ECM chemical cues with mechanical function providing an ideal 3D microenvironment for patient-specific bone tissue engineering and cell therapy applications. The implemented methodology in design of ECM-mimetic 3D matrix capable of immobilizing BMP2 to improve seeding efficiency of customized scaffolds can be exploited for other bioactive molecules.