Combining adipose-derived stem cells, resorbable scaffolds and growth factors: an overview of tissue engineering

Interdependency exists between adipogenesis, the formation of fat tissue from stem cells, and osteogenesis, the formation of bone from its stem-cell precursors. Adipose-derived adult mesenchymal stem cells may be useful for future bone regeneration and tissue engineering. This review discusses potential future relationships between autogenous adipose-derived stem cells, growth factors and resorbable scaffolds for future tissue-engineering applications.     

Construction of tissue-engineered bone with differentiated osteoblasts from adipose-derived stem cell and coral scaffolds at an ectopic site

Cell sheets from bone marrow mesenchymal stem cells (BMSC) have been widely used in the field of bone tissue engineering, although their source remains a challenging issue. In this study, adipose-derived stem cells (ADSC) were induced to differentiate into osteoblasts, and the incorporation of coral scaffolds with ADSC sheets for bone formation at an ectopic site was also investigated. First, ADSC isolated from inguinal adipose tissue of New Zealand rabbits were cultured for two weeks without passaging under osteogenic induction, and the microstructures of cell sheets were analysed by histological and scanning electron microscope (EM) observation. In addition, the activity of alkaline phosphatase (ALP) and alizarin red staining was also measured to detect their osteogenic ability. Subsequently, ADSC were proved to be able to proliferate well when seeded on the coral scaffolds. Next, coral scaffolds were wrapped in cell sheets to prepare sheet-coral complexes, which were implanted into subcutaneous pockets in nude mice. At eight weeks after implantation, gross examination, microcomputed tomography (MicroCT), and histological analysis were investigated to assess new bone formation. MicroCT scanning and histological analysis showed that there was more highly dense tissue formed in the complex group than control group (p=0.0004). These results indicated that osteoblastic ADSC sheets could be used to construct engineered bone and the incorporation of coral scaffolds with ADSC sheets significantly improved bone formation, providing a newly approach for bone tissue engineering.     

Biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs)

The aim is to investigate in vitro biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs) in terms of proliferation, morphological appearance, cell viability, and expression of mesenchymal stem cell markers. Silk fibroin 3D scaffolding materials may represent promising suitable scaffolds for their application in regenerative endodontic therapy approaches. SHEDs were cultured in silk fibroin 3D scaffolds. Then, cell numbers were counted and the Alamar blue colorimetric assay was used to analyse cell proliferation after 24, 48, 72, and 168 h of culture. The morphological features of SHEDs cultured on silk fibroin scaffolds were evaluated by scanning electron microscopy (SEM). Finally, cell viability and the expression of mesenchymal stem cell markers were analysed by flow cytometry. One-way analysis of variance (ANOVA) followed by a Bonferroni post-test was performed (P < 0.05). At 24 and 48 h of culture, SHED proliferation on scaffolds was modest compared to the control although still significant (p < 0.05). However, cell proliferation progressively increased from 72 to 168 h compared with the control (p < 0.001; p < 0.01). In addition, flow cytometry analysis showed that the culture of SHEDs on silk fibroin scaffolds did not significantly alter the level of expression of the mesenchymal markers CD73, CD90, or CD105 up to 168 h; in addition, cell viability in silk fibroin was similar to than obtained in plastic. Moreover, SEM studies revealed a suitable degree of proliferation, cell spreading, and attachment, especially after 168 h of culture. The findings from the current study suggest that silk fibroin 3D scaffolds had a favourable effect on the biological responses of SHEDs. Further in vivo investigations are required to confirm these results.     

Cotransplantation of mesenchymal stromal cells and endothelial cells on calcium carbonate and hydroxylapatite scaffolds in vivo

This study investigated the cotransplantation of bone marrow mesenchymal stromal cells (BMSC) and human umbilical cord endothelial cells (HUVEC), and evaluated their contribution to vascular and bone tissue engineering in vivo.To evaluate the success of osteogenic differentiation and timely vascularization of different osteoconductive scaffolds in vivo, we transferred BMSC and HUVEC pre-cultivated calcium carbonate (CaCO₃) and hydroxylapatite (HA) matrices into immunocompromised RNU-rats, and analyzed mineralization, expression of osteopontin, and vascular integration via new vessel formation.After in vivo transplantation, pre-cultivated scaffolds demonstrated overall improved mineralization of 44% for CaCO₃ (p = 0.01, SD ± 14.3) and 34% for HA (p = 0.001, SD ± 17.8), as well as improved vascularization of 5.6 vessels/0.1 mm² on CaCO₃ (p < 0.0001, SD ± 2.0) and 5.3 vessels/0.1 mm² on HA (p < 0.0001, SD ± 2.4) compared with non-pre-cultivated controls. However, no significant differences between the implantation of BMSC-only, HUVEC-only, or BMSC + HUVEC cocultures could be observed.There is an increasing demand for improved bone regeneration in tissue engineering. Cotransplantation of mesenchymal stromal cells and endothelial cells often demonstrates synergistic improvements in vitro. However, the benefits or superiority of cotransplantation was not evident in vivo and so will require further investigation.     

BMP-2对大鼠骨髓间充质干细胞成骨作用的影响

目的:携带骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)基因的慢病毒载体感染大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)后,观察BMP-2对BMSCs成骨作用的影响。方法:原代培养大鼠BMSCs并进行传代及鉴定,用携带BMP-2的慢病毒感染第3代BMSCs后,进行骨向诱导,通过茜素红染色观察钙沉积。观察细胞黏附能力及对成骨标志分子骨桥蛋白(OPN)、骨钙素(OCN)、胶原蛋白Ⅰ(Col-Ⅰ)及smad(drosophila mothersagainst decapentaplegic protein)表达的影响。采用SPSS11.0软件包对数据进行统计学分析。结果:①原代培养出大鼠骨髓间充质干细胞并进行鉴定;②携带BMP-2和EGFP的慢病毒感染第3代BMSCs后,免疫荧光检测和RT-PCR及WB检测感染成功;③大鼠骨髓间充质干细胞感染慢病毒后进行骨向诱导,钙沉积明显增加;④BMSCs细胞黏附能力增强;⑤OPN、OCN、Col-Ⅰ及smad表达增加。结论:BMP-2通路增强了细胞黏附及相关成骨分化因子的表达,可明显促进大鼠...     

大鼠脂肪干细胞体外培养及成骨诱导分化研究

目的:观察SD大鼠脂肪干细胞(adipose-derived stem cells,ASCs)体外培养的基本生物学特性、鉴定其诱导成骨分化潜能。方法:无菌条件下取6周龄SD大鼠腹股沟处脂肪组织,0.1%I型胶原酶消化,分离培养。显微镜下观察细胞形态;采用MTT比色法绘制细胞生长曲线;用流式细胞仪检测细胞表面标志物;取第3代细胞行成骨和成脂诱导培养,分别用碱性磷酸酶(ALP)、茜素红染色和油红O染色鉴定成骨诱导效果。结果:从SD大鼠脂肪组织中分离获得的ASCs呈长梭形或多角形,呈集落生长;细胞生长曲线呈"S"型,具有较强的增殖能力;流式细胞仪分析示:CD29、CD90高表达,CD34、CD45低表达;成骨诱导后ALP染色及茜素红染色阳性,成脂诱导后油红O染色阳性。结论:分离培养的细胞为ASCs,具有成骨潜能。     

脂肪干细胞在骨组织工程中的应用进展

脂肪干细胞具有组织来源丰富,取材方便且创伤小等优点.利用外源性因子、基因修饰、支架材料诱导等方式,能成功诱导脂肪干细胞成骨性分化.本文介绍了脂肪干细胞生物学特点、各种成骨性分化诱导方法的研究现状,并讨论了年龄、获取部位等因素对其的影响.目前体外或体内实验虽然证明了脂肪干细胞的成骨潜力,但其分化成骨效能仍有待进一步提高....     

脂肪来源干细胞诱导分化为成牙本质样细胞的体外培养研究

目的:探讨大鼠脂肪来源干细胞(ADSCs)向成牙本质样细胞分化的可能性.方法:SD仔4只,处死后取腹股沟处脂肪组织.采用细胞体外共培养的方法,在牙胚细胞条件培养液(TGC-CM)的诱导下,通过形态学观察、免疫组化、RT-PCR等方法,观察脂肪来源干细胞向成牙本质样细胞分化的过程.结果:大鼠脂肪来源干细胞在诱导培养基中生长良好.培养7 d,在诱导组中细胞出现核极化,有很长的突起,细胞平行排列.抗牙本质涎蛋白(DSP)呈阳性反应.RT-PCR显示mRNA水平表达成牙本质细胞特异的牙本质涎磷蛋白(DSPP)和牙本质基质蛋白(DMP-1).结论:含有多种细胞因子的TGC-CM能提供较好的微环境,诱导大鼠脂肪来源干细胞向成牙本质样细胞分化,能为组织工程牙体再生提供种子细胞来源和微环境选择.     

Bone-plates and screws of bioabsorbable poly (L-lactide) An animal pilot study

Poly (L-lactide), a polymer of lactic acid (PLLA), with an extremely high molecular weight (Mv up to 1 x 10(6] has been synthesised under strictly controlled conditions resulting in a new microporous material with excellent mechanical properties. Bone-plates and screws machined from PLLA were used for fixation of two artificial mandibular fractures in sheep effected by a specially designed bone clamp. Fracture healing was uneventful without visible callus formation. Plates and screws of PLLA gave good stability over a sufficiently long period to enable normal fracture healing. Application in humans seems to be justified.     

内皮祖细胞对骨髓间充质干细胞－种植体复合物成骨能力的影响

目的：研究内皮祖细胞（EPCs）对骨髓间充质干细胞（BMSCs）膜片－种植体复合体成骨能力的影响。方法：分离培养大鼠 BMSCs 并采用细胞膜片技术培养细胞膜片;分离培养大鼠骨髓来源 EPCs;将 EPCs 加载于 BMSCs 细胞膜片上并检测相关基因;制作细胞膜片－种植体复合体并植入裸鼠皮下,8周取材,行 Micro-CT 检查及组织学检测。结果：体外实验显示EPCs 加载于 BMSCs 细胞膜片后成骨相关 Runx-2、ALP、BMP2和成血管相关 VEGF 的基因表达提高;裸鼠体内实验取材 Mi-cro-CT 检查显示加载 EPCs 组成骨量较高,HE 切片显示加载 EPCs 组成骨面积较多,免疫组化检测显示加载 EPCs 组 Runx2、BMP2表达较高。结论：加载 EPCs 能提高 BMSCs-种植体复合物的成骨能力。     

1,25（OH）2维生素D3矿化液对人牙髓干细胞成骨方向诱导作用的研究

目的研究维生素D3矿化液对人牙髓干细胞的成骨方向诱导是否有促进作用。方法从恒牙牙髓中分离培养成纤维样细胞并测试其间充质干细胞的特异性标记物Stro-1阳性率,用一定浓度的1,25（OH）2维生素D3、抗坏血酸、β-甘油磷酸钠的矿化液诱导牙髓干细胞,通过倒置相差显微镜、碱性磷酸酶染色、Vonkossa染色及骨钙素、Ⅰ型胶原基因表达观察和检测矿化液诱导后细胞形态变化及矿化基质分泌情况,以未诱导组为对照。结果维生素D3矿化液连续诱导21d后,诱导组的细胞碱性磷酸酶染色阳性、Ⅰ型胶原和骨钙素的基因表达阳性,并可见明显钙结节形成。结论维生素D3矿化液可以诱导人牙髓干细胞向成骨样细胞分化并促进其产生矿化基质。     

脂肪间充质干细胞在颅颌面修复重建中的应用

颅颌面软硬组织缺损是临床常见病和多发病,对患者容貌和功能均有严重妨碍。其修复重建是涉及多学科的综合性临床难题,目前仍有不少问题亟待解决。组织工程的发展为颅颌面修复重建带来了新的思路,而种子细胞来源是组织工程研究的首要问题。近年来,脂肪间充质干细胞因其具有来源广泛、取材方便、诱导条件下多向分化、扩增能力强等优点而成为较为理想的种子细胞。本文就脂肪间充质干细胞在颅颌面修复重建中的应用进展作一综述。     

Resorbable poly(L-lactide) plates and screws for the fixation of zygomatic fractures

Ten patients with unstable zygomatic fractures were treated with resorbable poly(L-lactide) (PLLA) plates and screws. The results show that this method of fixation gives good stability over a sufficiently long period to enable undisturbed fracture healing.     

聚乳酸-羟基乙酸在骨组织工程支架材料中的应用

因外伤、肿瘤等多种原因造成的骨组织缺损需要良好的骨组织替代物修复。随着骨组织工程的不断发展,为骨缺损的修复带来了新思路。学者们一直致力于研发与改性各种骨组织工程支架材料以更好地帮助修复骨缺损。聚乳酸-羟基乙酸因具有良好的生物降解性、生物相容性等优点而备受关注。本文仅对近年来在骨组织工程中聚乳酸-羟基乙酸支架材料的改性与设计进行简要综述。     

Surface modification of poly (L-lactide) by atmospheric pressure plasma treatment and cell response

This study investigated the influence of atmospheric pressure plasma treatment on the surface properties and cell response of poly(L-lactide) (PLLA) samples. The samples were analyzed by means of X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), micro- and nanosurface roughness, water contact angle, and zeta potential. Furthermore, cell adhesion assay and cell proliferation assay on the samples were carried out using MC3T3-E1 cells. Plasma treatment significantly increased the oxygen content of the samples and decreased the contact angle and zeta potential of the samples, resulting in hydrophilic surfaces. Further, plasma treatment of the samples also enhanced the number and growth of adhering MC3T3-E1 cells. These results therefore indicate that plasma treatment is effective for surface modification and cell responses.     

Comparison of strains produced by titanium and poly D, L-lactide Acid plating systems to in vitro forces.

PURPOSE: To determine if a specific resorbable plating system provides similar fixation, in terms of strain distribution under load, to a titanium system when the Champy technique is applied for the treatment of a mandibular angle fracture. MATERIALS AND METHODS: A formalin-fixed cadaver mandible was harvested just before the study. A bicortical osteotomy was then made using a diamond disc extending in an oblique direction in the area of the angle. It was then passively fixated with a 4-hole 2.0-mm miniplate. Two stacked rosette strain gauges were bonded to the mandible on either side of the fracture. Each rosette had 3 strain gauges arranged in specific degrees relative to each other. The mandible was then placed on a dynanometer and 30 lb loads were delivered on the ipsilateral molar. Static resistance was placed in the condylar neck region to simulate the glenoid fossa. Loading was repeated 10 times with a period of 3 minutes between loads. Measurements were recorded for each strain gauge after loads were in place for 30 seconds. The same process was repeated using a 4-hole 2.1-mm resorbable miniplate. The strains were then used to calculate the maximum and minimum strains for each rosette. Hooke's law was used to calculate the principal stresses. RESULTS: Differences were observed between the strain gauges for each individual plating system. There was variability within the resorbable plate measurements as shown by the standard deviation. Using the REML ANOVA test, a significant difference was found between the 2 materials. CONCLUSION: In this in vitro study, there were significant biomechanical differences observed between a 2.0-mm titanium miniplate and a 2.1-mm resorbable miniplate when used to treat a mandibular angle fracture following Champy's principles. Based on our finding, both systems cannot be used interchangeably for the treatment of mandibular angle fractures under the same clinical conditions.     

Erythropoietin (rhEPOa) promotes endothelial transdifferentiation of stem cells of the apical papilla (SCAP)

ObjectiveMesenchymal stem cells (MSCs) have attracted worldwide attention for their capacity to repair damaged tissue, immunosuppression, ability to differentiate into several cell types and their secretome. Earlier studies have demonstrated their angiogenic potential in vitro and in vivo. However, little is known regarding pro-angiogenic inducers of stable endothelial transdifferentiation of MSCs. Here, we employed human MSCs from the Apical Papilla (SCAP) and investigated whether recombinant human erythropoietin-alpha (rhEPOa) could act as such inducer.DesignCultured SCAP cells were exposed to rhEPOa and assessed for cell growth kinetics, viability and morphology, as well as their capacity to form capillary tubule structures in selected microenvironments. RT-PCR was used to monitor endothelial markers and activation of EPO/EPOR pathway signaling components; while gelatin zymographies to assess activation of MMP-2.ResultsrhEPOa treatment initially (48 h) accelerated cell proliferation and allowed SCAP to sprout micro-tubular structures. Morphological and biochemical differentiation was accompanied by activation of MMP-2 and upregulation of PECAM-1, VEGFR2, vWF and VE-cadherin/CDH5. SCAP expressed the cognate EPO-R, while rhEPOa-treated SCAP exhibited higher expression of molecules involved in EPO/EPOR pathway (EPOR and JAK2).ConclusionrhEPOa is capable of promoting endothelial transdifferentiation of SCAP which may be of clinical value in treating of ischemic disorders.     

Influence of resorbable poly(L-lactide) bone plates and screws on the dose distributions of radiotherapy beams

Metallic bone plates have been shown to affect radiation in vitro. Although no damage has ever been demonstrated in vivo these plates may cause dose enhancements and shielding of the adjacent tissue. Resorbable high molecular weight as-polymerized poly(L-lactide) (PLLA) bone plates have recently been used for reconstruction in the maxillofacial area. To determine their influence on dose distribution, a 4-hole bone plate and screws of PLLA were exposed to electron and photon beams. A tissue-equivalent phantom of perspex was irradiated and measurements were made with LiF thermoluminescent dosimeters. No significant changes in dose deviations could be determined when the dose distribution in the homogeneous phantom was compared with that of the phantom in which the PLLA material was placed. From this study it can be concluded that the PLLA material can be regarded as tissue-equivalent and can, thus, be safely used for fracture fixation of bone fragments when postoperative irradiation is anticipated.     

microRNA-145调控大鼠骨髓间充质干细胞成骨分化的实验研究

目的探讨microRNA-145在大鼠骨髓间充质干细胞成骨分化过程中的作用。方法取3周龄雄性大鼠胫骨骨髓间充质干细胞,分为阴性对照组与microRNA-145下调组,分别感染慢病毒,观察microRNA-145对大鼠骨髓间充质干细胞在细胞增殖、细胞周期、凋亡和成骨分化方面的影响。结果 microRNA-145下调表达组(p Lv3-(anti)miR-145)与阴性对照组(p Lv3-Negative control)相比,大鼠骨髓间充质干细胞的增殖、细胞周期和凋亡无统计学差异(P>0.05);microRNA-145下调表达组成骨相关基因Sema3A、OPG、Runx2及Osterix/SP7表达均明显高于阴性对照组,且microRNA-145下调组茜素红染色范围也大于阴性对照组。结论 microRNA-145下调表达可以促进大鼠骨髓间充质干细胞的成骨分化,但对其细胞增殖、细胞周期和凋亡无明显影响。