Genipin‐crosslinked decellularized annulus fibrosus hydrogels induces tissue‐specific differentiation of bone mesenchymal stem cells and intervertebral disc regeneration

Biomaterial‐based therapy that can restore annulus fibrosus (AF) function in early stage and promote endogenous repair of AF tissues is a promising approach for AF tissue repair. In this study, we established a genipin‐crosslinked decellularized AF hydrogels (g‐DAF‐G) that are injectable and could manifest better in situ formability than noncrosslinked decellularized AF hydrogel, while preserving the capacity of directing differentiation of human bone mesenchymal stem cells (hBMSCs) towards AF cells. Hematoxylin and eosin staining, 4',6‐diamidino‐2‐phenylindole staining, and so forth showed that the majority of cellular components were removed, whereas extracellular matrix and microstructure were largely preserved. The storage modulus increased from 465.5 ± 9.4 Pa to 3.29 ± 0.24 MPa after 0.02% genipin crosslinking of decellularized AF hydrogels (DAF‐G) to form g‐DAF‐G. AF‐specific genes (COL1A1, COL5A1, TNMD, IBSP, FBLN1) were significantly higher in DAF‐G and g‐DAF‐G groups than that in control group after 21 days of culturing. g‐DAF‐G significantly restored nucleus pulposus water content and preserved intervertebral structure in vivo. Summarily, we produced a novel AF regeneration biomaterial, g‐DAF‐G, which exhibited well biocompatibility, great bioactivity, and much higher mechanical strength than DAF‐G. This study will provide an easy and fast therapeutic alternative to repair AF injuries or tears.     

大鼠椎间盘纤维环细胞的培养和鉴定

背景:许多学者认为椎间盘退变必有其细胞学改变,而探讨椎间盘退变机制必须有可靠的细胞模型.目的;尝试建立大鼠椎间盘纤维环细胞体外培养体系,并对其表型进行鉴定.设计、时间及地点:以细胞为观察对象的随机分组实验,于2005-09/2006-05在解放军第三军医大学新桥医院实验中心完成.材料:清洁级1月龄SD大鼠30只,体质量100g左右,雌雄不拘.方法:采用酶消化法分离大鼠椎间盘纤维环细胞,进行单层培养.主要观察指标:观察其形态结构和生物学特性,通过甲苯胺蓝、免疫细胞化学和碱性磷酸酶染色等方法对其细胞表型进行鉴定.结果:成功进行细胞单层培养,细胞形态为圆形或多角形,胞质含有大量的粗面内质网;原代细胞生长较慢,第2代细胞生长加快;细胞具有甲苯胺蓝异染性;有Ⅰ型胶原和Ⅱ型胶原的阳性表达;细胞碱性磷酸酶染色阳性.结论:采用酶消化法可成功培养大鼠椎间盘纤维环细胞,表型鉴定符合纤维环细胞特征.     

Enhancing tissue repair in annulus fibrosus defects of the intervertebral disc: analysis of a bio‐integrative annulus implant in an in‐vivo ovine model

Annulus fibrosus repair techniques for the intervertebral disc (IVD) address the unsolved problem of reherniation after IVD herniation and might facilitate the development of nucleus pulposus replacement techniques for IVD diseases. This study investigates the suitability of a bio‐integrative annulus implant.Standardized box defects were applied to the annulus L3/4 and L4/5 of 16 sheep, followed by randomized insertion of the textile polyglycolic acid/polyvinylidene fluoride annulus implant in one of the defects. Explantation was conducted after 2, 6 and 12 weeks, followed by provocative pressure testing and histological analysis. At 2 weeks’ follow‐up, all specimens of the control defect group demonstrated uncontained herniated nucleus pulposus tissue in the annulus defects. For the treated specimens, the annulus implant consistently provided an effective barrier for herniating nucleus pulposus tissue, with no implant dislocation at all time‐points. After 2 weeks, a homogeneous cell infiltration of the annulus implant was observed, leading to a progressive directional matrix build‐up. Repair tissue thickness was significantly stronger with the annulus implant at all follow‐ups (p < 0.01). No pronounced foreign body reaction and no difference in the amount of supra‐annular scar tissue over the defect sites were observed. The implantation procedure inflicted annulus damage adjacent to the defect. At later time‐points, however, no difference in comparison with the control defect group was evident. The investigated biointegrative annulus implant showed promising results with regard to biointegration, enhancement of repair tissue and function as a mechanical barrier in an ovine model. © 2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

经皮纤维环穿刺与经肌间隙纤维环刀刺建立兔椎间盘退变模型

背景：有研究表明椎间盘退变模型的建立可为椎间盘退变治疗提供实验载体,但目前尚缺乏公认的最佳实验动物模型。 目的：比较经皮纤维环穿刺法和经肌间隙纤维环刀刺法建立兔椎间盘退变模型的差异。 方法：将新西兰大白兔分别采用经皮纤维环穿刺法和经肌间隙纤维环刀刺法建立兔椎间盘退变模型,穿刺后4,8,16周通过磁共振和组织病理学检查观察腰椎间盘髓核变性及组织病理情况。 结果与结论：穿刺后4周,两组兔椎间盘髓核内T2加权像信号降低、变暗,椎间隙高度下降,但经皮纤维环穿刺组T2信号强度评分较经肌间隙纤维环刀刺组低（P〈0.05）;穿刺后8周,两组T2信号强度评分均增高,经皮纤维环穿刺组T2信号强度评分较经肌间隙纤维环刀刺组低（P〈0.05）;穿刺后16周,两组兔椎间盘髓核内T2信号强度评分达最高且差异无显著性意义,两组椎间隙均明显变窄,椎间盘均亮度变黑,两组差异不明显;经皮纤维环穿刺组手术时间少于经肌间隙纤维环刀刺组（P〈0.05）,经肌间隙纤维环刀刺组感染率为5.6%,而经皮纤维环穿刺组无感染。结果证实,经皮纤维环穿刺法建立兔椎间盘退变模型建模时间短,感染率低,效果优于经肌间隙纤维环刀刺法造模。     

兔纤维环穿刺及终板下椎骨缺血诱导下椎间盘退变模型的建立

目的通过在同一新西兰兔体内建立两种腰椎间盘退变程度不同的模型,为今后运用磁共振T_2 mapping成像定量动态观察椎间盘退变过程提供实验基础。方法每只新西兰兔(n=6)均选L4/5及L7/S1椎间盘为空白对照组,L5/6椎间盘为终板下椎骨缺血组,L6/7椎间盘为纤维环穿刺组。在X线透视导引下经皮穿刺新西兰兔(n=6)L6/7椎间盘纤维环,同时经皮穿刺兔L5/6椎间盘两侧终板下椎骨并注射平阳霉素。造模前、造模后第4、7、10及13周使用1.5 T磁共振成像仪进行新西兰兔腰椎矢状位T_2WI扫描检查。用生物化学方法检测椎间盘组织蛋白多糖含量。结果纤维环穿刺组的腰椎间盘T_2WI信号在造模后第4周开始降低,而终板下椎骨缺血组的腰椎间盘T_2WI信号在造模后第13周才发生下降。造模术后第13周新西兰兔两个空白对照组(L4/5、L7/S1)椎间盘之间的腹、背侧纤维环及髓核组织的蛋白多糖含量无统计学差异(P0.05)。造模术后第13周终板下骨缺血组的椎间盘腹、背侧纤维环及髓核组织的蛋白多糖低于两个空白对照组(L4/5和L7/S1)(P0.01),明显高于纤维环穿刺组(P0.01)。结论采用微创方法可在新西兰兔体内同时构建纤维环穿刺或终板下椎骨缺血诱导的腰椎间盘退变模型。与纤维环穿刺模型相比,终板下椎骨缺血模型的椎间盘呈缓慢的退变过程。     

Evaluation of cross‐linked chitosan microparticles for bone regeneration

The objective of this preliminary study was to evaluate the applying of chitosan (CS)‐based microparticles (MPs) in bone regeneration in vivo. The CS MPs were fabricated using our scale‐up method, as previously described. Mesenchymal stem cells (MSC) were harvested from the femora and tibiae of Dark Agouti (DA) rats and seeded on CS MPs. An in vitro MSCs attachment experiment was conducted by trypsinizing the cells attached to the MPs at 5, 10, 20 and 30 h. Fluorescence images of MSCs attached to the MPs were taken at 24 and 48 h, using a LIVE/DEAD cell assay. The MSC/osteoblasts (OB) seeded on MPs were then cultured in vitro using osteogenic media and implanted into partial thickness bone defects in rat femurs. There were two groups of rats, including experimental animals and controls, for the in vivo studies. The experimental group were implanted with MSC‐seeded MPs and observed at 4 and 8 weeks. The control group of rats did not receive any implant material except the stainless steel plate to support the defect. Four rats per group were used for the study. The femurs were extracted at 4 and 8 weeks post‐implantation and bone formation at the defect site was analysed using radiography, microcomputed tomography (µCT) and histology. Among all groups, a significant increase in bone formation was observed in the experimental group at 8 weeks implantation. The results of this study suggested that CS MPs prove to be a successful biomaterial for bone regeneration. Copyright © 2010 John Wiley & Sons, Ltd.     

Enhancing human nucleus pulposus cells for biological treatment approaches of degenerative intervertebral disc diseases: a systematic review

Intervertebral disc (IVD) degeneration has been described as an aberrant, cell‐mediated, age‐ and genetics‐dependent molecular degeneration process, which can be accelerated by nutritional, mechanical and toxic factors. Collective involvement of these factors can result in structural failures, which are often associated with pain. Current treatment approaches are restricted to symptomatic therapies, not addressing options of restoring structural or biological deterioration of the IVD as the underlying problem. Therapeutic potentials of IVD cell transplantation, biomaterials, inhibiting or activating bioactive factors, including gene‐therapeutic approaches, have been shown in vitro or in small animal models. Since human degenerative IVD cells display distinctive features with regard to cell biology and regenerative potential, we attempted a systematic review, investigating the in vitro response of human nucleus pulposus cells to different stimuli. Therefore, we conducted an electronic database search on Medline through July 2011 to identify, compare and discuss publications concerning the effects of cell–cell stimulation, bioactive factors, biomaterials and combinations thereof in terms of cell isolation, proliferation, differentiation and matrix protein synthesis. This survey and discussion might serve as a source for designing future biological treatment strategies for the human IVD. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of high-intensity focused ultrasound on the intervertebral disc: a potential therapy for disc herniations

PURPOSE: To determine the potential application of high-intensity focused ultrasound for the minimally invasive treatment of herniated intervertebral discs by developing a probe that produces sufficiently high temperature locally to shrink collagen fibers (65-75 degrees Celsius). MATERIALS AND METHODS: A 5-mm ultrasound probe was produced with a geometric focal length of 15 mm. The probe produced 2.5 W of acoustic power and was operated at a frequency of 4.1 MHz. Measurements of temperature increase were performed in discs from bovine tails. In vivo experiments were performed to assess histologic changes in the disc as well as in nerve root and muscle. RESULTS: Sufficient temperature increase to produce collagen shrinkage was observed close to the focus of the ultrasound. Temperature measurements in vertebral end plates showed a temperature increase of only 4 degrees Celsius after 60-second exposure of the disc. In vivo experiments revealed histologic changes in the disc consistent with collagen shrinkage, with no adverse effects seen in surrounding tissues. CONCLUSIONS: The experiments demonstrated the feasibility of high-intensity focused ultrasound in the treatment of contained herniated discs. This technique has several advantages over other thermal treatment modalities.     

三氧化二砷对人肝癌SMMC-7721细胞的体内外作用及可能的机制

目的观察三氧化二砷（As203）在体内外对人肝癌SMMC-7721细胞抗肿瘤作用。方法不同浓度的As203作用于SMMC-7721细胞48h,采用MTT法测定细胞的存活率;荧光显微镜观察细胞形态变化;FITC-Annexinv／P1双标记检测SMMC-7721细胞的凋亡;比色法检测Caspase．3活性变化。用As203在SMMC-7721肝癌细胞荷瘤裸鼠的瘤体内进行注射治疗,观察肿瘤生长变化,12d后处死裸鼠,摘除瘤体,称瘤重,并通过免疫组化法检测Bcl-2、Bax和Caspase-3的表达。结果As203能显著抑制SMMC-7721细胞的增殖,半数抑制率浓度为（18．17±2．10）μmol／L;显微镜下可见有典型的细胞凋亡形态学改变,As203处理组SMMC-7721细胞凋亡率与对照组相比显著增加,As203可提高SMMC-7721细胞的Caspase-3活性。As203瘤内注射肝癌细胞株SMMC-7721裸鼠移植瘤后,能显著抑制肿瘤生长,瘤重的抑制率可达52．37％,免疫组化结果显示As203能明显上调与细胞凋亡相关因子Bax和Caspase．3的表达和下调Bcl-2的表达。结论As203能抑制肝癌SMMC-7721细胞的生长,诱导SMMC-7721细胞凋亡;As203能抑制SMMC-7721细胞的体内致瘤能力。     

人正常椎间盘髓核细胞体外不同培养代次的生物学性状分析:适应于组织工程椎间盘种子细胞的选择

背景:目前用作组织工程椎间盘研究的种子细胞主要来源于体外培养的髓核细胞,而体外培养的髓核细胞传至一定代次后即出现老化现象,老化的细胞失去生长能力不适于组织工程研究.目的:比较不同代次正常髓核细胞的形态学、增殖特性以及细胞的主要细胞外基质成分基因表达情况.方法:以酶消化法分离、培养人正常椎间盘髓核细胞,对不同代次髓核细胞采用光镜、电镜等形态学方法进行大体形态和超微结构观察,采用XTT实验检测不同代次细胞间的生长动力学差异,测定髓核细胞的活力和细胞Ⅱ型胶原及糖胺多糖mRNA表达.结果与结论:传3代之前细胞形态饱满,胞浆丰富,细胞呈多角形、短梭形,细胞内细胞器丰富,形态正常,线粒体含量少,核糖体含量多,说明细胞代谢旺盛,增殖能力强,生长曲线和XTT实验也提示传3代之前髓核细胞活性高,增殖能力强,细胞活力高,细胞外基质分泌旺盛.从传4代起,部分细胞形态逐渐向长梭形演化,逐渐表现为成纤维细胞的形态,说明细胞开始出现"返祖"现象,也称为老化现象,细胞的生长趋缓,传六七代后细胞均表现为长梭形,粗面内质网扩张,线粒体增多,细胞活力降低,细胞外基质明显减少,生长处于停滞状态.实验结果提示,体外培养条件下,人髓核细胞前3代细胞形态良好,增殖能力强,适合作为组织工程椎间盘的种子细胞,而细胞传4代后增殖能力逐渐减弱,不宜作为组织工程椎间盘的种子细胞.     

Labelling dendritic cells with SPIO has implications for their subsequent in vivo migration as assessed with cellular MRI

An optimized non‐invasive imaging modality capable of tracking and quantifying in vivo DC migration in patients would provide clinicians with valuable information regarding therapeutic DC‐based vaccine outcomes. Superparamagnetic iron oxide (SPIO) nanoparticles were used to label bone marrow‐derived DC. In vivo DC migration was tracked and quantified non‐invasively using cellular magnetic resonance imaging (MRI) in a mouse model. Labelling DC with SPIO reflects the kinetics of DC migration in vivo but appears to reduce overall DC migration, in part due to nanoparticle size. Magnetic separation of SPIO‐labelled (SPIO⁺) DC from unlabelled (SPIO^?) DC prior to injection improves SPIO⁺ DC migration to the lymph node. Corresponding MR image data better correlate with the presence of DC in vivo; an improved immunological response is also seen. Cellular MRI is a viable, non‐invasive imaging tool that can routinely track DC migration in vivo. Consideration should be given to optimizing MRI contrast agent‐labelling of clinical‐grade DC in order to accurately correlate DC fate to immunological outcomes in patients. Copyright © 2011 John Wiley & Sons, Ltd.     

In vivo instruction of suppressor commitment in naive T cells

The induction of antigen-specific tolerance in the mature immune system of the intact organism has met with limited success. Therefore, nonspecific immunosuppression has been the treatment of choice to prevent unwanted immunity. Here, it is shown that prolonged subcutaneous infusion of low doses of peptide by means of osmotic pumps transforms mature T cells into CD4+25+ suppressor cells that can persist for long periods of time in the absence of antigen and confer specific immunologic tolerance upon challenge with antigen. The described procedure resembles approaches of tolerance induction used decades ago, induces tolerance in the absence of immunity, and holds the promise to become an effective means of inducing antigen-specific tolerance prospectively, whereas its power to suppress already ongoing immune responses remains to be determined.     

间充质干细胞的体外标记方法及其体内分布研究新进展

间充质干细胞(mesenchymal stem cells,MSC)是一种具有高度自我更新能力和多向分化潜能的非造血成体干细胞,由于其取材方便、体外能大量扩增,近年成为干细胞领域的研究热点.MSC作为细胞治疗和基因治疗的种子载体细胞,已经广泛应用于心血管系统、神经系统、呼吸系统和创伤修复等方面的基础研究,部分成果已用于临床.但是关于MSC的许多生物学特性及分子调控机制尚不十分清楚,其研究和应用尚处于初级阶段,有待于进一步探索.经体外分离、培养的MSC是一类具有多向分化能力的细胞,对于其体内横向分化潜能还不确定.是否存在基因突变、植入机体后是否有癌变的可能,其有效性和安全性等问题仍有待进一步观察.深入研究其归巢特点、机制及其影响因素也有助于MSC的临床应用研究,也只有研究清楚MSC在体内特异微环境下的分化命运,MSC才可能更好地应用于临床.因此,体外如何稳定高效标记MSC,从而对其在体内的存活、迁移、分布及增殖、分化进行监测是亟待解决的关键问题.本文综述目前国内外关于MSC体外标记方法的最新研究成果,探讨不同体外标记方法的优缺点及其应用的适宜条件.     

Functionalized single‐walled carbon nanotubes containing traces of iron as new negative MRI contrast agents for in vivo imaging

Single‐walled carbon nanotubes (SWCNTs) containing traces of iron oxide were functionalized by noncovalent lipid‐PEG or covalent carboxylic acid function to supply new efficient MRI contrast agents for in vitro and in vivo applications. Longitudinal (r₁) and transversal (r₂) water proton relaxivities were measured at 300 MHz, showing a stronger T₂ feature as an MRI contrast agent (r₂/r₁ = 190 for CO₂H functionalisation). The r₂ relaxivity was demonstrated to be correlated to the presence of iron oxide in the SWNT‐carboxylic function COOH, in comparison to iron‐free ones. Biodistribution studies on mice after a systemic injection showed a negative MRI contrast in liver, suggesting the presence of the nanotubes in this organ until 48 h after i.v. injection. The presence of carbon nanotubes in liver was confirmed after ex vivo carbon extraction. Finally, cytotoxicity studies showed no apparent effect owing to the presence of the carbon nanotubes. The functionalized carbon nanotubes were well tolerated by the animals at the dose of 10 µg g^?1 body weight. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo conversion of astrocytes to myelinating cells by miR‐302/367 and valproate to enhance myelin repair

Enhancement of repair potential for degenerative brain diseases has been a research priority during recent years. Considering recent advancements in the field of direct transdifferentiation, conversion of astrocytes as a prominent component of glial scars to the progenitor cells that contribute to the repair mechanisms seems interesting. Recently, we have reported miR‐302/367‐mediated in vivo conversion of astrocytes into neuroblasts and neurons. In the current study, we used miR‐302/367 and valproate (VPA) to show the possibility of conversion of astrocytes to oligodendrocyte progenitor cells and myelinating cells in a cuprizone (CPZ)‐induced model of demyelination. Evaluation of behavioural impairment following CPZ and consequent to the treatments showed functional recovery from impairments. Enhanced remyelination was detected by luxol fast blue staining and immunostaining against two mature myelin markers, myelin basic protein and proteolipid protein. Tracing of transduced cells with green fluorescent protein showed their contribution toward generation of new myelinating cells. These findings have suggested that in vivo specific targeting of astrocytes for forced expression of the miR‐302/367 cluster and VPA administration may increase the brain's potential for repairing myelin insults by the generation of oligodendroglia from astrocytes. This finding may open a new avenue for enhancement of brain repair in neurodegenerative diseases such as multiple sclerosis. Copyright © 2017 John Wiley & Sons, Ltd.     

Osteogenic differentiation of two distinct subpopulations of human adipose-derived stem cells: an in vitro and in vivo study

The first stem cells considered for the reconstruction of bone were bone marrow mesenchymal stem cells (BMSCs). Subsequently, cells with similar marker expression panel and differentiation potential were found in new sources of cells, such as adipose tissue. This source of stem cells has a promising future in tissue-engineering applications, considering the abundance of this tissue in the human body, the easy harvesting and the high number of stem cells that are available from such a small amount of tissue. The isolation of the adipose stem cells is generally performed by means of enzymatic digestion of the tissues, followed by a natural selection of the stem cells based on their capacity to adhere to the culture flasks, leading to a quite heterogeneous population. This constitutes a major drawback for the use of these cells, since the heterogeneity of the cell culture obtained can compromise their proliferation and differentiation potential. In the present study we have analysed the in vitro and in vivo behaviour of two selected subpopulations with high osteogenic potential. For this purpose, ASCs(CD29+) and ASCs (STRO-1+)subpopulations were isolated and in vitro cultured onto a biodegradable polymeric scaffold, using osteogenic medium, before implantation in a nude mice model. The biodegradable polymeric scaffold used is a fibre-mesh structure based on a blend of starch and polycaprolatone (SPCL) that has been successfully used in several bone tissue-engineering studies. The implanted ASCs-scaffold constructs promoted the formation of new bone tissue in nude mice. However, the results obtained show differences in the behaviour of the two ASCs subpopulations under study, particularly regarding their potential to differentiate into the osteogenic lineage, and allowed the indentification of ASCs (STRO-1+) as the best subpopulation for bone tissue-engineering applications.     

Imaging modalities for the in vivo surveillance of mesenchymal stromal cells

Bone marrow stromal cells exist as mesenchymal stromal cells (MSCs) and have the capacity to differentiate into multiple tissue types when subjected to appropriate culture conditions. This property of MSCs creates therapeutic opportunities in regenerative medicine for the treatment of damage to neural, cardiac and musculoskeletal tissues or acute kidney injury. The prerequisite for successful cell therapy is delivery of cells to the target tissue. Assessment of therapeutic outcomes utilize traditional methods to examine cell function of MSC populations involving routine biochemical or histological analysis for cell proliferation, protein synthesis and gene expression. However, these methods do not provide sufficient spatial and temporal information. In vivo surveillance of MSC migration to the site of interest can be performed through a variety of imaging modalities such as the use of radiolabelling, fluc protein expression bioluminescence imaging and paramagnetic nanoparticle magnetic resonance imaging. This review will outline the current methods of in vivo surveillance of exogenously administered MSCs in regenerative medicine while addressing potential technological developments. Furthermore, nanoparticles and microparticles for cellular labelling have shown that migration of MSCs can be spatially and temporally monitored. In vivo surveillance therefore permits time‐stratified assessment in animal models without disruption of the target organ. In vivo tracking of MSCs is non‐invasive, repeatable and non‐toxic. Despite the excitement that nanoparticles for tracking MSCs offer, delivery methods are difficult because of the challenges with imaging three‐dimensional systems. The current advances and growth in MSC research, is likely to provide a wealth of evidence overcoming these issues. Copyright © 2014 John Wiley & Sons, Ltd.     

The role of plasminogen in angiogenesis in vivo

Summary.  Plasminogen, by virtue of its role in the degradation of extracellular matrix proteins and by facilitation of cell migration, may contribute to angiogenesis. Objective : the purpose of this study was to evaluate the contribution of plasminogen to angiogenesis in vivo . Methods : Angiogenesis was assessed in gene-targeted mice with deficiencies of plasminogen, urokinase plasminogen activator (uPA), and urokinase receptor (uPAR) in a mouse corneal model. In wild-type mice, female and young mice showed a trend toward increased angiogenesis compared to males and old mice. Because of this influence of age and gender on angiogenesis, young, female mice (6–13 weeks of age) were used for this study. Results : In response to angiogenic stimulation by basic fibroblast growth factor (bFGF), uPA deficient mice exhibited a decrease in new vessel formation as reflected by vessel length (0.47 in control vs. 0.33 mm in uPA^−/− mice, P  = 0.043), but new vessel formation was not altered ( P  = 0.107) in the uPAR deficient mice compared to control mice. A significantly decreased angiogenic response of new vessel formation to both vascular endothelial growth factor (VEGF) ( P  < 0.02) and bFGF ( P  < 0.007) was observed in Plg deficient (Plg^−/−) mice (VEGF – 0.36 mm, bFGF – 0.67 mm) compared to Plg^+/+ mice (VEGF – 0.56 mm, bFGF – 0.85 mm). Conclusions : These results demonstrate the importance of plasminogen, as well as of uPA, in angiogenesis in vivo.     

Multimodal imaging of micron‐sized iron oxide particles following in vitro and in vivo uptake by stem cells: down to the nanometer scale

In this study, the interaction between cells and micron‐sized paramagnetic iron oxide (MPIO) particles was investigated by characterizing MPIO in their original state, and after cellular uptake in vitro as well as in vivo. Moreover, MPIO in the olfactory bulb were studied 9 months after injection. Using various imaging techniques, cell–MPIO interactions were investigated with increasing spatial resolution. Live cell confocal microscopy demonstrated that MPIO co‐localize with lysosomes after in vitro cellular uptake. In more detail, a membrane surrounding the MPIO was observed by high‐angle annular dark‐field scanning transmission electron microscopy (HAADF‐STEM). Following MPIO uptake in vivo, the same cell–MPIO interaction was observed by HAADF‐STEM in the subventricular zone at 1 week and in the olfactory bulb at 9 months after MPIO injection. These findings provide proof for the current hypothesis that MPIO are internalized by the cell through endocytosis. The results also show MPIO are not biodegradable, even after 9 months in the brain. Moreover, they show the possibility of HAADF‐STEM generating information on the labeled cell as well as on the MPIO. In summary, the methodology presented here provides a systematic route to investigate the interaction between cells and nanoparticles from the micrometer level down to the nanometer level and beyond. Copyright © 2014 John Wiley & Sons, Ltd.     

脂肪间充质干细胞复合血小板凝胶修复兔椎间盘退变

背景:富含血小板的血浆凝胶作为三维支架使其中干细胞可以呈立体生长,同时富含血小板的血浆凝胶又释放大量生长因子,促进脂肪间充质干细胞增殖及分化.目的:探讨脂肪间充质干细胞-富含血小板的血浆凝胶复合体注入兔椎间盘退变模型后的修复作用.方法:取兔动脉血采用二次离心法制备自体富血小板血浆,取兔肩胛间区脂肪分离培养脂肪间充质干细胞,制备脂肪间充质干细胞-富含血小板的血浆凝胶复合体.新西兰大白兔随机分为对照组、模型组、富含血小板的血浆凝胶组和脂肪间充质干细胞-富含血小板的血浆凝胶复合体组,后3组以穿刺法制备椎间盘退变模型,退变模型制备完成2周后,富含血小板的血浆凝胶组和脂肪间充质干细胞-富含血小板的血浆凝胶复合体组分别对退变间盘中注射相应材料.结果与结论:兔椎间盘退变后,间隙明显降低,髓核信号明显降低,髓核内基质高,密度染色较深;而经富含血小板的血浆凝胶和脂肪间充质干细胞-富含血小板的血浆凝胶复合体治疗后,上述症状明显改善,且脂肪间充质干细胞-富含血小板的血浆凝胶复合体的治疗效果更好.提示对退变椎间盘内注射富含血小板的血浆凝胶支架及脂肪间充质干细胞-富含血小板的血浆凝胶复合体均有利于减少退变对椎间盘的影响,其中脂肪间充质干细胞-富含血小板的血浆凝胶复合体注射效果更为突出.