急性淋巴细胞白血病患儿骨髓基质细胞的粘附行为

目的和方法:用MTT方法检测了急性淋巴细胞白血病(ALL)患儿骨髓基质细胞(BMSC)对正常骨髓造血细胞和淋巴瘤Raji细胞的粘附行为,用流式细胞仪检测了BMSC表面的粘附分子ICAM-1和VCAM-1的表达。结果:急性期组ALLBMSC对骨髓造血细胞的粘附能力明显下降。长期缓解组ALLBMSC对肿瘤细胞的粘附能力明显增高。急性期组ALLBMSC表面ICAM-1表达明显降低。结论:ALL患儿BMSC对骨髓造血细胞和肿瘤细胞的粘附行为的异常,BMSC粘附行为的变化与细胞表面粘附分子的表达异常有关。     

Influence of the pore generator on the evolution of the mechanical properties and the porosity and interconnectivity of a calcium phosphate cement

Porosity and interconnectivity are important properties of calcium phosphate cements (CPCs) and bone-replacement materials. Porosity of CPCs can be achieved by adding polymeric biodegradable pore-generating particles (porogens), which can add porosity to the CPC and can also be used as a drug-delivery system. Porosity affects the mechanical properties of CPCs, and hence is of relevance for clinical application of these cements. The current study focused on the effect of combinations of polymeric mesoporous porogens on the properties of a CPC, such as specific surface area, porosity and interconnectivity and the development of mechanical properties. CPC powder was mixed with different amounts of PLGA porogens of various molecular weights and porogen sizes. The major factors affecting the properties of the CPC were related to the amount of porogen loaded and the porogen size; the molecular weight did not show a significant effect per se. A minimal porogen size of 40 μm in 30 wt.% seems to produce a CPC with mechanical properties, porosity and interconnectivity suitable for clinical applications. The properties studied here, and induced by the porogen and CPC, can be used as a guide to evoke a specific host-response to maintain CPC integrity and to generate an explicit bone ingrowth.     

RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration

Calcium phosphate cement scaffold (CPC) has been widely used as bone graft substitutes, but undesirable osteoinductivity and slow degradability greatly hamper their clinic application. To address these problems, a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium silicate/calcium phosphate cement scaffold (CSPC) with hierarchical pores was developed in this study. The CSPC scaffold with both interconnected macropores on the order of 200–500 μm and micropores of 2–5 μm was synthesized from CPC and calcium silicate (CS) by a NaCl particulate-leaching method. In vitro cell culture with C2C12 model cells, in vivo ectopic bone formation and rabbit femur cavity defect repair were performed to evaluate the osteogeneic capacity of the CSPC/rhBMP-2 scaffold. CPC, CSPC and CPC/rhBMP-2 scaffolds were parallelly investigated for comparison. The results demonstrated that the hierarchical macro/microporous structure, whether in presence of CS or rhBMP-2, highly favored the adhesion of C2C12 cells and bone in-growth into the CPC-based scaffolds. But, in comparison to the CPC-based scaffolds with CS or rhBMP-2 alone, the CSPC/rhBMP-2 scaffold strongly promoted osteogenic differentiation in vitro and osteogenetic efficacy in vivo. Further studies demonstrated that Si ions derived from CSPC contributed mainly to maintain the conformation of rhBMP-2 and thus stimulate the synergistic action of CS and rhBMP-2 in osteogenic differentiation and osteoinductivity. Additionally, the incorporation of CS was also beneficial for the dissolution of the scaffold. Those results suggest that the CSPC has superior properties for incorporation of rhBMP-2 and our developed CSPC/rhBMP-2 scaffold have great potential for future use in bone tissue regeneration.     

Calcium phosphate ceramics in bone tissue engineering: A review of properties and their influence on cell behavior

Calcium phosphate ceramics (CPCs) have been widely used as biomaterials for the regeneration of bone tissue because of their ability to induce osteoblastic differentiation in progenitor cells. Despite the progress made towards fabricating CPCs possessing a range of surface features and chemistries, the influence of material properties in orchestrating cellular events such as adhesion and differentiation is still poorly understood. Specifically, questions such as why certain CPCs may be more osteoinductive than others, and how material properties contribute to osteoinductivity/osteoconductivity remain unanswered. Therefore, this review article systematically discusses the effects of the physical (e.g. surface roughness) and chemical properties (e.g. solubility) of CPCs on protein adsorption, cell adhesion and osteoblastic differentiation in vitro. The review also provides a summary of possible signaling pathways involved in osteoblastic differentiation in the presence of CPCs. In summary, these insights on the contribution of material properties towards osteoinductivity and the role of signaling molecules involved in osteoblastic differentiation can potentially aid the design of CPC-based biomaterials that support bone regeneration without the need for additional biochemical supplements.     

体外模拟和体内植入实验分析磷酸钙骨水泥的降解活性

目的 研究CPC及CPC／BMP复合人工骨降解性能，寻找加快CPC降解的有效途径，方法将CPC作为BMP的载体制成CPC／BMP复合物，在体外模拟生理环境进行CPC和CPC／BMP复合物的溶解试验。通过植入小鼠肌袋和犬桡骨植入试验，观察材料在体内的降解情况和降解规律。结果BMP促进了CPC的体外溶解。肌肉内植入CPC／BMP可以异位诱导新骨形成。植入骨缺损后CPC／BMP可以诱导新骨形成，有效地修复骨缺损。新骨形成的同时，材料出现了较快的降解。CPC不能异位诱导新骨形成，骨缺损修复能力较弱，降解缓慢。结论CPC／BMF，生物活性人工骨具有理想的降解性能和成骨能力，可望成为新型的骨缺损修复材料。     

活性氧促进中性粒细胞与骨髓基质细胞黏附及机制初探

目的:探讨活性氧对中性粒细胞与骨髓基质细胞(BMSCs)黏附的影响及其机制。方法:采用密度梯度离心法从小鼠胫骨和股骨分离骨髓中性粒细胞,并用DMSO诱导HL60细胞分化为成熟中性粒细胞(d HL60细胞)。利用分光光度法检测CFDA-SE标记的小鼠骨髓中性粒细胞和d HL60细胞在H_2O_2刺激下与BMSCs的黏附。利用荧光显微成像技术和Western blot检测携带去谷胱甘肽化酶谷氧还蛋白1(Grx1)表达载体的慢病毒感染的d HL60细胞中Grx1的表达。PCR检测Grx1敲除小鼠的基因型。结果:(1) Diff-Quick染色显示,分离的骨髓中性粒细胞纯度高于90%; H_2O_2处理后,中性粒细胞与BMSCs黏附率显著增加(P 0. 01)。(2)荧光显微镜观察和Western blot结果表明,Grx1稳转株d HL60细胞中Grx1的表达水平较空载体稳转株细胞显著增加;体外黏附实验表明过表达Grx1的d HL60细胞较对照组d HL60细胞,在相同H_2O_2浓度下,黏附到BMSCs程度显著下降(P 0. 01)。(3)经PCR鉴定,实验所用基因敲除小鼠在全基因水平上未见Grx1;且敲除Grx1的小鼠较野生型小鼠,在相同浓度H_2O_2刺激下,其骨髓中性粒细胞与BMSCs黏附率显著升高。(4)血管细胞黏附分子1(VCAM-1)抗体预处理BMSCs后,由H_2O_2引起的d HL60与BMSCs之间的黏附增强回复到了静息水平。结论:活性氧促进骨髓中中性粒细胞与BMSCs的黏附,其可能的机制是通过诱导VCAM-1黏附信号的S-谷胱甘肽化。     

An injectable,biodegradable calcium phosphate cement containing poly lactic-co-glycolic acid as a bone substitute in ex vivo human vertebral compression fracture and rabbit bone defect models

Purpose/Aim of the study: To evaluate the biomechanical characteristics and biocompatibility of an injectable, biodegradable calcium phosphate cement (CPC) containing poly lactic-co-glycolic acid (PLGA). Materials and methods: A vertebral compression fracture model was established using 20 human cadaveric vertebrae (T11-L3) divided into CPC/PLGA composite versus PMMA groups for biomechanical testing. In addition, 35 New Zealand rabbits were used to evaluate biodegradability and osteoconductive properties of CPC/PLGA using a bone defect model. In vitro cytotoxicity was evaluated by culturing with L929 cells. Results: The CPC/PLGA composite effectively restored vertebral biomechanical properties. Compared with controls, the maximum load and compression strength of the CPC/PLGA group were lower, and stiffness was lower after kyphoplasty (all p <.05). Degradation was much slower in the control CPC compared with CPC/PLGA group. The bone tissue percentage in the CPC/PLGA group (44.9 ± 23.7%) was significantly higher compared with control CPC group (25.7 ± 10.9%) (p <.05). The viability of cells cultured on CPC/PLGA was greater than 70% compared with the blanks. Conclusions: Our biodegradable CPC/PLGA composite showed good biomechanical properties, cytocompatibility, and osteoconductivity and may represent an ideal bone substitute for future applications.     

A novel strontium(II)-modified calcium phosphate bone cement stimulates human-bone-marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation in vitro

In the present study, the in vitro effects of novel strontium-modified calcium phosphate bone cements (SrCPCs), prepared using two different approaches on human-bone-marrow-derived mesenchymal stem cells (hMSCs), were evaluated. Strontium ions, known to stimulate bone formation and therefore already used in systemic osteoporosis therapy, were incorporated into a hydroxyapatite-forming calcium phosphate bone cement via two simple approaches: incorporation of strontium carbonate crystals and substitution of Ca²⁺ by Sr²⁺ ions during cement setting. All modified cements released 0.03–0.07 mM Sr²⁺ under in vitro conditions, concentrations that were shown not to impair the proliferation or osteogenic differentiation of hMSCs. Furthermore, strontium modification led to a reduced medium acidification and Ca²⁺ depletion in comparison to the standard calcium phosphate cement. In indirect and direct cell culture experiments with the novel SrCPCs significantly enhanced cell proliferation and differentiation were observed. In conclusion, the SrCPCs described here could be beneficial for the local treatment of defects, especially in the osteoporotic bone.     

新型可注射可降解磷酸钙骨水泥的生物相容性

背景：体外实验已证实新型磷酸钙骨水泥有良好的可注射性、力学性能、抗溃散性及体外降解性能。 目的：验证新型可注射、可降解磷酸钙骨水泥的生物相容性。 方法：①急性毒性实验：分别向昆明小鼠尾静脉可注射新型磷酸钙骨水泥浸提液与生理盐水。②热源实验：在新西兰兔耳缘静脉注射新型磷酸钙骨水泥浸提液。③溶血实验：在兔抗凝血分别加入新型磷酸钙骨水泥浸提液、生理盐水及双蒸水。④迟发型超敏反应实验：在豚鼠肩胛骨内侧部位分别注射可注射新型磷酸钙骨水泥浸提液与生理盐水,并进行敷贴激发实验。⑤体外细胞毒性实验：在L929系小鼠成纤维细胞株培养液中分别加入可注射新型磷酸钙骨水泥浸提液、聚乙烯浸提液及苯酚溶液。⑥微核实验：分别在昆明小鼠腹腔注射可注射新型磷酸钙骨水泥浸提液、生理盐水与环磷酰胺。⑦肌肉植入实验：将新型磷酸钙骨水泥植入新西兰兔脊柱两侧肌肉内。 结果与结论：新型可注射磷酸钙骨水泥无毒,无刺激性及致敏性,无热源反应,具有良好的血液相容性,植入动物肌肉后为非组织刺激物,具有良好的生物相容性,因而具有较好的生物安全性。     

Influence of a novel radiopacifier on the properties of an injectable calcium phosphate cement

An injectable calcium phosphate cement (CPC) with excellent radiopacity was proposed by introducing a novel radiopacifier, strontium carbonate, into the powder phase of CPC. The results showed that the cement showed improved radiopacity even when the content of strontium carbonate was only 8 or 12wt.%. The addition of 8 or 12wt.% strontium carbonate clearly improved the injectability and compressive strength of the cement. Furthermore, the addition of strontium carbonate influenced the pore distribution in the cement. An injectable CPC containing 8 or 12wt.% strontium carbonate has the potential for use in procedures such as vertebroplasty and kyphoplasty.     

The in vitro behavior of as-prepared and pre-immersed RF-sputtered calcium phosphate thin films in a rat bone marrow cell model

In this paper we focus on the behavior of radio frequency (RF)-sputtered calcium phosphate (CaP) thin films in a rat bone marrow (RBM) cell model. Two issues are addressed. Firstly, we benchmarked the in vitro cell behavior of these CaP coatings by comparing their proliferation, differentiation and mineralization behavior and the structure of the formed interface to similar coatings of alumina and titania. We found that the CaP coatings showed reduced proliferation, enhanced early differentiation and enhanced activity of mature osteoblasts compared to the alumina coatings. Enhanced production of mineralized extracellular matrix (ECM) was seen for both CaP and titania. Two types of CaP precipitates could be observed, one directly bonded CaP layer at the coating interface and one of globular accretions associated with the ECM. The directly bonded layer was not observed on the alumina coatings. Further, no thin film effects were found. Secondly, the effect of pre-immersion of the CaP coatings in SBF2 was explored. We found that the early formation of a directly bonded CaP layer is obstructed by the absence of CaP nuclei. After approximately 8 days, cell activity induces the nucleation of CaP crystals on both the surface and the ECM, and growth is enhanced. By initially providing these coatings with CaP crystals, growth of the directly bonded CaP layer is immediate. Hence, the formation of the interfacial CaP layer and the matrix-associated CaP accretions can effectively be decoupled.     

Response of astrocytes and microglia/macrophages to brain injury after bone marrow stromal cell transplantation: a quantitative study

The aim of the present study was to investigate the effects of intracerebral and intravenous administration of bone marrow stromal cells (BMSCs) on cellular activity in the injured brain. Female Wistar rats were subjected to cerebral cortex injury followed by the injection of BMSCs or saline, directly to the injured site or to the tail vein. Lectin histochemistry and glial fibrillary acidic protein immunohistochemistry were used to analyze the number of microglia/macrophages and astrocytes in the injured cerebral cortex, respectively. BMSC treatment affected cell response to brain injury. The effects of BMSC action were dependent on the site of their administration. The intravenous injection of BMSCs noticeably increased the number of microglia/macrophages in the injured brain. Intracerebral transplantation of BMSCs significantly enhanced the number of astrocytes, and in a less degree caused changes in the number of microglia/macrophages. The results suggest that BMSCs can affect the restorative processes in the injured brain through stimulation of the cellular response to injury.     

Granulocyte-colony-stimulating factor stimulation of bone marrow mesenchymal stromal cells promotes CD34+ cell migration via a matrix metalloproteinase-2-dependent mechanism

Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models.     

The effect of a polyurethane-based reverse thermal gel on bone marrow stromal cell transplant survival and spinal cord repair

Cell therapy for nervous tissue repair is limited by low transplant survival. We investigated the effects of a polyurethane-based reverse thermal gel, poly(ethylene glycol)–poly(serinol hexamethylene urethane) (ESHU) on bone marrow stromal cell (BMSC) transplant survival and repair using a rat model of spinal cord contusion. Transplantation of BMSCs in ESHU at three days post-contusion resulted in a 3.5-fold increase in BMSC survival at one week post-injury and a 66% increase in spared nervous tissue volume at four weeks post-injury. These improvements were accompanied by enhanced hindlimb motor and sensorimotor recovery. In vitro, we found that ESHU protected BMSCs from hydrogen peroxide-mediated death, resulting in a four-fold increase in BMSC survival with two-fold fewer BMSCs expressing the apoptosis marker, caspase 3 and the DNA oxidation marker, 8-oxo-deoxyguanosine. We argue that ESHU protected BMSCs transplanted is a spinal cord contusion from death thereby augmenting their effects on neuroprotection leading to improved behavioral restoration. The data show that the repair effects of intraneural BMSC transplants depend on the degree of their survival and may have a widespread impact on cell-based regenerative medicine.     

Effects of 5-fluorouracil and gemcitabine on a breast cancer cell line (MCF-7) via the JAK/STAT pathway

Aberrant activation of the JAK/STAT pathway may predispose to malignancy as a consequence of the deregulation of cell proliferation, differentiation or apoptosis such as in cancer of the blood, head and neck, and breast. In our study we aimed to investigate the effects of 5-fluorouracil (5-FU) and gemcitabine on a breast cancer cell line (MCF-7 cells) via the JAK/STAT pathway. Distribution of JAK1, JAK2, JAK3 and STAT2, STAT3, STAT4, STAT5 were evaluated on MCF-7 cells following gemcitabine and 5-FU treatment and in the absence of drug treatment by an indirect immunohistochemical method. It was observed that JAK1, JAK3, STAT5 and particularly STAT2 activation were more effective than the other JAK/STATs in breast cancer progression. Following treatment with 5-FU, JAK1 and STAT5 immunoreactivities were decreased in MCF-7 cells in comparison with both gemcitabine-treated and non-treated groups. These results suggest that the JAK/STAT pathway plays an important role in breast cancer pathogenesis and may be more affected after 5-FU treatment rather than gemcitabine. Drugs which block STAT5 may provide a novel therapeutic approach for the treatment of breast cancer.