Effects of Poly(L-lysine), Poly(acrylic acid) and Poly(ethylene glycol) on the Adhesion,Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

Microenvironments, composed of many kinds of cytokines and growth factors plus extracellular matrices with diverse electrostatic properties, play key roles in controlling cell functions in vivo. In this study, three kinds of water-soluble polymers, positively charged poly(L-lysine) (PLL), negatively charged poly(acrylic acid) (PAAc) and neutral poly(ethylene glycol) (PEG), were compared based on their effects on the adhesion, spread, proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). The MSCs were seeded and cultured in the presence of polymers of different concentrations applied by methods using coating, mixing or covering. The effects of the water-soluble polymers depended on their electrostatic properties and method of application. The methods were in the order of coating, mixing and covering in terms of high to low influence. A low concentration of PLL promoted MSC adhesion, spread, proliferation and chondrogenic differentiation, while a high concentration of PLL was toxic. The PEG-coated surface facilitated cell aggregation and spheroid formation by inhibiting cell adhesion. A high concentration of mixed PEG (10 μg/ml) promoted cell proliferation in serum-free medium. PAAc showed no obvious effects on MSC adhesion, spread, proliferation, or chondrogenic differentiation.     

人工颈椎间盘不同涂层影响骨髓间充质干细胞的黏附及分化

背景：钛合金人工颈椎间盘具有良好的生物相容性,但钛合金存在生物活性差、与骨结合强度低、在生理环境下易造成金属离子释放等问题。 目的：观察国产人工颈椎间盘钛合金终板不同涂层对大鼠骨髓间充质干细胞黏附、分化的影响。 方法：将第3代Wistar大鼠骨髓间充质干细胞,接种于含羟基磷灰石涂层、钛粉复合羟基磷灰石涂层的钛合金板及裸钛合金板的24孔板内,培养的第24,48小时后分别终止培养,扫描电镜观察细胞生长情况;接种24 h后加成骨诱导剂进行诱导,并于第3,5,7天各收集细胞裂解后离心的上清,检测碱性磷酸酶表达。 结果与结论：骨髓间充质干细胞与材料复合培养48 h后,羟基磷灰石组和钛粉复合羟基磷灰石组表面的细胞呈多角形,并伸出细长伪足伸入到材料的微孔内,与材料表面紧密黏附;而裸钛合金组骨髓间充质干细胞分化较差且黏附率低。随时间延长,各组碱性磷酸酶表达均增加,羟基磷灰石组和钛粉复合羟基磷灰石组各时间点的碱性磷酸酶表达均较裸钛合金组明显增高(P < 0.05)。表明羟基磷灰石、钛粉复合羟基磷灰石涂层可有促进大鼠骨髓间充质干细胞的黏附和分化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Glycoengineering of HCELL, the human bone marrow homing receptor: sweetly programming cell migration

The successful clinical implementation of adoptive cell therapeutics, including bone marrow transplantation and other stem cell-based treatments, depends critically on the ability to deliver cells to sites where they are needed. E-selectin, an endothelial C-type lectin, binds sialofucosylated carbohydrate determinants on its pertinent ligands. This molecule is expressed in a constitutive manner on bone marrow and dermal microvascular endothelium, and inducibly on post-capillary venules at all sites of tissue injury. Engagement of E-selectin with relevant ligand(s) expressed on circulating cells mediates initial “tethering/rolling” endothelial adhesive interactions prerequisite for extravasation of blood-borne cells at any target tissue. Most mammalian cells express high levels of a transmembrane glycoprotein known as CD44. A specialized glycoform of CD44 called “Hematopoietic Cell E-/L-selectin Ligand” (HCELL) is a potent E-selectin ligand expressed on human cells. Under native conditions, HCELL expression is restricted to human hematopoietic stem/progenitor cells. We have developed a technology called “Glycosyltransferase-Programmed Stereosubstitution” (GPS) for custom-modifying CD44 glycans to create HCELL on the surface of living cells. GPS-based glycoengineering of HCELL endows cell migration to endothelial beds expressing E-selectin. Enforced HCELL expression targets human mesenchymal stem cell homing to marrow, licensing transendothelial migration without chemokine signaling via a VLA-4/VCAM-1-dependent “Step 2-bypass pathway.” This review presents an historical framework of the homing receptor concept, and will describe the discovery of HCELL, its function as the bone marrow homing receptor, and how enforced expression of this molecule via chemical engineering of CD44 glycans could enable stem cell-based regenerative medicine and other adoptive cell therapeutics.     

Engineering of cell membranes with a bisphosphonate-containing polymer using ATRP synthesis for bone targeting

The field of polymer-based membrane engineering has expanded since we first demonstrated the reaction of N-hydroxysuccinimide ester-terminated polymers with cells and tissues almost two decades ago. One remaining obstacle, especially for conjugation of polymers to cells, has been that exquisite control over polymer structure and functionality has not been used to influence the behavior of cells. Herein, we describe a multifunctional atom transfer radical polymerization initiator and its use to synthesize water-soluble polymers that are modified with bisphosphonate side chains and then covalently bound to the surface of live cells. The polymers contained between 1.7 and 3.1 bisphosphonates per chain and were shown to bind to hydroxyapatite crystals with kinetics similar to free bisphosphonate binding. We engineered the membranes of both HL-60 cells and mesenchymal stem cells in order to impart polymer-guided bone adhesion properties on the cells. Covalent coupling of the polymer to the non-adherent HL-60 cell line or mesenchymal stem cells was non-toxic by proliferation assays and enhanced the binding of these cells to bone.     

Viability and proliferation of rat MSCs on adhesion protein-modified PET and PU scaffolds

In 2011, the first in-man successful transplantation of a tissue engineered trachea-bronchial graft, using a synthetic POSS-PCU nanocomposite construct seeded with autologous stem cells, was performed. To further improve this technology, we investigated the feasibility of using polymers with a three dimensional structure more closely mimicking the morphology and size scale of native extracellular matrix (ECM) fibers. We therefore investigated the in?vitro biocompatibility of electrospun polyethylene terephthalate (PET) and polyurethane (PU) scaffolds, and determined the effects on cell attachment by conditioning the fibers with adhesion proteins. Rat mesenchymal stromal cells (MSCs) were seeded on either PET or PU fiber-layered culture plates coated with laminin, collagen I, fibronectin, poly-d-lysine or gelatin. Cell density, proliferation, viability, morphology and mRNA expression were evaluated. MSC cultures on PET and PU resulted in similar cell densities and amounts of proliferating cells, with retained MSC phenotype compared to data obtained from tissue culture plate cultures. Coating the scaffolds with adhesion proteins did not increase cell density or cell proliferation. Our data suggest that both PET and PU mats, matching the dimensions of ECM fibers, are biomimetic scaffolds and, because of their high surface area-to-volume provided by the electrospinning procedure, makes them per se suitable for cell attachment and proliferation without any additional coating.     

Solution technique to incorporate polyethylene oxide and other water-soluble polymers into surfaces of polymeric biomaterials.

A simple solution technique was used to incorporate polyethylene oxide (PEO, of 5000, 10,000, 18,500, and 100,000 g/mol) and other water-soluble polymers such as polyvinylpyrrolidone and polyethyl oxazoline into the surfaces of commonly used biomedical polymers such as polyethylene terephthalate, a polyurethane (Pellethane 2363-80AE), and polymethylmethacrylate. The presence of the water-soluble polymers on these surfaces was verified by using contact angle analysis and ESCA. Protein adsorption studies, fibroblast adhesion assays, and whole blood perfusions over these polymers showed that the surface modified with PEO 18,500 was the most effective in reducing all the tested biological interactions. It was concluded that PEO 18,500 had a chain length that was optimal, using this technique for surface incorporation, to reduce protein adsorption and hence prevent protein-mediated biological interactions.     

Chemically coupled hydroxyapatite-polyethylene composites: structure and properties

Silanation of hydroxyapatite and acrylic acid grafting of polyethylene were employed to improve bonding between hydroxyapatite and polyethylene. The structure and properties of chemically coupled hydroxyapatite reinforced high-density polyethylene (HA/HDPE) composites were subsequently investigated using various techniques. It was shown that there existed a silicon-containing interphase between the hydroxyapatite particle and the polyethylene matrix. The silane coupling agent used promoted chemical adhesion between hydroxyapatite particles and the polymer. The use of the silane coupling agent also facilitated the penetration of polymer into cavities in individual ceramic particles, which resulted in enhanced mechanical interlocking at the matrix-reinforcement interface. With a strong bond (both mechanical and chemical) being formed between hydroxyapatite and polyethylene, chemically coupled HA/HDPE composites possessed improved mechanical properties and fracture behaviour.     

Osteoprogenitor response to semi-ordered and random nanotopographies

In bone tissue engineering, it is desirable to use materials to control the differentiation of mesenchymal stem cell populations in order to gain direct bone apposition to implant materials. It has been known for a number of years that microtopography can alter cell adhesion, proliferation and gene expression. More recently, the literature reveals that nanotopography is also of importance. Here, the reaction of primary human osteoprogenitor cell populations to nanotopographies down to 10 nm in size is considered. The topographies were originally produced by colloidal lithography and polymer demixing on silicon and then embossed (through an intermediate nickel shim) into polymethylmethacrylate. The biological testing considered cell morphology (image analysis of cell spreading and scanning electron microscopy), cell cytoskleton and adhesion formation (fluorescent staining of actin, tubulin, vimentin and vinculin) and then subsequent cell growth and differentiation (fluorescent staining of osteocalcin and osteopontin). The results demonstrated that the nanotopographies stimulated the osteoprogenitor cell differentiation towards an osteoblastic phenotype.     

Human mesenchymal stem cell adhesion and proliferation in response to ceramic chemistry and nanoscale topography

Modification of the chemistry and surface topography of nanophase ceramics was used to provide biomaterial formulations designed to direct the adhesion and proliferation of human mesenchymal stem cells (HMSCs). HMSC adhesion was dependent upon both the substrate chemistry and grain size, but not on surface roughness or crystal phase. Specifically, cell adhesion on alumina and hydroxyapatite was significantly reduced on the 50 and 24 nm surfaces, as compared with the 1500 and 200 nm surfaces, but adhesion on titania substrates was independent of grain size. HMSC proliferation was minimal on the 50 and 24 nm substrates of any chemistry tested, and thus significantly lower than the densities observed on either the 1500 or 200 nm surfaces after 3 or more consecutive days of culture. Furthermore, HMSC proliferation was enhanced on the 200 nm substrates, compared with results obtained on the 1500 nm substrates after 7 or more days of culture. HMSC proliferation was independent of both substrate surface roughness and crystal phase. Rat osteoblast and fibroblast adhesion and proliferation exhibited similar trends to that of HMSCs on all substrates tested. These results demonstrated the potential of nanophase ceramic surfaces to modulate functions of HMSCs, which are pertinent to biomedical applications such as implant materials and devices.     

DNA去甲基化对大鼠骨髓间充质干细胞端粒酶反转录酶的调节

背景：DNA去甲基化是一种重要的表观遗传修饰,对肿瘤细胞的端粒酶具有重要调节作用,而对骨髓间充质干细胞端粒酶活性有何影响尚不清楚。 目的：观察DNA去甲基化对骨髓间充质干细胞增殖及端粒酶反转录酶蛋白表达的影响。 方法：全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞;按照下列分组加入5-杂氮胞苷,使各组5-杂氮胞苷终浓度分别为0,3,6,12,24 μmol/L。加入5-杂氮胞苷后第1,2,3,5,7天进行指标检测。 结果与结论：与对照组相比,5-杂氮胞苷干预24 h,各浓度组均显著促进细胞增殖活性(P < 0.05);干预48 h,6,12,24 μmol/L组显著促进细胞增殖活性(P < 0.05);干预72 h,12,24 μmol/L组显著抑制细胞增殖活性(P < 0.05);干预120,168 h,对照组与各浓度组间差异均无显著性意义(P > 0.05)。5-杂氮胞苷干预48 h,6,12,24 μmol/L组端粒酶反转录酶蛋白IA值较对照组显著增加(P < 0.05)。提示在一定浓度范围及一定作用时间内,5-杂氮胞苷可以促进骨髓间充质干细胞增殖与端粒酶反转录酶蛋白的表达。     

Bone tissue engineering with porous hydroxyapatite ceramics

The main principle of bone tissue engineering strategy is to use an osteoconductive porous scaffold in combination with osteoinductive molecules or osteogenic cells. The requirements for a scaffold in bone regeneration are: (1) biocompatibility, (2) osteoconductivity, (3) interconnected porous structure, (4) appropriate mechanical strength, and (5) biodegradability. We recently developed a fully interconnected porous hydroxyapatite (IP-CHA) by adopting the “form-gel” technique. IP-CHA has a three-dimensional structure with spherical pores of uniform size that are interconnected by window-like holes; the material also demonstrated adequate compression strength. In animal experiments, IP-CHA showed superior osteoconduction, with the majority of pores filled with newly formed bone. The interconnected porous structure facilitates bone tissue engineering by allowing the introduction of bone cells, osteotropic agents, or vasculature into the pores. In this article, we review the accumulated data on bone tissue engineering using the novel scaffold, focusing especially on new techniques in combination with bone morphogenetic protein (BMP) or mesenchymal stem cells.     

In vitro culture of mesenchymal cells onto nanocrystalline hydroxyapatite-coated Ti13Nb13Zr alloy

In this study we coated a new biocompatible, nanostructured titanium alloy, Ti13Nb13Zr, with a thin layer of hydroxyapatite nanocrystals and we investigated the response of human bone-marrow-derived mesenchymal cells. The coating was realized using a slightly supersaturated CaP solution, which provokes a fast deposition of nanocrystalline hydroxyapatite. A thin layer of deposition is appreciable on the etched Ti13Nb13Zr substrates after just 1.5 h soaking in the CaP solution, and it reaches a thickness of 1-2 mum after 3 h soaking. The coating seems thinner than that deposited on Ti6Al4V, which was examined for comparison, likely because of the different roughness profiles of the two etched alloys, and it is constituted of elongated HA nanocrystals, with a mean length of about 100 nm. Mesenchymal stem cells were seeded onto coated and uncoated Ti alloys and cultured for up to 35 days. Cell morphology, proliferation and differentiation were evaluated. The cells display good adhesion and proliferation on the uncoated substrates, whereas the presence of hydroxyapatite coating slightly reduces cell proliferation and induces differentiation of MSCs towards a phenotypic osteoblastic lineage, in agreement with the increase of the expression of osteopontin, osteonectin and collagen type I, evaluated by means of rt-PCR. Type I collagen expression is higher in Ti13Nb13Zr MSC culture compared to Ti6Al4V, standing for a more efficient extracellular matrix deposition.     

A new hypothesis on the role of c-erbB2 oncogene in the progress of breast cancer

Proliferation and the state of adhesion molecules (E-cadherin, galectin-3) and estrogen and progesterone receptors were immunohistochemically analyzed in breast cancer biopsy specimens under conditions of c-erbB2 overexpression with and without gene amplification. It was hypothesized that c-erbB2 overexpression without gene amplification led to suppression of proliferation and “conservation” of tumor cell. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 106–109, July, 2006     

聚醚醚酮/纳米羟基磷灰石生物材料对MG-63细胞增殖作用

目的制备PEEK/HA复合材料,研究在不同时间和不同浓度条件下对MG-63细胞增殖的作用,评价其细胞毒性。测定该材料对MG-63细胞黏附率。方法合成聚醚醚酮,与一定含量的纳米羟基磷灰石共混,制备PEEK/HA复合材料。通过倒置荧光显微镜观察材料对细胞形态影响,采用MTT法研究在不同时间和不同浓度条件下对细胞增殖的作用,评价其细胞毒性。结果 PEEK/10%HA、PEEK/20%HA对细胞形态影响显示,都有大量活细胞,PEEK/20%HA中活细胞多于PEEK/10%HA,说明PEEK/20%HA对细胞增殖作用比PEEK/10%HA强,均有黏附现象。各组材料细胞毒性级别均在0或Ⅰ级,说明各组材料对细胞无毒性。在8mg/ml浓度下材料对细胞增殖有明显促进作用,随着浓度的增加,对细胞生长有明显的抑制作用,随着浓度进一步增加,材料对细胞增殖作用逐渐减小,曲线呈下降趋势,当浓度为64mg/ml时,PEEK复合材料对细胞增殖作用最小。PEEK/HA复合材料随着HA含量的增加,细胞黏附率增加,HA可以改善材料黏附性能。结论 PEEK/HA材料对MG-63细胞无毒性。     

An Evaluation Regarding the Current Situation of Stem Cell Studies in Turkey

Turkey is in a parallel state to that of other countries for developments in stem cell research and practices. Nevertheless, Turkish law has no regulations for stem cell practices. To define a legal framework for stem cell research, rules of general content should be used as the starting point. In 2005 and 2006, a general regulation and guidelines on stem cell research were published by Turkish Ministry of Health. Thus, the ministry, based on this first general regulation, stopped “Embryonic Stem Cell Research”, while allowing “Adult Stem Cell Research” by a second general regulation. The method of such research was regulated with the addendum of ‘Guidelines for Clinical Research on Non-embryonic stem Cell’. With the latest regulation, clinical stem cell research in Turkey has been based on ‘Regulations for Clinical Research’, which was legislated in 2009. However, the aforementioned regulations by the ministry are still legally binding. In addition, other regulations such as Medical Deontology Regulations of 1960 and Patients’ Rights Regulations of 1998 are to be consulted for stem cell research in Turkey. While it is especially important that research that is still at experimental level not provide an opportunity for trade of hope in patients and their relatives, ethics discussions are enlightening in developing regulations and critical evaluation of current practices.     

The use of a shape-memory poly(ε-caprolactone)dimethacrylate network as a tissue engineering scaffold

Shape-memory polymers produced from many natural or synthetic raw polymers are able to undergo a shape transformation after exposure to a specific external stimulus. This feature enables their use in minimal-invasive surgery with a small, compact starting material switching over to a more voluminous structure in the body. The use of biomaterials in modern medicine calls for compatibility tests with cell types, encountering the biomaterial during a short-term or long-term in vivo application. We analysed the cell behaviour of L929 mouse fibroblasts, human mesenchymal stem cells, human mesothelial cells and rat mesothelial cells on a biodegradable shape-memory polymer network to assess its suitability for medical applications. Further, we investigated the differentiation capacity of mesenchymal stem cells into osteoblasts and adipocytes on the polymer and we analysed the influence of the shape-memory effect on adherent cells. The polymer was cytocompatible for all tested cell types, supporting cell viability and proliferation. The differentiation capacity of mesenchymal stem cells was supported by the polymer and shape-memory effect activation did not affect the majority of adherent cells.     

Human mesenchymal stem cell culture on heparin-based hydrogels and the modulation of interactions by gel elasticity and heparin amount

Human adipose-derived stem cells (hADSCs) are a promising cell source for tissue engineering and regenerative medicine with no ethnical issue and easy access of large quantities. Conventional surfaces for hADSC culture, such as tissue culture plates (TCPs), do not provide optimal environmental cues, leading to limited expansion, loss of pluripotency and undesirable differentiation of stem cells. The present study demonstrated that heparin-based hydrogels without additional modification provided an excellent surface for adhesion and proliferation of hADSCs, which were further tunable by both the amount of heparin (in a positive way) and the elasticity of hydrogel (in a negative way). The optimized heparin-based hydrogel could selectively modulate the adhesion of hADSCs and human bone marrow stem cells (but not all kinds of cells), and resulted in a significant increase in cell proliferation compared to TCP. Furthermore, in terms of the maintenance of pluripotency and specific differentiation, heparin-based hydrogel was much superior to TCP. The selective binding and proliferation of human mesenchymal stem cells on heparin-based hydrogel over other hydrogels were largely mediated by integrin β1 and selectin, and these superior characteristics were observed regardless of the presence of serum proteins in the culture medium. Consequently, heparin-based hydrogel could be a powerful platform for cultivation of mesenchymal stem cells in various applications.     

Improved bioactivity of PAN-based carbon nanofibers decorated with bioglass nanoparticles

Composite nanofibers composed of polyacrylonitrile (PAN)-based carbon nanofibers and bioactive glass (BG) nanoparticles have been prepared by electrospinning and in situ sintering. Morphology observation showed that the BG nanoparticles of size 20–50?nm were uniformly distributed on the surface of composite nanofibers with 350?nm average diameter after carbonization. Biological mineralization indicated the formation of apatite-like layer on the surface of composite nanofibers, in which the composition of carbonate hydroxyapatite was proved by FTIR and XRD analysis. Cell growth dynamics according to cellular morphology, CCK-8 assay, and alkaline phosphatase activity assay exhibited better cell adhesion, proliferation, and osteogenic induction of bone marrow-derived mesenchymal stem cells cultured on the composite nanofibers, which suggested the higher bioactivity of composite nanofibers compared to pure PAN-based carbon nanofibers.     

Synthesis, characterization, and osteocompatibility evaluation of novel alanine-based polyphosphazenes

This study deals with the synthesis and in vitro osteocompatibility evaluation of two novel alanine-containing biodegradable ester polyphosphazenes as candidates to form self-setting composites with hydroxyapatite (HAp) precursors. The two novel biodegradable polyphosphazenes synthesized were poly[(ethyl alanato)1.0(ethyl oxybenzoate)1.0 phosphazene] (PN-EA/EOB) and poly[(ethyl alanato)1.0(propyl oxybenzoate)1.0 phosphazene] (PN-EA/POB). The polymers were characterized by multinuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and gel permeation chromatography (GPC). Biodegradability and percentage water absorption of the polymers were evaluated by following the mass change in phosphate buffer (pH 7.4) at 37 degrees C. PN-EA/POB underwent faster degradation and showed higher water absorption compared to PN-EA/EOB. Both polymers became insoluble in common organic solvents following hydrolysis presumably due to crosslinking reactions accompanying the degradation process. Osteoblast cell adhesion and proliferation on PN-EA/EOB and PN-EA/POB was followed by scanning electron microscopy (SEM) and by using a biochemical assay. Both PN-EA/EOB and PN-EA/POB supported the adhesion and proliferation of primary rat osteoblast cells in vitro. Furthermore, the enzymatic activity of the osteoblast cells cultured on the polymers was confirmed by the alkaline phosphatase activity. Thus, these biodegradable amino-acid-based polyphosphazenes are promising new materials for forming self-setting bone cements.     

多发性骨髓瘤患者骨髓间充质干细胞冻存前后的生物学特性

背景：多发性骨髓瘤患者的骨髓间充质干细胞具有多向分化、免疫调节和支持造血作用,但是这些功能是否受冻存的影响目前尚不清楚。 目的：探讨冻存对多发性骨髓瘤患者骨髓间充质干细胞生物学特性的影响。 方法：采用细胞贴壁法获取多发性骨髓瘤患者骨髓间充质干细胞,将传代后的细胞用IMDM细胞冻存液(含10%的二甲基亚砜和体积分数40%的胎牛血清)保存在-196 ℃液氮中。检测短期(1个月)和长期(12个月)冻存复苏后间充质干细胞的活性和增殖能力;将冻存后多发性骨髓瘤患者骨髓间充质干细胞作为滋养层,应用甲基纤维素半固体培养,检测其支持造血的能力;混合淋巴细胞反应检测冻存后多发性骨髓瘤患者骨髓间充质干细胞调控免疫能力。 结果与结论：经过短、长期冻存后多发性骨髓瘤患者骨髓间充质干细胞的细胞活性分别为(92.9±7.5)%和(86.7±9.2)%;短、长期冻存后细胞的增殖能力与冻存前间充质干细胞相似;冻存后多发性骨髓瘤患者骨髓间充质干细胞仍具有支持造血祖细胞生长的作用和抑制T淋巴细胞增殖的能力,与冻存前相比,没有明显差别。说明冻存可以降低多发性骨髓瘤患者骨髓间充质干细胞的细胞活性,但是并不影响间充质干细胞的增殖、支持造血和免疫调节的能力。