Heparinized collagen scaffolds with and without growth factors for the repair of diaphragmatic hernia

A regenerative medicine approach to restore the morphology and function of the diaphragm in congenital diaphragmatic hernia is especially challenging because of the position and flat nature of this organ, allowing cell ingrowth primarily from the perimeter. Use of porous collagen scaffolds for the closure of surgically created diaphragmatic defects in rats has been shown feasible, but better ingrowth of cells, specifically blood vessels and muscle cells, is warranted. To stimulate this process, heparin, a glycosaminoglycan involved in growth factor binding, was covalently bound to porous collagenous scaffolds (14%), with or without vascular endothelial growth factor (VEGF; 0.4 µg/mg scaffold), hepatocyte growth factor (HGF; 0.5 µg/mg scaffold) or a combination of VEGF + HGF (0.2 + 0.5 µg/mg scaffold). All components were located primarily at the outside of scaffolds. Scaffolds were implanted in the diaphragm of rats and evaluated after 2 and 12 weeks. No herniations or eventrations were observed, and in several cases, growth factor-substituted scaffolds showed macroscopically visible blood vessels at the lung site. The addition of heparin led to an accelerated ingrowth of blood vessels at 2 weeks. In all scaffold types, giant cells and immune cells were present primarily at the liver side of the scaffold, and immune cells and individual macrophages at the lung side; these cell types decreased in number from week 2 to week 12. The addition of growth factors did not influence cellular response to the scaffolds, indicating that further optimization with respect to dosage and release profile is needed.     

Electrospun Scaffold of Collagen and Polycaprolactone Containing ZnO Quantum Dots for Skin Wound Regeneration

Nanofibers(NFs)have been widely used in tissue engineering such as wound healing.In this work,the antibacterial ZnO quantum dots(ZnO QDs)have been incorporated into the biocompatible poly(ε-caprolactone)/collagen(PCL/Col)fibrous scaffolds for wound healing.The as-fabricated PCL-Col/ZnO fibrous scaffolds exhibited good swelling,antibacterial activity,and biodegradation behaviors,which were beneficial for the applications as a wound dressing.Moreover,the PCL-Col/ZnO fibrous scaffolds showed excellent cytocompatibility for promoting cell proliferation.The resultant PCL-Col/ZnO fibrous scaffolds containing vascular endothelial growth factor(VEGF)also exhibited promoted wound-healing effect through promoting expression of transforming growth factor-β(TGF-β)and the vascular factor(CD31)in tissues in the early stages of wound healing.This new electrospun fibrous scaffolds with wound-healing promotion and antibacterial property should be convenient for treating wound healing.     

Expression of Matrix Metalloproteinase-9, Type IV Collagen and Vascular Endothelial Growth Factor in Adamantinous Craniopharyngioma

To explore the expression of matrix metalloproteinase 9 (MMP-9), type IV collagen (Col IV) and vascular endothelial growth factor (VEGF) in adamantinomatous craniopharyngioma (ACP) and analyze the correlation between the level of these markers and adamantimous craniopharyngiomas recurrence. Expressions of MMP-9, Col IV and VEGF were tested by immunohistochemistry (IHC) in 40 cases of ACP, including 24 cases of primary group and 16 cases of recurred group. The expression level of MMP-9 and VEGF in recurred group were significantly higher than primary group (93.7% vs. 41.7%, P < 0.05, 87.5% vs. 45.8%, P < 0.05, respectively). The expression of Col IV in the recurred group was significant different from the primary group (Z = −2.619, P < 0.05). MMP-9, Col IV and VEGF may be the potential specific bio-marker related to the recurrence of ACP.     

A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col,PLLA/HA,and PLLA/Col for Tissue Engineering Application

A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200–950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.     

Supercritical carbon dioxide generated vascular endothelial growth factor encapsulated poly(DL-lactic acid) scaffolds induce angiogenesis in vitro

The ability to deliver, over time, biologically active vascular endothelial growth factor-165 (VEGF) through tailored designed scaffolds offers tremendous therapeutic opportunities to tissue-engineered therapies. Porous biodegradable poly(DL-lactic) acid (PLA) scaffolds encapsulating VEGF have been generated using supercritical CO2 (scCO2) and the kinetic release and angiogenic activity of these scaffolds examined in vitro and in an ex vivo chick chorioallantoic membrane (CAM) angiogenesis model. After processing through scCO2, VEGF maintained its angiogenic activity as assessed by increased tubule formation of human umbilical vein endothelial cells (HUVEC) cultured on Matrigel (VEGF = 1937 +/- 205 microm; scCO2-VEGF = 2085 +/- 234 microm; control = 1237 +/- 179 microm). VEGF release kinetics from scCO2-VEGF incorporated PLA monolith scaffolds showed a cumulative release of VEGF (2837 +/- 761 rhog/ml) over a 21 day period in culture. In addition, VEGF encapsulated PLA scaffolds increased the blood vessel network in the CAM compared to controls; control, 24.8 +/- 9.6; VEGF/PLA, 44.1 +/- 12.1 (vessels/field). These studies demonstrate that the controlled release of growth factors encapsulated into three-dimensional PLA scaffolds can actively stimulate the rapid development of therapeutic neovascularisation to regenerate or engineer tissues.     

Critical effect of VEGF in the process of endothelial cell apoptosis induced by high glucose

The underlying molecular mechanism whereby hyperglycemia causes endothelial cell apoptosis is not well understood. This study aims to elucidate the role of survival factor VEGF involved in the apoptosis of endothelial cells induced by elevated glucose. The present study confirmed that high concentration of glucose (25 mmol/l) significantly increased the apoptotic cell number in cultured primary human umbilical vein endothelial cells (HUVEC). Up-regulation of Bax/Bcl-2 ratio and activation of caspase-3 induced by high glucose suggested that mitochondria apoptosis pathway was involved. High glucose significantly reduced VEGF expression in HUVEC both at mRNA and protein levels. p42/44 MAPK phosphorylation was transitory attenuated when exposed to high glucose and preceded VEGF reduction, thus suggesting down-regulation of VEGF through inhibition of p42/44 MAPK. Addition of VEGF prevented HUVEC apoptosis from high glucose exposure. Moreover, elevated reactive oxygen species (ROS) generation, calcium overload, Bax/Bcl-2 ratio, caspase-3 activation in HUVEC induced by high glucose were reversed by pre-challenge with VEGF. This may represent a mechanism for the anti-apoptotic effect of VEGF. These results suggest that down-regulation of VEGF plays a critical role in apoptosis of endothelial cells induced by high glucose and restoration of VEGF might have benefits in the early stage of diabetic endothelial dysfunction. Zhonghan Yang, Xuehua Mo, and Qing Gong have contributed equally to this study.     

Combining electrospun scaffolds with electrosprayed hydrogels leads to three-dimensional cellularization of hybrid constructs

A common problem in the design of tissue engineered scaffolds using electrospun scaffolds is the poor cellular infiltration into the structure. To tackle this issue, three approaches to scaffold design using electrospinning were investigated: selective leaching of a water-soluble fiber phase (poly ethylene oxide (PEO) or gelatin), the use of micron-sized fibers as the scaffold, and a combination of micron-sized fibers with codeposition of a hyaluronic acid-derivative hydrogel, Heprasil. These designs were achieved by modifying a conventional electrospinning system with two charged capillaries and a rotating mandrel collector. Three types of scaffolds were fabricated: medical grade poly(epsilon-caprolactone)/collagen (mPCL/Col) cospun with PEO or gelatin, mPCL/Col meshes with micron-sized fibers, and mPCL/Col microfibers cosprayed with Heprasil. All three scaffold types supported attachment and proliferation of human fetal osteoblasts. However, selective leaching only marginally improved cellular infiltration when compared to meshes obtained by conventional electrospinning. Better cell penetration was seen in mPCL/Col microfibers, and this effect was more pronounced when Heprasil regions were present in the structure. Thus, such techniques could be further exploited for the design of cell permeable fibrous meshes for tissue engineering applications.     

Induction of Angiogenesis by Matrigel Coating of VEGF-Loaded PEG/PCL-Based Hydrogel Scaffolds for hBMSC Transplantation

hBMSCs are multipotent cells that are useful for tissue regeneration to treat degenerative diseases and others for their differentiation ability into chondrocytes, osteoblasts, adipocytes, hepatocytes and neuronal cells. In this study, biodegradable elastic hydrogels consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(ε-caprolactone) (PCL) scaffolds were evaluated for tissue engineering because of its biocompatibility and the ability to control the release of bioactive peptides. The primary cultured cells from human bone marrow are confirmed as hBMSC by immunohistochemical analysis. Mesenchymal stem cell markers (collagen type I, fibronectin, CD54, integrin1β, and Hu protein) were shown to be positive, while hematopoietic stem cell markers (CD14 and CD45) were shown to be negative. Three different hydrogel scaffolds with different block compositions (PEG:PCL=6:14 and 14:6 by weight) were fabricated using the salt leaching method. The hBMSCs were expanded, seeded on the scaffolds, and cultured up to 8 days under static conditions in Iscove’s Modified Dulbecco’s Media (IMDM). The growth of MSCs cultured on the hydrogel with PEG/PCL= 6/14 was faster than that of the others. In addition, the morphology of MSCs seemed to be normal and no cytotoxicity was found. The coating of the vascular endothelial growth factor (VEGF) containing scaffold with Matrigel slowed down the release of VEGF in vitro and promoted the angiogenesis when transplanted into BALB/c nude mice. These results suggest that hBMSCs can be supported by a biode gradable hydrogel scaffold for effective cell growth, and enhance the angiogenesis by Matrigel coating.     

Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu²⁺-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu²⁺-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu²⁺-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu²⁺-doped BG scaffolds release Cu²⁺, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches.     

Ⅰ型胶原/聚己内酯/凹凸棒石复合支架材料体外诱导成骨的研究

目的:研究Ⅰ型胶原(ColⅠ)/聚己内酯(PCL)/凹凸棒石(ATP)复合支架材料的生物相容性及体外骨诱导性。方法:采用溶液浇铸-粒子滤沥法制备三种不同ATP含量(0% wt、10% wt、30% wt)的ColⅠ/PCL/ATP复合支架材料;将D1细胞与三种支架材料共培养,扫描电镜、鬼笔环肽和H&E染色、CCK-8法评价支架材料的生物相容性;D1细胞复合三种支架材料培养7天、14天、21天后RT-qPCR检测其成骨相关基因(Runx-2、Osterix、ALP、Col I、OPN、OC)的相对表达量,分别评价比较三种支架材料的成骨诱导效应。结果:SEM、鬼笔环肽和H&E染色显示D1细胞在三种支架材料表面均呈现良好的黏附;CCK-8结果显示,细胞在ATP含量30% wt的支架材料上增殖率显著高于其他两组,RT-qPCR检测结果显示,与0% wt、10% wt ATP相比,30% wt ATP组的Runx-2相对表达量在7天时显著升高, 14天、21天降低;ALP相对表达量在14天时显著升高,21天时显著降低;Osterix、Col I、OPN、OC的相对表达量随时间和ATP剂量的增加显著上调(P<0.05)。结论:ColⅠ/PCL/ATP复合支架材料具有良好的生物相容性及骨诱导性,有望成为一种理想的骨组织工程支架材料。     

In Vitro Study of a Novel Nanogold-Collagen Composite to Enhance the Mesenchymal Stem Cell Behavior for Vascular Regeneration

Novel nanocomposites based on type I collagen (Col) containing a small amount (17.4, 43.5, and 174 ppm) of gold nanoparticles (AuNPs, approximately 5 nm) were prepared in this study. The pure Col and Col-AuNP composites (Col-Au) were characterized by the UV-Vis spectroscopy (UV-Vis), surface-enhanced raman spectroscopy (SERS) and atomic force microscopy (AFM). The interaction between Col and AuNPs was confirmed by infrared (IR) spectra. The effect of AuNPs on the biocompatibility of Col, evaluated by the proliferation and reactive oxygen species (ROS) production of mesenchymal stem cells (MSCs) as well as the activation of monocytes and platelets, was investigated. Results showed that Col-Au had better biocompatibility than Col. Upon stimulation by vascular endothelial growth factor (VEGF) and stromal derived factor-1α (SDF-1α), MSCs expressed the highest levels of αvβ3 integrin/CXCR4, focal adhesion kinase (FAK), matrix metalloproteinase-2 (MMP-2), and Akt/endothelial nitric oxide synthase (eNOS) proteins when grown on the Col-Au (43.5 ppm) nanocomposite. Taken together, Col-Au nanocomposites may promote the proliferation and migration of MSCs and stimulate the endothelial cell differentiation. These results suggest that Col-Au may be used to construct tissue engineering scaffolds for vascular regeneration.     

Culture of endothelial cells by transfection with plasmid harboring vascular endothelial growth factor

Vascular endothelial cells (ECs) are usually difficult to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement (ECGS). The expression of VEGF by HUVEC tansfected with VEGF gene was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS., However, when the culture medium was supplied with 2.5 ng/mL of basic fibroblast growth factor (bFGF), a synergistic effect of VEGF and bFGF was observed. In this case, the final cell density was recovered up to about 78% of maxium value.     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

In situ gelable interpenetrating double network hydrogel formulated from binary components: thiolated chitosan and oxidized dextran

In situ gelable interpenetrating double-network hydrogels composed of thiolated chitosan (Chitosan-NAC) and oxidized dextran (Odex), completely devoid of potentially cytotoxic small molecule cross-linkers and that do not require complex maneuvers or catalysis, have been formulated. The interpenetrating network structure is created by Schiff base formations and disulfide bond inter-cross-linkings through exploiting the disparity of their reaction times. Compared with the autogelable thiolated chitosan hydrogels that typically require a relatively long time span for gelation to occur, the Odex/Chitosan-NAC composition solidifies rapidly and forms a well-developed 3D network in a short time span. Compared with typical hydrogels derived from natural materials, the Odex/Chitosan-NAC hydrogels are mechanically strong and resist degradation. The cytotoxicity potential of the hydrogels was determined by an in vitro viability assay using fibroblast as a model cell, and the results reveal that the hydrogels are noncytotoxic. In parallel, in vivo results from subdermal implantation in mice models demonstrate that this hydrogel is not only highly resistant to degradation but also induces very mild tissue response.     

5-Substituted pyrido[2,3-d]pyrimidine,an inhibitor against three receptor tyrosine kinases

NP506, the 3-{2,4-dimethyl-5-[2-oxo-5-(N′-phenylhydrazinocarbonyl)-1,2-dihydro-indol-3-ylidenemethyl]-1H-pyrrol-3-yl}-propionic acid, was designed as FGF receptor 1 inhibitor by computational study and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation than SU6668 with minimum effective dose of 10 μM. NP506 inhibited the tyrosine phosphorylation in FGF, VEGF, and PDGF receptors and the activation of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal-kinase (JNK) and AKT after the rhFGF-2 stimulation. The introduction of the phenyl hydrazide motif to the position 5 of the pyrido[2,3-d]pyrimidine scaffold led to the inhibitory effect in two signaling pathways: inhibition of AKT activation in the phosphatidyl inositol 3′-kinase (PI13K)/AKT signaling pathway and the inhibition of ERK and JNK activation in MAPK pathway.     

Covalently grafted VEGF(165) in hydrogel models upregulates the cellular pathways associated with angiogenesis

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF(165)). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF(165) incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF(165) and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF(165) into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.     

Bioactive/Natural Polymeric Scaffolds Loaded with Ciprofloxacin for Treatment of Osteomyelitis

Local delivery of antibiotic into injured bone is a demand. In this work, different scaffolds of chitosan (C) with or without bioactive glass (G) were prepared using the freeze-drying technique in 2:1, 1:1, and 1:2 weight ratios. Chitosan scaffolds and selected formulas of chitosan to bioglass were loaded with ciprofloxacin in 5%, 10%, and 20% w/w. Scaffold morphology showed an interconnected porous structure, where the glass particles were homogeneously dispersed in the chitosan matrix. The kinetic study confirmed that the scaffold containing 1:2 weight ratio of chitosan to glass (CG12) showed optimal bioactivity with good compromise between Ca and P uptake capacities and Si release rate. Chitosan/bioactive glass scaffolds showed larger t ₅₀ values indicating less burst drug release followed by a sustained drug release profile compared to that of chitosan scaffolds. The cell growth, migration, adhesion, and invasion were enhanced onto CG12 scaffold surfaces. Samples of CG12 scaffolds with or without 5% drug induced vascular endothelial growth factor (VEGF), while those containing 10% drug diminished VEGF level. Only CG12 induced the cell differentiation (alkaline phosphatase activity). In conclusion, CG12 containing 5% drug can be considered a biocompatible carrier which would help in the localized osteomyelitis treatment.     

Effect of selenium-enriched malt on VEGF and several relevant angiogenic cytokines in diethylnitrosamine-induced hepatocarcinoma rats

One hundred and ninety-three Sprague–Dawley (SD) rats (average body weight 100–120 g) were randomly divided into five groups (I–V). Groups I and II rats served as the negative and positive controls respectively and both received 0.1 mg/kg Se from sodium selenite supplemented diets for the 18-week experimental period. Groups III–V rats were fed Se from SEM supplemented diets (0.3, 1 and 3 mg/kg respectively). To induce hepatocarcinoma, groups II–V rats received diethylnitrosamine solution (100 mg/L) at the dosage of 10 mg/kg body weight in drinking water daily for 16 weeks, followed by sterilized water for a further 2 weeks. Group I rats received sterilized water throughout. At weeks 4, 8, 12 and 16 five rats in each group were sacrificed by cervical decapitation. At the termination of the study, at week 18, the surplus rats were sacrificed by cervical decapitation. Feed was withheld from the rats for 12  h before sampling. The following items including TNF-α, IGF-II, NO and T-NOS levels in plasma were tested using kit techniques. At the same time the expression of vascular endothelial growth factor (VEGF) in tumor tissue was analyzed by immunohistochemistry using the envision two-step methods with a kit. The results indicated that SEM could increase the levels of TNF-α in the early stages of hepatocarcinoma formation, however there was a decrease in the later stage of hepatocarcinogenesis. SEM could also significantly decrease the levels of IGF-II and NO, and inhibit the expression of VEGF in tumor tissue. SEM delayed the development of hepatocarcinoma in rats and that could be partially attributed to inhibition of angiogenesis.     

Electrospinning of carboxymethyl chitin/poly(vinyl alcohol) nanofibrous scaffolds for tissue engineering applications

A novel fibrous membrane of carboxymethyl chitin (CMC)/poly(vinyl alcohol) (PVA) blend was successfully prepared by electrospinning technique. The concentration of CMC (7%) with PVA (8%) was optimized, blended in different ratios (0–100%) and electrospun to get nanofibers. Fibers were made water insoluble by chemical followed by thermal cross-linking. In vitro mineralization studies identified the ability of formation of hydroxyapatite deposits on the nanofibrous surfaces. Cytotoxicity of the nanofibrous scaffold was evaluated using human mesenchymal stem cells (hMSCs) by the MTT assays. The cell viability was not altered when these nanofibrous scaffolds were pre-washed with phosphate buffer containing saline (PBS) before seeding the cells. The SEM images also revealed that cells were able to attach and spread in the nanofibrous scaffolds. Thus our results indicate that the nanofibrous CMC/PVA scaffold supports cell adhesion/attachment and proliferation and hence this scaffold will be a promising candidate for tissue engineering applications.     

Influence of biological matrix and artificial electrospun scaffolds on proliferation,differentiation and trophic factor synthesis of rat embryonic stem cells

Two-dimensional vs three-dimensional culture conditions, such as the presence of extracellular matrix components, could deeply influence the cell fate and properties. In this paper we investigated proliferation, differentiation, survival, apoptosis, growth and neurotrophic factor synthesis of rat embryonic stem cells (RESCs) cultured in 2D and 3D conditions generated using Cultrex® Basement Membrane Extract (BME) and in poly-(l-lactic acid) (PLLA) electrospun sub-micrometric fibres. It is demonstrated that, in the absence of other instructive stimuli, growth, differentiation and paracrine activity of RESCs are directly affected by the different microenvironment provided by the scaffold. In particular, RESCs grown on an electrospun PLLA scaffolds coated or not with BME have a higher proliferation rate, higher production of bioactive nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) compared to standard 2D conditions, lasting for at least 2 weeks. Due to the high mechanical flexibility of PLLA electrospun scaffolds, the PLLA/stem cell culture system offers an interesting potential for implantable neural repair devices.