Promotion of cell affinity of porous PLLA scaffolds by immobilization of RGD peptides via plasma treatment

In the present work, RGDS (Arg-Gly-Asp-Ser) was immobilized on PLLA scaffolds with plasma treatment. The amount of immobilization, determined by HPLC, was confirmed to be in the effective order. Results from the culture of rat osteosarcoma (ROS), osteoblastic-like cells, demonstrate that the immobilization of RGDS could effectively enhance the attachment of ROS cells on PLLA and increase the cell density in PLLA scaffolds. In addition, experiments of in vitro mineralization indicate that there were more cells and mineralization focci in the RGDS-immobilized scaffolds, suggesting a tendency to form bone-like tissues, compared with the unmodified PLLA scaffold. On the other hand, the PLLA scaffolds immobilized with RGES (Arg-Gly-Glu-Ser) were much less effective in promotion of ROS attachment, suggesting that the enhancement on cell attachment was mainly due to the recognition of RGDS by the adhesion receptors on the cell membrane. The results presented in this work demonstrate that RGDS could be successfully immobilized on PLLA scaffolds with plasma treatment and such modification can make PLLA scaffolds more suitable for culture of osteoblast-like cells and for generation of bone-like tissues.     

明胶/聚乳酸复合纤维膜的制备、表征及作为角膜细胞载体的评价

利用静电纺丝技术制备了明胶与聚乳酸的复合纤维膜, 研究了组分配比对复合膜的表面性能、孔隙结构和力学性能的影响, 并以复合膜为组织工程支架进行兔角膜上皮细胞的体外培养. 采用扫描电子显微镜、免疫荧光染色和噻唑蓝四氮唑溴化物(MTT)比色法综合评价了细胞在支架表面的黏附与增殖能力. 结果表明, 纺丝溶液的组分对纤维的直径分布和表面亲水性有显著影响, 不同组分配比的复合纤维膜均具有高孔隙率的通孔结构; 以明胶为基材可维持复合膜的细胞黏附性; 与聚乳酸复合可以明显提高复合膜的力学性能.     

Response of Human Mesenchymal Stem Cells to Patterned and Randomly Oriented Poly(Vinyl Alcohol) Nano-fibrous Scaffolds Surface-Modified with Arg-Gly-Asp (RGD) Ligand

The aim of this study was to obtain a better insight of how nano-fibrous scaffolds can affect human mesenchymal stem cells responses. Therefore, in this study, using electrospinning technique, poly(vinyl alcohol) (PVA) nano-fibers with two different patterns were prepared. In the first structure, PVA nano-fibers were oriented randomly and in the second structure, nano-fibers were electrospun in such a way that a special pattern was obtained. In order to enhance their stability, scaffolds were cross-linked using glutaraldehyde vapor. RGD immobilization was used to improve cell adhesion properties of the scaffolds. SEM micrographs demonstrated that the cell adhesion was effectively enhanced after RGD immobilization and higher cell densities were observed on RGD-modified scaffolds. Randomly oriented nano-fibers showed better cell adhesion compared to patterned structure. Patterned structure also revealed slightly lower cell viability compared to random nano-fibers. Finally, it was assumed that randomly oriented nano-fibers provide a more favorable surface for cells.     

Surface Modification of Multiple Bioactive Peptides to Improve Endothelialization of Vascular Grafts

Endothelialization is an effective approach to prevent thrombus formation and enhance vascular graft survival. Surface modification of biomolecules has been proved to be effective in regulating endothelial cell behaviors. In this study, several peptides including YIGSR, RGD, and REDV sequences are covalently immobilized on the surface of electrospun silk fibroin scaffolds and the effects of combined application of these peptides on cell behaviors are studied. The results show that, compared with the scaffolds modified with single peptides, the scaffolds modified with dual peptides (YIGSR+RGD) could significantly enhance the proliferation of human umbilical vein endothelial cells (HUVECs). However, the combination of REDV+RGD or YIGSR+REDV does not promote the adhesion or proliferation of HUVECs. Notably, YIGSR‐modified scaffolds improved HUVEC migration significantly in comparison to REDV‐ or RGD‐modified groups. Moreover, its combination with either of these two peptides also presents excellent effect on cell migration. Thus, all the data suggest that the combined application of peptides might be a promising method to enhance the endothelialization of small‐diameter vascular grafts.     

Cellulose nanocrystals as biobased nucleation agents in poly-l-lactide scaffold: Crystallization behavior and mechanical properties

The mechanical strength of polymer scaffold is closely related to its crystallinity. In this work, cellulose nanocrystals (CNC) were incorporated into poly-l-lactide (PLLA) scaffold which was fabricated by selective laser sintering, aiming to improve the mechanical properties. CNC possesses numerous hydroxyl groups which might form hydrogen bond with PLLA molecular chains. The hydrogen bond induces the ordered arrangement of PLLA chain by using CNC as heterogeneous nucleating agent, thereby increasing crystallization rate and crystallinity. Results showed that PLLA scaffolds with 3 wt% CNC resulted in 191%, 351%, 34%, 83.5%, 56% increase in compressive strength, compressive modulus, tensile strength, tensile modulus and Vickers hardness, respectively. Encouragingly, with the incorporation of hydrophilic CNC, the PLLA/CNC scaffolds showed not only better hydrophilicity, but also faster degradation than PLLA. In vitro cell culture studies proved that the PLLA/CNC scaffolds were biocompatible and capable of supporting cell adhesion, proliferation and differentiation. The above results indicated that the PLLA/CNC scaffolds may therefore be a potential replacement in bone repair.     

The accumulation of phytoalexin in cucumber plant after stress

During the course of pathogens penetrating the plant cell, besides of chemical secretion, the pathogens may cause mechanical signal by the physical pressure on the plant cell. In the current study, we use the pressure as the stress signal to study the induction in plant resistance and the effect of accumulation of phytoalexin. We found that stress can induce the resistance in cucumber seeding significantly. Peptides contained RGD motif can specific block the adhesion between plant cell wall and plasma membrane. When breaking the plant cell wall and plasma membrane by using RGD peptides, the stress induction effect is almost absolutely eliminated. The results of assay with TLC and HPLC showed that stress stimulation could increase the accumulation of cucumber seeding phytoalexin. So, we can conclude that the accumulation of phytoalexin is one possible reason of improve the stress induced resistance. When block the adhesion between plant cell wall and plasma membrane by RGD, there are only part of accumulation of phytoalexin. The results suggest that stress induced resistance and accumulation of phytoalexin of plant is required for the adhesion of plant cell wall–plasma membrane.     

Improved efficacy of bio‐mineralization of human mesenchymal stem cells on modified PLLA nanofibers coated with bioactive materials via enhanced expression of integrin α2β1

Current therapeutic interventions in bone defects are mainly focused on finding the best bioactive materials for inducing bone regeneration via activating the related intracellular signaling pathways. Integrins are trans‐membrane receptors that facilitate cell‐extracellular matrix (ECM) interactions and activate signal transduction. To develop a suitable platform for supporting human bone marrow mesenchymal stem cells (hBM‐MSCs) differentiation into bone tissue, electrospun poly L‐lactide (PLLA) nanofiber scaffolds were coated with nano‐hydroxyapatite (PLLA/nHa group), gelatin nanoparticles (PLLA/Gel group), and nHa/Gel nanoparticles (PLLA/nHa/Gel group) and their impacts on cell proliferation, expression of osteoblastic biomarkers, and bone differentiation were examined and compared. MTT data showed that proliferation of hBM‐MSCs on PLLA/nHa/Gel scaffolds was significantly higher than other groups (P < .05). Alkaline phosphatase activity was also more increased in hBM‐MSCs cultured under osteogenic media on PLLA/nHa/Gel scaffolds compared to others. Gene expression evaluation confirmed up‐regulation of integrin α2β1 as well as the osteogenic genes BGLAP, COL1A1, and RUNX2. Following use of integrin α2β1 blocker antibody, the protein level of integrin α2β1 in cells seeded on PLLA/nHa/Gel scaffolds was decreased compared to control, which confirmed that most of the integrin receptors were bound to gelatin molecules on scaffolds and could activate the integrin α2β1/ERK axis. Collectively, PLLA/nHa/Gel scaffold is a suitable platform for hBM‐MSCs adhesion, proliferation, and osteogenic differentiation in less time via activating integrin α2β1/ERK axis, and thus it might be applicable in bone tissue engineering.     

Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns

Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.     

Engineering of Covalently Immobilized Gradients of RGD Peptides on Hydrogel Scaffolds: Effect on Cell Behaviour

Summary: The aim of this study has been to design a system for the preparation of Polyethylene-glycol (PEG) based hydrogels with a controlled spatial distribution of covalently immobilised RGD adhesion signals in order to control and guide cell response for tissue engineering application. Gradients of immobilised RGD peptides were characterized by confocal microscopy analysis. Moreover, the effect of RGD spatial distribution on cell behaviour was evaluated by using mouse embryo fibroblasts NIH3T3. In particular, we observed cell adhesion and migration of fibroblasts seeded on RGD gradient compared to cells on control hydrogels having an uniform distribution of RGD. Our data suggest that a linear gradient of covalently immobilised adhesion signals affects cell behaviour. In particular, cells feel RGD gradient and oriented themselves and move along gradient direction.     

Fabrication of porous 3-D structure from poly(l-lactide)-based nano-composite foams. Effect of foam structure on enzymatic degradation

To understand the effect of the foam structure on the enzymatic degradation and porous structure development, we have examined the enzymatic degradation of a poly(l-lactide) (PLLA)-based nano-composite foam having different cell density (microcellular and nanocellular), using proteinase-K as a degrading agent at 37 °C. The surface and cross-sectional morphologies of the foam recovered after enzymatic hydrolysis for different intervals were investigated by using scanning electron microscopic and mercury porosimetric analyses. The fabrication of porous three-dimensional structure for tissue engineering scaffolds and the degradation performance in nano-composite foams were discussed.     

Thick Soft Tissue Reconstruction on Highly Perfusive Biodegradable Scaffolds

The lack of a vascular network and poor perfusion is what mostly prevents three‐dimensional (3D) scaffolds from being used in organ repair when reconstruction of thick tissues is needed. Highly‐porous scaffolds made of poly(L ‐lactic acid) (PLLA) are prepared by directional thermally induced phase separation (dTIPS) starting from 1,4‐dioxane/PLLA solutions. The influence of polymer concentration and temperature gradient, in terms of imposed intensity and direction, on pore size and distribution is studied by comparison with scaffolds prepared by isotropic TIPS. The processing parameters are optimized to achieve an overall porosity for the 3D scaffolds of about 93% with a degree of interconnectivity of 91%. The resulting pore network is characterized by the ordered repetition of closely packed dendrite‐like cavities, each one showing stacks of 20 µm large side lamellar branches departing from 70 µm diameter vertical backbones, strongly resembling the vascular patterns. The in vitro biological responses after 1 and 2 weeks are evaluated from mesenchymal (bone marrow stromal) cells (MSC) static culturing. A novel vacuum‐based deep‐seeding method is set up to improve uniform cell penetration down to scaffold thicknesses of over 1 mm. Biological screenings show significant 3D scaffold colonization even after 18 h, while cellular retention is observed up to 14 d in vitro (DIV). Pore architecture‐driven cellular growth is accompanied by cell tendency to preserve their multi‐potency towards differentiation. Confluent tissues as thick as 1 mm were reconstructed taking advantage of the large perfusion enhanced by the highly porous microstructure of the engineered scaffolds, which could successfully serve for applications aimed at vascular nets and angiogenesis.

     

Cellular Adhesion Tripeptide RGD Inhibits Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Induces Apoptosis

The tripeptide,Arg-Gly-Asp(RGD)motif is an integrin-recognition site found in adhesive proteins present in extracellular matrices(ECM)and in the blood.HCT-8 cells were treated with cellular adhesion tripeptide RGD at various concentrations.MTT assay was performed to examine the growth and proliferation of HCT-8 cells after treatment with RGD for 48 h.Haematoxylin and Eosin(HE)staining and electromicroscope were used to observe the morphology of apoptotic cells.Survivin and flow cytometry were also used to analyze the HCT-8 apoptosis.Cellular adhesion tripeptide RGD significantly inhibits the growth and proliferation of HCT-8 cells in a dose-dependent manner and induces apoptosis of HCT-8.These results indicate that cellular adhesion tripeptide RGD inhibits the growth and proli-feration of tumor HCT-8 cell,probably by the aid of inducing apoptosis of HCT-8 cell.     

Acceleration of osteogenic differentiation by sustained release of BMP2 in PLLA/graphene oxide nanofibrous scaffold

After about three decades of experience, tissue engineering has become one of the most important approaches in reconstructive medical research to treat non‐self‐healing bone injuries and lesions. Herein, nanofibrous composite scaffolds fabricated by electrospinning, which containing of poly(L‐lactic acid) (PLLA), graphene oxide (GO), and bone morphogenetic protein 2 (BMP2) for bone tissue engineering applications. After structural evaluations, adipose tissue derived mesenchymal stem cells (AT‐MSCs) were applied to monitor scaffold's biological behavior and osteoinductivity properties. All fabricated scaffolds had nanofibrous structure with interconnected pores, bead free, and well mechanical properties. But the best biological behavior including cell attachment, protein adsorption, and support cells proliferation was detected by PLLA‐GO‐BMP2 nanofibrous scaffold compared to the PLLA and PLLA‐GO. Moreover, detected ALP activity, calcium content and expression level of bone‐related gene markers in AT‐MSCs grown on PLLA‐GO‐BMP2 nanofibrous scaffold was also significantly promoted in compression with the cells grown on other scaffolds. In fact, the simultaneous presence of two factors, GO and BMP2, in the PLLA nanofibrous scaffold structure has a synergistic effect and therefore has a promising potential for tissue engineering applications in the repair of bone lesions.     

Cell adhesive and growth behavior on electrospun nanofibrous scaffolds by designed multifunctional composites

Nanostructured biocomposite scaffolds of poly(l-lactide) (PLLA) blended with collagen (coll) or hydroxyapatite (HA), or both for tissue engineering application, were fabricated by electrospinning. The electrospun scaffolds were characterized for the morphology, chemical and tensile properties by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), Fourier transform infrared (FTIR) measurement, and tensile testing. Electrospun biocomposite scaffolds of PLLA and collagen or (and) HA in the diameter range of 200-700 nm mimic the nanoscale structure of the extracellular matrix (ECM) with a well-interconnection pore network structure. The presence of collagen in the scaffolds increased their hydrophility, and enhanced cell attachment and proliferation, while HA improved the tensile properties of the scaffolds. The biocompatibility of the electrospun scaffolds and the viability of contacting cells were evaluated by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and by fluorescein diacetate (FDA) and propidium iodide (PI) double staining methods. The results support the conclusion that 293T cells grew well on composite scaffolds. Compared with pure PLLA scaffolds a greater density of viable cells was seen on the composites, especially the PLLA/HA/collagen scaffolds.     

Gelatin/PLLA sponge-like scaffolds allow proliferation and osteogenic differentiation of human mesenchymal stromal cells

Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].     

Preparation of HMWCNT/PLLA nanocomposite scaffolds for application in nerve tissue engineering and evaluation of their physical,mechanical and cellular activity properties

This study was aimed to prepare biodegradable and porous nanocomposite scaffolds with microtubular orientation structure as a model for nerve tissue engineering by thermally induced phase separation (TIPS) method using dioxane as the solvent, crystalline poly (L‐lactic acid) (PLLA) and multi‐walled carbon nanotubes (MWCNTs). In order to overcome dispersion of MWCNTs in the PLLA matrix, heparinization of MWCNTs was performed. Solvent crystallization, oriented structure, the mean pore diameter and porosity percentage of the scaffolds were controlled by fundamental system parameters including temperature‐gradient of the system, polymer solution concentration and carbon nanotube content. Scanning Electron Microscopy (SEM), ImageJ, software and dynamic mechanical thermal analysis (DMTA) were used to investigate the structural and mechanical properties. TEM observation was carried out for characterization of nanotube dispersion in PLLA. It was found that the scaffolds containing heparinized multi‐walled carbon nanotubes (HMWCNTs) exhibited higher storage modulus, better carbon nanotube (CNT) dispersion and tubular orientation structure than those with non heparinized MWCNTs. In‐vitro studies were also conducted by using murine P₁₉ cell line as a suitable model system to analyze neuronal differentiation over a 2‐week period. Immunofluorescence and DAPI staining were used to confirm the cells' attachment and differentiation on the PLLA/HMWCNT nanocomposite scaffolds. Based on the results, we can conclude that the PLLA/HMWCNT scaffolds enhanced the nerve cell differentiation and proliferation, and therefore, acted as a positive cue to support neurite outgrowth. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced Cell Penetration and Pluripotency Maintenance of hiPSCs in 3D Natural Chitosan Scaffolds

Human-induced pluripotent stem cells (hiPSCs) cultured in 3D matrices hold great promise in disease modeling, drug discovery, and tissue regeneration. Uniform cell distribution in a 3D structure is critical to the growth and function of hiPSCs, yet cell seeding in 3D matrices often remains superficial, leading to limited cell proliferation and compromised pluripotency. Here, an approach to improve cell penetration depth of hiPSCs in 3D scaffolds modified with hiPSCs conditioned medium (CM) is reported. It is shown that extracellular matrix components are successfully deposited onto the scaffold wall surface after CM treatment and promoted homogeneous cell adhesion during initial seeding. Compared to plain, unmodified scaffolds, the CM treated scaffold improves spatial cell distribution uniformity and upregulates pluripotency markers. Notably, the expression of 29 genes associated with 11 signaling pathways participated in the pluripotency maintenance of hiPSCs exhibits >2-fold change in hiPSCs grown in the CM treated scaffolds than 2D counterparts, demonstrating that CM treated scaffolds can support a more primitive and undifferentiated phenotype of hiPSCs. This study introduces a simple and effective method to enhance cell penetration and maintain cell pluripotency in 3D matrices.     

Characterization of drawn and undrawn poly-L-lactide films by differential scanning calorimetry

Poly-L-lactic acid (PLLA) is an optically active, biocompatible and biodegradable polymer that has been widely investigated as an artificial cell scaffold material. In its most crystalline form, PLLA is highly anisotropic and is one of the most piezoelectric polymers known. Conversely, amorphous PLLA exhibits little, if any, piezoelectric behavior. Compression molded PLLA films can be endowed with varying amounts of crystalline character and piezoelectricity by uniaxially stretching the polymer in a hot air bath. Understanding the precise crystalline architecture of PLLA that results from tensile drawing is important for constructing cell scaffolds that have highly tailored biodegradation and cell guiding properties. In our work here, we investigate the changes in the thermal properties of PLLA at draw ratios between 1.0 and 5.5 using differential scanning calorimetry (DSC). The crystallinity of the compression molded undrawn starting material is characterized using X-ray diffraction. Our DSC results show an increase in percent crystallinity with increasing draw up to a draw ratio of 4.0. At greater draw ratios, there is a decrease in the crystalline character exhibited by PLLA.This revised version was published online in November 2005 with corrections to the Cover Date.     

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.     

Self-assembly and bioactivity of a polymer/peptide conjugate containing the RGD cell adhesion motif and PEG

The self-assembly and bioactivity of the peptide–polymer conjugate DGRFFF–PEG3000 containing the RGD cell adhesion motif has been examined, in aqueous solution. The conjugate is designed to be amphiphilic by incorporation of three hydrophobic phenylalanine residues as well as the RGD unit and a short poly(ethylene glycol) (PEG) chain of molar mass 3000 kg mol⁻¹. Above a critical aggregation concentration, determined by fluorescence measurements, signals of β-sheet structure are revealed by spectroscopic measurements, as well as X-ray diffraction. At high concentration, a self-assembled fibril nanostructure is revealed by electron microscopy. The fibrils are observed despite PEG crystallization which occurs on drying. This suggests that DGRFFF has an aggregation tendency that is sufficiently strong not to be prevented by PEG crystallization. The adhesion, viability and proliferation of human corneal fibroblasts was examined for films of the conjugate on tissue culture plates (TCPs) as well as low attachment plates. On TCP, DGRFFF–PEG3000 films prepared at sufficiently low concentration are viable, and cell proliferation is observed. However, on low attachment surfaces, neither cell adhesion nor proliferation was observed, indicating that the RGD motif was not available to enhance cell adhesion. This was ascribed to the core–shell architecture of the self-assembled fibrils with a peptide core surrounded by a PEG shell which hinders access to the RGD unit.