Bilayer porous scaffold based on poly-(?-caprolactone) nanofibrous membrane and gelatin sponge for favoring cell proliferation

Electrospun poly-(?-caprolactone) (PCL) nanofibers has been widely used in the medical prosthesis. However, poor hydrophilicity and the lack of natural recognition sites for covalent cell-recognition signal molecules to promote cell attachment have limited its utility as tissue scaffolds. In this study, Bilayer porous scaffolds based on PCL electrospun membranes and gelatin (GE) sponges were fabricated through soft hydrolysis of PCL electrospun followed by grafting gelatin onto the fiber surface, through crosslinking and freeze drying treatment of additional gelatin coat and grafted gelatin surface. GE sponges were stably anchored on PCL membrane surface with the aid of grafted GE molecules. The morphologies of bilayer porous scaffolds were observed through SEM. The contact angle of the scaffolds was 0°, the mechanical properties of scaffolds were measured by tensile test, Young's moduli of PCL scaffolds before and after hydrolysis are 66–77.3 MPa and 62.3–75.4 MPa, respectively. Thus, the bilayer porous scaffolds showed excellent hydrophilic surface and desirable mechanical strength due to the soft hydrolysis and GE coat. The cell culture results showed that the adipose derived mesenchymal stem cells did more favor to adhere and grow on the bilayer porous scaffolds than on PCL electrospun membranes. The better cell affinity of the final bilayer scaffolds not only attributed to the surface chemistry but also the introduction of bilayer porous structure.     

Role of reactive gas in atmospheric plasma for cell attachment and proliferation on biocompatible poly ?-caprolactone film

This paper reports the surface modification of a biocompatible poly ?-caprolactone (PCL) film treated by atmospheric cold plasma (ACP) with reactive gases. The change in wettability and surface morphology of the PCL film after the plasma treatment with the reactive gases (Ar, H₂, N₂ and O₂) were determined using contact angle and surface roughness measurements. The chemical bonding states and molecular vibration modes of the activated organic groups on the polymer surface were examined by X-ray photoelectron spectroscopy and Fourier-transformation infrared techniques. The surface of the ACP-treated PCL films was also examined for their in vitro cell attachment and proliferation using human prostate epithelial cells (HPECs). The increase in the hydrophobicity of the Ar + H₂ plasma-treated PCL film resulted in a lower cell loading in the initial step of cell culture as well as a decrease in the level of cell attachment and proliferation compared with the pristine film. However, the hydrophilic properties of the Ar + N₂, Ar and Ar + O₂ plasma-treated PCL film improved the adhesion properties. Therefore, the Ar + N₂, Ar and Ar + O₂ plasma-treated PCL films showed a better cell distribution and growth than that of the pristine PCL film. The ACP-treated PCL film is potentially useful as a suitable scaffold in biophysics and bio-medical engineering applications.     

Functionally graded PCL/β-TCP biocomposites in a multilayered structure for bone tissue regeneration

Functionally graded (FG) composites consisting of polycaprolactone (PCL) and beta-tricalcium phosphate (β-TCP) particles were fabricated with a multilayered structure using a melt plotter with a two-heating-barrel system. Using this process, the concentration of β-TCP particles varied in each layered strut. Scanning electron microscopy (SEM) and energy dispersive spectroscopy mapping of calcium on the fabricated scaffolds indicated that the β-TCP particles were well distributed in each PCL strut, according to conceptual design. By incorporating β-TCP, the FG-PCL/β-TCP scaffolds had meaningful increases in water absorption (30 % increase) and showed good mechanical properties, although the mechanical properties are slightly low compared to pure PCL/β-TCP composite. We performed biological assessments to evaluate the capability of these FG scaffolds to act as a biomaterial for bone tissue regeneration with osteoblast-like cells (MG63). SEM images of cell-seeded FG scaffolds showed that the concentrated β-TCP struts were affected as good cell attachment/proliferation sites. Additionally, calcium deposition on the FG scaffolds was higher than that of normal scaffolds after 14 days. In particular, we observed high levels of mineralization in the highly concentrated β-TCP struts in the FG scaffolds. Based on these results, we believe that the FG scaffolds having various spatially designed structures with graded properties will be widely applicable for hard tissue engineering applications.     

Preparation and Properties of Polyurethane Hydrogels Based on Hexamethylene Diisocyanate/Polycaprolactone-Polyethylene Glycol

Hydrogels are considered an optimum material for controlled release drug systems and tissue engineering scaffolds since they are tri-dimensional networks. In this work hexamethylene diisocyanate (HMDI), polycaprolactone (PCL) and polyethylene glycol (PEG) were used to prepare polyurethane prepolymers using diethylene glycol (DEG) as a chain-extender. Then the prepolymer was used to fabricate the HMDI/PCL-PEG/DEG polyurethane hydrogels by free radical polymerization using benzoyl peroxide (BPO) as a cross-linking agent. The influences of the ratio of polyol on the contact angle, swelling ratio, morphology and cytotoxicity in-vitro of the HMDI/PCL-PEG/DEG polyurethane hydrogel were investigated. The biological behavior of the polyurethane hydrogels was analyzed by studying the cell behavior using the standard biological MTT (3–4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) test. The Fourier transform infrared (FTIR) spectra results showed that the polyurethane hydrogels were successfully synthesized. The change of the molar the ratio of the polyhydric alcohols (PEG and PCL) played important roles in the swelling degree, the contact angle and the pore size. The HMDI/PCL-PEG/DEG polyurethane hydrogel (PCL/PEG = 1:3) was hydrophilic with many more large pores while the polyurethane hydrogel with PCL/PEG = 3:1 had a dense structure. The fibroblastic cell proliferation improved with decreasing relative PEG content; however, there were insignificant differences (P > 0.05) on all days of observation of the samples with various PEG contents compared with the negative control group. The MTT assays revealed that the cells were able to grow and proliferate quite quickly in the extracts of the HMDI/PCL-PEG/DEG polyurethane hydrogels as well as the extract of the negative control.     

Poly(ɛ-caprolactone) Electrospun Scaffolds Filled with Nanoparticles. Production and Optimization According to Taguchi's Methodology

Polycaprolactone (PCL) scaffolds were produced by electrospinning. Polymeric solutions in a mix of dichloromethane (DCM) and dimethylformamide were electrospun to form fibers in the sub-micron range. Physical properties of the PCL solutions were characterized with respect to density, viscosity, conductivity and surface tension. Processing was optimized following Taguchi's methodology to select the set of processing parameters that resulted in producing fibers with the smallest diameters, minimum number of defects and with the narrowest distribution of fiber diameter. Morphology of electrospun fibers was qualitatively and quantitatively analyzed for the different sets of processing parameters. The optimum conditions found to electrospun PCL were used to process PCL solutions containing nanoparticles of hydroxyapatite (HA) or bioactive glass (BG). Bioactivity of nanocomposite electrospun membranes in simulated body fluid (SBF) was analyzed and biological response was tested by assessing proliferation and viability of MT3C3-E1 preosteoblasts cultured on PCL and its nanocomposite membranes.     

Contribution of power on cell adhesion using atmospheric dielectric barrier discharge (DBD) plasma system

This study examined the effect of the treatment power on the enhanced cell attachment and proliferation on poly ε-caprolactone (PCL) films treated with atmospheric plasma using a dielectric barrier discharge method (AP-DBD). The peak intensities of the –CH, CO, –OH and –COO vibration modes, and binding energies of carbon and oxygen of the AP-DBD treated-PCL film increased with increasing plasma treatment power. The surfaces of the AP-DBD treated-PCL films were also examined for their in vitro cell adhesion properties using human prostate epithelial cells. The results showed that the level of cell attachment and proliferation on the AP-DBD treated-PCL film was ten times better than that observed on the untreated-PCL films.     

The influence of polymer concentrations on the structure and mechanical properties of porous polycaprolactone-coated hydroxyapatite scaffolds

Polycaprolactone (PCL)-coated porous hydroxyapatite (HA) composite scaffolds were prepared by combining polymer impregnating method with dip-coating method. Three different PCL solution concentrations were used in dip-coating process to improve the mechanical properties of porous HA scaffolds. The results indicated that as the concentration of PCL solution increases the compressive strength significantly increased from 0.09 MPa to 0.51 MPa while the porosity decreased from 90% to 75% for the composite scaffolds. An interlaced structure was found inside the pore wall for all composite scaffolds due to the penetration of PCL. The porous HA/PCL composite scaffolds dip-coated with 10% PCL exhibited optimal combination of mechanical properties and pore interconnectivity, and may be a potential bone candidate for the tissue engineering applications.     

Preparation and Characterization of Gelatin–Poly(L-lactic) Acid/Poly(hydroxybutyrate-co-hydroxyvalerate) Composite Nanofibrous Scaffolds

Poly(L-lactic) acid (PLLA) scaffolds, prepared by electrospinning technology, have been suggested for use in tissue engineering. They remain a challenge for application in biological fields due to PLLA's slow degradation and hydrophobic nature. We describe PLLA, PLLA/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), and PLLA/PHBV/gelatin (Gt) composite nanofiberous scaffolds (Gt–PLLA/PHBV) electrospun by changing the electrospinning technology. The morphologies and hydrophilicity of these fibers were characterized by scanning electron microscopy (SEM) and water contact angle measurement. The results showed that the addition of PHBV and Gt resulted in a decrease in the diameters and their distribution and greatly improved the hydrophilicity. The in-vitro degradation test indicated that GT–PLLA/PHBV composite scaffolds exhibited a faster degradation rate than PLLA and PLLA/PHBV scaffolds. Dermal fibroblasts viabilities on nanofibrous scaffolds were characterized by [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (MTT) assay and cell morphologies after 7 days culture. Results indicated that the GT–PLLA/PHBV composite nanofibers showed the highest bioactivity among the three scaffolds and increased with increasing time. The SEM images of cells/scaffolds composite materials showed the GT–PLLA/PHBV composite nanofibers enhanced the dermal fibroblasts's adhesion, proliferation, and spreading. It is suggested that the nanofibrous composite scaffolds of GT–PLLA/PHBV composites would be a promising candidate for tissue engineering scaffolds.     

In vitro mineralization of surface-modified porous polycaprolactone scaffolds in simulated body fluid

Porous polycaprolactone (PCL) scaffolds were fabricated by combination of porogen-leaching and freeze-drying processes. Ice particulates were used as porogen materials. The porous PCL scaffolds were modified by potassium hydroxide solution with concentration of 1 mol/L at room temperature for 8 h, subsequently biomineralized in simulated body fluid for 2 h and 8 h, respectively. The microstructure and characteristics of the PCL scaffolds were investigated by scanning electron microscope (SEM) and EDS. The results showed (1) PCL scaffolds had high degree of connectivity and different pore sizes. (2) Plate-like apatite was observed on the surface of the scaffolds after being immersed into SBF for 8 h.     

Adhesion comparison of human bone marrow stem cells on a gradient wettable surface prepared by corona treatment

Corona discharge treatment was applied to modify the surface of polyethylene (PE). The wettability of PE surface was gradually increased by power increase of a corona treatment along the PE length, indicating that the hydrophilicity of PE surface increased gradually. The adhesion and proliferation behavior of human bone marrow stem cells (hBMSCs) on the gradient PE surface was evaluated. We found that hBMSCs were adhered to and proliferated on better highly hydrophilic than hydrophobic surfaces. The plot of proliferation rate vs. the water contact angles was parabolic. These results indicate that surface wettability plays an important role in the cell attachment and proliferation.     

Pluripotency of adult stem cells derived from human and rat pancreas

Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases. PACS 87.17.-d; 87.18.Ed; 81.17.Ez; 87.18.-h     

Cerebrospinal fluid promotes survival and astroglial differentiation of adult human neural progenitor cells but inhibits proliferation and neuronal differentiation

Background  

Neural stem cells (NSCs) are a promising source for cell replacement therapies for neurological diseases. Growing evidence suggests an important role of cerebrospinal fluid (CSF) not only on neuroectodermal cells during brain development but also on the survival, proliferation and fate specification of NSCs in the adult brain. Existing in vitro studies focused on embryonic cell lines and embryonic CSF. We therefore studied the effects of adult human leptomeningeal CSF on the behaviour of adult human NSCs (ahNSCs).     

Highly porous 3D nanofibrous scaffolds processed with an electrospinning/laser process

Electrospinning has been widely used to produce micro/nanosized fibres. Although the method is very simple, easy, and effective for obtaining nanosized material, the fabrication of three dimensional (3D) shapes comprised of micro/nanofibres has been a major obstacle for use in tissue engineering. In this study, a new electrospinning method to fabricate controllable 3D micro/nanofibrous structure (with thickness over 3 mm) is suggested. The fabricated 3D fibrous structure was fully porous and successfully consisted of submicron-sized fibres. However, the pores in the 3D fibrous structure were too small (5–10 μm), so we used a femtosecond laser process to achieve enough cell infiltration and proliferation in the thickness direction of the 3D structure. By controlling appropriate processing conditions, we can successfully fabricate a highly porous 3D micro/nanofibrous structure with various pore sizes ranging from 189 ± 28 μm to 380 ± 21 μm. The fabricated 3D fibrous scaffolds were assessed for in vitro biological capabilities by culturing osteoblast like cells (MG63). Compared with the rapid-prototyped PCL scaffold, the 3D fibrous scaffold exhibited significantly higher biological activities (initial cell attachment and cell proliferation) due to the topographical structure of micro/nanofibres.     

Morphological and surface compositional changes in poly(lactide-co-glycolide) tissue engineering scaffolds upon radio frequency glow discharge plasma treatment

Chemical functionalisation of polymeric scaffolds with functional groups such as amine could provide optimal conditions for loading of signalling biomolecules over the entire volume of the porous scaffolds. Three-dimensional (both surface and bulk) functionlisation of large volume scaffolds is highly desirable, but preferably without any change to the basic morphological, structural and bulk chemical properties of the scaffolds. In this work, we have carried out and compared treatments of poly(lactide-co-glycolide) tissue engineering scaffolds by two methods, that is, a wet chemical method using ethylenediamine and a glow discharge plasma method using heptylamine as a precursor. The samples thus prepared were analysed by scanning electron microscopy and X-ray photoelectron spectroscopy. The plasma treatment generated amide and protonated amine (NH⁺) groups which were present in the bulk and on the surface of the scaffold. Amination also occurred for the wet chemical treatments but the structural and chemical integrity were adversely affected.     

Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation

Background  

The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.     

Relationship between adsorbed fibronectin and cell adhesion on a honeycomb-patterned film

Substratum surface morphology plays a vital roles in cellular behavior. Here, we characterized adsorption of fibronectin (Fn) as a typical cell adhesion protein onto honeycomb-patterned films made of poly(ε-caprolactone) (PCL) by using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). In order to determine how cells adhere to a honeycomb-patterned film, focal adhesion of cardiac myocytes (CMYs) and endothelial cells (ECs) on the films were studied by using fluorescence labeling of vinculin. Fn adsorbs around the pore edges to form ring-shaped structures. CMYs and ECs adhere onto the honeycomb-patterned films at focal contact points localized around pore edges distributed over the entire cellular surface. The focal contact points on the honeycomb-patterned films correspond well with the adsorption sites of Fn. We suggest that the cell response to honeycomb-patterned films is associated with the adsorption pattern of Fn on the film.     

Effect of the internal microstructure in rapid-prototyped polycaprolactone scaffolds on physical and cellular properties for bone tissue regeneration

Biomedical scaffolds should be designed to optimize their inter-microstructure to enable cell infiltration and nutrient/waste transport. To acquire these properties, several structural parameters, such as pore size, pore shape, porosity, pore interconnectivity, permeability, and tortuosity are required. In this study, we explored the effect of tortuosity on the viable cell proliferation and mineralization of osteoblast-like-cells (MG63) in polycaprolactone scaffolds. For analysis, we designed four different scaffolds of various tortuosities ranging from 1.0 to 1.3 under the same porosity (56?%) and 100?% pore interconnectivity. The pore size of the scaffolds was set as 150 and 300?μm, and a mixture of these sizes. We found that despite the porosity being same, the elastic modulus was dependent on the pore size of the scaffolds due to the distributed stress concentration. In addition, the relative water movement within scaffolds was also related to the internal microstructure. Cell viability and Ca²⁺ deposition of the cell-seeded scaffolds showed that the proliferation of viable cells and mineralization in the scaffolds with appropriate tortuosity (1.2) was relatively high compared to those of the scaffolds displaying low (1.05 and 1.1) or high (1.3) tortuosity. Our findings indicated that the internal microstructure of the scaffolds may influence not only the physical properties, but in addition the cellular behavior.     

Plasticity of human dental pulp stromal cells with bioengineering platforms: A versatile tool for regenerative medicine

In recent years, human dental pulp stromal cells (DPSCs) have received growing attention due to their characteristics in common with other mesenchymal stem cells, in addition to the ease with which they can be harvested. In this study, we demonstrated that the isolation of DPSCs from third molar teeth of healthy individuals allowed the recovery of dental mesenchymal stem cells that showed self-renewal and multipotent differentiation capability. DPSCs resulted positive for CD73, CD90, CD105, STRO-1, negative for CD34, CD45, CD14 and were able to differentiate into osteogenic and chondrogenic cells. We also assayed the angiogenic potential of DPSCs, their capillary tube-like formation was assessed using an in vitro angiogenesis assay and the uptake of acetylated low-density lipoprotein was measured as a marker of endothelial function. Based on these results, DPSCs were capable of differentiating into cells with phenotypic and functional features of endothelial cells. Furthermore, this study investigated the growth and differentiation of human DPSCs under a variety of bioengineering platforms, such as low frequency ultrasounds, tissue engineering and nanomaterials. DPSCs showed an enhanced chondrogenic differentiation under ultrasound application. Moreover, DPSCs were tested on different scaffolds, poly(vinyl alcohol)/gelatin (PVA/G) sponges and human plasma clots. We showed that both PVA/G and human plasma clot are suitable scaffolds for adhesion, growth and differentiation of DPSCs toward osteoblastic lineages. Finally, we evaluated the interactions of DPSCs with a novel class of nanomaterials, namely boron nitride nanotubes (BNNTs). From our investigation, DPSCs have appeared as a highly versatile cellular tool to be employed in regenerative medicine.     

Effect of frequency on the structure and cell response of Ca- and P-containing MAO films

The Ca- and P-containing MAO films were prepared on titanium substrate at different frequencies (100-5000 Hz) and were characterized by SEM, XRD, XPS and contact angle goniometer. For in vitro test, the rat bone marrow mesenchymal stem cells (rMSCs) were seeded on the films. The fluorescence microscopy and the PicoGreen assay were used to determine the cell initial adhesion and proliferation. It shows that the frequency of the MAO affected the crystallinity, composition, morphology and wetting ability of the oxidation film. At a high frequency, the crystallinity decreased, and the content of Ca and P increased. The structure formed at a high frequency - there were many smaller pores on the wall of the larger ones and many inner pores in the film - could improve the connectivity of the film. The wetting ability of the film was also improved by increasing the frequency. The mechanism of how the frequency of the MAO process could influence the oxidation film was discussed. It could be explained by the theory of electron avalanche and the phenomenon of secondary breakdown. In vitro test showed that the film formed at 5000 Hz was more favorable for the initial cell attachment and proliferation.     

Mechanism of adhesion between protein-based hydrogels and plasma treated polypropylene backing

We studied the mechanism of adhesion between N₂ plasma treated polypropylene (PP/N₂) backing and a hybrid hydrogel (HG) produced by chemical crosslinking between poly(ethylene glycol) and soy albumin. The work of adhesion, measured by peel testing, was found to be 25 times higher for PP/N₂ compared to untreated PP (≈5.0 J/m² versus ≈0.2 J/m²). In order to understand the adhesion mechanism, we performed a detailed analysis of the surface chemical composition of PP and PP/N₂ using X-ray photoelectron spectroscopy (XPS), chemical derivatization and attenuated total reflectance infra-red (ATR-IR) measurements. The results confirm incorporation of different nitrogen- (amine, amide,…) and oxygen- (hydroxyl, carboxyl,…) containing chemical groups on the PP/N₂ surface. The derivatized functions were primary amine, hydroxyl, carboxyl and carbonyl groups. Chemical derivatization reactions validated the XPS results (except for carbonyl groups), and they clearly underlined the essential role of primary amine groups in the adhesion process. In fact, after derivatization of the amine functions, the work of adhesion was found to be 0.41 ± 0.12 J/m². Participation of amine groups in the formation of covalent bonds at the interface between PP/N₂ and HG was directly confirmed by ATR-IR measurements.