Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) based tissue engineering matrices

In this study, the aim was to produce tissue-engineered bone using osteoblasts and a novel matrix material, poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV). In order to prepare a porous PHBV matrix with uniform pore size, sucrose crystals were loaded in the foam and then leached leaving pores behind. The surface of the PHBV matrix was treated with rf-oxygen plasma to increase the surface hydrophilicity. SEM examination of the PHBV matrices was carried out. Stability of PHBV foams in aqueous media was studied. The pH decrease is an indication of the degradation extent. The weight and density were unchanged for a period of 120 days but then a significant decrease was observed for the rest of the study. Osteoblast cells were then isolated from rat bone marrow and seeded onto PHBV matrices. The metabolization and proliferation on the foams was determined with MTS assay which showed that osteoblasts proliferated on PHBV. It was also found that cells proliferated better on large pore size foams (300–500 m) than on the small pore size foams (75–300 m). Production of ALP was measured spectrophotometrically. The present study demonstrated that PHBV matrices are suitable substrates for osteoblast proliferation and differentiation.     

Design of Plasma Surface‐Activated,Electrospun Polylactide Non‐Wovens with Improved Cell Acceptance

Electrospinning is a versatile technique to generate tissue engineering matrices possessing structural features similar to the extracellular matrix. Biodegradable polylactides are well suited for processing by this technique, but their innate hydrophobicity impairs initial protein adsorption and cell adhesion. In this work, therefore, electrospun poly(L ‐lactide‐co‐D,L ‐lactide) (70/30) non‐wovens are modified with an ultrathin plasma‐polymerized allylamine (PPAAm) coating. Using scanning electron microsocopy (SEM), it is shown that the fiber structure of the non‐woven is not affected by the plasma treatment. X‐ray photoelectron spectroscopy (XPS) and contact angle measurements of PPAAm‐coated non‐wovens confirm the presence of nitrogen and oxygen‐functional groups in the coating and a hydrophilic nature of the coated non‐woven surface. Cell experiments in vitro demonstrate that the PPAAm‐coated surface promotes occupancy of the non‐woven by human MG‐63 osteoblasts accompanied by improved initial cell spreading and filopodia formation along and between the electrospun polylactide fibers. Overall, plasma‐assisted incorporation of amino groups into electrospun polylactone non‐wovens represents a promising approach to tissue engineering scaffolds with improved cell–material interfaces.     

Plasma surface treatments of poly(l-lactic acid) (PLLA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV)

The surfaces of poly(l-lactic acid) (PLLA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) were modified by oxygen and nitrogen plasma treatments. The physical and chemical surface characteristics were evaluated by contact angle tests, scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). The plasma treatments caused an increase in both contact angle and roughening, altered the surface morphology, inserted polar groups, and, consequently, enhanced the hydrophilicity for both PLLA and PHBV polymers.     

Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.     

In vitro biocompatibility assessment of PHBV/Wollastonite composites

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing bone tissue engineering scaffolds, the in vitro biocompatibility of degradable and bioactive composites consisting of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and wollastonite (W) was studied by culturing osteoblasts on the PHBV/W substrates, and the cell adhesion, morphology, proliferation, and alkaline phosphatase (ALP) activity were evaluated. The results showed that the incorporation of wollastonite benefited osteoblasts adhesion and the osteoblasts cultured on the PHBV/W composite substrates spread better as compared to those on the pure PHBV after culturing for 3 h. In the prolonged incubation time, the osteoblasts cultured on the PHBV/W composite substrates revealed a higher proliferation and differentiation rate than those on the pure PHBV substrates. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composites increased from 10 to 20 wt%. All of the results showed that the addition of wollastonite into PHBV could stimulate osteoblasts to proliferate and differentiate and the PHBV/W composites with wollastonite up to 20 wt% were more compatible than the pure PHBV materials for bone repair and bone tissue engineering.     

Zwitterionic PMCP‐Modified Polycaprolactone Surface for Tissue Engineering: Antifouling,Cell Adhesion Promotion,and Osteogenic Differentiation Properties

Biodegradable polycaprolactone (PCL) has been widely applied as a scaffold material in tissue engineering. However, the PCL surface is hydrophobic and adsorbs nonspecific proteins. Some traditional antifouling modifications using hydrophilic moieties have been successful but inhibit cell adhesion, which is not ideal for tissue engineering. The PCL surface is modified with bioinspired zwitterionic poly[2‐(methacryloyloxy)ethyl choline phosphate] (PMCP) via surface‐initiated atom transfer radical polymerization to improve cell adhesion through the unique interaction between choline phosphate (CP, on PMCP) and phosphate choline (PC, on cell membranes). The hydrophilicity of the PCL surface is significantly enhanced after surface modification. The PCL‐PMCP surface reduces nonspecific protein adsorption (e.g., up to 91.7% for bovine serum albumin) due to the zwitterionic property of PMCP. The adhesion and proliferation of bone marrow mesenchymal stem cells on the modified surface is remarkably improved, and osteogenic differentiation signs are detected, even without adding any osteogenesis‐inducing supplements. Moreover, the PCL‐PMCP films are more stable at the early stage of degradation. Therefore, the PMCP‐functionalized PCL surface promotes cell adhesion and osteogenic differentiation, with an antifouling background, and exhibits great potential in tissue engineering.     

Synthesis and characterization of cholesterol-poly(ethylene glycol)-poly(D,L-lactic acid) copolymers for promoting osteoblast attachment and proliferation

A novel cholesterol-poly(ethylene glycol)-poly(D,L-lactic acid) copolymer (CPEG-PLA) has been synthesized as a potential surface additive for promoting osteoblast attachment and proliferation. The gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR) results indicated the product had expected structure with low polydispersities in the range of 1.1–1.5. By blending the poly(D,L-lactic acid) (PLA) with CPEG-PLA, the surface of modified PLA membrane was investigated by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle. The results revealed the enrichment of PEG chain on the surface. Osteoblast cell line (MC3T3) was chosen to test the cell behavior on modified PLA membranes. The osteoblast test about cell attachment, proliferation, cell viability and cell morphology investigation on CPEG-PLA modified PLA substrates showed the CPEG-PLA with 15 and 5 ethylene glycol units promoted osteoblast attachment and growth, while the CPEG-PLA with 30 ethylene glycol units prevent osteoblast adhesion and proliferation. This simple surface treatment method may have potentials for tissue engineering and other biomedical applications.     

Plasma-modified and polyethylene glycol-grafted polymers for potential tissue engineering applications

Modified and grafted polymers may serve as building blocks for creating artificial bioinspired nanostructured surfaces for tissue engineering. Polyethylene (PE) and polystyrene (PS) were modified by Ar plasma and the surface of the plasma activated polymers was grafted with polyethylene glycol (PEG). The changes in the surface wettability (contact angle) of the modified polymers were examined by goniometry. Atomic Force Microscopy (AFM) was used to determine the surface roughness and morphology and electrokinetical analysis (Zeta potential) characterized surface chemistry of the modified polymers. Plasma treatment and subsequent PEG grafting lead to dramatic changes in the polymer surface morphology, roughness and wettability. The plasma treated and PEG grafted polymers were seeded with rat vascular smooth muscle cells (VSMCs) and their adhesion and proliferation were studied. Biological tests, performed in vitro, show increased adhesion and proliferation of cells on modified polymers. Grafting with PEG increases cell proliferation, especially on PS. The cell proliferation was shown to be an increasing function of PEG molecular weight.     

Surface modification of poly(ethylene terephthalate) by plasma polymerization of poly(ethylene glycol)

Poly(ethylene glycol) (PEG) was ‘polymerized’ onto poly(ethylene terephthalate) (PET) surface by radio frequency (RF) plasma polymerization of PEG (average molecular weight 200 Da) at a monomer vapour partial pressure of 10 Pa. Thin films strongly adherent onto PET could be produced by this method. The modified surface was characterized by infra red (IR) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), cross-cut test, contact angle measurements and static platelet adhesion studies. The modified surface, believed to be extensively cross-linked, however showed all the chemical characteristics of PEG. The surface was found to be highly hydrophilic as evidenced by an interfacial free energy of about 0.7 dynes/cm. AFM studies showed that the surface of the modified PET became smooth by the plasma polymerized deposition. Static platelet adhesion studies using platelet rich plasma (PRP) showed considerably reduced adhesion of platelets onto the modified surface by SEM. Plasma ‘polymerization’ of a polymer such as PEG onto substrates may be a novel and interesting strategy to prepare PEG-like surfaces on a variety of substrates since the technique allows the formation of thin, pin-hole free, strongly adherent films on a variety of substrates.     

Electrospinning of aligned biodegradable polymer fibers and composite fibers for tissue engineering applications

Fibrous membranes of aligned poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) fibers have been made through electrospinning. A high-speed rotating drum was used as the fiber collector while the electric field was manipulated by using five knife-edged auxiliary electrodes. It was found that a high drum rotating speed of 3000 rpm could lead to a nearly perfect alignment of PHBV fibers during electrospinning. Multilayered fibrous structures with each layer having a different direction of fiber alignment could also be constructed through electrospinning. The electrospun PHBV fibers were further modified by incorporating carbonated hydroxyapatite (HA) nanospheres (up to 20% of HA) in the fibers. The fibrous membranes made of aligned PHBV fibers and made of HA/PHBV composite fibers should be very useful for the tissue engineering of different human body tissues.     

改性淫羊藿苷共价结合壳聚糖/聚羟基丁酸酯-羟基戊酸酯构建组织诱导型骨修复支架材料

为构建一种诱导型骨修复支架材料,将淫羊藿苷（Ica）经过氨基化改性得到Ica-NH₂,并对Ica-NH₂进行FTIR、XRD和TG-DTA表征。以Ica-NH₂共价结合壳聚糖（CS）为诱导因子添加方式,聚羟基丁酸酯-羟基戊酸酯（PHBV）和CS为基材,经过两相混合急速冷冻/冷冻干燥成型技术制备了Ica-NH₂-CS/PHBV骨组织工程支架。随后,对支架材料进行了SEM、体外药物缓释、力学性能、细胞相容性及细胞增殖评价。结果显示:氨基化改性Ica的FTIR谱图在3 371、3 328 cm-1处出现2个中等强度、尖锐的N—H伸缩振动峰,在1 689 cm-1处出现N—H变角振动峰;XRD谱图显示其衍射增强且整体左移,证明Ica被成功氨基化改性;支架材料显微结构呈网络状串珠状,并均匀分布着3~10 μm的微孔,其体外药物缓释效果良好,力学强度介于硬质骨与松质骨之间,复合细胞培养7 d 后表现出良好的贴附与增殖,且细胞在Ica-NH₂-CS/PHBV支架材料上的增殖增长率显著高于CS/PHBV。研究表明所构建的Ica-NH₂-CS/PHBV支架材料可作为一种良好的诱导型骨修复材料。      

Plasma surface modification of polylactic acid to promote interaction with fibroblasts

In this work, medium pressure plasma treatment of polylactic acid (PLA) is investigated. PLA is a biocompatible aliphatic polymer, which can be used for bone fixation devices and tissue engineering scaffolds. Due to inadequate surface properties, cell adhesion and proliferation are far less than optimal and a surface modification is required for most biomedical applications. By using a dielectric barrier discharge (DBD) operating at medium pressure in different atmospheres, the surface properties of a PLA foil are modified. After plasma treatment, water contact angle measurements showed an increased hydrophilic character of the foil surface. X-ray photoelectron spectroscopy (XPS) revealed an increased oxygen content. Cell culture tests showed that plasma modification of PLA films increased the initial cell attachment both quantitatively and qualitatively. After 1 day, cells on plasma-treated PLA showed a superior cell morphology in comparison with unmodified PLA samples. However, after 7 days of culture, no significant differences were observed between untreated and plasma-modified PLA samples. While plasma treatment improves the initial cell attachment, it does not seem to influence cell proliferation. It has also been observed that the difference between the 3 discharge gases is negligible when looking at the improved cell-material interactions. From economical point of view, plasma treatments in air are thus the best choice.     

Surface modification of nanoporous alumina membranes by plasma polymerization

The deposition of plasma polymer coatings onto porous alumina (PA) membranes was investigated with the aim of adjusting the surface chemistry and the pore size of the membranes. PA membranes from commercial sources with a range of pore diameters (20, 100 and 200?nm) were used and modified by plasma polymerization using n-heptylamine (HA) monomer, which resulted in a chemically reactive polymer surface with amino groups. Heptylamine plasma polymer (HAPP) layers with a thickness less than the pore diameter do not span the pores but reduce their diameter. Accordingly, by adjusting the deposition time and thus the thickness of the plasma polymer coating, it is feasible to produce any desired pore diameter. The structural and chemical properties of modified membranes were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM) and x-ray electron spectroscopy (XPS). The resultant PA membranes with specific surface chemistry and controlled pore size are applicable for molecular separation, cell culture, bioreactors, biosensing, drug delivery, and engineering complex composite membranes.     

Change in cell adhesion property on cytocompatible interface using phospholipid polymer grafted with poly( , -lactic acid) segment for tissue engineering

Tissue engineering is a multi-disciplinary science that utilizes basic principles from materials engineering and molecular biology to reconstruct tissues from polymer matrices and cellular components. Artificial skins were well known as one of the concrete examples. Technological innovation of the tissue engineering must be contributed to improve quality of life. From the viewpoint, design of cytocompatible materials for tissue engineering would be the most important candidate to reconstruct tissue. 2-Methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and polylactic acid (PLA) macromonomer were polymerized for the preparation of cytocompatible interface. The polymer may involve following novel properties: (i) cytocompatibility by phospholipid groups, and (ii) enhancement of cell adhesion by PLA segment. The results of X-ray photoelectron spectroscopy showed the MPC unit and PLA segment on the membrane, which was prepared by dip coating. The surface mobility by contacting water was estimated with static contact angle measurement. The contact angle by water decreased after contact with water due to the chain rearrangement of hydrophilic MPC unit. Fibroblast cells adhesion and protein adsorption on the membranes were studied. The number of cell adhesion and cell proliferation on the membrane was well correlated with each other. Furthermore, the number of cell adhesion was proportional to the PLA macromonomer (MaPLA) composition. The adherent cell morphology showed round shape, because of the existence of MPC unit. However, the cell morphology would be spread after the cell proliferation. These findings suggest that the change in the polymer composition by combination of MPC and MaPLA could regulate the number of cell adhesion and the morphology.     

Nanofibrous modification on ultra-thin poly(e-caprolactone) membrane via electrospinning

Poly(e-caprolactone) (PCL) is a favorable material for tissue engineering. PCL was successfully fabricated into less than 10 μm thin membranes using a 2-roll-heated-mill and biaxial stretching process. However, PCL is known for its poor cellular adhesion and surface modifications are needed for any tissue engineering applications. This paper reports on a novel surface modification technique of the PCL membrane by coating with electrospun nanofibers. The purpose was to mimic the architecture of the natural extracellular matrix and create nanotopography for enhanced cellular attachment. The surfaces were characterized by scanning electron microscopy (SEM), water contact angle and atomic force microscopy. The results showed that uniform nanofibrous topology were successfully achieved on the surface of the PCL membrane, with increased roughness (more than 17 times) and surface area. This nanofibrous topology induced capillary effects after sodium hydroxide (NaOH) treatment, causing the water contact angle to drop to almost zero. Scratch tests revealed a strong interaction of PCL nanofiber coating on the PCL membrane. AlamarBlue assay indicated that 3T3 fibroblast cells proliferated well on the nanofibrous membrane. Confocal Laser Scanning Microscope revealed better cell attachment onto the nanofibrous membranes than the untreated membranes. Results from SEM showed that the cells' spindle-shaped morphology on the NaOH-treated fibrous surface was evident while they remained in isolated spherical shaped entities in the non-treated fibrous surfaces.     

Change in cell adhesion property on cytocompatible interface using phospholipid polymer grafted with poly(D,L-lactic acid)segment for tissue engineering

Tissue engineering is a multi-disciplinary science that utilizes basic principles from materials engineering and molecular biology to reconstruct tissues from polymer matrices and cellular components. Artificial skins were well known as one of the concrete examples. Technological innovation of the tissue engineering must be contributed to improve quality of life. From the viewpoint, design of cytocompatible materials for tissue engineering would be the most important candidate to reconstruct tissue. 2-Methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and polylactic acid (PLA) macromonomer were polymerized for the preparation of cytocompatible interface. The polymer may involve following novel properties: (i) cytocompatibility by phospholipid groups, and (ii) enhancement of cell adhesion by PLA segment. The results of X-ray photoelectron spectroscopy showed the MPC unit and PLA segment on the membrane, which was prepared by dip coating. The surface mobility by contacting water was estimated with static contact angle measurement. The contact angle by water decreased after contact with water due to the chain rearrangement of hydrophilic MPC unit. Fibroblast cells adhesion and protein adsorption on the membranes were studied. The number of cell adhesion and cell proliferation on the membrane was well correlated with each other. Furthermore, the number of cell adhesion was proportional to the PLA macromonomer (MaPLA) composition. The adherent cell morphology showed round shape, because of the existence of MPC unit. However, the cell morphology would be spread after the cell proliferation. These findings suggest that the change in the polymer composition by combination of MPC and MaPLA could regulate the number of cell adhesion and the morphology.     

Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

The electrospun scaffolds are potential application in vascular tissue engineering since they can mimic the nano-sized dimension of natural extracellular matrix (ECM). We prepared a fibrous scaffold from polycarbonateurethane (PCU) by electrospinning technology. In order to improve the hydrophilicity and hemocompatibility of the fibrous scaffold, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto the fiber surface by surface-initiated atom transfer radical polymerization (SI-ATRP) method. Although SI-ATRP has been developed and used for surface modification for many years, there are only few studies about the modification of electrospun fiber by this method. The modified fibrous scaffolds were characterized by SEM, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). The scaffold morphology showed no significant difference when PEGMA was grafted onto the scaffold surface. Based on the water contact angle measurement, the surface hydrophilicity of the scaffold surface was improved significantly after grafting hydrophilic PEGMA (P = 0.0012). The modified surface showed effective resistance for platelet adhesion compared with the unmodified surface. Activated partial thromboplastin time (APTT) of the PCU-g-PEGMA scaffold was much longer than that of the unmodified PCU scaffold. The cyto-compatibility of electrospun nanofibrous scaffolds was tested by human umbilical vein endothelial cells (HUVECs). The images of 7-day cultured cells on the scaffold surface were observed by SEM. The modified scaffolds showed high tendency to induce cell adhesion. Moreover, the cells reached out pseudopodia along the fibrous direction and formed a continuous monolayer. Hemolysis test showed that the grafted chains of PEGMA reduced blood coagulation. These results indicated that the modified electrospun nanofibrous scaffolds were potential application as artificial blood vessels.     

Reduced fibroblast adhesion and proliferation on plasma-modified titanium surfaces

Soft tissue complications are clinically relevant problems after osteosynthesis of fractures. The goal is to develop a method for reduction of fibroblast adhesion and proliferation on titanium implant surfaces by plasma polymerisation of the organo-silicon monomer hexamethyldisiloxane (HMDSO). HMDSO was deposited under continuous wave conditions in excess oxygen (ppHMDSO surface) and selected samples were further modified with an additional oxygen plasma (ppHMDSO + O₂ surface). Surface characterization was performed by scanning electron microscopy, profilometry, water contact angle measurements, infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy. In our experimental setup the mechanical properties, roughness and topography of the titanium were preserved, while surface chemistry was drastically changed. Fibroblast proliferation was assessed by alamarBlue assay, cell morphology by confocal microscopy visualization of eGFP-transducted fibroblasts, and cell viability by Annexine V/propidium iodide assay. Both modified surfaces, non-activated hydrophobic ppHMDSO and activated hydrophilic ppHMDSO + O₂ were able to dramatically reduce fibroblast colonization and proliferation compared to standard titanium. However, this effect was more strongly pronounced on the hydrophobic ppHMDSO surface, which caused reduced cell adhesion and prevented proliferation of fibroblasts. The results demonstrate that plasma modifications of titanium using HMDSO are valuable candidates for future developments in anti-adhesive and anti-proliferative coatings for titanium fracture implants.     

Surface modification of three-dimensional Ca-P/PHBV nanocomposite scaffolds by physical entrapment of gelatin and its in vitro biological evaluation

The properties of bone tissue engineering scaffolds such as architecture, porosity, mechanical properties and surface properties have significant effects on cellular response and play an important role in bone regeneration. In this study, three-dimensional nanocomposite scaffolds consisting of calcium phosphate (Ca-P) nanoparticles and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) copolymer with controlled external and internal architectures were successfully produced via selective laser sintering (SLS), one of the versatile rapid prototyping techniques. The Ca-P/PHBV nanocomposite scaffolds had a porosity of (61.75±1.24)%, compressive strength of (2.16±0.21) MPa and Young’s modulus of (26.98±2.29) MPa. The surface modification of scaffolds by gelatin was achieved through physical entrapment. The amount of entrapped gelatin could be controlled by varying the solvent composition and reaction time. The surface modification improved the hydrophilicity of scaffolds but did not significantly affect the surface morphology and mechanical properties. Osteoblast-like cells (SaOS-2) were cultured on scaffolds with and without gelatin surface modification. The majority of SaOS-2 cells were viable and proliferated in both types of scaffolds for up to 14 d in culture, as indicated by MTT assay and live and dead assay. Surface modification significantly increased cell proliferation for surface modified scaffolds, which could be due to the improvement in hydrophilicity of the scaffolds.