Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells

A major challenge to the effective treatment of injured cardiovascular tissues is the promotion of endothelialization of damaged tissues and implanted devices. For this reason, there is a need for new biomaterials that promote endothelialization to enhance vascular repair. The goal of this work was to develop antibody-modified polysaccharide-based hydrogels that could selectively capture endothelial progenitor cells (EPCs). We showed that CD34 antibody immobilization on hyaluronic acid (HA) hydrogels provides a suitable surface to capture EPCs. The effect of CD34 antibody immobilization on EPC adhesion was found to be dependent on antibody concentration. The highest level of EPC attachment was found to be 52.2 cells per mm(2) on 1% HA gels modified with 25 μg mL(-1) antibody concentration. Macrophages did not exhibit significant attachment on these modified hydrogel surfaces compared to the EPCs, demonstrating the selectivity of the system. Hydrogels containing only HA, with or without immobilized CD34, did not allow for spreading of EPCs 48 h after cell seeding, even though the cells were adhered to the hydrogel surface. To promote spreading of EPCs, 2% (w/v) gelatin methacrylate (GelMA) containing HA hydrogels were synthesized and shown to improve cell spreading and elongation. This strategy could potentially be useful to enhance the biocompatibility of implants such as artificial heart valves or in other tissue engineering applications where formation of vascular structures is required.     

The synergistic effect of aligned nanofibers and hyaluronic acid modification on endothelial cell behavior for vascular tissue engineering

The endothelialization of tissue-engineered vascular grafts (TEVGs) is considered to be an effective strategy to prevent the coagulation and restenosis of small-diameter vascular grafts. In this study, we fabricated well aligned nanofibrous scaffolds with PCL using a high speed rotating collector, modified those surfaces with hyaluronic acid (HA) and studied the synergistic effect of the scaffolds on the endothelial cells behavior in vitro. The well-aligned oriented architecture was observed by SEM images in the nanofibrous scaffolds. The contact angle measurements and FTIR-ATR evidenced that HA was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was increased after HA coating. The results of adhesion and morphology of human umbilical vein endothelial cells (HUVECs) showed that the HA-coating aligned PCL (HA-aPCL) nanofibrous scaffolds could highly promote attachment and guide HUVECs bipolar spread with the parallel aligned nanofibers. Furthermore, HUVECs on the HA-aPCL formed a confluent monoendothelial cell layer and exhibited superior protein expression levels of von Willebrand factor (vWF). This study suggested that the combination of aligned nanostructure and HA modification was more capable of promoting the regeneration of functional endothelium for vascular tissue engineering than individual use.     

Stem cell differentiation on electrospun nanofibrous substrates for vascular tissue engineering

Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and requirements in vascular tissue regeneration. In our study, poly-l-lactide (PLLA) and hybrid PLLA/collagen (PLLA/Coll) nanofibers (3:1 and 1:1) with fiber diameters of 210 to 430 nm were fabricated by electrospinning. Their morphological, chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR), and tensile instrument, respectively. Bone marrow derived mesenchymal stem cells (MSCs) seeded on electrospun nanofibers that are capable of differentiating into vascular cells have great potential for repair of the vascular system. We investigated the potential of MSCs for vascular cell differentiation in vitro on electrospun PLLA/Coll nanofibrous scaffolds using endothelial differentiation media. After 20 days of culture, MSC proliferation on PLLA/Coll(1:1) scaffolds was found 256% higher than the cell proliferation on PLLA scaffolds. SEM images showed that the MSC differentiated endothelial cells on PLLA/Coll scaffolds showed cobblestone morphology in comparison to the fibroblastic type of undifferentiated MSCs. The functionality of the cells in the presence of ‘endothelial induction media’, was further demonstrated from the immunocytochemical analysis, where the MSCs on PLLA/Coll (1:1) scaffolds differentiated to endothelial cells and expressed the endothelial cell specific proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and Von Willebrand factor (vWF). From the results of the SEM analysis and protein expression studies, we concluded that the electrospun PLLA/Coll nanofibers could mimic the native vascular ECM environment and might be promising substrates for potential application towards vascular regeneration.     

Polysaccharide-based magnetically responsive polyelectrolyte hydrogels for tissue engineering applications

Polysaccharide-based bionanocomposite hydrogels with functional nanomaterials were used in biomedical applications.Self-organization of xanthan gum and chitosan in the presence of iron oxide magnetic nanoparticles(Fe_3O_4MNPs)allowed us to form magnetically responsive polyelectrolyte complex hydrogels(MPECHs)via insitu ionic complexation using D-(+)-glucuronic acid?-lactone as a green acidifying agent.Characterization confirmed the successful formation of(and structural interactions within)the MPECH and good porous structure.The rheological behavior and compressive properties of the PECH and MPECH were measured.The results indicated that the incorporation of Fe_3O_4MNPs into the PECH greatly improved mechanical properties and storage modulus(G’).In vitro cell culture of NIH3T3 fibroblasts on MPECHs showed improvements in cell proliferation and adhesion in an external magnetic field relative to the pristine PECH.The results showed that the newly developed MPECH could potentially be used as a magnetically stimulated system in tissue engineering applications.     

Design, synthesis and properties of polyurethane hydrogels for tissue engineering

Due to their similarity to natural soft tissues, water-swellable polymeric materials (hydrogels) are, in principle, ideal candidates for scaffolds/matrices in tissue engineering. Polyurethanes (PU), hydrophilic but water-insoluble, can be obtained by the incorporation of hydrophilic soft segments, e.g. poly(ethylene oxide) (PEO). These materials possess the favorable characteristics of the family of PUs as well as the ability to mimic soft tissues. In this work, new crosslinked PU-hydrogels were prepared in a one-step bulk polymerization process using an aliphatic diisocyanate, PEO, a low molecular weight diol, and a tri-functional crosslinking agent. A porous structure was also obtained by air-incorporation under mechanical stirring at a controlled high speed during the polymerization. Structural characteristics of the compact (PU-HyC) and the porous (PU-HyP) material were investigated. Molecular weight between cross-links, m¯_c, and crosslinking density, _x, were typical of a low crosslinking degree. A homogeneous distribution of non-interconnecting pores (100 m) was observed in PU-HyP. Both materials showed a high water adsorption. The swelling behavior and weight loss in water was affected by porosity. For their mechanical behavior in the swollen state, the novel PU hydrogels can be considered for biomedical applications where good mechanical properties are required (i.e. 3D scaffold for tissue engineering).     

Novel crosslinked alginate/hyaluronic acid hydrogels for nerve tissue engineering

Artificial tissue engineering scaffolds can potentially provide support and guidance for the regrowth of severed axons following nerve injury. In this study, a hybrid biomaterial composed of alginate and hyaluronic acid (HA) was synthesized and characterized in terms of its suitability for covalent modification, biocompatibility for living Schwann cells and feasibility to construct three dimensional (3D) scaffolds. Carbodiimide mediated amide formation for the purpose of covalent crosslinking of the HA was carried out in the presence of calcium ions that ionically crosslink alginate. Amide formation was found to be dependent on the concentrations of carbodiimide and calcium chloride. The double-crosslinked composite hydrogels display biocompatibility that is comparable to simple HA hydrogels, allowing for Schwann cell survival and growth. No significant difference was found between composite hydrogels made from different ratios of alginate and HA. A 3D BioPlotter^TM rapid prototyping system was used to fabricate 3D scaffolds. The result indicated that combining HA with alginate facilitated the fabrication process and that 3D scaffolds with porous inner structure can be fabricated from the composite hydrogels, but not from HA alone. This information provides a basis for continuing in vitro and in vivo tests of the suitability of alginate/HA hydrogel as a biomaterial to create living cell scaffolds to support nerve regeneration.     

A novel porous scaffold fabrication technique for epithelial and endothelial tissue engineering

Porous scaffolds have the ability to minimize transport barriers for both two- (2D) and three-dimensional tissue engineering. However, current porous scaffolds may be non-ideal for 2D tissues such as epithelium due to inherent fabrication-based characteristics. While 2D tissues require porosity to support molecular transport, pores must be small enough to prevent cell migration into the scaffold in order to avoid non-epithelial tissue architecture and compromised function. Though electrospun meshes are the most popular porous scaffolds used today, their heterogeneous pore size and intense topography may be poorly-suited for epithelium. Porous scaffolds produced using other methods have similar unavoidable limitations, frequently involving insufficient pore resolution and control, which make them incompatible with 2D tissues. In addition, many of these techniques require an entirely new round of process development in order to change material or pore size. Herein we describe “pore casting,” a fabrication method that produces flat scaffolds with deterministic pore shape, size, and location that can be easily altered to accommodate new materials or pore dimensions. As proof-of-concept, pore-cast poly(ε-caprolactone) (PCL) scaffolds were fabricated and compared to electrospun PCL in vitro using canine kidney epithelium, human colon epithelium, and human umbilical vein endothelium. All cell types demonstrated improved morphology and function on pore-cast scaffolds, likely due to reduced topography and universally small pore size. These results suggest that pore casting is an attractive option for creating 2D tissue engineering scaffolds, especially when the application may benefit from well-controlled pore size or architecture.     

Human vascular endothelial cells in primary cell culture for the evaluation of nanoparticle bioadhesion

Nanoparticles (NP) are employed in various therapeutic approaches for innovative drug delivery strategies. Among them, there is drug delivery to the brain and sustained release forms for intravenous drug delivery. In order to optimize drug carriers and to elucidate involved mechanisms such as bioadhesion and cellular uptake, NP were surface modified and analyzed for their interaction with human endothelial cells in cell culture. Fluorescently labeled NP of different diameters (50 to 1000 nm) were surface modified either by simple adsorption of chitosan or by covalent binding to the lectin ulex europaeus agglutinin and thereafter applied to human endothelial cells for different incubation periods. After incubation with NP the binding of NP was quantified directly by the fluorescence emission signals from the cell layers. In order to visualize the binding behaviour, NP were localized three-dimensionally in the cell layer by confocal laser scanning microscopy. Cell binding experiments in phosphate buffer were observed to be particle size dependent with the 50 nm NP showing the highest binding percentage over all experiments. Binding decreased with increasing particle diameter and shorter incubation interval. The adhesion was further enhanced by NP surface modifications in the order blank < chitosan < lectin. The presence of plasma proteins enhanced the adhesiveness of chitosan coated NP, while the binding of lectin coated NP was inhibited. Experiments at 4 degrees C indicated the involvement of an active process in the binding of NP to endothelial cells.     

Human osteoprogenitor responses to orthopaedic implant: mechanism of cell attachment and cell adhesion

Cell culture models are becoming prevalent in the investigation of tissue responses to implant materials. Cellular attachment and cell adhesion studies can aid in the development of more effective orthopaedic and dental implants. Cell attachment was studied on extracellular matrix proteins (type I, IV collagen, peptide solubilized elastin (PSE), fibronectin laminin). Human osteoprogenitor cells responded differently to these collagenous and non-collagenous proteins. PSE and type I or type IV collagen are the most effective proteins in cellular attachment and cell spreading. Cell behaviour was measured in the presence of macroporous materials (Porites astreoïdes from the West Indies and a bovine hydroxyapatite ceramic ENDOBON^®) and bioartificial connective matrices comprising hydroxyapatite, peptide solubilized elastin, collagen, fibronectin and chondroïtin-6-sulfate, components of the extracellular matrix (ECM). Human osteoprogenitor cells responded differently to the materials tested according to the content of components of ECM. About 40% of attached cells were obtained on the composite materials PSE, collagen, fibronectin and chondroïtin-6-sulfate, and about 10% on the macroporous materials, whatever their porosity and their chemical components. These results demonstrate a need for more effective surface treatment to promote cell attachment, cell spreading and cell growth.     

Alginate hydrogels crosslinked with different strontium-calcium ratios as injectable scaffolds for bone tissue engineering

The easy loss of crosslinking ions in alginate can result in a structural collapse of the physiological environment, thereby losing its characteristics as a bone scaffold. Meanwhile, alginate lacks osteoconductive properties, which are necessary for ideal bone scaffolds. In this study, strontium (Sr) in combination with calcium (Ca) at different ratios were used as a crosslinking agent for the alginate to investigate the effect of Ca–Sr ratio on the physicochemical properties and biological preformation of alginate hydrogel. Here, Ca and Sr in different weight ratios (4:0, 3:1, 2:2, 1:3, and 0:4) were employed as crosslinking agents. The physicochemical properties of hydrogels, including pore size, elastic modulus, degradation rate and swelling ratio, could be effectively tuned by controlling the amount of Sr. The ion release experiment revealed a burst release of Sr²⁺ in the first day after crosslinking. However, after 3 days, the amount of Sr²⁺ release had significantly declined and was proportional to the total strontium initially introduced into the alginate. Meanwhile, the live/dead results exhibited higher cell viability for alginate with 2:2 Ca–Sr weight ratio. The alginate with 2:2 Ca–Sr ratio not only improved osteoblastic attachment, but also up-regulated the alkaline phosphatase activity, the expression of osteogenic marker genes, and relative growth factors. These findings indicate that alginate with 2:2 Ca–Sr ratio might be a promising scaffold for bone tissue engineering.

Graphical abstract

     

壳聚糖涂层聚乳酸细胞微载体的制备和性能

采用氨解技术在聚乳酸微球表面引入自由氨基,再利用戊二醛将氨基转化为醛基,最后采用接枝涂层技术将壳聚糖固定到聚乳酸微球表面,制备了壳聚糖表面改性的聚乳酸细胞微载体.分别采用茚三酮法和乙酰丙酮-对二甲氨基苯甲醛法测定了聚乳酸微球表面的氨基和壳聚糖含量.发现氨基的量初始随氨解时间的延长而增大,达到最大(2.94×10-7mol/mg)后保持不变.与空白聚乳酸微球相比,软骨细胞在壳聚糖改性聚乳酸微球表面能够更有效地粘附和生长,分布更为均匀.     

A high strength semi-degradable polysaccharide-based hybrid hydrogel for promoting cell adhesion and proliferation

Traditionally, practical applications of polysaccharide hydrogels have been limited for their weak mechanical properties under physiological conditions. In this study, we constructed a novel polysaccharide-based semi-degradable hydrogel whose network was constructed by chemical cross-linking of glycidyl methacrylate-modified laminarin and the hydrogen bonded physical cross-linking of poly(N-acryloyl glycinamide). In addition, the introduction of 1-vinyl-1,2,4-triazole content could increase the equilibrium water content of hydrogels and endow hydrogels with anti-bacterial and anti-inflammatory abilities. The prepared hydrogels exhibited comprehensive high mechanical properties up to 0.63 MPa tensile strength, 650% stretchability, and maximum 3.2 MPa compressive strength at swelling equilibrium state. The hydrogen bond interactions could well support the three-dimensional network of hydrogel after the degradation of modified laminarin. Meanwhile, the content of laminarin could facilitate cell adhesion and proliferation on the surface of hydrogel. It is anticipated that this high strength semi-degraded hydrogel may find a promising application as articular cartilage replacement.     

A 3D biodegradable protein based matrix for cartilage tissue engineering and stem cell differentiation to cartilage

A protein based 3D porous scaffold is fabricated by blending gelatin and albumin. The biomimetic biodegradable gelatin, promoted good cell adhesion and its hydrophilic nature enabled absorption of culture media. Albumin is proposed to serve as a nontoxic foaming agent and also helped to attain a hydrophobic-hydrophilic balance. The hydrophobic-hydrophilic balance and appropriate crosslinking of the scaffold avoided extensive swelling, as well as retained the stability of scaffold in culture medium for long period. The scaffold is found to be highly porous with open interconnected pores. The adequate swelling and mechanical property of the scaffold helped to withstand the loads imparted by the cells during in vitro culture. The scaffold served as a nontoxic material to monolayer of fibroblast cells and is found to be cell compatible. The suitability of scaffold for chondrocyte culture and stem cell differentiation to chondrocytes is further explored in this work. The scaffold provided appropriate environment for chondrocyte culture, resulting in deposition of cartilage specific matrix molecules that completely masked the pores of the porous scaffold. The scaffold promoted the proliferation and differentiation of mesenchymal stem cells to chondrocytes in presence of growth factors. The transforming growth factor, TGFbeta3 promoted better chondrogenic differentiation than its isoform TGFbeta1 in this scaffold.     

Evaluation of alginate hydrogels under in vivo–like bioreactor conditions for cartilage tissue engineering

Alginate hydrogels in forms of discs and packed beds of microbeads (~800 μm) were tested in a novel bioreactor at 10% strain using two regimes: at a loading rate of 337.5 μm/s and at sequential increments of 50 μm displacement every 30 min. Compressive strength increased with the increase in alginate concentration (1.5 vs. 2% w/w) and the content of guluronic residues (38.5 vs. 67%). Packed beds of microbeads exhibited significantly higher (~1.5–3.4 fold) compression moduli than the respective discs indicating the effects of gel form and entrapped water. Short-term cultivation of microbeads with immobilized bovine calf chondrocytes (1.5% w/w, 33 × 10⁶ cells/ml) under biomimetic conditions (dynamic compression: 1 h on/1 h off, 0.42 Hz, 10% strain) resulted in cell proliferation and bed compaction, so that the compression modulus slightly increased. Thus, the novel bioreactor demonstrated advantages in evaluation of biomaterial properties and cell-biomaterial interactions under in vivo–like settings.     

Preparation and characterization of polyvinyl alcohol hydrogels crosslinked by biodegradable polyurethane for tissue engineering of cartilage

Polyurethane was prepared from hexamethylene diisocyanate (HMDI) and polycaprolactone diol (PCL) with stoichiometry ratio of two in a reactor to form prepolymer. Polyvinyl alcohol (PVA) at PVA/prepolymer ratios of 8, 4, 2 and 1 was crosslinked with the former degradable polyester polyurethane. Fourier transform infrared (FTIR) was employed to confirm polyurethane formation during the course of reactions. FTIR spectrum revealed bands at 1729–1733 cm^? 1 and 3347–3340 cm^? 1 which indicates carbonyl and NH of amine groups, respectively. Polyurethane formation was also confirmed by the absence of the isocyanate peaks (NCO) at 2270 cm^? 1. Dynamic mechanical thermal analysis (DMTA) showed that by increasing prepolymer concentration glass transition temperature decreases from 26 °C for PVA to 19 °C for sample with PVA/prepolymer ratio of 4 and then it rises up to 31 °C. Water uptake measurements illustrated about four fold reduction in swelling ratio of PVA after crosslinking and the sample with equal amounts of PVA and PPU had water uptake of 100%, close to that of a natural cartilage and much less than PVA (425%). All samples had compressive modulus in the range of the articular cartilage (1.9–14.4 MPa). The morphology of the isolated cells on the samples was evaluated by scanning electron microscopy (SEM) and revealed cell attachment and proliferation. The cell viability (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) and GAG expression (dimethylmethylene blue, DMMB) assays with human chondrocytes on the sample with PVA/prepolymer ratio of one showed about 14 and 33% increase in cell viability and GAG expression after 14 days of culture compare to the PVA, respectively.