Solid Free-Form Fabrication-Based PCL/HA Scaffolds Fabricated with a Multi-head Deposition System for Bone Tissue Engineering

In this study, we fabricated polycaprolactone/hydroxyapatite (PCL/HA) scaffolds with a multi-head deposition system, a solid free-form fabrication technology that was developed in our previous study. The bone regeneration potential of the scaffolds was compared with that of PCL scaffolds fabricated with the same system. The fabricated scaffolds had a pore size of 400 μm and a porosity of 66.7%. The PCL/HA scaffolds had higher mechanical strength and modulus than the PCL scaffolds. To compare the osteogenic potential, the two types of scaffolds were seeded with rat osteoblasts and cultured in vitro or implanted subcutaneously into athymic mice. The cells cultured on PCL/HA scaffolds expressed higher levels of osteopontin and osteonectin, both of which are osteogenic proteins. The PCL/HA scaffolds resulted in larger bone area and calcium deposition in the implants compared to the PCL scaffolds.     

A First-in-Human Evaluation of a Novel Mesh-Covered Stent for Treatment of Carotid Stenosis in Patients at High Risk for Endarterectomy: 30-Day Results of the SCAFFOLD Trial

Objectives

The primary purpose of this study was the composite of major adverse events through 30 days post-index procedure or ipsilateral stroke from 30 days to 1 year (365 days). Presented here is the composite of death, stroke, and myocardial infarction (MI) through 30 days.

A novel construct as a cell carrier for tissue engineering

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.     

Electrospun Silk Fibroin–Hydroxybutyl Chitosan Nanofibrous Scaffolds to Biomimic Extracellular Matrix

Silk fibroin (SF)–hydroxybutyl chitosan (HBC) blend nanofibrous scaffolds were fabricated using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and trifluoroacetic acid (TFA) as solvents to biomimic the native ECM by electrospinning. SEM results showed that the average nanofibrous diameter increased when the content of HBC was raised from 20% to 100%. Whereas water contact angle measurements confirmed that SF/HBC nanofibrous scaffolds with different weight ratios were of good hydrophilicity. Both the tensile strength and the elongation at break were improved obviously when the weight ratio of SF to HBC was 20:80. ¹³C-NMR clarified that SF and HBC molecules existed in H-bond interactions, but HBC did not induce SF conformation to transform from random coil form to β-sheet structure. Moreover, the use of genipin vapour not only induced conformation of SF to convert from random coil to β-sheet structure but also acted as a cross-linking agent for SF and HBC. Cell viability studies demonstrated that SF/HBC nanofibrous scaffolds presented good cellular compatibility. Thus, electrospun SF/HBC blended nanofibres may provide an ideal biomimic tissue-engineering scaffold.     

Multiphase blends from poly(L-lactide) and poly(methyl mathacrylate)

Melt processing of poly(L-lactide) (PLLA) and poly(methyl methacrylate) (PMMA) was conducted over a targeted range of compositions with PLLAs of 118 and 316 kDa in molecular mass to identify morphologies and the phase relationships in these blends. These blends are of interest for use in biomaterials and the morphologies are critical for tissue-engineering studies where biodegradability, pore connectivity and surface texture control tissue viability and adhesion. Simple extrusion of the two polymers produced multiphase blends with an average domain size near 25 μm. Scanning electron microscopy and dynamic mechanical analysis demonstrated that these blends are immiscible, at least in a metastable sense, and regions of co-continuous structures were identified. Such co-continuous, which occurred generally in accordance with rheology prediction models, exhibit a fine interconnected structure that appears effective for fabricating certain biomaterials. A broad and unexpected transition appears in these blends, as measured by modulated differential scanning calorimetry between 70 and 100°C, which may be the glass transition temperature of an alloy phase. The magnitude of this transition is greatest in the fine-structured co-continuous composition region of blends, suggesting the presence of a complex or other derivative of the two primary phases.     

In Vitro Evaluation of Elastic Multiblock Co-polymers as a Scaffold Material for Reconstruction of Blood Vessels

There is a need to create cell- and histocompatible implant materials, which might temporarily replace the mechanical function of a native tissue for regenerative therapies. To match the elastic behavior of the native tissue two different multiblock co-polymers were investigated: PDC, consisting of poly(p-dioxanone) (PPDO)/poly(ε-caprolactone) (PCL), and PDD, based on PPDO/poly((adipinate-alt-1,4-butanediol)-co-(adipinate-alt-ethylene glycol)-co-adipinate-alt-diethylene glycol) (Diorez). PDC is capable of a shapememory effect. Both multiblock co-polymers show an improved elasticity compared to materials applied in established vascular prosthesis. PDD is softer than PDC at 20°C, while PDC maintains its elasticity at 37°C. Thermodynamic characteristics indicate a more polar surface of PDD. Low cell adhesion was found on surfaces with low molar free energy of hysteresis (ΔG) derived from contact angle measurements in wetting and dewetting mode and high cell adhesion on high-ΔG surfaces. An increasing content of PCL in PDC improved cell adhesion and spreading of human umbilical vein endothelial cells. The prothrombotic potential of PDD is higher than PDC. Finally, it is concluded that PDC is a promising material for vascular tissue engineering because of its improved elastic properties, as well as balanced prothrombotic and anti-thrombotic properties with endothelial cells.     

Homogeneous Chitosan/Poly(L-Lactide) Composite Scaffolds Prepared by Emulsion Freeze-Drying

The combination between chitosan (CS)-based hydrophilic extracellular matrix polysaccharide and polylactide (PLA)-based hydrophobic biodegradable aliphatic polyester is a challenge in the biomaterials field. This study investigated the formation of homogeneous chitosan/poly(L-lactide) (CS/PLLA) porous composite scaffold using a novel emulsion freeze-drying technique. An oil-in-water (O/W) emulsification system was used in the presence of surfactant Tween-80, in which CS solution was used as the water phase and PLLA solution was used as the oil phase. The composite scaffolds showed well interconnected pore structures and homogenous distribution of CS and PLLA when the PLLA volume fraction was not higher than 50%. Once the PLLA content increased to 75%, SEM micrographs demonstrated that the two components present phase separation region. FT-IR analysis revealed that there are strong hydrogen bond interactions between CS and PLLA components. The porosity of the CS/PLLA composites was in the range of 85–90% and showed a slight decrease with increasing PLLA dose. The mechanical properties of the composites lay between that of the pure CS and the PLLA scaffold. The compressive strength increased from 0.17 to 0.21 MPa, while the compressive modulus increased from 2.37 to 3.38 MPa as the PLLA contents increased from 25 to 75%. In vitro cytotoxicity was evaluated by MTT assay. The results indicated that MC3T3-E1 cell viability and proliferation in the CS/PLLA scaffold were comparable to that in the CS scaffold, and much higher than that in the PLLA scaffold. The successful hydrophilic polysaccharide and hydrophobic polyester system offers a new delivery method of growth factors and a novel scaffold design for tissue engineering.     

Preparation of Interconnected Porous Chitosan Scaffolds by Sodium Acetate Particulate Leaching

For tissue-engineering applications, a 3D porous chitosan scaffold was simply prepared from a mixture of acidic chitosan solution and sodium acetate particles as the porogen by a salt-leaching method. Differences in the porous structure in terms of pore morphology and interconnectivity between the salt-leached chitosan scaffold and phase-separated scaffold as the control were examined by using scanning electron microscopy, protein release and enzymatic degradation tests. A fibroblast (NIH-3T3) cell culture was performed for cell affinity evaluation. The chitosan scaffold prepared by salt-leaching showed good interconnectivity and improved mechanical properties. Furthermore, the chitosan scaffolds showed a high initial cell adhesion after 4 h cell culture and increased cell proliferation than the control. Thus, salt-leached chitosan scaffolds can be used for various tissue-engineering applications.     

Effects of In Situ and Physical Mixing on Mechanical and Bioactive Behaviors of Nano Hydroxyapatite–Chitosan Scaffolds

Nano hydroxyapatite (HAP) was employed to intensify chitosan (CS) scaffolds by two methods. The first one is nano HAP crystallized in situ from the CS matrix by a biomimetic method (in situ scaffold). In the second method the sol–gel nano HAP powder was added directly to the CS solution (physical mixing scaffold). The distribution status of nano HAP was examined by scanning electron microscopy. The compressive performance was measured by a universal material testing machine. The in vitro study in stimulated body fluid was performed to evaluate the biological properties of both scaffolds. MTT testing and alkaline phosphatase activity from human bone mesenchymal stem cell culture showed differences in biocompatibility and bioactivity between the scaffolds. The results indicated that the in situ scaffold possessed more excellent mechanical and bioactive behaviors than that of the physical mixing scaffold.     

Effects of the Surface Characteristics of Nano-Crystalline and Micro-Particle Calcium Phosphate/Chitosan Composite Films on the Behavior of Human Mesenchymal Stem Cells In Vitro

Calcium phosphate (CaP) compounds, the main inorganic constituent of mammalian bone tissues, are believed to support bone precursor cell growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the present study, different scales of CaP crystals on chitosan membranes were prepared for culture of human mesenchymal stem cells (hMSCs) in vitro. A series of aqueous CaP suspensions with different concentrations were mixed with chitosan solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. With different weight ratios of CaP in chitosan solution, the various surface characteristics of nano-amorphous (C/CaP 0.1), nano-crystalline (C/CaP 0.5) and micro-particle (C/CaP 2) CaP compounds were examined by scanning electron microscopy and electron dispersion spectroscopy. X-ray diffraction on micro-particles of CaP indicated the formation of crystalline hydroxyapatite. The behavior of hMSCs, including proliferation, cell spreading and osteogenic differentiation, was studied on the C/CaP films. In basal culture medium, the incorporation of CaP into chitosan films could promote the proliferation of hMSCs. The C/CaP 0.5 film with connected CaP nano-crystals had better cellular viability. The fluorescence microscope images at 14 days of culture revealed extensive networks of F-actin filaments of hMSCs on chitosan, C/CaP 0.1 and C/CaP 0.5 films. The cellular morphology on C/CaP 2 film with discrete CaP micro-particles was partly restrained. In osteogenic medium, the alkaline phosphatase (ALP) activity of hMSCs increased and showed the process of osteogenic differentiation. The ALP levels on C/CaP 2 film were higher than those on C/CaP 0.1 and C/CaP 0.5 films. These results demonstrated that the crystallinity and topography of CaP on chitosan membranes could modulate the behaviors of cultured hMSCs in vitro.