Interconnectivity and permeability of supercritical fluid-foamed scaffolds and the effect of their structural properties on cell distribution

This study aims to investigate interconnectivity and permeability of scCO₂-foamed scaffolds and the influence of structural scaffold properties on cell distribution. Supercritical fluid technology was utilized to fabricated scaffolds from 37 kDa, 53 kDa and 109 kDa PLGA (85:15). Pore size, pore size distribution and porosity were quantified by MicroCT, and window sizes were measured using SEM. A novel interconnectivity algorithm allowed the quantification of scaffold interconnectivity in three space dimensions. To determine the permeability of porous materials direct perfusion experiments were performed, where a known flow rate was applied to measure the pressure differential across the scaffolds. The permeability was calculated using Darcy's law. Largest pore sizes, porosities, interconnectivities and permeabilities were obtained for scaffolds fabricated from 37 kDa PLGA. These scaffolds showed a heterogeneous pore structure and distribution, whereas homogeneous pore structure, smallest pore sizes, porosities, interconnectivities and permeabilities were observed for scaffolds fabricated from 109 kDa PLGA. The distribution of 3T3 fibroblasts through scCO₂-foamed scaffolds was investigated by MicroCT and MTT staining. Cells were further visualized by fluorescent imaging. Uniform cell distribution was observed on scaffolds fabricated from 109 kDa PLGA and an average of 10% of the total scaffold volume was covered with cells that had adhered onto them.     

Layered-lamination Preparing of Tissue Engineering Scaffolds via Emulsion Templates Method

Three-dimensional PHB porous scaffolds were prepared based on the mono-membrane fabricated by emulsion templates method. The key factors of the method affecting the pore size and porosity of the PHB scaffolds were studied. The surface of PHB scaffolds were investigated by scanning electron microscope (SEM), which showed the even pore size and regularly arranged pore. The transect of the PHB scaffolds prepared using the templates method was good. Moreover, the effects of variation of surfactant content (P%) and water content (R) on the pore size and porosity of PHB films were discussed. Preliminary studies showed that when P% is less than 20%, the pore size made by emulsion templates ranged from 5 µm to 30 µm with the value of P increasing. As P% is up to 20%, it was interesting to see that the scaffolds had multi-pore size distribution, i.e., median pore sizes were about 5 µm and inside the wall of pore, there existed numerous micro-pore sizes, which can be controlled from 100 nm to 500 nm only by adjusting the parameter R of the microemulsion. The degradation experiment indicated that the degradation of PHB scaffolds were accelerated by enzyme in vitro and the porous configuration was favorable to its degradation.     

Impact and biocompatibility studies on Mg2+-substituted Apatite-derived 3D porous scaffolds for hard tissue engineering

The objective of this study was to develop Mg²⁺-substituted Apatite scaffolds by slip-casting method. The Apatite scaffolds were prepared as engineering constructs with interconnected pore structure with a pore size of 128-194 μm range. The physicochemical properties such as crystalline phase, functional group, microstructure, pore size distribution, and elemental compositions of the scaffolds were characterized. The bioactivity of the developed porous scaffolds was investigated in Simulated Body Fluid (SBF) for various time periods (3 and 7 days). In vitro bioactivity results confirm the hydroxyl Apatite layer formation of the scaffolds and results suggest that the developed microporous scaffold could be used as suitable candidates in bone tissue engineering.     

Assessment of cell proliferation in knitting scaffolds with respect to pore‐size heterogeneity,surface wettability,and surface roughness

In this study, various types of poly(ε‐caprolactone) (PCL) knitting scaffolds were fabricated and analyzed to assess the cell‐culturing characteristics of knitting scaffolds with respect to pore‐size heterogeneity, surface wettability, and surface roughness. First, control knitting scaffolds were fabricated using 150‐µm‐diameter PCL monofilaments. Using chloroform and NaOH, PCL knitting scaffolds with varying roughness, pore‐size heterogeneity, and surface wettability were fabricated. Cell‐culture assessments were performed on these six types of PCL knitting scaffolds. Saos‐2 cells were used for cell assessments and cultured for 14 days on each scaffold. Consequently, heterogeneous pore‐size distribution and high surface wettability were found to enhance cell proliferation in knitting scaffolds. In addition, for highly hydrophobic knitting scaffolds exhibiting water contact angles greater than 110 degrees, smaller surface roughness was found to enhance cell proliferation. According to this study, in the case of knitting scaffold, NaOH‐treated knitting scaffold, without any control for the pore‐size homogenization, could be a candidate as the optimal knitting scaffold. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42566.     

Fabrication of porous scaffolds with a controllable microstructure and mechanical properties by porogen fusion technique

Macroporous scaffolds with controllable pore structure and mechanical properties were fabricated by a porogen fusion technique. Biodegradable material poly (d, l-lactide) (PDLLA) was used as the scaffold matrix. The effects of porogen size, PDLLA concentration and hydroxyapatite (HA) content on the scaffold morphology, porosity and mechanical properties were investigated. High porosity (90% and above) and highly interconnected structures were easily obtained and the pore size could be adjusted by varying the porogen size. With the increasing porogen size and PDLLA concentration, the porosity of scaffolds decreases, while its mechanical properties increase. The introduction of HA greatly increases the impact on pore structure, mechanical properties and water absorption ability of scaffolds, while it has comparatively little influence on its porosity under low HA contents. These results show that by adjusting processing parameters, scaffolds could afford a controllable pore size, exhibit suitable pore structure and high porosity, as well as good mechanical properties, and may serve as an excellent substrate for bone tissue engineering.     

Microwave processing of starch‐based porous structures for tissue engineering scaffolds

A novel microwave (MW) processing technique was used to produce biodegradable scaffolds for tissue engineering from different types of starch‐based polymers. Potato, sweet potato, corn starch, and nonisolated amaranth and quinoa starch were used to produce porous structures. Water and glycerol were used as plasticizers for the different types of starch. Characterization of the pore morphology of the scaffolds was carried out with scanning electron microscopy. Three‐dimensional structures with variable porosity and pore size distribution were obtained with the MW foaming technique. The amount of remaining water in the scaffolds and their corresponding densities showed important variations among the different types of starch. Compressive mechanical properties were assessed by indentation tests, and a strong dependence of the indentation stress on the average pore size was found. Studies in simulated body fluid were used to assess the in vitro bioactivity, degradability, and surface topology evolution in the scaffolds. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1332–1339, 2007     

Design and fabrication of 3D‐plotted polymeric scaffolds in functional tissue engineering

Regenerating the load‐bearing tissues requires 3D scaffolds that balance the temporary mechanical function with the biological requirements. In functional tissue engineering, designing scaffolds with biomimetic mechanical properties could promote tissue ingrowth since the cells are sensitive to their local mechanical environment. This work aims to design scaffolds that mimic the mechanical response of the biological tissues under physiological loading conditions. Poly(L ‐lactide) (PLLA) scaffolds with varying porosities and pore sizes were made by the 3D‐plotting technique. The scaffolds were tested under unconfined ramp compression to compare their stress profile under load with that of bovine cartilage. A comparison between the material parameters estimated for the scaffolds and for the bovine cartilage based on the biphasic theory enabled the definition of an optimum window for the porosity and pore size of these constructs. Moreover, the finite element prediction for the stress distribution inside the scaffolds, surrounded by the host cartilaginous tissue, demonstrated a negligible perturbation of the stress field at the site of implantation. The finite element modeling tools in combination with the developed methodology for optimal porosity/pore size determination can be used to improve the design of biomimetic scaffolds. POLYM. ENG. SCI., 47:608–618, 2007. © 2007 Society of Plastics Engineers.     

Relationship between pore fluid velocity distribution and pore size distribution

This study investigated the normalized velocity magnitude distribution and normalized pore volume distribution in different porous media with porosity between 13.5% and 85%, including sandstones, carbonates, synthetic silica, sphere packings and fiber scaffolds. It was found that both velocity magnitude and pore size follow the same distribution. These results allow the prediction of the velocity distribution in a porous medium when its pore structure is known or tuning the velocity by controlling the pore structure.     

Effects of pore size on the mechanical and biological properties of stereolithographic 3D printed HAp bioceramic scaffold

In this study, hydroxyapatite (HAp) scaffolds with the pore size of 400, 500, and 600 μm were prepared by stereolithographic 3D printing (SL-3DP). The effects of pore size on mechanical and biological properties of the HAp scaffolds were investigated. Firstly, the macro- and microstructure of the HAp scaffolds were observed. Then, the compressive strength of the HAp scaffolds were tested. Finally, the biological properties of the HAp scaffolds were further characterized in vitro by the synthetic body fluid (SBF) solution immersion testing, as well as by using the cell proliferation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. From this study, it was found that the HAp scaffold with a pore size of 600 μm had the most promising application prospect.     

Fabrication of a porous wall and higher interconnectivity scaffold comprising gelatin/chitosan via combination of salt-leaching and lyophilization methods

A novel gelatin/chitosan scaffold with higher porosity and interconnectivity was designed through salt-leaching/lyophilization (SLL) method. The properties of the fabricated scaffolds were compared with conventional scaffolds, which are obtained by thermally induced phase-separation (TIPS) method. The scaffolds made by phase-separation method have high tensile strength, but suffer from less channel interconnectivity, pore uniformity and also low surface porosity. The microstructure, porosity, phosphate-buffered saline (PBS) solution absorption and tensile strength of the prepared scaffolds by SLL method were studied. In this work, SLL as a two-step technique is introduced for creating porosity to improve both channel interconnectivity and pore uniformity for water-soluble polymers in comparison with the TIPS method. The SLL technique includes two mechanisms: the first, leaching of mixed sodium chloride crystals and particles created during recrystallization of the dissolved NaCl and the second, phase separation during lyophilization at the pore walls. These two steps in porosity formation lead to special pore morphology, which is more suitable for cell culturing because of higher interconnectivity and rich surface porosity in comparison with the phase-separated scaffolds. The prepared scaffolds, using this technique with different salt/polymer ratios and salt crystal size, have 91?C97% porosity and 94?C190???m mean pore size with tensile strength of 72?C215?kPa and PBS solution absorption between 12.4 and 19 times dry weight. The pore size of scaffolds prepared using the SLL method could be adjusted independently of polymer solution concentration. These scaffolds have a great potential in skin tissue engineering application.     

A review of bioceramic porous scaffolds for hard tissue applications: Effects of structural features

Tissue engineering has acquired remarkable attention as an alternative strategy to treat and restore bone defects during recent years. A scaffold is a fundamental component for tissue engineering, on which cells attach, proliferate and differentiate to form new desirable functional tissue. The composition, and structural features of scaffolds, including porosity and pore size, play a fundamental role in the success of tissue-engineered construct. This review summarizes the effect of porosity and pore size of bioceramic-based scaffolds on their mechanical properties and biological performances. The focus of this review is on scaffolds with porosities 40% and above. From the mechanical point of view, the degree of porosity is a more important factor than pore size and scaffolds with porosities greater than 40% were more likely to substitute trabecular bones. While for in vitro and in vivo performances, pore size appeared more influential feature and co-existence of macropores and micropores led to better bone formation.     

Poly(ε‐caprolactone) composite scaffolds loaded with gentamicin‐containing β‐tricalcium phosphate/gelatin microspheres for bone tissue engineering applications

In this study, novel poly(ε‐caprolactone) (PCL) composite scaffolds were prepared for bone tissue engineering applications, where gentamicin‐loaded β‐tricalcium phosphate (β‐TCP)/gelatin microspheres were added to PCL. The effects of the amount of β‐TCP/gelatin microspheres added to the PCL scaffold on various properties, such as the gentamicin release rate, biodegradability, morphology, mechanical strength, and pore size distribution, were investigated. A higher amount of filler caused a reduction in the mechanical properties and an increase in the pore size and led to a faster release of gentamicin. Human osteosarcoma cells (Saos‐2) were seeded on the prepared composite scaffolds, and the viability of cells having alkaline phosphatase (ALP) activity was observed for all of the scaffolds after 3 weeks of incubation. Cell proliferation and differentiation enhanced the mechanical strength of the scaffolds. Promising results were obtained for the development of bone cells on the prepared biocompatible, biodegradable, and antimicrobial composite scaffolds. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40110.     

Fabrication of Hydroxyapatite/Ethylene-Vinyl Acetate/Polyamide 66 Composite Scaffolds by the Injection-Molding Method

This research investigated the injection-molding techniques to produce hydroxyapatite (HA)/ethylene-vinyl acetate (EVA)/polyamide 66 (PA66) composite scaffolds. The effects of HA, EVA, azodicarbonamide (AC) content and shot size on the mechanical properties, pore morphology, porosity and crystallization behavior of the composite scaffolds were analyzed by XRD, DSC, SEM and mechanical test. The compressive modulus and strength of the HA/EVA/PA66 scaffolds with a pore size of 200–600 µm are close to the cancellous bone. Compared with common methods to fabricate scaffolds, this process makes the fabrication of composite scaffolds come true in a rapid and convenient manner.     

Pore Size Distribution and Blend Composition Affect In Vitro Prevascularized Bone Matrix Formation on Poly(Vinyl Alcohol)/Gelatin Sponges

This study aims at identifying compositional and architectural (pore size and distribution) parameters of biocompatible scaffolds, which can be best suitable for both osteoblasts and endothelial cells to produce optimized 3D cocultured constructs. Spongy scaffolds are prepared using poly(vinyl alcohol) (PVA) and gelatin (G) at different weight compositions (PVA/G range: 100/0–50/50, w/w) via emulsion and freeze‐drying. The higher the gelatin content, the larger is the volume occupied by higher size pores. Human umbilical vein endothelial cells and human mesenchymal stromal cells are independently differentiated on the scaffolds to select the best candidate for the coculture. The results of metabolic activity and histology on single platforms show both cell‐ and material‐type dependent outcomes. PVA/G 80/20 scaffolds are finally selected and allow the formation of mineralized matrix containing organized endothelial‐like structures. This study highlights the need for systematic investigations on multifactorial parameters of scaffolds to improve vascularized bone substitutes.     

Synthesis and cytocompatibility of collagen/hydroxyapatite nanocomposite scaffold for bone tissue engineering

Collagen/hydroxyapatite nanocomposite scaffolds were prepared by in situ precipitation and freeze‐drying approach. The synthesized collagen/hydroxyapatite nanocomposites were characterized using various modalities. It was revealed that the inorganic phase in the nanocomposite was carbonate‐substituted hydroxyapatite with low crystallinity. Morphology studies showed the uniform distribution of hydroxyapatite particles in the collagen hydrogel. In addition, hydroxyapatite particles were gradually becoming irregular enough and the surface morphology had more wrinkles with the increase of inorganic component. Morphology, mechanical properties and cell biocompatibility of the prepared nanocomposite scaffolds were evaluated. The scaffolds presented a well‐developed macropore structure with a pore size ranging from 100 to 200 μm and the pore size of scaffold can also be regulated by changing the organic/inorganic weight ratio. Furthermore, the growth of MG63 cells on scaffolds showed they could significantly promote the proliferation of cells and could be potential candidate for bone engineering applications. POLYM. COMPOS., 81–90, 2016. © 2014 Society of Plastics Engineers     

Fabrication of bioceramic scaffolds with pre-designed internal architecture by gel casting and indirect stereolithography techniques

A new technique of combining the gel casting and indirect rapid prototyping methods was utilized to fabricate macroporous β-tricalcium phosphate (β-TCP) scaffolds, which provided an excellent control over the internal architecture of scaffolds and enhanced their mechanical properties. A stereolithography apparatus was used to produce resin molds for ceramic gel casting. These molds were filled with a water based thermosetting ceramic slurry which solidifies inside the mold. After burning the resin mold and sintering, the β-TCP scaffolds with designed pore architecture were obtained. The pore morphology, size, and distribution of the resulting scaffolds were characterized using a scanning electron microscope. X-ray diffraction was used to determine the crystal structure and chemical composition of scaffolds. The mechanical measurements showed that the average compressive strength was 16.1 ± 0.8 MPa.     

Three-dimensional fibroin/collagen scaffolds derived from aqueous solution and the use for HepG2 culture

Although three-dimensional fibroin scaffolds have been prepared with freeze–drying method, these scaffolds still cannot meet the requirements of tissue engineering. In this article, a new process is described to form fibroin-based porous scaffolds with controllable structure and morphological features. When collagen was added to fibroin solution, the viscosity of the blend solution increased because of the interaction between fibroin and collagen, and then it restrained the unwanted fibroin leaf formation in freezing process that generally appeared in the previous fibroin scaffold preparation. With methanol treatment, the fibroin/collagen scaffolds became water-stable, following the transition from random and -helix to β-sheet conformation. The aqueous-fibroin porous scaffolds had highly homogeneous and interconnected pores with pore sizes ranging from 127 to 833 μm, depending on the fibroin concentration. The porosity of scaffolds was >90%, and the yield strength and modulus were up to 354±25 kPa and 30±0.1 MPa, respectively, when the blend solution, containing 20% collagen, maintained 4% fibroin concentration. Adhesion, spreading and proliferation of HepG2 cells on fibroin and fibroin/collagen blend scaffolds were also observed to investigate the biocompatibility. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the adding of collagen evidently facilitated HepG2 attachment and proliferation in vitro. These new fibroin based three-dimensional scaffolds provided much more excellent properties due to the greatly improved control of pore size, the uniform pore distribution, the hydrophility, the mechanical properties and the biocompatibility compared with those of reported three-dimensional fibroin scaffolds.     

Polycaprolactone‐hydroxy apatite composites for tissue engineering applications

In this study, scaffolds with polycaprolactone (PCL) and hydroxy apatite (HA) were produced. Their properties are not sufficient to be used alone. Oleic Acid (OA) and glycerol monooleate (GMO) as organic additives were selected for a homogeneous distribution of the ceramic material in the polymer matrix. Biocomposite materials were prepared with solvent casting‐salt leaching technique using dichloromethane as the solvent. Salt was used as the porosifier. Materials were kept in simulated body fluid (SBF) to determine the bioactivity in vitro conditions. FTIR and EDX analyses for chemical characterization, tensile and compressive tests for mechanical properties, SEM analyses for surface properties and BET analyses for pore sizes, total surface areas and total pore volumes of scaffolds were performed. FTIR, EDX, and SEM analyses were repeated after SBF treatment. Pore diameters were highly increased with 3 and 20 wt% HA addition. Small amount of GMO addition is more effective on pore size. Mechanical properties of scaffolds were suitable for soft tissue applications, as smooth muscle cells, skin and cancellous bone. The cytotoxicity and cell proliferation on scaffolds were studied with smooth muscle cells (SMC) and L929 fibroblastic cells in vitro. No cytotoxic effect was observed for the scaffolds in both cell types. J. VINYL ADDIT. TECHNOL., 24:248–261, 2018. © 2016 Society of Plastics Engineers     

Preparation of chitosan‐nanohydroxyapatite composite scaffolds by a supercritical CO2 assisted process

In this study, chitosan‐nanohydroxyapatite composite scaffolds were prepared by a supercritical fluid assisted process. For this purpose, different amounts of nanohydroxyapatite particles, that is, 0.25, 0.50, and 1.00 wt% were added to chitosan (deacetylation degree: DD 75–85%) solution (2%, w/v, in acetic acid). The gels were then frozen at −20°C, treated in acetone and dried in a supercritical fluid extractor under a constant CO₂ flow of 15 g/min at 35°C and 200 bar for 5 h to obtain porous scaffolds. Scanning electron microscope views showed that the drying of gels under supercritical CO₂ lead to the formation of microporous scaffolds with a pore size distribution of 30–150 μm. Addition of nanohydroxyapatite particles did not significantly affect the pore size distribution. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy and X‐Ray diffraction analyses supported the successful incorporation of nanohydroxyapatite particles in the scaffold. An increase in water uptake and mechanical strength were observed in composite scaffolds. The results obtained from this study indicated that chitosan‐nanohydroxyapatite scaffolds prepared by using supercritical CO₂ shall be considered as a potential candidate for bone tissue engineering applications. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers     

Solvent-Free Fabrication of Tissue Engineering Scaffolds With Immiscible Polymer Blends

A completely organic solvent-free fabrication method is developed for tissue engineering scaffolds by gas foaming of immiscible polylactic acid (PLA) and sucrose blends, followed by water leaching. PLA scaffolds with above 90% porosity and 25–200 µm pore size were fabricated. The pore size and porosity was controlled with process parameters including extrusion temperature and foaming process parameters. Dynamic mechanical analysis showed that the extrusion temperature could be used to control the scaffold strength. Both unfoamed and foamed scaffolds were used to culture glioblastoma (GBM) cells M059 K. The results showed that the cells grew better in the foamed PLA scaffolds. The method presented in the paper is versatile and can be used to fabricate tissue engineering scaffolds without any residual organic solvents.