Porogen Leaching Research Articles

Three-Dimensional Microfibrous Scaffold with Aligned Topography Produced via a Combination of Melt-Extrusion Additive Manufacturing and Porogen Leaching for In Vitro Skeletal Muscle Modeling.

Skeletal muscle tissue (SMT) has a highly hierarchical and anisotropic morphology, featuring aligned and parallel structures at multiple levels. Various factors, including trauma and disease conditions, can compromise the functionality of skeletal muscle. The in vitro modeling of SMT represents a useful tool for testing novel drugs and therapies. The successful replication of SMT native morphology demands scaffolds with an aligned anisotropic 3D architecture. In this work, a 3D PCL fibrous scaffold with aligned morphology was developed through the synergistic combination of Melt-Extrusion Additive Manufacturing (MEAM) and porogen leaching, utilizing PCL as the bulk material and PEG as the porogen. PCL/PEG blends with different polymer ratios (60/40, 50/50, 40/60) were produced and characterized through a DSC analysis. The MEAM process parameters and porogen leaching in bi-distilled water allowed for the development of a micrometric anisotropic fibrous structure with fiber diameters ranging from 10 to 100 µm, depending on PCL/PEG blend ratios. The fibrous scaffolds were coated with Gelatin type A to achieve a biomimetic coating for an in vitro cell culture and mechanically characterized via AFM. The 40/60 PCL/PEG scaffolds yielded the most homogeneous and smallest fibers and the greatest physiological stiffness. In vitro cell culture studies were performed by seeding C2C12 cells onto a selected scaffold, enabling their attachment, alignment, and myotube formation along the PCL fibers during a 14-day culture period. The resultant anisotropic scaffold morphology promoted SMT-like cell conformation, establishing a versatile platform for developing in vitro models of tissues with anisotropic morphology.

Characterization of PVA/Chitosan and Tuna Fish Bones CHA Scaffold for Bone Tissue Engineering

Bone tissue engineering has been used in the biomedical field to treat bone defects by implanting scaffolds into bone tissue. However, the currently developed scaffold still needs to be developed to obtain scaffold building materials with good compatible properties and can regenerate damaged bone cells. This study combines PVA/Chitosan polymer with CHA of tuna bone using the porogen leaching method at a calcination temperature of 100°C for 12 hours. The purpose of this study was to determine the physicochemical properties by characterizing XRD, SEM-EDX, FTIR, and the porosity of the scaffold. The results obtained from the results of the PVA/Chitosan/CHA XRD patterns are the formation of the PVA/Chitosan phase at 2θ(°)=19.68, the IR spectrum of the 43−group band and 2 stretching, the ratio mol Ca/P is 1.98, the pore diameter is 1.561 ± 0.07 μm and the porosity is 55.04%. These results indicate that the PVA/Chitosan/CHA scaffold is an amorphous calcium phosphate (ACP) that has the potential for bone tissue engineering.

Ground tire rubber/activated carbon/expanded graphite aerogels and foams as support material for the preparation of polyethylene glycol composite phase change materials for thermal energy storage applications

The 3 “Rs” of trash management, reduce, reuse, and recycle, lead to a theoretical topic. Techniques for waste management should reprocess these waste products into new functional materials. In the area of thermal energy storage, phase change material (PCM) is another hot topic due to its high latent heat. Unfortunately, it is constrained by issues such as simple leakage and poor heat conductivity. Herein, we integrate waste material and phase change material by preparing composite phase change material in which aerogels and foams serve as 3D support matrices that were made using ground tire rubber, activated carbon and expanded graphite additives to successfully overcome the aforementioned disadvantages of phase change material via Porogen leaching and freeze drying techniques. Experiment findings demonstrated that the CPCM has excellent shape stability. TAA5 and TAE5 had the highest melting enthalpies, at 146.21 and 148.23 J/g respectively. After 500 thermal test cycles and 25 photothermal test cycles, the prepared CPCMs exhibit strong thermal reliability as evidenced by a small decrease in enthalpy value. In addition, the thermal conductivity of TAE15 was remarkable at 1.18 W/(mK), which was 5.36 times greater than that of PEG 0.22 W/(mK). The use of AC and EG as additives boosted TAE15's photothermal conversion efficiency to 93 %. Hence, CPCMs offer a significant deal of potential in fields such as thermal energy storage, solar energy, and building energy savings.

Synthesis of bioactive heat cured PMMA/PEKK blend reinforced by nano titanium dioxide for bone scaffold applications

In bone tissue engineering (BTE), defects in large bones remain the greatest issue which can be addressed using bone scaffolds. In this work, blends of heat cured polymethylmethacrylate (HC PMMA) and various weight percentages of poly-ether-ketone-ketone (PEKK) (0, 2, 4, 8, and 10%) were made using a porogen leaching process. The blends were then subjected to tensile, compression and bending tests to select the optimum blend. Based on the results obtained, HC PMMA blended with 2 wt% PEKK was selected to produce the bio-porous blends. Here, the porosity was imparted using tartaric acid (C4H6O6) and sodium hydrogen carbonate (NaHCO3) as porogen leaching components. Porous blends resulted were then reinforced with a nano titanium dioxide powder (nTiO2) at different weight percentages of (0, 1, 3, and 5). The results showed that porous composites made of (HC PMMA: 2 wt% PEKK) blend reinforced with 5 wt % nTiO2 exhibit the highest strength values under various loadings.The FTIR identified the functional groups of the bone scaffold components. The mean pore size and pore depth were measured using atomic force microscopy (AFM) analysis and the values are 92.6 nm and 42.78 nm, respectively. The good distribution of the PEKK and nTiO2 within the HC PMMA and the uniform porous structure with multi-scale pores between 535 nm and 1.187 mm were confirmed by the AFM data and SEM images, respectively. This research expected that the porous composite (HC PMMA: 2% PEKK: 5% nTiO2) is a good candidate for bone scaffold applications.

Porous scaffold hydroxyapatite from sand lobster shells (Panulirus homarus) using polyethylene oxide/chitosan as polymeric porogen for bone tissue engineering.

The hydroxyapatite (HAp; Ca10 (PO4 )6 (OH)2 )) has good biocompatibility, bioactivity, and osteoconductivity as a bone implant because the main inorganic mineral of human bone is HAp. The use of scaffold HAp from biogenic resources that contain high calcium and polymer as a pore forming agent to support bone growth is a longstanding area of interest. In this study, porous scaffolds based on HAp were synthesized from sand lobster (SL; Panulirus homarus) shells as a source of calcium using the porogen leaching method with polyethylene oxide (PEO) and chitosan (Chs) as polymeric porogen. The present study aims to synthesize HAp derived from SL shells and evaluate the effect variations of PEO on the physicochemical properties of the scaffold and cytotoxicity in cell viability assay. Briefly, the SL shell powder was calcinated with temperature variations of 600°C, 800°C, and 1000°C for 6 h. Based on the characterization, it was shown that 1000°C was the optimum calcination temperature for SL shells to synthesize HAp using the precipitation method. The characterization results of HAp using energy dispersive x-ray (EDX) revealed that the molar ratio of Ca/P was 1.67. The Fourier transform infrared (FTIR) and x-ray diffractometer (XRD)spectral patterns indicated that HAp had been successfully synthesized with minor β-tricalcium phosphate (β-TCP), a calcium phosphate with high biocompatibility. Porous scaffolds were synthesized by varying the concentration of PEO at 0, 5, 10, and 15 wt %. Physicochemical analysis revealed that a higher concentration of PEO affected decreased crystallinity and compressive strength, but on the other hand, the porosity and pore sizes increased. Based on the physicochemical analysis, the synthesized porous scaffold showed that HAp/PEO/Chs 15 wt % had the most potential as a scaffold for biomedical applications. MTT Assay, after 24 h incubation, revealed that the scaffold was safe for use at low concentrations on the MC3T3E1 osteoblast cells, with a percentage of cell viability of 83.23 ± 3.18% at 23.4375 μg/mL. Although the cell viability decreased at higher concentrations, the HAp/PEO/Chs 15 wt % scaffold was cytocompatible with the cells. Thus, in the present study, HAp/PEO/Chs 15 wt % was the best scaffold based on pore structure, chemical composition, mechanical and crystalographic properties and cell viability.

Versatile, elastomeric and degradable polyHIPEs of poly(glycerol sebacate)-methacrylate and their application in vascular graft tissue-engineering

Polymer scaffolds are an important enabling technology in tissue engineering. A wide range of manufacturing techniques have been developed to produce these scaffolds, including porogen leaching, phase separation, gas foaming, electrospinning and 3D printing. However, all of these techniques have limitations. Delivering suitable scaffold porosity, small feature sizes and macroscopic geometry remain challenging.Here, we present the development of a highly versatile scaffold fabrication method utilising emulsion templating to produce polymerised high internal phase emulsions (polyHIPEs) of the polymer poly(glycerol sebacate) methacrylate (PGS-M). PGS-M is biocompatible, degradable and highly elastic, with tunable mechanical properties. PGS-M was formulated into an emulsion using solvents and surfactants and then photocured into polyHIPE structures. The porosity, degradation behaviour, mechanical properties and biocompatibility of the PGS-M polyHIPEs was investigated.The versatility of the PGS-M polyHIPEs was demonstrated with the production of various complex tubular scaffold shapes, using injection moulding. These shapes were designed for applications in vascular graft tissue engineering and included straight tubes, bends, branches, functioning valves, and a representative aortic arch. The PGS-M polyHIPE scaffolds supported vascular smooth muscle cells (SMCs) in 3D cell culture in a bioreactor.

4D printing of biocompatible, hierarchically porous shape memory polymeric structures

Conventional implants tend to have significant limitations, as they are one-size-fits-all, require monitoring, and have the potential for immune rejection. However, 4D Printing presents a method to manufacture highly personalized, shape-changing, minimally invasive biomedical implants. Shape memory polymers (SMPs) with a glass transition temperature (Tg) between room and body temperature (20–38 °C) are particularly desirable for this purpose, as they can be deformed to a temporary shape before implantation, then undergo a shape change within the body. Commonly used SMPs possess either an undesirable Tg or lack the biocompatibility or mechanical properties necessary to match soft biological tissues. In this work, Poly(glycerol dodecanoate) acrylate (PGDA) with engineered pores is introduced to solve these issues. Pores are induced by porogen leaching, where microparticles are mixed with the printing ink and then are dissolved in water after 3D printing, creating a hierarchically porous texture to improve biological activity. With this method, highly complex shapes were printed, including overhanging structures, tilted structures, and a “3DBenchy”. The porous SMP has a Tg of 35.6 °C and a Young's Modulus between 0.31 and 1.22 MPa, comparable to soft tissues. A one-way shape memory effect (SME) with shape fixity and recovery ratios exceeding 98 % was also demonstrated. Cultured cells had a survival rate exceeding 90 %, demonstrating cytocompatibility. This novel method creates hierarchically porous shape memory scaffolds with an optimal Tg for reducing the invasiveness of implantation and allows for precise control over elastic modulus, porosity, structure, and transition temperature.

Microporous/Macroporous Polycaprolactone Scaffolds for Dental Applications.

This study leverages the advantages of two fabrication techniques, namely, melt-extrusion-based 3D printing and porogen leaching, to develop multiphasic scaffolds with controllable properties essential for scaffold-guided dental tissue regeneration. Polycaprolactone-salt composites are 3D-printed and salt microparticles within the scaffold struts are leached out, revealing a network of microporosity. Extensive characterization confirms that multiscale scaffolds are highly tuneable in terms of their mechanical properties, degradation kinetics, and surface morphology. It can be seen that the surface roughness of the polycaprolactone scaffolds (9.41 ± 3.01 µm) increases with porogen leaching and the use of larger porogens lead to higher roughness values, reaching 28.75 ± 7.48 µm. Multiscale scaffolds exhibit improved attachment and proliferation of 3T3 fibroblast cells as well as extracellular matrix production, compared with their single-scale counterparts (an approximate 1.5- to 2-fold increase in cellular viability and metabolic activity), suggesting that these structures could potentially lead to improved tissue regeneration due to their favourable and reproducible surface morphology. Finally, various scaffolds designed as a drug delivery device were explored by loading them with the antibiotic drug cefazolin. These studies show that by using a multiphasic scaffold design, a sustained drug release profile can be achieved. The combined results strongly support the further development of these scaffolds for dental tissue regeneration applications.

Highly Porous Gas-Blown Hydrogels for Direct Cell Encapsulation with High Cell Viability.

Cell transplant therapies show potential as treatments for a large number of diseases. The encapsulation of cells within hydrogels is often used to mimic the extracellular matrix and protect cells from the body's immune response. However, cell encapsulation can be limited in terms of both scaffold size and cell viability due to poor nutrient and waste transport throughout the bulk of larger volume hydrogels. Strategies to address this issue include creating prevascularized or porous structured materials. For example, cell-laden hydrogels can be formed by porogen leaching or three-dimensional printing, but these techniques involve the use of multiple materials, long preparation times, and/or specialized equipment. Postfabrication cell seeding in porous scaffolds can result in inconsistent cell density throughout scaffold volumes and typically requires a bioreactor to ensure even cell distribution. In this study, we developed a highly cytocompatible direct cell encapsulation method during the rapid fabrication of porous hydrogels. Using sodium bicarbonate and citric acid as blowing agents, we employed photocurable polymers to produce highly porous materials within a matter of minutes. Cells were directly encapsulated within methacrylated poly(vinyl alcohol), poly(ethylene glycol), and gelatin hydrogels at viabilities as high as 93% by controlling solution variables, such as citric acid content, viscosity, pH, and curing time. Cell viability within the resulting porous constructs was high (>80%) over 14 days of analysis with multiple cell types. This work provides a simple, versatile, and tunable method for cell encapsulation within highly porous constructs that can be built upon in future work for the delivery of cell-based therapies. Impact Statement This simple method to obtain cell-laden hydrogel foams allows direct cell encapsulation within biomaterials without the need for porogens or microcarriers, while maintaining high cell viability. The successful encapsulation of multiple cell types into gas-blown hydrogels with varied chemistries shows the versatility of this method. While this work focuses on photocrosslinkable polymers, any quick gelling material could be used for foam fabrication in expansion of this work. The potential future impact of this work on the treatment of diseases and injuries that utilize cell therapies is wide-ranging.

Fabrication of meso/macroporous TiO2/PCL composite scaffolds by direct ink writing: The effects of porogen content on the compressive modulus and in vitro behavior

In this paper, TiO2/PCL composite scaffolds with different pore sizes were fabricated by a combination of sol-gel, porogen leaching, and direct ink writing techniques. The effects of porogen content and leaching time on the compressive modulus and porosity percentage were investigated. The central composite design was employed for statistical modeling and prediction of the compressive modulus and porosity percentage for deferent porogen contents and leaching time. The results showed that with increasing the porogen content (in the printing ink) from 30 to 70 wt%, the compressive modulus decreased from 20.03 to 4.96 MPa and the porosity percentage increased from 74.56% to 80.02%. The optimum leaching time for porogen removal was around 7 days in deionized water. In vitro degradability of the scaffolds was enhanced by increasing the NaCl content in the printing ink and reached 15% for the samples containing 70 wt% NaCl. Furthermore, the hydrophilicity of the samples was also increased and the water contact angle decreased from 74.80 to 58.48 º. The suitability of the scaffolds for bone tissue engineering applications was proved by in vitro cytotoxicity (MTS) and Alkaline Phosphatase activity assays (performed on Human Bone Marrow Stromal cells). DAPI/Phalloidin cell staining was also employed to monitor the cell morphology on the scaffolds. The results indicated the existence of multiscale porosity in 3D-printed TiO2/PCL scaffolds which supplies a suitable structure for cell nesting, tissue regeneration, and potential drug delivery applications.

Multiscale characterisation of acoustic behaviour of a new bio-based porous material

This work aims at studying the mechanical and acoustic behaviours of a new bio-based porous epoxy resin obtained by a "green" adapted combination of the cationic photopolymerization and the porogen leaching technique. This new kind of material generally possesses interconnected complex morphology and it would be useful to consider this feature in a model. In this study, the effective properties of the material were estimated by using the asymptotic homogenization method. Four types of ordered pore arrangements together with systematic variations of the porosity and the pore size have been studied. Based on the results of these investigations, a subtle relation between the microstructure and mechanical/acoustic properties has been established. The estimated equivalent dynamic density and equivalent dynamic bulk modulus were compared with experimental results obtained by conducting the three-microphone impedance tube testing. The processing parameters of material elaboration could be adjusted so that the obtained porous material would possess the best sound absorption performance.

Manufacturing of scaffolds with interconnected internal open porosity and surface roughness.

Manufacturing of three-dimensional scaffolds with multiple levels of porosity are an advantage in tissue regeneration approaches to influence cell behavior. Three-dimensional scaffolds with surface roughness and intra-filament open porosity were successfully fabricated by additive manufacturing combined with chemical foaming and porogen leaching without the need of toxic solvents. The decomposition of sodium citrate, a chemical blowing agent, generated pores within the scaffold filaments, which were interconnected and opened to the external environment by leaching of a water-soluble sacrificial phase, as confirmed by micro-CT and buoyancy measurements. The additional porosity did not result in lower elastic modulus, but in higher strain at maximum load, i.e. scaffold ductility. Human mesenchymal stromal cells cultured for 24 h adhered in greater numbers on these scaffolds when compared to plain additive-manufactured ones, irrespectively of the scaffold pre-treatment method. Additionally, they showed a more spread and random morphology, which is known to influence cell fate. Cells cultured for a longer period exhibited enhanced metabolic activity while secreting higher osteogenic markers after 7 days in culture. STATEMENT OF SIGNIFICANCE: Inspired by the function of hierarchical cellular structures in natural materials, this work elucidates the development of scaffolds with multiscale porosity by combining in-situ foaming and additive manufacturing, and successive porogen leaching. The resulting scaffolds displayed enhanced mechanical toughness and multiscale pore network interconnectivity, combined with early differentiation of adult mesenchymal stromal cells into the osteogenic lineage.

Biocompatible carboxymethyl cellulose-based super-elastic hierarchical sponge via a novel templating and plasticizing method

Herein, a facile method to fabricate hierarchical super-elastic (SE) sponge using a water-soluble cellulose derivative, carboxymethyl cellulose (CMC), is reported. The method includes ice templating and porogen leaching steps which facilitate to generate macro-sized pores as well as pore wall structures that can dissipate stress effectively. By controlling the porogen content, the specific surface area and the morphology of the sponges can be tuned. Furthermore, a plasticizing method was used before vacuum drying to reduce the deformation of the inner structure. The derived hierarchical SE CMC sponges exhibit excellent fatigue resistance, fast shape recovery, high-water absorption, biosafeness, and fast degradation. Thus, our strategy provides a novel method for the construction of SE sponges which show great potential in green elastic wound dressing, tissue engineering, and absorbent materials.

In vitro and in vivo investigation of a zonal microstructured scaffold for osteochondral defect repair

Articular cartilage is comprised of zones that vary in architecture, extracellular matrix composition, and mechanical properties. Here, we designed and engineered a porous zonal microstructured scaffold from a single biocompatible polymer (poly [ϵ-caprolactone]) using multiple fabrication strategies: electrospinning, spherical porogen leaching, directional freezing, and melt electrowriting. With this approach we mimicked the zonal structure of articular cartilage and produced a stiffness gradient through the scaffold which aligns with the mechanics of the native tissue. Chondrocyte-seeded scaffolds accumulated extracellular matrix including glycosaminoglycans and collagen II over four weeks in vitro. This prompted us to further study the repair efficacy in a skeletally mature porcine model. Two osteochondral lesions were produced in the trochlear groove of 12 animals and repaired using four treatment conditions: (1) microstructured scaffold, (2) chondrocyte seeded microstructured scaffold, (3) MaioRegen™, and (4) empty defect. After 6 months the defect sites were harvested and analyzed using histology, micro computed tomography, and Raman microspectroscopy mapping. Overall, the scaffolds were retained in the defect space, repair quality was repeatable, and there was clear evidence of osteointegration. The repair quality of the microstructured scaffolds was not superior to the control based on histological scoring; however, the lower score was biased by the lack of histological staining due to the limited degradation of the implant at 6 months. Longer follow up studies (e.g., 1 yr) will be required to fully evaluate the efficacy of the microstructured scaffold. In conclusion, we found consistent scaffold retention, osteointegration, and prolonged degradation of the microstructured scaffold, which we propose may have beneficial effects for the long-term repair of osteochondral defects.

Simple Fabrication and Enhanced Bioactivity of Bioglass‐Poly(lactic‐co‐glycolic acid) Composite Scaffolds with Matrix Microporosity

AbstractScaffold porosity plays an important role in bone tissue engineering as macropores promote cell migration and micropores promote protein adsorption and cell adhesion. Currently, most methods use complex, multi‐step processes to create dual‐scale porosity in composite scaffolds, and no studies evaluate the effect of microporosity on the bioactivity of composite scaffolds with dual porosity. To fill this gap, a simple solvent casting and porogen leaching technique using paraffin microspheres as a porogen and CitriSolv as the leaching solvent to prepare macroporous Bioglass‐poly(lactic‐co‐glycolic acid) (Bg‐PLGA) scaffolds with intrinsic micropores (1–10 µm) in the PLGA matrix is proposed, and the effect of microporosity on the bioactivity of the scaffolds is analyzed. PLGA matrix microporosity induces larger apatite deposition upon immersion in simulated body fluid, as well as enhanced protein adsorption upon contact with serum, compared to non‐microporous scaffolds. Also, mesenchymal cells cultured on the microporous scaffolds show extensive matrix deposition. These results highlight this method as a simple and effective technique to produce dual‐porosity scaffolds that are excellent candidates for bone tissue engineering, as their enhanced bioactivity, protein adsorption, and the extensive matrix deposition observed in‐vitro are good indicators of fast bone integration upon implantation.

The cell viability assay analysis and physicochemical characterization of porous hydroxyapatite scaffold using honeycomb and paraffin wax as polymeric porogen for bone tissue engineering

To fabricate and characterize the porous hydroxyapatite-based scaffold, honeycomb as a natural polymer (HA/HCB) and paraffin wax (HA/Wax) were used. The fabrication of scaffold using the porogen leaching method was varied temperatures between 700, 900, and 1100 °C. Theoretically, the temperature of calcination influenced the morphology of the scaffold, crystallite size, functional group, and porosity. According to the previous study, the crystallite size of the polymer scaffold is less than 100 nm. The HA-based scaffold was analyzed by the Energy Dispersive X-Ray Spectroscopy (EDS), Scanning Electron Microscopy (SEM), X-Ray Diffractometer (XRD), Fourier Transform Infrared Spectroscopy (FTIR), and tested by the cell viability. According to the XRD results, the crystallite size of HA/HCB scaffold decreases, while scaffold HA/Wax crystallite size tends to decrease when calcination temperature increases. As calcination temperature increases, porosity tends to be small for both HA/HCB and HA/wax scaffolds. The scaffold HA/HCB 900 °C has interconnected pores, uniform, and small porosity. In contrast, the scaffold HA/Wax 900 °C has fewer interconnected pores and non-uniform particles. The FTIR result of the HA/HCB 900 °C has C-H functional group, affecting cell viability. Through MTT (3-(4,5-Dimethylthiazol-2-yl)−2,5 diphenyltetrazolium bromide) assays, the cell viability value of the HA/Wax 900 °C was greater than the HA/HCB 900 °C for 48 h incubated time. It is caused by the alkane chains on HA/HCB, causing the death of cells. Considering cell viability assay studies for the nanocomposite scaffold, the obtained results confirm the non-toxicity of the material.

Ginseng used for bone tissue scaffold

The scaffold based tissue engineering materialized for bone tissue therapy. Gelatin-glutaraldehyde cross linked scaffold was prepared by solvent casting -porogen leaching method. It was characterized by FTIR and SEM microphotograph analysis. Absence of peak at waves no. 1625 cm−1 in ATR-FTIR indicated formation of cross-linking. FE-SEM micrograph showed honeycomb pad like structure with high porosity. Methanolic extract of Withania somnifera (Ashwagandha) root extract induced MC3T3 E1 osteoblast cell adhesion and proliferation on porous gelatin scaffold. GC-MS analysis pointed out presence of 4-amino- 2-ethyl-3-methylquinoline, an active phyto-chemicals having tissue regeneration potential. High anti-oxidant capacity down regulates cell death mechanism by scavenging free radical. The biocompatible gelatin scaffold has RGD moiety that attune the MC3T3 E1 osteoblast cell adhesion. Withania somnifera root extract may boost up cell proliferation on scaffold. Therefore treatment with Withania somnifera root extract may be the new approaches for designing bone tissue scaffold for bone tissue therapy.

Chitosan-coated pore wall polycaprolactone three-dimensional porous scaffolds fabricated by porogen leaching method for bone tissue engineering: a comparative study on blending technique to fabricate scaffolds.

One of the significant challenges in the fabrication of scaffolds for tissue engineering lies in the direct interaction of bioactive agents with cells in the scaffolds matrix, which curbs the effectiveness of bioactive agents resulting in diminished cell recognition and attachment ability of the scaffolds. Here, three-dimensional porous scaffolds were fabricated using polycaprolactone (PCL) and chitosan, by two approaches, i.e., blending and surface coating to compare their overall effectiveness. Blended scaffolds (Chi-PCL) were compared with the scaffolds fabricated using surface coating technique, where chitosan was coated on the pore wall of PCL scaffolds (C-PCL). The C-PCL exhibited a collective improvement in bioactivities of the stem cell on the scaffold, because of the cell compatible environment provided by the presence of chitosan over the scaffolds interface. The C-PCL showed the enhanced cell attachment and proliferation behavior of the scaffolds along with two-fold increase in hemolysis compatibility compared to Chi-PCL. Furthermore, the compression strength in C-PCL increased by 24.52% and 8.62% increase in total percentage porosity compared to Chi-PCL was attained. Along with this, all the bone markers showed significant upregulation in C-PCL scaffolds, which supported the surface coating technique over the conventional methods, even though the pore size of C-PCL was compromised by 19.98% compared with Chi-PCL.

Fabrication of porous chitosan particles using a novel two-step porogen leaching and lyophilization method with the label-free multivariate spectral assessment of live adhered cells

Porous chitosan (CS) particles were fabricated using a novel two-step technique that employed a porogen leaching phase followed by lyophilization or freeze-drying. Poly(ethylene glycol) (PEG) was mixed as a porogen in two different quantities with the CS solution before particle synthesis via coacervation. After the PEG leached out into deionized (DI) water at an elevated constant temperature, the final freeze-dried CS particles revealed surface features that resembled pore pockets. A three-dimensional (3D) culture of murine osteoblast cell line (OB-6) was seeded on these particles to analyze the effect of the porous structure on the cell activity, as compared to a control group with no added porogen. The results highlighted an enhancement in cell adhesion and proliferation on the two porous sample groups. A Raman spectroscopy-based label-free technique for live cell biomarker analysis was applied using multivariate spectral analysis. Results of the spectral analysis in the molecular fingerprint region corresponding to the Raman shift between 900 cm−1 and 1700 cm−1inferred inter-group variations. The bands at 1005 cm−1 and 1375 cm−1 were assigned to the live cell biomarkers phenylalanine and glycosaminoglycan, respectively, and were assessed during the multivariate spectral analysis. The corresponding score plot and loading information generated from the Principal Component Analysis (PCA) of the Raman spectrum at day 7 and day 14, pointed at inter-group spectral variations related to cell adhesion and proliferation between the two porous CS particle groups and the control.

Vascular Network Formation on Macroporous Polydioxanone Scaffolds.

In this study, microvascular network structures for tissue engineering were generated on newly developed macroporous polydioxanone (PDO) scaffolds. PDO represents a polymer biodegradable within months and offers optimal material properties such as elasticity and nontoxic degradation products. PDO scaffolds prepared by porogen leaching and cryo-dried to achieve pore sizes of 326 ± 149.67 μm remained stable with equivalent values for Young's modulus after 4 weeks. Scaffolds were coated with fibrin for optimal cell adherence. To exclude interindividual differences, autologous fibrin was prepared out of human plasma-derived fibrinogen and proved a comparable quality to nonautologous commercially available fibrinogen. Fibrin-coated scaffolds were seeded with recombinant human umbilical vein endothelial cells expressing GFP (GFP-HUVECs) in coculture with adipose tissue-derived mesenchymal stem cells (AD-hMSCs) to form vascular networks. The growth factor content in culture media was optimized according its effect on network formation, quantified and assessed by AngioTool®. A ratio of 2:3 GFP-HUVECs/AD-hMSCs in medium enriched with 20 ng/mL vascular endothelial growth factor, basic fibroblast growth factor, and hydrocortisone was found to be optimal. Network structures appeared after 2 days of cultivation and stabilized until day 7. The resulting networks were lumenized that could be verified by dextran staining. This new approach might be suitable for microvascular tissue patches as a useful template to be used in diverse vascularized tissue constructs. Impact statement We consider this work as important for the current research in the field of tissue engineering and the development of new and functional tissue. The approach for the production of vascularized tissue patches, consisting of the biodegradable synthetic polymer polydioxanone and of the physiological, autologous, and patient-specific polymer fibrin, and seeded with endothelial cells and mesenchymal stem cells, displayed within this work, could be useful for the sustaining development of diverse and more complex tissue constructs. Therefore, these scaffolds could be used as a cornerstone for future tissue engineering approaches.