A simple and effective approach to produce tubular polysaccharide-based hydrogel scaffolds

The production of porous tubular scaffolds is of great interest in the field of tissue engineering, given the existence of several tubular structures in the human body. In this work, a methodology was developed for the fabrication of tubular-shaped scaffolds based on the casting of polymeric solutions by controlled crosslinking mediated by a semipermeable cast. The fabrication of hydrogel tubular scaffolds from chitosan–pectin polymeric mixtures (tCh-P, 3% w/v) was performed to attest the feasibility of the technique. Tubular structures with about 4.15 mm internal diameter and 1.55 mm wall thickness were produced. The structures are highly porous, presenting interconnected pores with average diameter of about 360 μm. Seeding of human smooth muscle cells on the material was successfully achieved by using collagen gel to facilitate cell migration and retention inside the structure of the scaffold. The methodology herein proposed was successfully validated for the production of tubular constructs, opening new perspectives for the fabrication of matrices based on polymers that are passive of crosslinking with small molecules. Besides being an interesting approach to produce tubular scaffolds, this methodology can be considered an useful platform to obtain materials for drug screening and diagnostic studies. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48510.     

In Situ Crosslinking of Highly Porous Chitosan Scaffolds for Bone Regeneration: Production Parameters and In Vitro Characterization

Various methods of chitosan scaffold production are reported in the literature so far. Here, in situ crosslinking with glutaraldehyde is reported for the first time. It combines pore formation and chitosan crosslinking in a single step. This combination allows incorporation of fragile molecules into 3D porous chitosan scaffolds produced by simple and gentle lyophilization. In this study, parameters of in situ crosslinking of porous chitosan scaffold formation as well as their effect on degradation and bioactivity of the scaffolds are examined. The scaffolds are characterized in the context of their prospective application as bone substitute material. The addition of calcium phosphate phases (hydroxyapatite, brushite) to the macroporous chitosan scaffolds allows manipulation of the bioactivity that is investigated by incubation in simulated body fluid (SBF). The bioactivity is significantly influenced by the modus of changing the fluid (static, daily‐, and twice‐a‐week change). Scaffolds are morphologically characterized by means of scanning electron microscopy, and the mechanical stability is tested after incubation in SBF and phosphate‐buffered saline.     

Preparation of enhanced three-dimensional porous chitosan scaffolds by acetylation and aqueous extraction

We have devised a method to prepare a 3-dimensional (3D) porous acetylated chitosan scaffold for use as a cell adhesion matrix in tissue engineering applications. The scaffold was prepared by molding a mixture of chitosan and gelatin (as porogen), then removing the uncomplexed porogen by aqueous extraction from the freeze-dried material prior to acetylation. The extent of chitosan acetylation according to the reaction time was observed by X-ray diffraction (XRD) analysis. The differences between the aqueous-extracted and control phase-separated chitosan scaffolds in terms of pore morphology and interconnectivity were examined by scanning electron microscopy (SEM), enzymatic degradation, and surface roughness tests. The fibroblast cell line NIH-3T3 was used to test relative cell affinities for the acetylated versus untreated (control) chitosan scaffolds. The acetylated 3D porous scaffolds showed high interconnectivity and improved biocompatibility properties. Thus, these scaffolds may be very useful for a variety of tissue engineering applications.     

Preparation and characterization of porous scaffold composite films by blending chitosan and gelatin solutions for skin tissue engineering

Biodegradable polymers have significant potential in biotechnology and bioengineering. However, for some applications, they are limited by their inferior mechanical properties and unsatisfactory compatibility with cells and tissues. In the present investigation blends of chitosan and gelatin with various compositions were produced as candidate materials for biomedical applications. Fourier transform infrared spectral analysis showed good compatibility between these two biodegradable polymers. The composite films showed improved tensile properties, highly porous structure, antimicrobial activities, low water dissolution, low water uptake and high buffer uptake compared to pure chitosan or gelatin films. These enhanced properties could be explained by the introduction of free ? OH, ? NH₂ and ? NHOCOCH₃ groups of the amorphous chitosan in the blends and a network structure through electrostatic interactions between the ammonium ions (? NH³⁺) of the chitosan and the carboxylate ions (? COO^?) of the gelatin. Scanning electron microscopy images of the blend composite films showed homogeneous and smooth surfaces which indicate good miscibility between gelatin and chitosan. The leafy morphologies of the scaffolds indicate a large and homogeneous porous structure, which would cause increased ion diffusion into the gel that could lead to an increase in stability in aqueous solution, buffer and temperature compared to the gelatin/chitosan system. In vivo testing was done in a Wistar rat (Rattus norvegicus) model and the healing efficiencies of the scaffolds containing various compositions of chitosan were measured. The healing efficiencies in Wistar rat of composites with gelatin to chitosan ratios of 10:3 and 10:4 were compared with that of a commercially available scaffold (Eco‐plast). It was observed that, after 5 days of application, the scaffold with a gelatin to chitosan ratio of 10:3 showed 100% healing in the Wistar rat; however, the commercial Eco‐plast showed only a little above 40% healing of the dissected rat wound. Copyright © 2012 Society of Chemical Industry     

Fabrication and characterization of natural origin chitosan‐ gelatin‐alginate composite scaffold by foaming method without using surfactant

A natural origin tripolymer scaffold from chitosan, gelatin, and alginate was fabricated by applying foaming method without adding any foam stabilizing surfactant. Previously, in foaming method of scaffold fabrication, toxic surfactants were used to stabilize the foam, but in this work, the use of surfactant has been avoided strictly, which can provide better environment for cellular response and viability. In foaming method, stable foam is produced simply by agitating the polymer (alginate‐gelatin) solution, and the foam is crosslinked with CaCl₂, glutaraldehyde, and chitosan to produce tripolymer alginate‐gelatin‐chitosan composite scaffold. Microscopic images of the composite scaffold revealed the presence of interconnected pores, mostly spread over the entire surface of the scaffold. The scaffold has a porosity of 90% with a mean pore size of 57 μm. Swelling and degradation studies of the scaffold showed that the scaffold possesses excellent properties of hydrophilicity and biodegradability. In vitro cell culture studies by seeding L929 mouse fibroblast cells on scaffold revealed excellent cell viability, proliferation rate and adhesion as indicated by MTT assay, DNA quantification, and phase contrast microscopy of cell‐scaffold construct. The natural origin composite scaffold fabricated by the simplest method i.e., foaming method, but without adding any surfactant, is cheap, biocompatible, and it might find potential applications in the field of tissue engineering. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Preparation of biomimetic three-dimensional gelatin/montmorillonite–chitosan scaffold for tissue engineering

A novel gelatin/montmorillonite–chitosan (Gel/MMT–CS) nanocomposite scaffold was prepared via the intercalation process and the freeze-drying technique, using the ice particulates as the porogen materials. Properties including pore structure, water adsorption content, in vitro degradation and tensile strength were investigated. It was demonstrated that the introduced intercalation structure endowed the Gel/MMT–CS scaffold with good mechanical properties and a controllable degradation rate. Scanning of the electron microscope images revealed that the scaffold obtained was highly porous and suitable for the implanted cells to adhere and grow. The mitochondrial activity assay provided good evidences of cells viability on the Gel/MMT–CS membranes, giving an indication of possible application as a matrix for tissue engineering.     

仿生型壳聚糖-明胶/溶胶凝胶生物玻璃复合支架的制备及其矿化性能研究

利用冷冻干燥法制备出用于骨和软骨组织工程的壳聚糖-明胶/溶胶凝胶生物玻璃(CS-Gel/SGBG)仿生型复合多孔支架,并进行了孔隙率的测定和显微形貌的观察;探讨了各组分不同用量对CS-Gel/SGBG复合支架显微结构的影响以及复合支架在模拟生理体液中的仿生矿化性能。研究表明,通过调节各组分的不同用量,可以制备出三维连通的复合多孔支架,且孔隙率达到90%以上;在模拟生理体液中浸泡后发现CS-Gel/SGBG支架表面有大量结晶态类骨碳酸羟基磷灰石生成,表明复合支架有良好的生物矿化性能。     

Fabrication of gelatin/chitosan nanofibrous scaffold: process optimization and empirical modeling

Electrospinning is a very useful technique for producing polymeric nanofibers by applying electrostatic forces. This study reports on the modeling and optimization of the electrospinning process of gelatin/chitosan, using response surface methodology. The individual and the interaction effects of the gelatin/chitosan blend ratio (50/50, 60/40 and 70/30), applied voltage (20, 25 and 30 kV) and feeding rate (0.2, 0.4 and 0.6 mL h^?1) on the mean fiber diameter and standard deviation of the fiber diameter were investigated on optimization section, using scanning electron microscopy. To fabricate the nanofibrous gelatin/chitosan blend, trifluoroacetic acid/dichloromethane was selected as the solvent system. The model obtained for the mean fiber diameter has a quadratic relationship with applied voltage and feeding rate. The interaction between applied voltage and flow rate were found significant but the interactions of blend ratio and flow rate and also blend ratio and applied voltage were negligible. A quadratic relationship was obtained for applied voltage and flow rate with standard deviation of the fiber diameter and there was no interaction between the parameters in the model. The optimum condition for electrospinning of gelatin/chitosan was also introduced using the model obtained in this study. Scanning electron micrographs of human dermal fibroblast cells on the nanofibrous structures show good attachment and proliferation on the fabricated scaffold surface. Electrospun gelatin/chitosan nanofibrous mats have great potential for use as a scaffold for skin tissue engineering. © 2014 Society of Chemical Industry     

Investigation of porous polydimethylsiloxane structures with tunable properties induced by the phase separation technique

This article reports the fabrication and characterization of porous polydimethylsiloxane (PDMS) structures developed by the solvent evaporation-induced phase separation technique. Ternary systems containing water/tetrahydrofuran (THF)/PDMS with various concentrations are produced to form a stable solution. The porous PDMS structures are formed by removing the solvent (THF) and nonsolvent (water) phases during the stepping heat treatment procedure. The analytical ternary phase diagram is constructed based on the thermodynamic equilibrium state in the polymer solution to explain the stable/unstable formulations and the possible composition change path. The results show that the isolated pores with the adjustable pore size ranging from 330 to 1900 μm are obtained by tuning the water to the THF ratio. The mechanical properties of the porous PDMS structures are determined by conducting the tensile tests on the prepared dog bone-shaped specimens. A wide range of elastic modulus ranging between 0.49 and 1.05 MPa was achieved without affecting the density of the porous sample by adjusting the solvent and non-solvent content in the solution. It is shown that the flexibility of the porous structures can be improved by reducing the ratio of water to THF and decreasing the PDMS content. The porosity measurements reveal that the PDMS concentration is the major phase controlling the porosity of the structure, while the effect of water/THF is negligible.     

A study on the fabrication of porous chitosan/gelatin network scaffold for tissue engineering

A novel porous chitosan/gelatin scaffold for tissue engineering was prepared via polyelectrolyte complex formation, freeze drying and post‐crosslinking with glutaraldehyde. The porosity and mean pore diameters could be controlled within 30∼100 µm by varying the original water content and the freezing conditions. Dipping the scaffolds in poly(lactic acid) provided good mechanical properties making it a promising candidate towards tissue engineering. © 2000 Society of Chemical Industry     

Design,Fabrication, and Characterization of Novel Porous Conductive Scaffolds for Nerve Tissue Engineering

Highly conductive polypyrrole/graphene (PYG) nanocomposite was synthesized with chemical oxidation process via emulsion polymerization and used for the preparation of novel porous conductive gelatin/chitosan-based scaffolds. The effect of PYG loading on various properties of scaffolds was investigated. The obtained results indicated that by introducing PYG into the polymeric matrix, the porosity and swelling capacity decreased while electrical conductivity and Young's modulus demonstrated increasing trend. The in vitro biodegradation test revealed that pure gelatin/chitosan matrix lost 80% of its weight after six weeks in the presence of lysozyme whilst the biodegradation rate was significantly lower for the conductive scaffolds. Furthermore, Schwann cell attachment and proliferation were evaluated by MTT assay and SEM image and the results revealed significant cell biocompatibility of the conductive scaffold with low amount of PYG. The results confirmed the potential of gelatin/chitosan/PYG compounding as a suitable biomaterial for using in nerve tissue engineering applications in which electrical stimulation plays a vital role.     

Biofabrication of Gelatin Tissue Scaffolds with Uniform Pore Size via Microbubble Assembly

The control of pore size and uniform porosity remains as an important challenge in gelatin scaffolds. The precise control in building blocks of tissue scaffolds without any additional porogen is possible with costly equipment and techniques, though some pre‐requirements for polymeric material, such as photo‐polymerizability or sintering ability, may be needed prior to construction. Herein, a method for the fabrication of gelatin scaffolds with homogenous porosity using simple T‐junction microfluidics is described. The size of the microbubbles is precisely controlled with 5% deviation from the average. Porous gelatin scaffolds are obtained by building‐up the monodispersed microbubbles in dilute cross‐linker solutions. The effect of cross‐linker density on pore diameter is also investigated. After cross‐linking, pore size of the resultant five scaffold groups are precisely controlled as 135 ± 11, 193 ± 11, 216 ± 9, 231 ± 5, and 250 ± 12 µm. Porosity ratios above 65% are achieved in every sample group. According to the cell culture experiments, structures support high cell adhesion, viability, and migration through the porous network via interconnectivity. This study offers a practical and economical approach for the preparation of porous gelatin scaffolds with homogenous porosity which can be utilized in diverse tissue engineering applications.     

Suture Fiber Reinforcement of a 3D Printed Gelatin Scaffold for Its Potential Application in Soft Tissue Engineering

Gelatin has excellent biological properties, but its poor physical properties are a major obstacle to its use as a biomaterial ink. These disadvantages not only worsen the printability of gelatin biomaterial ink, but also reduce the dimensional stability of its 3D scaffolds and limit its application in the tissue engineering field. Herein, biodegradable suture fibers were added into a gelatin biomaterial ink to improve the printability, mechanical strength, and dimensional stability of the 3D printed scaffolds. The suture fiber reinforced gelatin 3D scaffolds were fabricated using the thermo-responsive properties of gelatin under optimized 3D printing conditions (−10 °C cryogenic plate, 40–80 kPa pneumatic pressure, and 9 mm/s printing speed), and were crosslinked using EDC/NHS to maintain their 3D structures. Scanning electron microscopy images revealed that the morphologies of the 3D printed scaffolds maintained their 3D structure after crosslinking. The addition of 0.5% (w/v) of suture fibers increased the printing accuracy of the 3D printed scaffolds to 97%. The suture fibers also increased the mechanical strength of the 3D printed scaffolds by up to 6-fold, and the degradation rate could be controlled by the suture fiber content. In in vitro cell studies, DNA assay results showed that human dermal fibroblasts’ proliferation rate of a 3D printed scaffold containing 0.5% suture fiber was 10% higher than that of a 3D printed scaffold without suture fibers after 14 days of culture. Interestingly, the supplement of suture fibers into gelatin biomaterial ink was able to minimize the cell-mediated contraction of the cell cultured 3D scaffolds over the cell culture period. These results show that advanced biomaterial inks can be developed by supplementing biodegradable fibers to improve the poor physical properties of natural polymer-based biomaterial inks.     

Porous organosilicone modified gelatin hybrids with controllable and homogeneous in vitro degradation behaviors for potential application as skin regeneration scaffold

This work aims at investigating intensively the effects of organosilicone species and their dosage on the physicochemical and particularly the in vitro degradation properties of gelatin hybrids. We prepared various porous organosilicone modified gelatin hybrids with epoxy‐polydimethylsiloxane (PDMS) and/or glycidoxypropyltrimethoxysilane (GPTMS) and further systematically investigated their degradation behaviors in simulated physiological environments. It was found that the chemical composition, thermal stability, crosslinking degree, mechanical properties and porous structure of the gelatin hybrids could be tuned by adjusting the amount of PDMS and GPTMS. More importantly, degradation rates of the gelatin hybrids were reduced with increasing content of GPTMS, implying that the degradation behaviors could be controlled by tailoring the chemical interaction between the gelatin and organosilicone moieties. In addition, gelatin hybrids modified with both PDMS and GPTMS (PGs‐GE) were demonstrated as a homogeneous hybridization, and their maximum weight losses met the typical healing period of a normal skin wound. Noticeably, the P1G1‐GE hybrid with PDMS to GPTMS molar ratio 1:1 exhibited appropriate weight loss, integrity of pore structure and synchronous dissolution of silicon and protein during the degradation process, indicating a homogeneous degradation behavior. Furthermore, both the original and degraded P1G1‐GE hybrid exhibited favorable cytocompatibility in vitro. The findings will be helpful for further insight into the in vivo degradation of gelatin hybrids, suggesting their potential application as skin regeneration scaffolds. © 2019 Society of Chemical Industry     

壳聚糖对烟草抗菌活性研究

本试验通过对不同浓度、不同种类酸溶解和不同脱乙酰度的壳聚糖对烟草中主要霉菌的抑菌活性,以及加入卷烟烟丝中对其内在质量、焦油、烟碱等的影响研究,结果表明：酸溶解壳聚糖对烟草霉变微生物具有抑制作用,醋酸作溶剂较柠檬酸效果好,且施用于卷烟上对其内在品质有所改善,其主流烟气焦油、烟碱释放量也有所降低。     

In vitro characterization of porous calcium phosphate scaffolds capped with crosslinked hydrogels to avoid inherent brittleness

Ductile composite scaffolds can avoid being crushed during filling processes in clinical applications. Thus, this study aimed to modify brittle porous ceramic scaffolds into ductile scaffolds through hydrogel capping. The surfaces of calcium phosphate (CaP) ceramic scaffolds were effectively capped with alginate/gelatin hydrogels. The composite scaffolds were then crosslinked, subjected to vacuum drainage, and washed prior to lyophilization. The detailed controlled approach in this study was proposed with the aim to develop biocompatible composites with anisotropic open pores through the formation of a thin, homogenous, hydrogel film coating on porous ceramic scaffold surfaces. The performances of the hydrogel/CaP composite scaffolds were evaluated on the basis of their morphological characteristics, compressive strengths, and cell viabilities. Results showed that strength, toughness, and specimen integrity after cracking are strongly related to the concentration of the hydrogel cap. Strength testing results showed that the use of 50 vol% alcohol as the crosslinker removal solution yielded scaffolds with high toughness and ductility. Moreover, the cracked specimen dipped in 50 vol% alcohol possessed better integrity than that dipped in water only. This study successfully identified the optimal hydrogel quantity for the fabrication of biocompatible scaffolds with open connective pores. The advantages of the fabricated scaffolds indicate the relevance of the proposed method to clinical applications, such the production of fillers for successful alveolar bone augmentation.     

Development and Evaluation of Biomimetic 3D Coated Composite Scaffold for Application as Skin Substitutes

Skin injuries are an urgent health issue, which raises a great concern in the clinic. Although numerous strategies have been proposed to fabricate skin substitutes for treatment of wounds over the past several decades, fabricating an ideal skin substitute to replace the damaged one can still be a problem. In this study, a novel biomimetic 3D composite skin scaffold is fabricated by combining electrohydrodynamic (EHD) jetting, electrospinning, and coating techniques. Here, the first polycaprolactone (PCL) porous structure is produced by the EHD jetting. Next, the second polylactic acid (PLA) membrane consisted of nanoscale fibers is prepared on the PCL porous structure via the electrospinning. The PCL porous structure and PLA fibers membrane can mimic the dermis and epidermis layer, respectively. Furthermore, gelatin is used as coating solution to enhance the biocompatibility of the scaffold. The structure and morphology of the fabricated scaffolds are analyzed, and the mechanical properties are investigated as well. Moreover, the in vitro and in vivo experiments demonstrate the biocompatibility of the materials and the fabrication process. In conclusion, these results demonstrate that the composite scaffold is effective and holds great potential for skin regeneration in the clinic.     

Porous chitin matrices for tissue engineering: Fabrication and in vitro cytotoxic assessment

A series of porous chitin matrices were fabricated by freezing and lyophilization of chitin gels cast from a 5% N,N-dimethylacetamide (DMAc)/lithium chloride (LiCl) solvent system. The porous chitin matrices were found to have uniform pore structure in the micron range. Scanning electron microscopy (SEM) revealed that the pore size of the porous chitin matrices varied according to the freezing method prior to lyophilization. By subjecting the chitin gels to dry-ice/acetone (−38 °C), the final porous chitin matrix gave pore dimensions measuring 200–500 μm with 69% porosity. A smaller pore dimension of 100–200 μm with 61% porosity was produced when the chitin gels were frozen by liquid nitrogen (−196 °C) and 10 μm pores with 54% porosity were produced when the gels were placed in a freezer (−20 °C) for 20 min. In comparison, lower porosity structures of ca. 10% porosity were obtained from both air-dried and critical point dried chitin gels. Furthermore, a low gel concentration (< 0.5%, w/w) also produced porous morphology by vacuum drying without any freezing and lyophilization. The resulting pore properties influenced the water uptake profile of the materials in water. These porous chitin matrices are found to be non-cytotoxic and to hold promise as potential structural scaffolds for cell growth and proliferation in vitro.     

Fabrication of 3D melt electrowritting multiphasic scaffold with bioactive and osteoconductivite functionalities for periodontal regeneration

This work aimed to design a multifunctional biphasic 3D scaffold for periodontal tissue regeneration. A 3D fibrous scaffold made from medical-grade poly (ε-caprolactone) (PCL) with high porosity (>90%) and well-oriented fibres was fabricated by a custom design melt electrowriting (MEW) device. A biomimetic process was employed to form a bioactive calcium phosphate (CaP) layer with nanostructure (nanoflakes-like) morphology onto the 3D MEW fibrous surface to stimulate rapid bone formation. Primary human osteoblasts (hOBs) were seeded within the coated 3D fibrous scaffolds for 28 days to acquire the bone compartment of the tissue-engineered construct (TEC). The biphasic construct was obtained by placing an established in vitro periodontal ligament (PDL) cell sheet onto the surface of the bone compartment. Subsequently, a decellularized multiphasic TEC by exploiting a lyophilization approach was obtained. Laser scanning confocal microscopy and scanning electron microscopy confirmed the retention of a functional extracellular matrix within the PDL and bone compartments following scaffold decellularization and lyophilization processes. These findings suggest that lyophilized decellularized biphasic 3D constructs with high porosity constitute a viable ‘off the shelf’ strategy for developing an extracellular matrix-based product to facilitate periodontal regeneration.     

Chitosan: Application in tissue engineering and skin grafting

Tissue Engineering and skin grafting, an essential part of regenerative medicine is one of the fastest growing biomedical fields which could offer an important therapeutic strategy for management of hard to heal wounds. 2D and 3D polymeric scaffolds are prerequisites in this field to promote cell adhesion, proliferation and tissue regeneration. Convergence of technology and research has successfully unveiled unknown properties of Chitosan as a bioactive polymer. Natural abundance, cost effectiveness, biodegradability, biocompatibility and wound healing capabilities of chitosan and its derivatives has drawn the attention of many researchers for its use as an alternative for fabrication of a scaffold in tissue engineering and skin graft. However lower mechanical strength and solubility has limited its application in the biomedical field. It has been found that the derivatization and combination with other polymers can successfully overcome these limitations. This review focuses on the applicability of chitosan and its derivatives in combination with other polymers in tissue engineering and skin grafting along with the novel scaffold fabrication techniques. Studies so far have demonstrated the potential of chitosan and its derivative as a scaffold in the field of regenerative medicine. However, even if the promising results obtained from in-vitro and preclinical studies prove the efficacy of chitosan scaffolds it still has a long way to go to be used in clinical set ups.