Polyelectrolyte Complex Scaffolds Research Articles

Numerical analysis of scaffold degradation in cryogenic environment: impact of cell migration and cell apoptosis

The analysis of degradation in the presence of cell death and migration is a critical aspect of research in various biological fields, such as tissue engineering, regenerative medicine, and disease pathology. In present study, numerical study of degradation of scaffold were performed in present of cells, cell apoptosis and cell migration. A poly electrolyte complex (PEC) silk fibroin scaffold was used for degradation study. Degradation study in the presence of cells and migration were performed at fixed pH concentration 7.2. Similarly, degradation study of scaffold were performed at different pH cell apoptosis. A transient analysis of scaffold was evaluated in COMSOL 5.5 in presence of cryogenic temperature at different temperature gradient. The parameters; temperature, stress, strain tensor and deformation gradient associated with the degradation of polyelectrolyte complex scaffold were evaluated. Result shows that in both geometries minimum temperature had been achieved as 230.051 K at point P4 in series view and parallel view and at a point P3 for cell migration study for −5 k min−1 and −1 k min−1, respectively. The maximum stress had been generated for 5.57 × 107 N m−2 for the temperature gradient of −2 K min−1 at T cycle in the case of cell migration study. In contrast in series view the maximum stress 2.9 × 107 N m−2 were observed at P4 which was higher as compare to P3. Similarly, for a parallel view, maximum stress (3.93 × 107 N m−2) was obtained for point P3. It had been observed that the maximum strain tensor 5.21 × 10−3, 5.15 × 10−3 and 5.26 × 10−3 was generated in series view at 230 k on a point P3 for − 1, −2 and −5 K min−1, respectively. Similarly, the maximum strain tensor 8.16 × 10−3, 8.09 × 10−3 and 8.09 × 10−3 was generated in parallel view at 230 k on a point P3 for −1, −2 and −5 K min−1, respectively. In the presence of cells, at a point P4 for temperature gradient of −1 and −2 K min−1, it had been closed to the scaffold wall, which had a different temperature profile than the point P3 and scaffold comes to the contact with the cells. The analysis of PEC scaffold degradation in the presence of cells, including cell apoptosis and migration, offers significant insights into the relationship between scaffold properties, cell behaviour, and tissue regeneration.

Fabrication and investigation of physicochemical and biological properties of3Dprinted sodium alginate‐chitosan blend polyelectrolyte complex scaffold for bone tissue engineering application

AbstractIn tissue engineering technique, a biological scaffold with appropriate composition and structure for promoting growth and differentiation of cells thereby regenerating damaged tissue, is a prime necessity. In this paper, 3D‐printed scaffolds comprising sodium alginate (SA) and chitosan (CH) biopolymers of natural origin are reported. Bioinks with varying ratios of SA and CH were prepared and scaffolds were fabricated by 3D printing. The fabricated scaffolds possess many desired properties that are vital for tissue regeneration purposes. The scaffolds possess open pore microstructures with interconnected pores and desired pore size as revealed by scanning electron microscopic image analysis. The polyelectrolyte complex formation (PEC) between SA and CH as revealed by Fourier‐transform‐infrared spectroscopic analysis is favorable as it offers a better surface for cell attachment and proliferation, and an ideal microenvironment for bone regeneration. Among the scaffolds, SA/CH with 60:40 showed controlled swelling and degradation behavior, with higher tensile strength of 0.387 ± 0.015 MPa. In vitro‐biomineralization showed superior apatite layer deposition ability over the SA/CH: 60/40 scaffold surface. The fabricated SA/CH scaffolds are hydrophilic and biocompatible as evident from the contact angle, protein adsorption, MTT assay, and cell attachment studies. However, SA/CH: 60/40 is shown to have superior biological properties compared with the other SA/CH compositions. Thus, it is concluded that 3Dv printed SA/CH: 60/40 scaffold having some superior properties and bioactivity can be used as a suitable matrix for future bone tissue regeneration applications.

Controllable fabrication of alginate/poly-L-ornithine polyelectrolyte complex hydrogel networks as therapeutic drug and cell carriers

Polyelectrolyte complex (PEC) hydrogels are advantageous as therapeutic agent and cell carriers. However, due to the weak nature of physical crosslinking, PEC swelling and cargo burst release are easily initiated. Also, most current cell-laden PEC hydrogels are limited to fibers and microcapsules with unfavorable dimensions and structures for practical implantations. To overcome these drawbacks, alginate (Alg)/poly-L-ornithine (PLO) PEC hydrogels are fabricated into microcapsules, fibers, and bulk scaffolds to explore their feasibility as drug and cell carriers. Stable Alg/PLO microcapsules with controllable shapes are obtained through aqueous electrospraying technique, which avoids osmotic shock and prolongs the release time. Model enzyme and nanosized cargos are successfully encapsulated and continuously released for more than 21 days. Alg/PLO PEC fibers are then prepared to encapsulate brown adipose progenitors (BAPs) and Jurkat T cells. The electrostatic interactions between Alg and PLO are found to facilitate the printability and self-support ability of Alg/PLO bioinks. Alg/PLO PEC fibers and scaffolds support cell proliferation, differentiation, and functionalization. These results demonstrate new options for biocompatible PEC hydrogel preparation and improve the understanding of PEC hydrogels as drug and cell carriers. Statement of significanceIn this study, the concept of polyelectrolyte complex hydrogel networks as drug and cell carriers has been demonstrated. Their feasibility to achieve sustained drug release and cell functionality was explored, from microcapsules to fibers to three-dimension printed scaffolds. PEC microcapsules with controllable shapes were obtained. Therapeutic drugs can be encapsulated and continuously release for more than 21 days. Benefiting from the dynamic interactions of physically crosslinked PEC, self-healing fibers were achieved. Besides, the electrostatic interactions between polyelectrolytes were found to facilitate the printability and self-support ability of PEC bioinks. The PEC fibers and scaffolds with controllable structure supported cell proliferation, differentiation, and function. The outcome of current research promotes design of new biocompatible PEC hydrogels and potential drug and cell carriers.

Graphene oxide-reinforced pectin/chitosan polyelectrolyte complex scaffolds

Three-dimensional (3D) porous scaffolds based on graphene oxide (GO) incorporated pectin/chitosan polyelectrolyte complex (PCGO) were prepared by the freeze-drying technique. The chemical composition and microstructure of the prepared PCGO scaffolds were studied by FTIR and XRD analysis. The presence of GO and its uniform distribution within the polymer matrix was confirmed by Raman spectroscopy and confocal Raman mapping analysis, respectively. TGA analysis revealed that the addition of GO improves the thermal stability of the pectin/chitosan complex. SEM analysis confirmed the uniform pore distribution of PCGO scaffolds. Moreover, it showed that the pore size of the scaffolds was decreased with the increase in GO content. Among the developed PCGO scaffolds, the scaffolds with 1 wt.% of GO presented the improved hydrophilicity by exhibiting the water swelling degree of 2004%, water retention capacity of 1101% and water contact angle (WCA) of 21°. In addition, these scaffolds presented better compressive strength (∼283 kPa) and resistance towards lysozyme-mediated degradation. The PCGO scaffolds presented an acceptable level of bio-and hemocompatibility and GO concentration-dependent cell attachment ability. These results demonstrate the suitability of PCGO scaffolds for tissue engineering.

Hydroxyapatite Nanoparticles Fortified Xanthan Gum-Chitosan Based Polyelectrolyte Complex Scaffolds for Supporting the Osteo-Friendly Environment.

Nanoparticle-reinforced polymer-based scaffolding matrices as artificial bone-implant materials are potential suitors for bone regenerative medicine as they simulate the native bone. In the present work, a series of bioinspired, osteoconductive tricomposite scaffolds made up of nano-hydroxyapatite (NHA) embedded xanthan gum-chitosan (XAN-CHI) polyelectrolyte complex (PEC) are explored for their bone-regeneration potential. The Fourier transform infrared spectroscopy studies confirmed complex formation between XAN and CHI and showed strong interactions between the NHA and PEC matrix. The X-ray diffraction studies indicated regulation of the nanocomposite (NC) scaffold crystallinity by the physical cues of the PEC matrix. Further results exhibited that the XAN-CHI/NHA5 scaffold, with a 50/50 (polymer/NHA) ratio, has optimized porous structure, appropriate compressive properties, and sufficient swelling ability with slower degradation rates, which are far better than those of CHI/NHA and other XAN-CHI/NHA NC scaffolds. The simulated body fluid studies showed XAN-CHI/NHA5 generated apatite-like surface structures of a Ca/P ratio ∼1.66. Also, the in vitro cell-material interaction studies with MG-63 cells revealed that relative to the CHI/NHA NC scaffold, the cellular viability, attachment, and proliferation were better on XAN-CHI/NHA scaffold surfaces, with XAN-CHI/NHA5 specimens exhibiting an effective increment in cell spreading capacity compared to XAN-CHI/NHA4 and XAN-CHI/NHA6 specimens. The presence of an osteo-friendly environment is also indicated by enhanced alkaline phosphatase expression and protein adsorption ability. The higher expression of extracellular matrix proteins, such as osteocalcin and osteopontin, finally validated the induction of differentiation of MG-63 cells by tricomposite scaffolds. In summary, this study demonstrates that the formation of PEC between XAN and CHI and incorporation of NHA in XAN-CHI PEC developed tricomposite scaffolds with robust potential for use in bone regeneration applications.

Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate

Chitosan microparticles were mixed with chitosan and carboxymethyl cellulose solution to achieve a good binding between the microparticles. Three different compositions of scaffolds were made by varying the calcium phosphate (CaP) amount: 0%, 10%, and 20%. Potassium chloride was used as salt, to make pores inside the scaffolds after leaching out when immersed in phosphate buffer saline (PBS). Compressive strength and compressive modulus of both non-porous (before leaching out), and porous (after leaching out) scaffolds were measured according to the ASTM standards. The highest compressive strength of 27 MPa was reported on 10% CaP scaffolds while 20% CaP scaffolds showed the lowest. The increasing CaP content reduces the compressive strength of the scaffolds. The highest wet state compressive strength was reported on 0% CaP scaffolds with 0.36 MPs and 0.40 MPa at day 1 and day 3 respectively. In vitro cell culture studies showed good cell adhesion and cell proliferation on 10% CaP scaffolds.

Fabrication and Characterization of Chitosan-Hyaluronic Acid Scaffolds with Varying Stiffness for Glioblastoma Cell Culture.

The invasive and recurrent nature of glioblastoma multiforme (GBM) is linked to a small subpopulation of cancer cells, which are self-renewing, resistant to standard treatment regimens, and induce formation of new tumors. Matrix stiffness is implicated in the regulation of cell proliferation, drug resistance, and reversion to a more invasive phenotype. Therefore, understanding the relationship between matrix stiffness and tumor cell behavior is vital to develop appropriate in vitro tumor models. Here, chitosan-hyaluronic acid (CHA) polyelectrolyte complex scaffolds are fabricated with statistically significant stiffness variances to characterize the effect of scaffold stiffness on morphology, proliferation, drug resistance, and gene expression in human glioblastoma cells (U-87 MG). All scaffolds support GBM proliferation over a 12-day culture period, yet larger spheroids are observed in scaffolds with higher stiffness. Additionally, GBM cells cultured in stiffer CHA scaffolds prove significantly more resistant to the common chemotherapeutic temozolomide. Moreover, the stiffer 8% CHA scaffolds exhibit an increase in expression of drug resistance and invasion related genes compared to 2D culture. CHA scaffolds present a tunable microenvironment for enhanced tumor cell malignancy and may provide a valuable in vitro microenvironment for studying tumor progression and screening anticancer therapies.

Immobilization of β-galactosidase in Chitosan-Alginate composite scaffolds and optimization of lactose hydrolysis

The immobilization of lactase (s-galactosidase) enzyme was achieved by entrapping it in chitosan-alginate composite scaffolds, which were formed by ionotropic pre-gelation followed by a pH increase to synthesize polyelectrolyte complex scaffolds, and subsequent cross-linking and activation by glutaraldehyde. The operational stability upon storage of the enzyme entrapped was checked by performing batch hydrolysis of lactose concentrations ranging from 1mg/ml to 5mg/ml and the resulting glucose produced in every reaction was determined using the glucose oxidase-peroxidase assay. This study also investigates the stability of the free and immobilized enzymes at altered sets of pH and temperature at a fixed concentration of enzyme for the reaction medium. Morphology and structure characterization of lyophilized composite scaffolds were examined using scanning electron microscope and Fourier transform infra-red spectra, respectively.

Understanding the molecular mechanism of improved proliferation and osteogenic potential of human mesenchymal stem cells grown on a polyelectrolyte complex derived from non-mulberry silk fibroin and chitosan

The development of engineered bone tissue, as a promising alternative to conventional bone grafts, has so far not proven successful and still remains challenging. Thus, attempts have been made in the present study to synthesize polyelectrolyte complex (PEC) scaffolds by blending chitosan (CS) to silk fibroin (SF) derived from the non-mulberry silkworm (Antheraea pernyi) at three different pH values (5.0, 6.0, and 7.0), and to characterize them in terms of morphology, ultrastructure and mechanical properties with scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, x-ray diffraction and tensile strength analyses. The prepared PEC scaffolds showed a mean pore size of 130 μm, as revealed by SEM analysis, and a comparatively higher compressive strength. The findings of in vitro cytocompatibility, in vivo biocompatibility and osteogenic marker (genes/proteins) analysis suggest that the PECs blended at pH 7.0 showed greater stability and enhanced growth and an osteogenic differentiation capability of human mesenchymal stem cells (MSCs). To aid our understanding of protein–polyion binding mechanisms, we employed a molecular docking and simulation study of SF macrodomains and CS oligomer using Schrödinger 14 and GROMACS (Groningen Machine for Chemical Simulations) software. The study involved analytical techniques for macromolecular solution characterization and theoretical simulations based on molecular dynamics. The computational studies confirmed the presence of an integral RGD sequence that played a vital role in superior cell-attachment, proliferation and osteogenic differentiation of MSCs grown on the developed SF–CS PEC scaffolds.

Restoration of Epidural Fat with Poly(L-glutamic acid) and Chitosan Polyelectrolyte Complex Scaffold Seeded with Adipose Derived Stem Cells

Restoration of Epidural Fat with Poly(L-glutamic acid) and Chitosan Polyelectrolyte Complex Scaffold Seeded with Adipose Derived Stem Cells

Chitosan–hyaluronic acid polyelectrolyte complex scaffold crosslinked with genipin for immobilization and controlled release of BMP-2

Polyelectrolyte complex (PEC) is formed when polymers with opposite charges are combined in solution. PECs are recently gaining attention as carriers for controlled release of drugs and proteins. Herein, bone morphogenetic protein-2 (BMP-2) was immobilized in a PEC of natural polymers, chitosan and hyaluronic acid. Charge-to-charge stoichiometry of the formed PEC was estimated based on turbidity of combined chitosan and hyaluronic acid solutions. Free amino groups in chitosan were crosslinked with different amounts of genipin. The degree of crosslinking, consequently its effects in vitro in terms of swelling, degradation and cytocompatibility were analyzed. Immobilization of three different amount of BMP-2 in chitosan–hyaluronic acid PEC scaffold resulted sustained release of the growth factor for more than 30 days. Immobilization efficacies varied from 61% to 76% depending on the amount of BMP-2. Finally effects in osteogenic differentiation of the PEC with BMP-2 to MC3T3-E1 cells were determined by reverse transcriptase PCR.

Osteogenic activity of chitosan-based hybrid scaffold prepared by polyelectrolyte complex formation with alginate

A chitosan (CS)/alginate (AG) hybrid scaffold was prepared through solid state polyelectrolyte complex (PEC) formation. The PEC formation was confirmed by Fourier-transformed infrared spectroscopy. Positive von Kossa staining of calcium depositions appeared in a large area of the CS/AG PEC scaffold cultured with osteoblastlike KUSA-A1 cells. The alkaline phosphatase activity of the cells cultured in the CS/AG scaffold was higher than that of the cells in the CS/hydroxyapatite (HA) scaffold. The osteogenic activity of the CS/AG PEC scaffold was considered comparable to that of the CS/HA scaffold. These findings suggest that modification of CS-based scaffold by PEC formation have a significant effect on the cell behavior towards osteogenic differentiation and bone tissue regeneration.

Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(l-glutamic acid) and chitosan

As a synthetic polypeptide water-soluble poly(l-glutamic acid) (PLGA) was designed to fabricate scaffolds for cartilage tissue engineering. Chitosan (CHI) has been employed as a physical cross-linking component in the construction of scaffolds. PLGA/CHI scaffolds act as sponges with a swelling ratio of 760±45% (mass%), showing promising biocompatibility and biodegradation. Autologous adipose-derived stem cells (ASCs) were expanded and seeded on PLGA/CHI scaffolds, ASC/scaffold constructs were then subjected to chondrogenic induction in vitro for 2weeks. The results showed that PLGA/CHI scaffolds could effectively support ASC adherence, proliferation and chondrogenic differentiation. The ASCs/scaffold constructs were then transplanted to repair full thickness articular cartilage defects (4mm in diameter, to the depth of subchondral bone) created in rabbit femur trochlea. Histological observations found that articular defects were covered with newly formed cartilage 6weeks post-implantation. After 12weeks the regenerated cartilage had integrated well with the surrounding native cartilage and subchondral bone. Toluidine blue and immunohistochemical staining confirmed similar accumulation of glycosaminoglycans and type II collagen in engineered cartilage as in native cartilage 12weeks post-implantation. The result was further supported by quantitative analysis of extracellular matrix deposition. The compressive modulus of the engineered cartilage increased significantly from 30% of that of normal cartilage at 6weeks to 83% at 12weeks. Cyto-nanoindentation also showed analogous biomechanical behavior of the engineered cartilage to that of native cartilage. The results of the present study thus demonstrate the potentiality of PLGA/CHI scaffolds in cartilage tissue engineering.

Chitosan-based polyelectrolyte complex scaffolds with antibacterial properties for treating dental bone defects

The aim of this study was to develop an antibacterial polyelectrolyte complex (PEC) scaffold for treating dental bone defects. The PEC scaffold was composed of chitosan (CS), γ-polyglutamic acid (γ-PGA), and carboxy-methyl-cellulose (CMC). The resulted network structures formed via electrostatic crosslinking were characterized by using FTIR, gel content, equilibrium swelling ratio, volume change, and SEM test. The antibacterial property, cell cytotoxicity, and in vivo biocompatibility tests were conducted according to an agar diffusion method, ISO10993-5, and ISO 10993-6, respectively. The resulted specimens showed an interconnected pore structure with pore sizes ranging 100–500 μm. The equilibrium swelling ratio, volume change, and antibacterial property were inversely proportional to the gel content. The PEC-2 scaffold composed of 8 wt.% CS, and 2 wt.% γ-PGA + 2 wt.% CMC had more-suitable gel properties (gel content of 55.3 ± 1.1 wt.% and volume change of 97.7 ± 1.4 v/v%) with inhibition zones of 14.4 ± 0.3 mm for Escherichia coli and 13.0 ± 0.7 mm for Staphylococcus aureus. The cytotoxicity and cell attachment tests of the PEC scaffolds showed satisfactory cell compatibility. Moreover, the in vivo biocompatibility test of the PEC scaffolds revealed little foreign body reaction. For this reason, the newly developed antibacterial PEC scaffold may be a good alternative for dental applications.

Influence of processing parameters on pore structure of 3D porous chitosan–alginate polyelectrolyte complex scaffolds

Fabrication of porous polymeric scaffolds with controlled structure can be challenging. In this study, we investigated the influence of key experimental parameters on the structures and mechanical properties of resultant porous chitosan-alginate (CA) polyelectrolyte complex (PEC) scaffolds, and on proliferation of MG-63 osteoblast-like cells, targeted at bone tissue engineering. We demonstrated that the porous structure is largely affected by the solution viscosity, which can be regulated by the acetic acid and alginate concentrations. We found that the CA PEC solutions with viscosity below 300 Pa.s yielded scaffolds of uniform pore structure and that more neutral pH promoted more complete complexation of chitosan and alginate, yielding stiffer scaffolds. CA PEC scaffolds produced from solutions with viscosities below 300 Pa.s also showed enhanced cell proliferation compared with other samples. By controlling the key experimental parameters identified in this study, CA PEC scaffolds of different structures can be made to suit various tissue engineering applications.

Sodium hyaluronate/chitosan polyelectrolyte complex scaffolds for dental pulp regeneration: Synthesis and characterization

In the present study, small-sized porous scaffolds were obtained from the freeze-drying of sodium hyaluronate/chitosan polyelectrolyte complexes. The obtained materials were characterized by a set of techniques including attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, swelling determination and weight loss studies. The morphology of the scaffolds was observed using scanning electron microscopy. Thermal characterization of the scaffolds was also performed by dynamic mechanical thermal analysis and thermogravimetric analysis. Finally, the cytotoxic profile of the prepared scaffolds was evaluated in vitro, using mesenchymal stem cells. The results obtained showed that cells adhered to scaffolds and proliferated. This study also confirmed that the degradation by-products of sodium hyaluronate/chitosan scaffold are noncytotoxic, which is fundamental for its application in the biomedical field.

Silk fibroin protein and chitosan polyelectrolyte complex porous scaffolds for tissue engineering applications

In this study, we examined the porous polyelectrolyte complex scaffolds of silk fibroin and amino polysaccharide chitosan. The blended scaffolds were fabricated and characterized. The fabricated scaffolds showed pore sizes in the range of 100–160 μm as revealed by scanning electron microscope, good interconnectivity and high porosity. Fourier transform infrared and X-ray diffraction results demonstrated the blend formation. The addition of silk fibroin reduced the degradation of chitosan containing scaffolds in lysozyme solution. The blended scaffolds showed a higher compressive strength and modulus than the individual components. Chitosan incorporation had an antibacterial effect when incorporated at the higher levels in the blends. In vitro cytocompatibility results demonstrated that the blended scaffolds supported the growth and adhesion of feline fibroblasts. Taken together, the silk fibroin/chitosan scaffolds might be a promising biohybrid material for tissue engineering.

Development of a chitosan–polyglutamate based injectable polyelectrolyte complex scaffold

In this study, the chitosan–polyglutamate hydrogel was developed as injectable polyelectrolyte complex (PEC) scaffold for bone regeneration. In practice, oppositely charged polyelectrolytes were mixed using a static injection mixer, the gelation time was less than 1 min. The resorbable PEC scaffold can be in situ-formed as a scaffold for osteoblast ingrowth. The canine alveolar defect model was used to evaluate the efficacy of PEC implants. The PEC scaffold was highly effective in enhancing bone regeneration. Moreover, the addition of CaSO 4·2H 2O (CSD) to PEC scaffold can further enhance the early stage healing of bone regeneration.

Preparation and chemical and biological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications

In this work, porous scaffolds obtained from the freeze-drying of pectin/chitosan polyelectrolyte complexes were prepared and characterized by FTIR, SEM and weight loss studies. Additionally, the cytotoxicity of the prepared scaffolds was evaluated in vitro, using human osteoblast cells. The results obtained showed that cells adhered to scaffolds and proliferated. The study also confirmed that the degradation by-products of pectin/chitosan scaffold are noncytotoxic.

Alginate–chitosan/hydroxyapatite polyelectrolyte complex porous scaffolds: Preparation and characterization

Porous scaffolds composed of alginate (AG) and chitosan (CS) were fabricated by combining the formation of polyelectrolyte complex (PEC) with freeze-drying. The AG scaffold was used as a framework with uniformly distributed and interconnected pore structure. The chitosan or chitosan/hydroxyapatite (CS/HA) composite solution was introduced into the pores of AG scaffold to form PEC scaffolds. FT-IR, XRD and XPS analysis confirmed that CS or CS/HA was coated on the AG scaffold surface. Microstructure, porosity, mechanical strength and thermal stability of PEC scaffolds were also investigated. The AG–CS PEC scaffold and Ca 2+ crosslinked AG scaffold showed smaller average pore diameter and lower porosity than those of uncrosslinked AG scaffold. Moreover, compared with Ca 2+ crosslinked AG scaffold, AG–CS PEC scaffold exhibited higher mechanical strength and better thermal stability.