Evaluation of biodegradation and biocompatibility of collagen/chitosan/alkaline phosphatase biopolymeric membranes

The aim of this study was to develop a new variant of membranes based on collagen (COL), chitosan (CHI) and alkaline phosphatase (ALP) immobilized and cross-linking with glutaraldehyde (GA) at different concentrations. The biodegradation in the presence of collagenase was investigated. Biocompatibility was evaluated by MTT assay using a mouse fibroblast cell culture type NCTC (clone 929). Non-cross-linked samples were biocompatible and membranes cross-linked with low concentrations of GA (0.04, 0.08%) were also biocompatible. However, high concentrations of GA lead to a decreased biocompatibility. The adsorption behaviour of Ca²⁺ ions to all membranes were evaluated using the Freundlich isotherms. Haemolytic studies were performed in order to consider their applications in biomineralization process. By the addition of collagen and ALP to chitosan, the haemolytic index decreases, the COL–CHI–ALP membrane being in the non-haemolytic domain, while the COL–CHI–ALP–GA membrane has a haemolytic index greater than 2, and is slightly haemolytic.     

Development of poly (lactic-co-glycolic acid)-collagen scaffolds for tissue engineering

Collagen as an important extra-cellular matrix (ECM) in many tissues is weakly antigenic and the structure of collagen sponges is highly porous with interconnected pores effective for cell infiltration and mass transfer of oxygen and nutrients. Its application as a scaffold is limited by poor mechanical strength and rapid biodegradation. In this paper, we attempt to graft hydrolyzed PLGA fiber surfaces with collagen by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), and then embed these collagen-grafted PLGA fibers in collagen sponge to form a hybrid PLGA-collagen scaffold. For further stability, we cross-linked the collagen in the scaffold and used it in rat liver cell cultivation. The scaffold was characterized by mechanical micro-tester, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Results showed that (1) the scaffolds exhibited isotropic and interconnected porous structure; (2) the compression modulus of this scaffold was enhanced 50 fold compared to the collagen scaffolds. The cell attachment and cytotoxicity of this scaffold were studied. Cell attachment was improved remarkably and the cytotoxicity of the hybrid PLGA-collagen scaffold was lower than that of the un-grafted PLGA-collagen scaffolds using alamarBlue™ assay normalized to the DNA content in each scaffold. This new hybrid scaffold has potential applications for tissue engineering.     

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds. This work was performed in the National Centre for Biomedical Engineering Science, Orbsen Building, National University of Ireland, Galway, Ireland.     

Microstructure and characteristic properties of gelatin/chitosan scaffold prepared by a combined freeze-drying/leaching method

A combined freeze-drying and particulate leaching method for scaffold synthesis showed an improvement in the horizontal microstructure of the gelatin/chitosan scaffolds. Type and concentration of the cross-linking agent, freezing temperature, concentration of the polymeric solution and gelatin/chitosan weight ratio were the variables affecting the scaffold properties. Assessment of the tensile properties of the scaffolds revealed that for a scaffold with 50% chitosan, glutaraldehyde, as a cross-linking agent, created much tighter polymeric network compared to N,N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide (EDC). However, in the case of gelatin scaffolds, EDC was identified as the stronger cross-linker. Compressive behavior of the scaffolds satisfied formulations obtained from the theoretical modeling of the low-density, elastomeric foams. The investigation of the scaffold degradation indicated that the increase in the mechanical strength of the scaffolds would not always reduce their degradation rate.     

Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.     

Optimization strategies on the structural modeling of gelatin/chitosan scaffolds to mimic human meniscus tissue

Meniscus lesions are frequently occurring injuries with poor ability to heal. Typical treatment procedure includes removal of damaged regions, which can lead to sub-optimal knee biomechanics and early onset of osteoarthritis. Some of the drawbacks of current treatment approach present an opportunity for a tissue engineering solution. In this study, gelatin (G)/chitosan (Cs) scaffolds were synthesized via gel casting method and cross-linked with naturally derived cross-linker, genipin, through scaffold cross-linking method. Based on the characteristics of native meniscus tissue microstructure and function, three different layers were chosen to design the macroporous multilayered scaffolds. The multi-layered scaffolds were investigated for their ability to support human-derived meniscus cells by evaluating their morphology and proliferation using MTT assay at various time points. Based on structural, mechanical and cell compatibility considerations, laminated scaffolds composed of G60/Cs40, G80/Cs20 and G40/Cs60 samples, for the first, second and third layers, respectively, could be an appropriate combination for meniscus tissue engineering applications.     

Attachment of stem cells on porous chitosan scaffold crosslinked by Na5P3O10

In this study, porous chitosan scaffolds were prepared by freeze-dried method using Na₅P₃O₁₀ as a crosslinking agent. The three-dimensional pore structure of the scaffold was interconnected with a mean pore size about 40 to 100 μm. The remained weight of crosslinked scaffold was about 76% after being exposed to PBS for 30 days. Mouse embryonic stem (E. S.) cells could grow on these crosslinked scaffolds. E. S. cells differentiated to other cells after 21 days of culturing on the scaffolds. The growth rate of E. S. cells was improved by post surface treatment of the scaffolds with collagen. However, there was no significant increase in growth rate of E. S. cells when scaffolds were surface treated with argon plasma. These porous chitosan scaffolds present a promising approach for tissue engineering applications.     

In vitro gentamicin sustained and controlled release from chitosan cross-linked films

A novel wound dressing film was investigated for controlled and sustained delivery of gentamicin, while covering and protecting the wound. Chitosan was cross-linked with Hexamethylene 1,6-bis (aminocarboxysulfonate) to prepare the wound dressing films. Cross-linking reaction was required to control the water retention and release of encapsulated gentamicin. Cross-linked films swell less and became more hydrophilic than chitosan film itself. However, this behavior was inversely proportional to the degree of cross-linking. In vitro gentamicin release from the cross-linked films, at physiological conditions of pH and temperature, was studied for 2 weeks. The effects of gentamicin initial concentration and cross-linking ratio on the kinetics of gentamicin release were evaluated. Results showed that the diffusion rate was governed by initial concentration of gentamicin and degree of cross-linking, since higher gentamicin initial concentration and degree of cross-linking promoted the slower release, while lower gentamicin initial concentration and degree of cross-linking promoted the faster one.     

Preparation and characterization of oxidized alginate covalently cross-linked galactosylated chitosan scaffold for liver tissue engineering

Liver tissue engineering (LTE) requires a perfect extracellular matrix (ECM) for hepatocytes culture to maintain high level of liver-specific functions. Here, we reported a LTE scaffold derived from oxidized alginate covalently cross-linked galactosylated chitosan via Schiff base reaction, without employing any extraneous chemical cross-linking agent. The structure of galactosylated chitosan (GC) and oxidized alginate was confirmed by Fourier transformed infrared (FTIR) spectra, proton nuclear magnetic resonance (¹H-NMR) spectroscopy, X-ray diffraction (XRD) or thermogravimetric (TG) analysis. The structure and properties of a series of the scaffolds were characterized by FTIR, XRD, scanning electron microscopy (SEM), porosity, equilibrium swelling, mechanical properties, thermal stability and in vitro degradation. FTIR spectra confirmed the characteristic peak of Schiff base groups in the scaffolds and XRD indicated the scaffolds could be amorphous. SEM analysis showed that the scaffolds displayed highly porous surfaces with average pore size of 50-150 μm and interconnected pore structure in the internal structure with average pore size of 100-250 μm. Porosity measurement suggested the scaffolds had a porosity of about 70%. The compressive modulus of the scaffolds (hydrated) was in the range of 4.2-6.3 kPa. Further studies showed that, with the increase of the oxidized alginate content, the equilibrium swelling and in vitro degradation rate of the scaffolds decreased and the thermal stability slightly increased, which might mainly attribute to the difference of the degree of cross-linking and the nature properties of the raw materials. Additionally, the biocompatibility of the scaffolds was evaluated in vitro. The results showed that the hepatocytes cultured on the scaffolds had a typical spheroidal morphology, formed multi-cellular aggregates and presented perfect integration with the scaffolds, which suggested that the scaffolds may be potential candidates for LTE strategies.     

The effect of cross-linking on the microstructure,mechanical properties and biocompatibility of electrospun polycaprolactone–gelatin/PLGA–gelatin/PLGA–chitosan hybrid composite

Abstract

In this study, multilayered scaffolds composed of polycaprolactone (PCL)–gelatin/poly(lactic-co-glycolic acid) (PLGA)–gelatin/PLGA–chitosan artificial blood vessels were fabricated using a double-ejection electrospinning system. The mixed fibers from individual materials were observed by scanning electron microscopy. The effects of the cross-linking process on the microstructure, mechanical properties and biocompatibility of the fibers were examined. The tensile stress and liquid strength of the cross-linked artificial blood vessels were 2.3 MPa and 340 mmHg, respectively, and were significantly higher than for the non-cross-linked vessel (2.0 MPa and 120 mmHg). The biocompatibility of the cross-linked artificial blood vessel scaffold was examined using the MTT assay and by evaluating cell attachment and cell proliferation. The cross-linked PCL–gelatin/PLGA–gelatin/PLGA–chitosan artificial blood vessel scaffold displayed excellent flexibility, was able to withstand high pressures and promoted cell growth; thus, this novel material holds great promise for eventual use in artificial blood vessels.     

The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/β-glycerophosphate hydrogel: in vitro and in vivo

Type I collagen was added to the composite chitosan solution in a ratio of 1:2 to build a physical cross-linked self-forming chitosan/collagen/β-GP hydrogel. Osteogenic properties of this novel injectable hydrogel were evaluated. Gelation time was about 8 min which offered enough time for handling a mixture containing cells and the subsequent injection. Scanning electronic microscopy (SEM) observations indicated good spreading of bone marrow mesenchymal stem cells (BMSCs) in this hydrogel scaffold. Mineral nodules were found in the dog-BMSCs inoculated hydrogel by SEM after 28 days. After subcutaneous injection into nude mouse dorsum for 4 weeks, partial bone formation was observed in the chitosan/collagen/β-GP hydrogel loaded with pre-osteodifferentiated dog-BMSCs, which indicated that chitosan/collagen/β-GP hydrogel composite could induce osteodifferentiation in BMSCs without exposure to a continual supply of external osteogenic factors. In conclusion, the novel chitosan/collagen/β-GP hydrogel composite should prove useful as a bone regeneration scaffold.     

Production and in vitro characterization of 3D porous scaffolds made of magnesium carbonate apatite (MCA)/anionic collagen using a biomimetic approach

3D porous scaffolds are relevant biomaterials to bone engineering as they can be used as templates to tissue reconstruction. The aim of the present study was to produce and characterize in vitro 3D magnesium-carbonate apatite/collagen (MCA/col) scaffolds. They were prepared by using biomimetic approach, followed by cross-linking with 0.25% glutaraldehyde solution (GA) and liofilization. Results obtained with Fourier-transform infrared spectroscopy (FT-IR) confirmed the type-B carbonate substitution, while by X-ray diffraction (XRD), a crystallite size of ~ 10 nm was obtained. Optical and electron microscopy showed that the cylindrical samples exhibited an open-porous morphology, with apatite nanocrystals precipitated on collagen fibrils. The cross-linked 3D scaffolds showed integrity when immersed in culture medium up to 14 days. Also, the immersion of such samples into an acid buffer solution, to mimic the osteoclastic resorption environment, promotes the release of important ions for bone repair, such as calcium, phosphorus and magnesium. Bone cells (SaOs2) adhered, and proliferated on the 3D composite scaffolds, showing that synthesis and the cross-linking processes did not induce cytotoxicity.     

Fabrication and characterization of chitosan microspheres agglomerated scaffolds for bone tissue engineering

In the presented paper authors describe a method for bone scaffolds fabrication. The technique is based on the agglomeration of chitosan microspheres. The fabrication process is complex and consists of a few steps: chitosan spheres extrusion, scaffold formation by compression followed by the spheres agglomeration and bonding with cross-linking agent (STPP, sodium tripolyphosphate). The described method allows manufacturing of porous materials with controllable shape, pore size distribution and their interconnectivity. In this technique 3D scaffold porosity can be regulated by altering spheres diameter. Authors studied influence of cross-linker concentrations and time of cross-linking process on the scaffold morphology, mechanical properties, enzymatic degradation rate (in the presence of lysozyme) and human osteoblasts response. Surface morphology and topography were evaluated by SEM. Porosity and pore interconnectivity were observed via μCT scanning. Mechanical tests showed that chitosan scaffolds perform compression characteristic (Young Modulus) similar to natural bone. Cytotoxicity established by XTT assay confirmed that most of the developed composite materials do not show toxic properties. Osteoblast adhesion and morphology were analyzed by SEM and optical microscopy.     

双交联明胶-壳聚糖复合人工真皮支架的制备及其性能研究

采用1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和三聚磷酸钠(STPP)作为共价-离子双交联剂交联制备了明胶-壳聚糖(gel-CS)复合人工真皮支架。通过FT-IR和SEM对复合多孔支架结构及形貌进行表征,同时对复合多孔支架的孔隙率、平均孔径、溶胀性能和降解性能进行了研究。研究结果表明,明胶和壳聚糖两相间成功地发生了交联反应,形成了具有贯通孔结构和良好孔隙率的复合多孔支架。该支架平均孔径在136.1～182.9μm之间,且随着壳聚糖含量的增加,孔径及孔隙率随之降低;溶胀性能研究发现,相较于单一共价交联方法,采用双交联法制备的支架溶胀率有所降低,再次印证双交联法制备的支架其交联度较单一共价交联的高;酶降解实验表明,固定STPP含量,随着EDC含量增加,复合支架的降解性能越好,而EDC含量一定,则STPP含量为0.5%时,复合支架具有最好的降解性能。     

Investigation of cancer cell behavior on nanofibrous scaffolds

Tissue engineering and the use of nanofibrous biomaterial scaffolds offer a unique perspective for studying cancer development in vitro. Current in vitro models of tumorigenesis are limited by the use of static, two-dimensional (2D) cell culture monolayers that lack the structural architecture necessary for cell-cell interaction and three-dimensional (3D) scaffolds that are too simplistic for studying basic pathological mechanisms. In this study, two nanofibrous biomaterials that mimic the structure of the extracellular matrix, bacterial cellulose and electrospun polycaprolactone (PCL)/collagen I, were investigated as potential 3D scaffolds for an in vitro cancer model. Multiple cancer cell lines were cultured on each scaffold material and monitored for cell viability, proliferation, adhesion, infiltration, and morphology. Both bacterial cellulose and electrospun PCL/collagen I, which have nano-scale structures on the order of 100-500 nm, have been used in many diverse tissue engineering applications. Cancer cell adhesion and growth were limited on bacterial cellulose, while all cellular processes were enhanced on the electrospun scaffolds. This initial analysis has demonstrated the potential of electrospun PCL/collagen I scaffolds toward the development of an improved 3D in vitro cancer model.     

Properties of scaffolds based on chitosan and collagen with bioglass 45S5

Scaffolds based on chitosan (CTS), collagen (Coll) and glycosaminoglycans (GAG) mixtures cross‐linked by tannic acid (TA) with bioglass 45S5 addition were obtained with the use of the freeze‐drying method. The prepared scaffolds were characterised for morphology, mechanical strength and degradation rate. Moreover, cell viability on the obtained scaffolds was measured with and without the presence of ascorbic acid and dexamethasone. The main purpose of the research was to compare the effectiveness of bioglass 45S5 influence on the physicochemical and biological properties of scaffolds. The results demonstrated that the scaffolds based on the blends of biopolymers cross‐linked by TA are stable in an aqueous environment. Scanning electron microscope images allowed the observation of a porous scaffold structure with interconnected pores. The addition of bioglass nanoparticles improved the mechanical properties and decreased the degradation rate of composite materials. The biological properties were improved for 20% tannic acid addition compared to 5%. However, the addition of bioglass 45S5 did not change to cells response significantly.Inspec keywords: biomedical materials, drying, porous materials, freezing, tissue engineering, proteins, nanofabrication, bone, scanning electron microscopy, polymers, molecular biophysics, cellular biophysics, nanoparticles, porosityOther keywords: chitosan, collagen, glycosaminoglycans, bioglass 45S5 addition, freeze‐drying method, degradation rate, ascorbic acid, dexamethasone, physicochemical properties, biological properties, porous scaffold structure, bioglass nanoparticles, mechanical properties, tannic acid addition, scanning electron microscopy     

Effect of collagen II coating on mesenchymal stem cell adhesion on chitosan and on reacetylated chitosan fibrous scaffolds

The biocompatibility and biomimetic properties of chitosan make it attractive for tissue engineering but its use is limited by its cell adhesion properties. Our objectives were to produce and characterize chitosan and reacetylated-chitosan fibrous scaffolds coated with type II collagen and to evaluate the effect of these chemical modifications on mesenchymal stem cell (MSC) adhesion. Chitosan and reacetylated-chitosan scaffolds obtained by a wet spinning method were coated with type II collagen. Scaffolds were characterized prior to seeding with MSCs. The constructs were analyzed for cell binding kinetics, numbers, distribution and viability. Cell attachment and distribution were improved on chitosan coated with type II collagen. MSCs adhered less to reacetylated-chitosan and collagen coating did not improve MSCs attachment on those scaffolds. These findings are promising and encourage the evaluation of the differentiation of MSCs in collagen-coated chitosan scaffolds. However, the decreased cell adhesion on reacetylated chitosan scaffold seems difficult to overcome and will limit its use for tissue engineering.     

Preparation and characterization of malonic acid cross-linked chitosan and collagen 3D scaffolds: an approach on non-covalent interactions

The present study emphasizes the influence of non-covalent interactions on the mechanical and thermal properties of the scaffolds of chitosan/collagen origin. Malonic acid (MA), a bifuncitonal diacid was chosen to offer non-covalent cross-linking. Three dimensional scaffolds was prepared using chitosan at 1.0% (w/v) and MA at 0.2% (w/v), similarly collagen 0.5% (w/v) and MA 0.2% (w/v) and characterized. Results on FT-IR, TGA, DSC, SEM and mechanical properties (tensile strength, stiffness, Young’s modulus, etc.) assessment demonstrated the existence of non-covalent interaction between MA and chitosan/collagen, which offered flexibility and high strength to the scaffolds suitable for tissue engineering research. Studies using NIH 3T3 fibroblast cells suggested biocompatibility nature of the scaffolds. Docking simulation study further supports the intermolecular hydrogen bonding interactions between MA and chitosan/collagen.     

Central composite rotatable design for optimization of budesonide-loaded cross-linked chitosan–dextran sulfate nanodispersion: characterization,in vitro diffusion and aerodynamic study

Budesonide is a BCS class II drug with low water solubility (0.045?mg/mL) and low oral bioavailability (6–8%) due to high first pass effect. The aim is to prepare cross-linked chitosan–dextran sulfate nanoparticles and/or nanodispersion. Nebulizable cross-linked nanodispersion was prepared by the solvent evaporation technique and characterized through XRPD, FTIR, mean particle size (MPS), polydispersity index (PDI), zeta potential (ZP), drug loading, entrapment efficiency, SEM, % production yield, in vitro diffusion, aerodynamic and stability study. The optimization of formulation was done by using central composite rotatable design to study the effect of independent variables, concentration of chitosan (X1) and concentration dextran sulfate (X2) on the dependent variables, MPS (Y1), drug loading (Y2) and % CDR (% cumulative drug release) (Y3). The MPS, PDI, and ZP of budesonide-loaded nanoparticles were 160.8?±?0.27?nm, 0.36?±?0.04, and 13?±?0.894?mV, respectively. The percent drug loading of all the batches was found in range of 10–16%. The emitted drug in target region (alveoli) was measured by using HPLC and it was found to be 18.26%. It was found that, nanodispersion had the optimum in vitro aerodynamic behavior. Stability study results showed no significant change in MPS, PDI, ZP, and % CDR after three month storage. In conclusion, cross-linked chitosan–dextran sulfate nanoparticles had properties suitable for nebulizable dispersion of increased drug loading, in vitro drug release and avoiding the first pass effect.