In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study

Biodegradable polymers reinforced with an inorganic phase such as calcium phosphate glasses may be a promising approach to fulfil the challenging requirements presented by 3D porous scaffolds for tissue engineering. Scaffolds’ success depends mainly on their biological behaviour. This work is aimed to the in vitro study of polylactic acid (PLA)/CaP glass 3D porous constructs for bone regeneration. The scaffolds were elaborated using two different techniques, namely solvent-casting and phase-separation. The effect of scaffolds’ micro and macrostructure on the biological response of these scaffolds was assayed. Cell proliferation, differentiation and morphology within the scaffolds were studied. Furthermore, polymer/glass scaffolds were seeded under dynamic conditions in a custom-made perfusion bioreactor. Results indicate that the final architecture of the solvent-cast or phase separated scaffolds have a significant effect on cells’ behaviour. Solvent-cast scaffolds seem to be the best candidates for bone tissue engineering. Besides, dynamic seeding yielded a higher seeding efficiency in comparison with the static method.     

Synthesis and characterization of chitosan–multiwalled carbon nanotubes/hydroxyapatite nanocomposites for bone tissue engineering

Chitosan–multiwalled carbon nanotubes/hydroxyapatite nanocomposites were synthesized by a novel in situ precipitation method. The electrostatic adsorption between multiwalled carbon nanotubes and chitosan was investigated and explained by Fourier transform infrared spectroscopy analysis. Morphology studies showed that uniform distribution of hydroxyapatite particles and multiwalled carbon nanotubes in the polymer matrix was observed. In chitosan–multiwalled carbon nanotubes/hydroxyapatite nanocomposites, the diameters of multiwalled carbon nanotubes were about 10 nm. The mechanical properties of the composites were evaluated by measuring their compressive strength and elastic modulus. The elastic modulus and compressive strength increased sharply from 509.9 to 1089.1 MPa and from 33.2 to 105.5 MPa with an increase of multiwalled carbon/chitosan weight ratios from 0 to 5 %, respectively. Finally, the cell biocompatibility of the composites was tested in vitro, which showed that they have good biocompatibility. These results suggest that the chitosan–multiwalled carbon nanotubes/hydroxyapatite nanocomposites are promising biomaterials for bone tissue engineering.     

Novel mineralized heparin–gelatin nanoparticles for potential application in tissue engineering of bone

Nanoparticles (NPs) were prepared from succinylated gelatin (s-GL) cross-linked with aldehyde heparin (a-HEP) and used subsequently as a nano-template for the mineralization of hydroxyapatite (HAP). Gelatin was functionalized with succinyl groups that made it soluble at room temperature. Heparin was oxidized to generate aldehyde groups and then used as a cross-linker that can react with s-GL to form NPs via Schiff’s base linkage. The polymer concentrations, feed molar ratios and pH conditions were varied to fabricate NPs suspension. NPs were obtained with a spheroid shape of an average size of 196 nm at pH 2.5 and 202 nm at pH 7.4. These NPs had a positive zeta potential of 7.3 ± 3.0 mV and a narrow distribution with PDI 0.123 at pH 2.5, while they had a negative zeta potential of ?2.6 ± 0.3 mV and formed aggregates (PDI 0.257) at pH 7.4. The NPs prepared at pH 2.5 with a mean particle size of 196 nm were further used for mineralization studies. The mineralization process was mediated by solution without calcination at 37 °C. The HAP formed on NPs was analyzed by Fourier transform infrared spectroscopy and X-ray diffraction. HAP coated s-GL/a-HEP NPs developed in this study may be used in future as osteoinductive fillers enhancing the mechanical properties of injectable hydrogel or use as potential multifunctional device for nanotherapeutic approaches.     

In vitro degradation,hemolysis and MC3T3-E1 cell adhesion of biodegradable Mg–Zn alloy

In this study a kind of patent binary Mg–6 wt.%Zn magnesium alloy was investigated as degradable biomedical material. The results of in vitro degradation including electrochemical measurements and immersion tests in simulated body fluid (SBF) revealed that zinc could elevate both the corrosion potential and Faraday charge transfer resistance of magnesium and thus improve the corrosion resistance. XRD and EDS analysis proved that the corrosion products on the surface of Mg–Zn contained hydroxyapatite (HA), Mg(OH)₂ and other Mg/Ca phosphates, which could reduce the degradation rate. The degradation process of magnesium alloy and the mechanism of corrosion layer formation were also discussed in this work, i.e. the byproducts of degradation of magnesium, Mg²⁺ and OH^?, reacted with the phosphate and Ca²⁺ in the SBF, thus the corrosion layer containing HA, Mg(OH)₂ and other magnesium-substituted apatite precipitated in corrosion pits and covered the surface of magnesium alloy.The hemolysis test found that the hemolysis rate of Mg–Zn was 3.4%, which is lower than the safe value of 5% according to ISO 10993-4. For the cell culture experiments, after 2 h incubation the pre-osteoblastic cell MC3T3-E1 was able to adhere and spread on the corrosion layer of Mg–Zn alloy, indicating that despite the fluctuation of pH value of DMEM culture solution, Mg–Zn alloy could still support the earlier adhesion of pre-osteoblastic cells on the surface. Hemolysis and adhesion of cells display good biocompatibility of Mg–Zn alloy in vitro.     

Biofabrication and in vitro study of hydroxyapatite/mPEG–PCL–mPEG scaffolds for bone tissue engineering using air pressure-aided deposition technology

The aims of this study were to fabricate biopolymer and biocomposite scaffolds for bone tissue engineering by an air pressure-aided deposition system and to carry out osteoblast cell culture tests to validate the biocompatibility of fabricated scaffolds. A mPEG–PCL–mPEG triblock copolymer was synthesized as a biopolymer material. Biocomposite material was composed of synthesized biopolymer and hydroxyapatite (HA) with a mean diameter of 100 μm. The weight ratio of HA added to the synthesized biopolymer was 0.1, 0.25, 0.5 and 1. The experimental results show that the maximum average compressive strength of biocomposite scaffolds, made of weight ratio 0.5, with mean pore size of 410 μm (porosity 81%) is 18.38 MPa which is two times stronger than that of biopolymer scaffolds. Osteoblast cells, MC3T3-E1, were seeded on both types of fabricated scaffolds to validate the biocompatibility using methylthianzol tetrazolium (MTT) assay and cell morphology observation. After 28 days of in vitro culturing, the seeded osteoblasts were well distributed in the interior of both types of scaffolds. Furthermore, MTT experimental results show that the cell viability of the biocomposite scaffold is higher than that of the biopolymer scaffold. This indicates that adding HA into synthesized biopolymer can enhance compressive strength and the proliferation of the osteoblast cell.     

Sustained release of Semaphorin 3A from α-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

The reinforcement of calcium phosphate materials with silk fibroin (SF) has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/α-tricalcium phosphate composite (Sema3A CMs/SF/α-TCP) in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/α-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction (RT-qPCR) and activity of alkaline phosphatase (ALP) were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/α-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/α-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/α-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/α-TCP and SF/α-TCP composites. In conclusion, Sema3A CMs/SF/α-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.     

Silver - calcium titanate – titania decorated Ti6Al4V powders: An antimicrobial and biocompatible filler in composite scaffold for bone tissue engineering application

The present work describes a technique to develop silver nanoparticles decorated calcium titanate–titania (AgNPs-CT-TiO₂) nanostructured layer over Ti6Al4V alloy powder by simple chemical and heat treatment approach. The NaOH solution and heat treatment form a uniform porous nano-network morphology of sodium titanate and subsequent calcium nitrate treatment transforms it into calcium titanate along with rutile TiO₂. However, simultaneous treatment with calcium and silver nitrate solution allows the incorporation of both calcium and silver ions over the nano-network morphology leading to the formation of AgNPs decorated calcium titanate along with anatase TiO₂ (AgNPs-CT-TiO₂@Ti6Al4V) that collectively induces biocompatibility towards MG 63 osteoblast-like cells as well as antimicrobial activity against Staphylococcus aureus. Thus modified AgNPs-CT-TiO₂@Ti6Al4V alloy powders were subsequently blended with polymethyl methacrylate to develop a composite scaffold using NaCl as a porogen. This composite could be used as a scaffold or bone cement in various bone tissue engineering applications.     

Response of MC3T3-E1 osteoblast cells to the microenvironment produced on Co–Cr–Mo alloy using laser surface texturing

Osteoblast responses to Co–Cr–Mo alloy depend on not only the chemistry of alloy but also the physical properties of alloy surface, such as its microtopography and roughness. This study was undertaken to examine changes in cell adhesion, morphology, differentiation and gene expression during osteoblast interaction with different textured Co–Cr–Mo alloys. With laser surface texturing, three kinds of textured surfaces were fabricated. It showed that the microenvironment processed by laser surface texturing leads to an increase in surface roughness and DMEM contact angles of samples. Adhesion and differentiation tests demonstrated that osteoblast cells can discriminate surfaces with different roughness and surfaces with comparable roughness but different topographies such as triangle, circle and square textures. Morphological characteristics obtained by SEM imaging showed that osteoblast cells are elongated and are in polygonal shape on the textured surface. OPG/RANKL mRNA expression studies showed a significant and marked decrease in RANKL gene expression on the square-textured surface; by contrast, the ratio of OPG/RANKL showed a significant increase. These results indicate that Co–Cr–Mo surface textures affect osteoblast proliferation, morphology and gene expression; the microenvironment of implant should be considered in the future design.