Bone‐like apatite‐coated chitosan scaffolds: Characterization and osteoblastic activity

In this study, porous scaffolds were prepared from chitosan (2% w/v in acetic acid and deacetylation degree: DD > 85%) by freeze‐drying method, and freshly lyophilized scaffolds were stabilized with ethanol solutions. Bone‐like apatite formation on chitosan scaffolds was achieved by immersing the scaffolds into a novel concentrated simulated body fluid (10× SBF‐like solution) for different periods, i.e., 6 and 24 h. Scanning electron microscope views showed that the 6‐h treatment in 10× SBF‐like solution led to the formation of calcium phosphate nucleation sites on chitosan scaffolds, whereas the apatite particles showed characteristic cauliflower‐like morphology at the end of 24‐h treatment. X‐ray diffraction results supported the fact that mineral phase was made of hydroxyapatite. Osteogenic activities of untreated and SBF‐treated chitosan scaffolds were examined by preosteoblastic MC3T3 cell culture studies. The mitochondrial activity test showed that apatite‐coated scaffolds stimulated cell proliferation compared with uncoated scaffolds. Alkaline phosphatase and osteocalcine levels indicated that the differentiation of the cells on all scaffolds increased significantly from 15th day of culture to the 21th day of culture, especially for the cells on 24‐h SBF‐treated scaffolds. The results of this study indicated that 10× SBF‐like solution‐treated chitosan scaffolds may be evaluated for bone tissue engineering. POLYM. COMPOS., 31:1418–1426, 2010. © 2009 Society of Plastics Engineers     

Bioactive monetite‐containing whisker‐like fibers reinforced chitosan scaffolds

The aim of this work was to develop bioactive chitosan scaffolds reinforced with monetite‐containing whisker‐like fibers. The fibers synthesized by homogeneous precipitation were characterized as monetite/hydroxyapatite short fibers (MAFs), using XRD, FTIR and SEM. The pure chitosan and MAFs/chitosan composite scaffolds were produced by freeze‐drying, and characterized with respect to porosity, pore size, swelling behavior, compressive strength and modulus, and in vitro bioactivity. The incorporation of MAFs in chitosan matrices led to increase the pore size, according to the evaluation by FE‐SEM, and decrease the porosity of composite scaffolds. The swelling ratio decreased as MAFs content of scaffolds increased. The compressive strength and modulus of scaffolds were improved by an increase in MAFs content. The noncross‐linked scaffolds with a chitosan: MAFs weight ratio of 1:1 (CW3) showed a porosity of 75.5%, and the strength and modulus of 259 kPa and 2.8 MPa in dry state, respectively. The crosslinking by glutaraldehyde resulted in improved mechanical properties. The strength and modulus of cross‐linked CW3 scaffolds in wet state reached to 345 kPa and 1.8 MPa, respectively. The in vitro bioactivity of the reinforced scaffolds, evaluated by FE‐SEM/EDS, XRD, and ATR‐FTIR, was confirmed by the formation of a carbonated apatite layer on their surfaces when they soaked in simulated body fluid (SBF). The results of this initial study indicate that the monetite‐containing whisker‐like fibers may be an appropriate reinforcement of chitosan scaffolds.     

Integrated three‐dimensional fiber/hydrogel biphasic scaffolds for periodontal bone tissue engineering

Combining a tissue engineering scaffold made of a load‐bearing polymer with a hydrogel represents a powerful approach to enhancing the functionalities of the resulting biphasic construct, such as its mechanical properties or ability to support cellular colonization. This research activity was aimed at the development of biphasic scaffolds through the combination of an additively manufactured poly(?‐caprolactone) (PCL) fiber construct and a chitosan/poly(γ‐glutamic acid) polyelectrolyte complex hydrogel. By investigating a set of layered structures made of PCL or PCL/hydroxyapatite composite, biphasic scaffold prototypes with good integration of the two phases at the macroscale and microscale were developed. The biphasic constructs were able to absorb cell culture medium up to 10‐fold of their weight, and the combination of the two phases had a significant influence on compressive mechanical properties compared with hydrogel or PCL scaffold alone. In addition, due to the presence of chitosan in the hydrogel phase, biphasic scaffolds exerted a broad‐spectrum antibacterial activity. The developed biphasic systems appear well suited for application in periodontal bone regenerative approaches in which a biodegradable porous structure providing mechanical stability and a hydrogel phase functioning as absorbing depot of endogenous proteins are simultaneously required. © 2016 Society of Chemical Industry     

Synthesis and cytocompatibility of collagen/hydroxyapatite nanocomposite scaffold for bone tissue engineering

Collagen/hydroxyapatite nanocomposite scaffolds were prepared by in situ precipitation and freeze‐drying approach. The synthesized collagen/hydroxyapatite nanocomposites were characterized using various modalities. It was revealed that the inorganic phase in the nanocomposite was carbonate‐substituted hydroxyapatite with low crystallinity. Morphology studies showed the uniform distribution of hydroxyapatite particles in the collagen hydrogel. In addition, hydroxyapatite particles were gradually becoming irregular enough and the surface morphology had more wrinkles with the increase of inorganic component. Morphology, mechanical properties and cell biocompatibility of the prepared nanocomposite scaffolds were evaluated. The scaffolds presented a well‐developed macropore structure with a pore size ranging from 100 to 200 μm and the pore size of scaffold can also be regulated by changing the organic/inorganic weight ratio. Furthermore, the growth of MG63 cells on scaffolds showed they could significantly promote the proliferation of cells and could be potential candidate for bone engineering applications. POLYM. COMPOS., 81–90, 2016. © 2014 Society of Plastics Engineers     

Novel Fe2O3-doped glass /chitosan scaffolds for bone tissue replacement

In this study quaternary bioglass system (BG) SiO₂–CaO–Na₂O–P₂O₅ doped with Fe₂O₃ was prepared by the sol–gel method. Furthermore, 3D scaffolds were designed through blending Fe₂O₃ -doped bioglass with chitosan to obtain various compositions of scaffolds by the freeze-drying technique. The thermal behavior, morphological properties, porosity (%), mechanical properties and physicochemical properties of BG and scaffolds were evaluated by DSC/TGA, TEM, SEM, liquid displacement method, universal testing machine, XRD and FTIR. In addition, the in vitro bioactivity of the prepared scaffolds was studied in phosphate buffer saline (PBS) through the determination of PBS ions concentrations, as well as the degradation and the observation of precipitated calcium phosphate layer by SEM coupled with EDX and FTIR behavior. The cell viability of the prepared scaffolds was conducted against Baby Hamster Kidney fibroblasts (BHK-21) cell line. The presence of Fe₂O₃ decreased the T_g (from 513 to 390?°C) and the size decreased (from 20.89 to 50.81–13.92–27.87?nm). The scaffolds porosity (%) decreased upon Fe₂O₃ doping but the mechanical strength increased. Cell viability results for the designed scaffolds demonstrated acceptable cell viability compared with normal cells. Therefore, the designed scaffolds are promoted as regenerated materials that can be used for bone tissue replacement.     

Inorganic calcium filled bacterial cellulose based hydrogel scaffold: novel biomaterial for bone tissue regeneration

Abstract

This work emphasizes the structural, physio-chemical characterization and cell biological efficiency analysis of novel inorganic calcium (only calcium phosphate and in combination of calcium phosphate & CaCO₃) filled bacterial cellulose (BC) based hydrogel scaffolds. FTIR and TG analysis indicates the presence of BC and inorganic calcium within the hydrogel scaffolds. SEM establishes the porous structures (50–200 µm). Swelling study indicates significant swelling ability in both calcium phosphate filled and calcium phosphate & CaCO₃ filled hydrogel scaffolds. Compressive strength (0.24–0.60?MPa) of the calcium filled hydrogel scaffolds are similar like trabecular bone. Significant cell viability (Lep-3) was further noticed until 72,120 and 168?h.     

Synthesis of Antibacterial Hybrid Hydroxyapatite/Collagen/Polysaccharide Bioactive Membranes and Their Effect on Osteoblast Culture

Inspired by the composition and confined environment provided by collagen fibrils during bone formation, this study aimed to compare two different strategies to synthesize bioactive hybrid membranes and to assess the role the organic matrix plays as physical confinement during mineral phase deposition. The hybrid membranes were prepared by (1) incorporating calcium phosphate in a biopolymeric membrane for in situ hydroxyapatite (HAp) precipitation in the interstices of the biopolymeric membrane as a confined environment (Methodology 1) or (2) adding synthetic HAp nanoparticles (SHAp) to the freshly prepared biopolymeric membrane (Methodology 2). The biopolymeric membranes were based on hydrolyzed collagen (HC) and chitosan (Cht) or κ-carrageenan (κ-carr). The hybrid membranes presented homogeneous and continuous dispersion of the mineral particles embedded in the biopolymeric membrane interstices and enhanced mechanical properties. The importance of the confined spaces in biomineralization was confirmed by controlled biomimetic HAp precipitation via Methodology 1. HAp precipitation after immersion in simulated body fluid attested that the hybrid membranes were bioactive. Hybrid membranes containing Cht were not toxic to the osteoblasts. Hybrid membranes added with silver nanoparticles (AgNPs) displayed antibacterial action against different clinically important pathogenic microorganisms. Overall, these results open simple and promising pathways to develop a new generation of bioactive hybrid membranes with controllable degradation rates and antimicrobial properties.     

Hydroxyapatite/poly(L-lactic acid)-chitosan Porous Scaffolds Prepared by Phase Separation Method

Hydroxyapatite/poly(L-lactic acid)–chitosan and poly(L-lactic acid)/chitosan porous scaffolds were prepared by phase separation technique using heated acetic acid–water as a common solvent of poly(L-lactic acid) and chitosan. The results show that the distribution of hydroxyapatite in the scaffolds is good. The porosity of the hydroxyapatite/poly(L-lactic acid)–chitosan scaffolds is nearly 85%. The hydroxyapatite particles in the scaffolds are beneficial for improving the compression property of the scaffolds. The in vitro bioactivity test shows that there is a low crystallinity of carbonate hydroxyapatite coating formed on the surface of the scaffold after immersing in simulated body fluid for 14 days, indicating that the hydroxyapatite/poly(L-lactic acid)–chitosan scaffolds have a good bioactivity.     

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.     

Synthesis,characterization, and enzymatic degradation of chitosan/PEG hydrogel films

Poly(ethyleneglycol) (PEG)/tartaric acid (TA)‐crosslinked chitosan hydrogel (CPT) films were prepared, and the formation of the PEG/TA‐crosslinked structure was confirmed by Fourier transformed infrared (FTIR), nuclear magnetic resonance (NMR), and scanning electron microscope (SEM) measurements. The thermal properties of the crosslinked films were also determined with thermogravimetric analysis (TGA) and differential scanning calorimeter (DSC) analysis. The swelling properties of the films were investigated at different temperature and pH values. It was found that the swelling ratio increased with the decrease of pH value of the surrounding buffer solutions, amount of PEG, and with the increase of temperature. Swelling behavior of the PEG/TA‐crosslinked chitosan hydrogel films depended on pH and reversible with the temperature. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Physically crosslinked polyvinyl alcohol/chitosan/gum tragacanth hydrogels loaded with vitamin E for wound healing applications

For the healing process, in this study, an innovative polymeric hydrogel network including polyvinyl alcohol (PVA)/chitosan (CS)/gum tragacanth (GT) loaded with vitamin E (VE) was produced by the freeze–thaw approach. In order to investigate the characteristics of the prepared samples, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) analyzes were performed. Also, water vapor transmission rate (WVTR), swelling ratio, gel fraction and mechanical properties were measured. Then, to observe their cytocompatibility, MTT assay and cell adhesion studies were assessed. The results of FTIR confirmed the presence of PVA, CS, GT, and VE in hydrogel films. As well as, the SEM images showed the effect of the freezing and thawing method in creating a smooth surface with small and regular pores. It was found with adding the CS and GT to PVA improves swelling ratio, gel fraction, WVTR and elongation of hydrogel films. Further, in examining the adhesion and cytotoxicity of the samples, the non-toxic quiddity of the PVA/CS/GT hydrogel films was corroborated. In the end, the antibacterial properties revealed that the film containing GT and CS had the greatest antibacterial activity. According to the observed results, PVA/CS/GT hydrogel films loaded with VE can be good for wound healing applications.     

Zinc-substituted hydroxyapatite produced from calcium precursor derived from eggshells

The availability of natural calcium derived from biowaste materials is an inexpensive alternative to commercial calcium reagents used to produce calcium phosphate bioceramics. This approach is more ecofriendly, cost effective and sustainable owing to its abundant availability. In this study, zinc-substituted hydroxyapatite (ZnHA-Es) was synthesised using calcium precursor derived from eggshells through precipitation method. Zn concentration was varied from 1 mol% to 5 mol% during the synthesis, and the derived powders were calcined at 700 °C in air atmosphere. XRD analysis revealed that ZnHA-Es powders were highly crystalline and comprised biphasic mixtures of HA as the major phase and β-tricalcium phosphate as the minor phase. FTIR examination indicated that all ZnHA-Es samples had a similar structure to pure HA as evidenced by the presence of frequency bands corresponding to phosphate, carbonate and hydroxyl functional groups. The disappearance of vibrational bands of carbonate groups became more apparent with increased Zn concentration. Microstructure analysis revealed that the derived nanostructured particles were spherical (approximately 40–100 nm in diameter) regardless of Zn substitution.     

The fabrication and characterization of bioactive Akermanite/Octacalcium phosphate glass-ceramic scaffolds produced via PDC method

In the present study, a bioactive silicate-phosphate glass-ceramic scaffold was fabricated via the polymer-derived ceramics (PDC) method. K₂HPO₄ phosphate salt was used as the P₂O₅ precursor in this method. The effect of K₂HPO₄ wt% and heat treatment temperatures (900–1100 °C) was evaluated. It was observed that although increasing the wt% of K₂HPO₄ led to the formation of scaffolds with higher densities and strengths, it could also increase the formation of the calcium phase, which could result in improper release behavior of scaffolds. On the other hand, higher heat treatment temperatures enhanced the strength of the scaffolds but eliminated the bioactive octacalcium phosphate (OCP) phase. X-ray diffraction (XRD) analysis showed that the dissolution of the OCP phase in simulated body fluid (SBF) resulted in precipitation of hydroxyapatite (HA) on the scaffold surface which enhanced the bioactivity. Furthermore, based on microstructural studies by Scanning Electron Microscopy (SEM), the fabricated scaffold possessed a wide range of pore sizes, appropriate for osteointegration and bone formation. The optimum wt% of phosphate salt was less than 6 wt% and the optimum heat treatment temperature was 1000 °C. After the optimization of compositions and processing, Alamar Blue Assay was used to evaluate HOb cell cultures, showing a continuous proliferation for the optimized samples.     

Influence of the addition of carbonated hydroxyapatite and selenium dioxide on mechanical properties and in vitro bioactivity of borosilicate inert glass

Five nanocomposite samples containing different percentages of carbonated hydroxyapatite (CHA), selenium dioxide (SeO₂) and inert glass (IG) have been prepared using high-energy ball milling method with the aim of improving the in vitro bioactivity of these nanocomposites. Fourier transform infrared (FTIR) spectroscopy along with X-ray diffraction (XRD) technique was applied on both nanopowders and the sintered nanocomposites to record the structural changes and examine the resultant sintered phases. Mechanical properties were measured by ultrasonic non-destructive technique. In order to assess the bioactivity of the sintered specimens, they were soaked in simulated body fluid for 14 days and then, they were investigated by FTIR and scanning electron microscopy (SEM). Both FTIR and XRD spectra showed that the glasses encouraged the partial HA decomposition to tricalcium phosphate (TCP) and calcium silicate (CaSiO₃) phases. The formation of the latter phase along with the remainder HA contents was responsible for good bioactivity and appropriate mechanical properties of the investigated nanocomposites. The successive addition of selenium dioxide to these nanocomposites led to further improvement of their bioactivity without any recorded changes in the mechanical properties. Based on the abovementioned results, the prepared nanocomposites can be used in various tissue-engineering applications.     

Fabrication of 3D calcium phosphates based scaffolds using bacterial cellulose as template

Bacterial cellulose – calcium phosphates composite materials were synthesized by successive immersing of bacterial cellulose membranes prepared in our laboratory in precursor solutions under ultrasonic irradiation, which provides the necessary cations for the formation of calcium phosphate phases at the surface of bacterial cellulose fibers. The subsequent thermal treatment at 700 or 1000 °C of the hybrid materials previously described allowed the obtaining of 3D porous scaffolds with different morphologies, as a function of the number of immersing cycles and calcining temperature. All samples exhibit similar phase composition, mainly based on hydroxyapatite and buchwaldite (sodium calcium phosphate). In the case of the composites thermally treated at lower temperature, the microstructure is fluffy and composed of large grains and monocrystalline nanorods, while the masses calcined at higher temperatures have a trabecular appearance, looking like a natural bone. The biological properties of the resulting architectures were also investigated.     

Fabrication and characterization of chitosan/PVA with hydroxyapatite biocomposite nanoscaffolds

Nanofibrous biocomposite scaffolds of chitosan (CS), PVA, and hydroxyapatite (HA) were prepared by electrospinning. The scaffolds were characterized by FTIR, SEM, TEM, and XRD techniques. Tensile testing was used for the characterization of mechanical properties. Mouse fibroblasts (L929) attachment and proliferation on the nanofibrous scaffold were investigated by MTT assay and SEM observation. FTIR, TEM, and XRD results showed the presence of nanoHA in the scaffolds. The scaffolds have porous nanofibrous morphology with random fibers in the range of 100–700 nm diameters. The CS/PVA (90/10) fibrous matrix (without HA) showed a tensile strength of 3.1 ± 0.2 MPa and a tensile modulus 10 ± 1 MPa with a strain at failure of 21.1 ± 0.6%. Increase the content of HA up to 2% increased the ultimate tensile strength and tensile modulus, but further increase HA up to 5–10% caused the decrease of tensile strength and tensile modulus. The attachment and growth of mouse fibroblast was on the surface of nanofibrous structure, and cells' morphology characteristics and viability were unaffected. A combination of nanofibrous CS/PVA and HA that mimics the nanoscale features of the extra cellular matrix could be promising for application as scaffolds for tissue regeneration, especially in low or nonload bearing areas. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Chitosan graft copolymers‐HA/DBM biocomposites: Preparation,characterization, and in vitro evaluation

The combination of biopolymer with a bioactive component takes advantage of the osteoconductivity and osteoinductivity properties. The studies on composites containing hydroxyapatite (HA), demineralized bone matrix (DBM) fillers and chitosan biopolymer are still conducted. In the present study, the bioactive fillers were loaded onto p(HEMA‐MMA) grafted chitosan copolymer to produce a novel biocomposites having osteoinductive and osteoconductive properties. The produced composites were assessed by TGA, XRD, FTIR, and SEM techniques to prove the interaction between both matrices. In vitro behavior of these composites was performed in SBF to verify the formation of apatite layer onto their surfaces and its enhancement. The results confirmed the formation of thick apatite layer containing carbonate ions onto the surface of biocomposites especially these containing HA‐DBM mixture and pMMA having bone cement formation in their structure. These a novel biocomposites have unique bioactivity properties can be applied in bone implants and tissue engineering applications as scaffolds in future. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

生理模拟液中的磷酸钙微晶玻璃的表面变化

应用玻璃结晶法制备了以磷酸钙为主体的多孔微晶玻璃载体材料。在一定的条件下对该药物载体材料进行了生理模拟液的浸泡实验,并用Fourier红外光谱和扫描电镜对其表面进行了表征分析。试验结果表明：经模拟液浸泡后,在材料的表面沉积了一定量的类骨磷灰石(碳酸羟基磷灰石),其形貌为球状颗粒,并证实了载体材料的粗糙表面有利于碳酸羟基磷灰石晶体的形成。研究结果有助于分析碳酸羟基磷灰石的形成机理及了解磷酸钙微晶玻璃载体材料在体内的骨诱导机理。     

A Continuous Static‐Mixer‐Based Reactor for Preparing Calcium Phosphate Bioceramics

The common methods for synthesizing calcium phosphates include solid‐state reaction, hydrothermal, sol–gel, and wet precipitation. The purpose of this study was to prepare various calcium phosphate bioceramics through a continuous reactor equipped with a static mixer. The precursors, amorphous calcium phosphate (ACP), and poor crystal hydroxyapatite (PC‐HAp) were prepared using calcium and phosphate ion‐containing solutions by adjusting the pH and the [Ca]/[P] input ratio. The phase transformation from precursors to HAp, alpha‐tricalcium phosphates (α‐TCP), beta‐tricalcium phosphate (β‐TCP), or biphasic calcium phosphate (BCP) was dependent on the sintering temperature.     

Studies of Bone-Bonding Ability and Antibacterial Properties of Ag<Superscript>+</Superscript>, Cu<Superscript>2+</Superscript> or Zn<Superscript>2+</Superscript> ions Doping within Hench’s Bioglass and Glass-Ceramic Derivatives

Derived Hench’s bioglasses with specific ionic dopants Ag⁺, Cu²⁺, or Zn²⁺ have been prepared. The bone-boding ability or bioactivity behavior for the prepared glasses and their glass-ceramic derivatives has been investigated after immersion in phosphate solution for two weeks. Collective Fourier transform infrared absorption spectra (FTIR) and scanning electron microscopic (SEM) studies were conducted in order to study the in-vitro bioactivity behavior. X-ray diffraction (XRD) analysis was carried out to identify the crystallized phases upon thermal heat treatment through a two-step regime. The glasses and their glass-ceramic derivatives were tested to study their antibacterial or antifungal efficiency responding to the doped metal ions. FTIR spectra revealed the generation of two split peaks at about 560 and 605 cm^?1, after immersion in (0.2 M) sodium phosphate solution (Na₃PO₄), signifying the formation of a crystalline calcium phosphate phase, leading to hydroxyapatite formation. SEM examinations show characteristic rounded or nodular microcrystals for hydroxyapatite which support the FTIR data. X-ray diffraction analysis indicated crystallization of the main soda-lime silicate phase (1Na₂O.2CaO.3SiO₂) besides a secondary silicon phosphate phase (SiO₂.P₂O₅) in the studied glass ceramics. The route of crystallization is discussed on the basis of the presence of 6% P₂O_5; which facilitates the formation of phase separation and voluminous bulk crystallization of the main soda-lime silicate phase. The introduction of dopants is identified to cause no changes in the precipitated phases, with only minor changes in the percent of the crystalline phases. Experimental data indicate that the glass-ceramic samples are effective in bioactivity and antimicrobial efficiency.