Sol–gel derived nanoscale bioactive glass (NBG) particles reinforced poly(ε-caprolactone) composites for bone tissue engineering

This study investigated the effect of the addition of sol–gel derived nanoscale bioactive glass (NBG) particles on the mechanical properties and biological performances of PCL polymer, in order to evaluate the potential applications of PCL/NBG composites for bone tissue regeneration. Regardless of the NBG contents (10, 20, and 30 wt.%), the NBG particles, which were synthesized through the sol–gel process using polyethylene glycol (PEG) polymer as a template, could be uniformly dispersed in the PCL matrix, while generating pores in the PCL/NBG composites. The elastic modulus of the PCL/NBG composites increased remarkably from 89 ± 11 MPa to 383 ± 50 MPa with increasing NBG content from 0 to 30 wt.%, while still showing good ultimate tensile strength in the range of 15–19 MPa. The hydrophilicity, water absorption and degradation behavior of the PCL/NBG composites were also enhanced by the addition of the NBG particles. Furthermore, the PCL/NBG composite with a NBG content of 30 wt.% showed significantly enhanced in vitro bioactivity and cellular response compared to those of the pure PCL.     

Evaluation of 3D nano–macro porous bioactive glass scaffold for hard tissue engineering

Recently, nano–macro dual-porous, three-dimensional (3D) glass structures were developed for use as bioscaffolds for hard tissue regeneration, but there have been concerns regarding the interconnectivity and homogeneity of nanopores in the scaffolds, as well as the cytotoxicity of the environment deep inside due to limited fluid access. Therefore, mercury porosimetry, nitrogen absorption, and TEM have been used to characterize nanopore network of the scaffolds. In parallel, viability of MG 63 human osteosarcoma cells seeded on scaffold surface was investigated by fluorescence, confocal and electron microscopy methods. The results show that cells attach, migrate and penetrate inside the glass scaffold with high proliferation and viability rate. Additionally, scaffolds were implanted under the skin of a male New Zealand rabbit for in vivo animal test. Initial observations show the formation of new tissue with blood vessels and collagen fibers deep inside the implanted scaffolds with no obvious inflammatory reaction. Thus, the new nano–macro dual-porous glass structure could be a promising bioscaffold for use in regenerative medicine and tissue engineering for bone regeneration.     

Sol–gel derived composite from bioactive glass–polyvinyl alcohol

Investigation of novel biomaterials for bone engineering is based on the development of porous scaffolds, which should match the properties of the tissue that is to be replaced. These materials need to be biocompatible, ideally osteoinductive, osteoconductive, and mechanically well-matched. In the present paper, we report the preparation and characterization of hybrid macroporous scaffold of polyvinyl alcohol (PVA)/bioactive glass through the sol–gel route. Hybrids containing PVA (80, 70 and 60 wt%) and bioactive glass with composition 58SiO₂–33CaO–9P₂O₅ were synthesized by foaming a mixture of polymer solution and bioactive glass via sol–gel precursor solution. PVA with two different degree of hydrolysis (DH), 98.5% (high degree) and 80% (low degree) were also investigated, in order to evaluate the influence of residual acetate group present in polymer chain on the final structure and properties of 3D porous composite produced. The microstructure, morphology and crystallinity of the hybrid porous scaffolds were characterized by X-ray diffraction (XRD), Infrared Fourier Transform spectrometry (FTIR) and Scanning electron microscopy (SEM/EDX) analysis. In addition, specific surface area was assessed by B.E.T. nitrogen adsorption method and mechanical behavior was evaluated by compression tests. Preliminary cytotoxicity and cell viability were also performed by the MTT assay. VERO cell monolayers were grown in 96-well microtiter plates. The results have clearly showed that hybrid foams of polyvinyl alcohol/bioactive glass (PVA/BG) with interconnected macroporous 3D structure were successfully produced. All the tested hybrids of PVA/BG have showed adequate cell viability properties for potential biological applications.     

Freeze extrusion fabrication of 13–93 bioactive glass scaffolds for bone repair

A solid freeform fabrication technique, freeze extrusion fabrication (FEF), was investigated for the creation of three-dimensional bioactive glass (13–93) scaffolds with pre-designed porosity and pore architecture. An aqueous mixture of bioactive glass particles and polymeric additives with a paste-like consistency was extruded through a narrow nozzle, and deposited layer-by-layer in a cold environment according to a computer-aided design (CAD) file. Following sublimation of the ice in a freeze dryer, the construct was heated according to a controlled schedule to burn out the polymeric additives (below ~500°C), and to densify the glass phase at higher temperature (1 h at 700°C). The sintered scaffolds had a grid-like microstructure of interconnected pores, with a porosity of ~50%, pore width of ~300 μm, and dense glass filaments (struts) with a diameter or width of ~300 μm. The scaffolds showed an elastic response during mechanical testing in compression, with an average compressive strength of 140 MPa and an elastic modulus of 5–6 GPa, comparable to the values for human cortical bone. These bioactive glass scaffolds created by the FEF method could have potential application in the repair of load-bearing bones.     

Sol–gel method to fabricate CaP scaffolds by robocasting for tissue engineering

Highly porous calcium phosphate (CaP) scaffolds for bone-tissue engineering were fabricated by combining a robocasting process with a sol–gel synthesis that mixed Calcium Nitrate Tetrahydrate and Triethyl Phosphite precursors in an aqueous medium. The resulting gels were used to print scaffolds by robocasting without the use of binder to increase the viscosity of the paste. X-ray diffraction analysis confirmed that the process yielded hydroxyapatite and β-tricalcium phosphate biphasic composite powders. Thus, the scaffold composition after crystallization of the amorphous structure could be easily modified by varying the initial Ca/P ratio during synthesis. The compressive strengths of the scaffolds are ~6 MPa, which is in the range of human cancellous bone (2–12 MPa). These highly porous scaffolds (~73 vol% porosity) are composed of macro-pores of ~260 μm in size; such porosity is expected to enable bone ingrowth into the scaffold for bone repair applications. The chemistry, porosity, and surface topography of such scaffolds can also be modified by the process parameters to favor bone formation. The studied sol–gel process can be used to coat these scaffolds by dip-coating, which induces a significant enhancement of mechanical properties. This can adjust scaffold properties such as composition and surface morphology, which consequently may improve their performances.     

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.     

Biocompatible glass–ceramic materials for bone substitution

A new bioactive glass composition (CEL2) in the SiO₂–P₂O₅–CaO–MgO–K₂O–Na₂O system was tailored to control pH variations due to ion leaching phenomena when the glass is in contact with physiological fluids. CEL2 was prepared by a traditional melting-quenching process obtaining slices that were heat-treated to obtain a glass-ceramic material (CEL2GC) that was characterized thorough SEM analysis. Pre-treatment of CEL2GC with SBF was found to enhance its biocompatibility, as assessed by in vitro tests. CEL2 powder was then used to synthesize macroporous glass–ceramic scaffolds. To this end, CEL2 powders were mixed with polyethylene particles within the 300–600 μm size-range and then pressed to obtain crack-free compacted powders (green). This was heat-treated to remove the organic phase and to sinter the inorganic phase, leaving a porous structure. The biomaterial thus obtained was characterized by X-ray diffraction, SEM equipped with EDS, density measurement, image analysis, mechanical testing and in vitro evaluation, and found to be a glass–ceramic macroporous scaffold with uniformly distributed and highly interconnected porosity. The extent and size-range of the porosity can be tailored by varying the amount and size of the polyethylene particles.     

Bioglass®-based scaffolds with carbon nanotube coating for bone tissue engineering

Highly porous 45S5 Bioglass^®-based foam scaffolds were coated with multi-walled carbon nanotubes (CNT) by electrophoretic deposition (EPD) technique. By placing the scaffolds in between the two electrodes of the EPD cell, a CNT coating of up to 1 μm thickness was achieved on the surface throughout the whole three dimensional (3D) matrix. A 0.5 wt% CNT aqueous suspension was used and EPD was carried out at 2.8 V for 10 mins. The compression strength of this CNT/Bioglass^® composite was measured to be 0.70 MPa. Moreover the increased electrical conductivity of the composite with CNT coating was confirmed. The scaffolds have the potential for applications in bone tissue engineering due to the high bioactivity, nano-roughness in 3D and electrical conductivity provided by the addition of CNT.     

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.     

A novel bioactive three-dimensional β-tricalcium phosphate/chitosan scaffold for periodontal tissue engineering

The development of suitable bioactive three-dimensional scaffold for the promotion of cellular proliferation and differentiation is critical in periodontal tissue engineering. In this study,porous β-tricalcium phosphate/chitosan composite scaffolds were prepared through a freeze-drying method. These scaffolds were evaluated by analysis of microscopic structure, porosity, and cytocompatibility. The gene expression of bone sialoprotein (BSP) and cementum attachment protein (CAP) was detected with RT-PCR after human periodontal ligament cells (HPLCs) were seeded in these scaffolds. Then cell–scaffold complexes were implanted subcutaneously into athymic mice. The protein expression of alkaline phosphatase (ALP) and osteopontin (OPN) was detected in vivo. Results indicated that composite scaffolds displayed a homogeneous three-dimensional microstructure; suitable pore size (120 μm) and high porosity (91.07%). The composite scaffold showed higher proliferation rate than the pure chitosan scaffold, and up-regulated the gene expression of BSP and CAP. In vivo, HPLCs in the composite scaffold not only proliferated but also recruited vascular tissue ingrowth. The protein expression of ALP and OPN was up-regulated in the composite scaffold. Therefore, it was suggested that the composite scaffold could promote the differentiation of HPLCs towards osteoblast and cementoblast phenotypes.     

SBA-15 ordered mesoporous silica inside a bioactive glass–ceramic scaffold for local drug delivery

The paper reports the synthesis of an ordered silica mesostructure of the SBA-15 type inside a macroporous bioactive glass-ceramic scaffold of the type SiO(2)-CaO-K(2)O, to combine the bioactivity of the latter with the release properties of the former, in view of local drug delivery from implants designed for tissue engineering. The standard procedure for SBA-15 synthesis has been modified to minimize the damage to the scaffold caused by the acidic synthesis medium. The composite system has been characterized by means of Scanning Electron Microscopy (coupled with EDS analysis), Small Angle X-Ray Diffraction, Thermogravimetry analysis and Infrared Spectroscopy: the formation of a well ordered hexagonal mesostructure was confirmed. Ibuprofen has been chosen as model drug. The uploading properties have been investigated of the scaffold-mesoporous silica composite as compared with the scaffold as such, and a five-fold increase in the adsorbing properties toward ibuprofen was found, due to the presence of the ordered mesoporous silica. The ibuprofen release to a SBF solution in vitro is complete in 1 day. Retention of bioactivity from the glass-ceramic scaffold after the silica mesostructure incorporation has been observed.     

Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering

In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control.     

Physical and cytocompatibility properties of bioactive glass–polyvinyl alcohol–sodium alginate biocomposite foams prepared via sol–gel processing for trabecular bone regeneration

In the present work, biocomposite foams of bioactive glass along with polyvinyl alcohol and sodium alginate are designed and developed as a potential biomaterial for bone regeneration. These biocomposite foams have a low density of 0.92 g/cm³, providing desired property for bone tissue engineering applications. Biocomposite foams were prepared via surfactant foaming. Scanning electron microscopic characterization revealed pore size of 200–500 μm of the biocomposite foams. When these materials were incubated in simulated body fluid, hydroxyapatite layer formation was observed on the material surface. To confirm the cell viability and proliferation on these materials, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed with NIH 3T3 fibroblast cells and the results revealed good biocompatibility with the biocomposite foams. Cell adhesion studies further confirmed the biocompatibility of the scaffolds via cell attachment and ECM production. The optimally synthesized biocomposite foams had a good combination of physical properties with compressive strength of 1.64 MPa and elastic modulus of 18 MPa. In view of the favorable combination of physical and biological properties, the newly developed materials are considered to be suitable for regeneration of trabecular bone.     

Injectable calcium phosphate–alginate–chitosan microencapsulated MC3T3-E1 cell paste for bone tissue engineering in vivo

Osteoblasts or stem cells have been delivered into injectable calcium phosphate cement (CPC) to improve its effectiveness and biological function. However, the osteogenic potential of the new construct in vivo has been rarely reported, and there are no reports on alginate–chitosan microencapsulated osteoblasts mixed with CPC. This study aimed to develop alginate–chitosan microencapsulated mouse osteoblast MC3T3-E1 cells (AC-cells), evaluate the osteogenic potential of a calcium phosphate cement complex with these AC-cells (CPC-AC-cell), and trace the implanted MC3T3-E1 cells in vivo. MC3T3-E1 cells were embedded in alginate microcapsules, cultured in osteogenic medium for 7 days, and then covered with chitosan before mixing with a paste of β-tricalcium phosphate/calcium phosphate cement (β-TCP/CPC). The construct was injected into the dorsal subcutaneous area of nude mice. Lamellar-bone-like mineralization, newly formed collagen and angiogenesis were observed at 4 weeks. At 8 weeks, areas of newly formed collagen expanded; further absorption of β-TCP/CPC and osteoid-like structures could be seen. Cell tracing in vivo showed that implanted MC3T3-E1 cells were clearly visible at 2 weeks. These in vivo results indicate that the novel injectable CPC-AC-cell construct is promising for bone tissue engineering applications.     

Crosslinked collagen–gelatin–hyaluronic acid biomimetic film for cornea tissue engineering applications

Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents the crosslinked collagen (Col)–gelatin (Gel)–hyaluronic acid (HA) films developed by making use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the crosslinker. The test results on the physical and biological properties indicate that the CGH631 film (the mass ratio of Col:Gel:HA = 6:3:1) has appropriate optical performance, hydrophilicity and mechanical properties. The diffusion properties of the CGH631 film to NaCl and tryptophan are also satisfactory and the measured data are 2.43 × 10^? 6 cm²/s and 7.97 × 10^? 7 cm²/s, respectively. In addition, cell viability studies demonstrate that the CGH631 film has good biocompatibility, on which human corneal epithelial cells attached and proliferated well. This biocompatible film may have potential use in cornea tissue engineering.     

Composite bone substitute materials based on β-tricalcium phosphate and magnesium-containing sol–gel derived bioactive glass

In the present study, bioceramic composites with improved mechanical and biological properties were synthesized by sintering mixtures of β-tricalcium phosphate and SiO₂–CaO–MgO–P₂O₅ sol–gel derived bioactive glass at 1000–1200°C. The physical, mechanical, structural and biological properties of the composites were evaluated by appropriate experiments such as microhardness, bending strength, XRD, SEM and MTT. The results showed that 1000 and 1100°C were not appropriate temperatures for sintering the composites and in contrast, the microhardness, bending strength and bulk density significantly increased by increasing in quantity of bioglass phase when the samples were sintered at 1200°C. No significant difference was found between the fracture toughness of the composites and pure β-tricalcium phosphate. β-tricalcium phosphate was structurally stable up to 1200°C and did not transform to its alpha form even in the presence of the bioglass phase but migration of magnesium cations from the glass composition into its lattice structure was found by right-shift in XRD patterns, especially when the composite contained higher amount of bioglass component. Calcium silicate was also crystallized in the composition of the composites, which was more detectable in higher sintering temperatures. The results of the MTT test showed that proliferation of human osteosarcoma cells on the composites was considerably better than that of pure β-TCP.     

Biofunctionalization of porous titanium coatings through sol–gel impregnation with a bioactive glass–ceramic

In implant technology, open porous Ti coatings are applied as functional surface layers on prosthetic devices to improve osseointegration. Since a successful clinical performance strongly depends on the (initial) quality of bone ingrowth in the porous structure, surface functionalization of the porous Ti to incorporate an additional osteoconductive capacity is recommended. In this paper, a bioactive glass–ceramic coating is applied into the open porous network of Ti coatings with a pore throat size of 1–20 μm through a sol–gel process. Using an all-alkoxide precursor route, homogeneous amorphous powders of three- (SiO₂–CaO–P₂O₅) and four-component (SiO₂–CaO–Na₂O–P₂O₅) bioactive glass compositions are prepared. By sol impregnation followed by a heat treatment, it is possible to deposit a micrometer thin bioactive glass–ceramic layer on the walls of the internal pore surface, while the original porosity and the open pore structure of the Ti coatings are maintained. The tensile adhesion strength of the Ti/bioactive glass–ceramic composite coatings is 22 to 29 MPa, suggesting a good mechanical adhesion.     

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.     

Design and in vitro evaluation of novel γ-PGA/hydroxyapatite nanocomposites for bone tissue engineering

In order to improve biocompatibility and cell growth, poly-γ-glutamic acid (γ-PGA) was used as an additive, and a series of γ-PGA/hydroxyapatite (γ-PGA/HAp) nanocomposites were prepared. Then the morphology, water uptake and retention abilities, in vitro degradation properties in the simulated medium, and cytotoxicity of these γ-PGA/HAp nanocomposites were investigated. The results show that the γ-PGA/HAp nanocomposites have homogenous nano-sized grains, hydrophilicity, biocompatibility, and controlled in vitro degradation, suggesting that the γ-PGA/HAp nanocomposites are novel nanostructure composites with great potential application in the field of bone tissue engineering.     

Radiation synthesis of gelatin/CM-chitosan/β-tricalcium phosphate composite scaffold for bone tissue engineering

A series of biodegradable composite scaffolds was fabricated from an aqueous solution of gelatin, carboxymethyl chitosan (CM-chitosan) and β-tricalcium phosphate (β-TCP) by radiation-induced crosslinking at ambient temperature. Ultrasonic treatment on the polymer solutions significantly influenced the distribution of β-TCP particles. An ultrasonic time of 20 min, followed by 30 kGy irradiation induced a crosslinked scaffold with homogeneous distribution of β-TCP particles, interconnected porous structure, sound swelling capacity and mechanical strength. Fourier Transform Infrared Spectroscopy and X-ray Diffraction analysis indicated that β-TCP successfully incorporated with the network of gelatin and CM-chitosan. In vivo implantation of the scaffold into the mandible of beagle dog revealed that the scaffolds had excellent biocompatibility and the presence of β-TCP can accelerate bone regeneration. The comprehensive results of this study paved way for the application of gelatin/CM-chitosan/β-TCP composite scaffolds as candidate of bone tissue engineering material.