Hard Tissue Engineering Applications Research Articles

Mechanical properties and biocompatibility of porous titanium scaffolds for bone tissue engineering.

Synthetic scaffolds are a highly promising new approach to replace both autografts and allografts to repair and remodel damaged bone tissue. Biocompatible porous titanium scaffold was manufactured through a powder metallurgy approach. Magnesium powder was used as space holder material which was compacted with titanium powder and removed during sintering. Evaluation of the porosity and mechanical properties showed a high level of compatibility with human cortical bone. Interconnectivity between pores is higher than 95% for porosity as low as 30%. The elastic moduli are 44.2GPa, 24.7GPa and 15.4GPa for 30%, 40% and 50% porosity samples which match well to that of natural bone (4-30GPa). The yield strengths for 30% and 40% porosity samples of 221.7MPa and 117MPa are superior to that of human cortical bone (130-180MPa). In-vitro cell culture tests on the scaffold samples using Human Mesenchymal Stem Cells (hMSCs) demonstrated their biocompatibility and indicated osseointegration potential. The scaffolds allowed cells to adhere and spread both on the surface and inside the pore structures. With increasing levels of porosity/interconnectivity, improved cell proliferation is obtained within the pores. It is concluded that samples with 30% porosity exhibit the best biocompatibility. The results suggest that porous titanium scaffolds generated using this manufacturing route have excellent potential for hard tissue engineering applications.

Manufacturing of biocompatible porous titanium scaffolds using a novel spherical sugar pellet space holder

A new and highly biocompatible space holder material is proposed for manufacturing of porous titanium with open and interconnected pore morphologies through powder metallurgy techniques. Sugar pellets are compacted with titanium powder and then removed by dissolution in water before sintering. The morphology, pore structure and porosity were observed by optical microscopy, SEM and micro-CT. The porous titanium has highly spherical pore shapes, well-controlled pore sizes and high interconnectivity. The results suggest that porous titanium scaffolds generated using this manufacturing route have the potential for hard tissue engineering applications.

Preparation and Characterization of Polycaprolactone / Layered Double Hydroxide Nanocomposite for Hard Tissue Engineering Applications

Preparation and Characterization of Polycaprolactone / Layered Double Hydroxide Nanocomposite for Hard Tissue Engineering Applications

Transformation of brushite to hydroxyapatite and effects of alginate additives

Phase transformations are important processes during mineral formation in both in vivo and in vitro model systems and macromolecules are influential in regulating the mineralization processes. Calcium phosphate mineralized alginate hydrogels are potential candidates for hard tissue engineering applications and transformation of the resorbable calcium phosphate phases to apatitic bone mineral in vivo enhances the success of these composite materials. Here, the transformation of brushite to hydroxyapatite (HA) and the effects of alginate additives on this process are studied by the investigation of supersaturation profiles with HA-seeded and unseeded experiments. This experimental design allows for detailed kinetic interpretation of the transformation reactions and deduction of information on the nucleation stage of HA by evaluating the results of seeded and unseeded experiments together. In the experimental conditions of this work, transformation was controlled by HA growth until the point of near complete brushite dissolution where the growth and dissolution rates were balanced. The presence of alginate additives at low concentration were not highly influential on transformation rates during the growth dominated region but their retardant effect became more pronounced as the dissolution and growth rates reached an equilibrium where both reactions were effective on transformation kinetics. Decoupling of seeded and unseeded transformation experiments suggested that alginate additives retard HA nucleation and this was most evident in the presence of G-block oligomers.

Fabrication and evaluation of silica-based ceramic scaffolds for hard tissue engineering applications

In recent decades, bone scaffolds have received a great attention in biomedical applications due to their critical roles in bone tissue regeneration, vascularization, and healing process. One of the main challenges of using scaffolds in bone defects is the mechanical strength mismatch between the implant and surrounding host tissue which causes stress shielding or failure of the implant during the course of treatment. In this paper, space holder method was applied to synthesize diopside/forsterite composite scaffolds with different diopside content. During the sintering process, NaCl, as spacer agent, gradually evaporated from the system and produced desirable pore size in the scaffolds. The results showed that adding 10wt.% diopside to forsterite can enormously improve the bioactivity, biodegradability, and mechanical properties of the composite scaffolds. The size of crystals and pores of the obtained scaffolds were measured to be in the range 70–100nm and 100–250μm, respectively. Composite scaffolds containing 10wt.% diopside showed similar compressive strength and Young's modulus (4.36±0.3 and 308.15±7MPa, respectively) to that of bone.

Physicochemical and mechanical properties of freeze cast hydroxyapatite-gelatin scaffolds with dexamethasone loaded PLGA microspheres for hard tissue engineering applications

Hydroxyapatite (HA)-gelatin scaffolds incorporated with dexamethasone-loaded polylactic-co-glycolic acid (PLGA) microspheres were synthesized by freeze casting technique. Scanning electron microscopy (SEM) micrographs demonstrated a unidirectional microstructure and a decrease in the pore size as a function of temperature gradient. Higher amounts of HA resulted in a decrease in the pore size. According to the results, at lower cooling rates, the formation of a lamellar structure decreased the mechanical strength, but at the same time, enhanced the swelling ratio, biodegradation rate and drug release level. On the other hand, higher weight ratios of HA increased the compressive strength, and reduced the swelling ratio, biodegradation rate and drug release level. The results obtained by furrier transform infrared spectroscopy (FTIR) and bioactivity analysis illustrated that the interactions of the materials support the apatite formation in the simulated body fluid (SBF) solution. Based on the obtained results, the synthesized composite scaffolds have the necessary mechanical and physicochemical features to support the regeneration of defects and to maintain their stability during the neo-tissue formation.

Silane crosslinking of electrospun poly (lactic acid)/nanocrystalline cellulose bionanocomposite

Biodegradable nanofibrous mats fabricated by electrospinning are commonly used in tissue engineering, however, lack of essential mechanical properties of such nanofibers is a challenging issue. In this work, vinyltrimethoxysilane (VTMS) was grafted onto poly (lactic acid) (PLA) and the silane grafted PLA was subsequently applied in electrospinning process. Electrospun nanofibrous mats based on PLA/nanocrystalline cellulose (NCC) and PLA-g-silane/NCC nanocomposites were fabricated and immersed in hot water (70°C) for crosslinking of silane grafted PLA. It was found that introducing NCC to the samples cause to reduction in fiber diameter and the other hand the silane crosslinking of PLA increase the mean fiber diameter. DSC thermograms also revealed that silane grafting caused a reduction in mobility of polymer segments, and consequently reduction of crystallinity. On the contrary, the NCC in the PLA-g-silane samples effectively influenced the crystal nucleation, while in the PLA nanofibers the nucleation was lower. The impact of NCC on tensile strength enhancement of samples was notable. The results suggested that the chemical crosslinking remarkably improves the mechanical properties of PLA nanofibers. Furthermore, biocompatibility of such modified nanofibers was also evaluated through cytotoxicity results, therefore the modified PLA nanocomposite can be considered as a practical candidate for hard tissue engineering applications.

Preparation and characterization of wollastonite/titanium oxide nanofiber bioceramic composite as a future implant material

In this study, novel composites consisting of electrospun titanium dioxide (TiO2) nanofibers incorporated into high-purity wollastonite glass ceramics were prepared as materials for use in hard tissue engineering applications. These materials were characterized and investigated by means of physical, mechanical and in vitro studies. The proposed composite showed greater densification and better mechanical characteristics compared to pure wollastonite. The influence of densification temperature and TiO2 content was investigated. Typically, TiO2/wollastonite composites having 0, 10, 20 and 30wt% metal oxide nanofibers were sintered at 900, 1100 and 1250°C. The results indicated that increasing TiO2 nanofibers content leads to increase the bulk density, compressive strength and microhardness with negligible, high and moderate influence for the densification temperature, respectively. While porosity and water adsorption capacity decreased with increasing the metal oxide nanofibers with a considerable impact for the sintering temperature in both properties. Moreover, bone-like apatite formed on the surface of wollastonite and wollastonite/TiO2 nanofibers soaked in simulated body fluid (SBF). All these results show that the inclusion of TiO2 nanofibers improved the characteristics of wollastonite while preserving its in vitro bioactivity; hence, the proposed composite may be used as a bone substitute in high load bearing sites.

Editorial: Inorganic Biomaterials.

EDITORIAL article Front. Bioeng. Biotechnol., 21 January 2016Sec. Biomaterials Volume 4 - 2016 | https://doi.org/10.3389/fbioe.2016.00002

Engineered phage films as scaffolds for CaCO3 biomineralization.

M13 bacteriophages (phage) were exploited as CaCO3 mineralization scaffolds for hard tissue engineering applications. M13 phage was first self-assembled into biomimetic fibrous scaffolds, followed by CaCO3 biomineralization via the polymer-induced liquid precursor process. The phage scaffolds successfully incorporated calcium carbonate, facilitating nucleation and growth of spherulitically textured calcite. The Young's modulus of the scaffolds increased by an order of magnitude after mineralization while also supporting the growth of mouse fibroblasts. These findings demonstrate that phage-based biomaterials are a feasible platform for creating biomineralized hard tissue constructs, in support of future studies in hard tissue engineering and biomedical applications.

When 1+1>2: Nanostructured composites for hard tissue engineering applications.

Multicomponent, synergistic and multifunctional nanostructures have taken over the spotlight in the realm of biomedical nanotechnologies. The most prospective materials for bone regeneration today are almost exclusively composites comprising two or more components that compensate for the shortcomings of each one of them alone. This is quite natural in view of the fact that all hard tissues in the human body, except perhaps the tooth enamel, are composite nanostructures. This review article highlights some of the most prospective breakthroughs made in this research direction, with the hard tissues in main focus being those comprising bone, tooth cementum, dentin and enamel. The major obstacles to creating collagen/apatite composites modeled after the structure of bone are mentioned, including the immunogenicity of xenogeneic collagen and continuously failing attempts to replicate the biomineralization process in vitro. Composites comprising a polymeric component and calcium phosphate are discussed in light of their ability to emulate the soft/hard composite structure of bone. Hard tissue engineering composites created using hard material components other than calcium phosphates, including silica, metals and several types of nanotubes, are also discoursed on, alongside additional components deliverable using these materials, such as cells, growth factors, peptides, antibiotics, antiresorptive and anabolic agents, pharmacokinetic conjugates and various cell-specific targeting moieties. It is concluded that a variety of hard tissue structures in the body necessitates a similar variety of biomaterials for their regeneration. The ongoing development of nanocomposites for bone restoration will result in smart, theranostic materials, capable of acting therapeutically in direct feedback with the outcome of in situ disease monitoring at the cellular and subcellular scales. Progress in this research direction is expected to take us to the next generation of biomaterials, designed with the purpose of fulfilling Daedalus' dream - not restoring the tissues, but rather augmenting them.

Aligned poly(ε-caprolactone)/graphene oxide and reduced graphene oxide nanocomposite nanofibers: Morphological, mechanical and structural properties.

A number of studies have demonstrated that the mechanical properties of electrospun polymeric nanofibrous scaffolds are enhanced with the incorporation of graphene and its derivatives, thus developing their applications in hard tissue engineering. However, our understanding of the relationship between the microstructure and properties of these fibrous scaffolds and how they are influenced by graphene oxide (GO) and reduced graphene oxide (RGO) loading is much more limited. Thus, in this paper, poly(ε-caprolactone) (PCL)/GO and RGO nanocomposite nanofibers containing 0, 0.1, 0.5 and 1wt.% GO and RGO were prepared using an electrospinning technique. With the addition of 0.1wt.% of GO and RGO nanosheets in PCL, the tensile strength of PCL scaffolds increased over ~160 and 304% respectively and elastic modulus increased over 103 and 163% due to the good dispersion of the nanosheets and their interaction with the molecular chains of PCL. These were supported by the parallel increase in relaxation time and molecular orientation of PCL chains at the presence of nanosheets with a loading of 0.1wt.%. The enhancement effect of the nanosheets was weakened with an increase in GO and RGO loading up to 1wt.% in which it is connected to a partial exfoliation of the nanosheets.

Degradation and silicon excretion of the calcium silicate bioactive ceramics during bone regeneration using rabbit femur defect model.

The investigation of the bone regeneration ability, degradation and excretion of the grafts is critical for development and application of the newly developed biomaterials. Herein, the in vivo bone-regeneration, biodegradation and silicon (Si) excretion of the new type calcium silicate (CaSiO3, CS) bioactive ceramics were investigated using rabbit femur defect model, and the results were compared with the traditional β-tricalcium phosphate [β-Ca3(PO4)2, β-TCP] bioceramics. After implantation of the scaffolds in rabbit femur defects for 4, 8 and 12weeks, the bone regenerative capacity and degradation were evaluated by histomorphometric analysis. While urine and some organs such as kidney, liver, lung and spleen were resected for chemical analysis to determine the excretion of the ionic products from CS implants. The histomorphometric analysis showed that the bioresorption rate of CS was similar to that of β-TCP in femur defect model, while the CS grafts could significantly stimulate bone formation capacity as compared with β-TCP bioceramics (P<0.05). The chemical analysis results showed that Si concentration in urinary of the CS group was apparently higher than that in control group of β-TCP. However, no significant increase of the Si excretion was found in the organs including kidney, which suggests that the resorbed Si element is harmlessly excreted in soluble form via the urine. The present studies show that the CS ceramics can be used as safe, bioactive and biodegradable materials for hard tissue repair and tissue engineering applications.

Physically-strengthened collagen bioactive nanocomposite gels for bone: A feasibility study

Collagen hydrogel systems have been limited in their uses for hard tissue engineering due to their poor mechanical properties in spite of their excellent biocompatibility. Physical strengthening and incorporation of the inorganic substances are considered as promising ways to improve mechanical stability of the collagen gel, while providing effective biomimetic environment for cells. Here, we developed three-dimensional matrix by rolling up the plastically-compressed collagen hydrogel composites with mesoporous bioactive glass nanoparticle (BGn). Monodispersed BGn with a size of ~90 nm was well incorporated within the collagen matrix which has nanofibrillar structure. The mechanical properties of the composite hydrogels measured by dynamic mechanical analysis were significantly improved by the compression of the hydrogels and further improved by addition of BGn into hydrogels. Moreover, the proliferation rate and osteogenic differentiation of rat bone marrow derived mesenchymal stem cells cultured within the composite hydrogels were enhanced by incorporation of BGn. The results suggest that physically-strengthened nanocomposite collagen hydrogel would be useful in hard tissue engineering applications.

PCL/alginate composite scaffolds for hard tissue engineering: fabrication, characterization, and cellular activities.

Alginates have been used widely in biomedical applications because of good biocompatibility, low cost, and rapid gelation in the presence of calcium ions. However, poor mechanical properties and fabrication-ability for three-dimensional shapes have been obstacles in hard-tissue engineering applications. To overcome these shortcomings of alginates, we suggest a new composite system, consisting of a synthetic polymer, poly(ε-caprolactone), and various weight fractions (10-40 wt %) of alginate. The fabricated composite scaffolds displayed a multilayered 3D structure, consisting of microsized composite struts, and they provided a 100% offset for each layer. To show the feasibility of the scaffold for hard tissue regeneration, the composite scaffolds fabricated were assessed not only for physical properties, including surface roughness, tensile strength, and water absorption and wetting, but also in vitro osteoblastic cellular responses (cell-seeding efficiency, cell viability, fluorescence analyses, alkaline phosphatase (ALP) activity, and mineralization) by culturing with preosteoblasts (MC3T3-E1). Due to the alginate components in the composites, the scaffolds showed significantly enhanced wetting behavior, water-absorption (∼12-fold), and meaningful biological activities (∼2.1-fold for cell-seeding efficiency, ∼2.5-fold for cell-viability at 7 days, ∼3.4-fold for calcium deposition), compared with a pure PCL scaffold.

Design and development of potential tissue engineering scaffolds from structurally different longitudinal parts of a bovine-femur.

The complex architecture of the cortical part of the bovine-femur was examined to develop potential tissue engineering (TE) scaffolds. Weight-change and X-ray diffraction (XRD) results show that significant phase transformation and morphology conversion of the bone occur at 500–750°C and 750–900°C, respectively. Another breakthrough finding was achieved by determining a sintering condition for the nucleation of hydroxyapatite crystal from bovine bone via XRD technique. Scanning electron microscopy results of morphological growth suggests that the concentration of polymer fibrils increases (or decreases, in case of apatite crystals) from the distal to proximal end of the femur. Energy-dispersive analysis of X-ray, Fourier transform infrared, micro-computer tomography, and mechanical studies of the actual composition also strongly support our microscopic results and firmly indicate the functionally graded material properties of bovine-femur. Bones sintered at 900 and 1000°C show potential properties for soft and hard TE applications, respectively.

Enhanced cellular activities of polycaprolactone/alginate-based cell-laden hierarchical scaffolds for hard tissue engineering applications

Biomedical scaffolds have been widely investigated because they are essential for support and promotion of cell adhesion, proliferation and differentiation in three-dimensional (3D) structures. An ideal scaffold should be highly porous to enable efficient nutrient and oxygen transfer and have a 3D structure that provides optimal micro-environmental conditions for the seeded cells to obtain homogeneous growth after a long culture period. In this study, new hierarchical osteoblast-like cell (MG-63)-laden scaffolds consisting of micro-sized struts/inter-layered micro-nanofibres and cell-laden hydrogel struts with mechanically stable and biologically superior properties were introduced. Poly(ethylene oxide) (PEO) was used as a sacrificial component to generate pores within the cell-laden hydrogel struts to attain a homogeneous cell distribution and rapid cell growth in the scaffold interior. The alginate-based cell-laden struts with PEO induced fast/homogeneous cell release, in contrast to nonporous cell-laden struts. Various weight fractions (0.5, 1, 2, 3 and 3.5wt%) of PEO were used, of which 2wt% PEO in the cell-laden strut resulted in the most appropriate cell release and enhanced biological activities (cell proliferation and calcium deposition), compared to nonporous cell-laden struts.

Collagen hydrogels incorporated with surface-aminated mesoporous nanobioactive glass: Improvement of physicochemical stability and mechanical properties is effective for hard tissue engineering

Collagen (Col) hydrogels have poor physicochemical and mechanical properties and are susceptible to substantial shrinkage during cell culture, which limits their potential applications in hard tissue engineering. Here, we developed novel nanocomposite hydrogels made of collagen and mesoporous bioactive glass nanoparticles (mBGns) with surface amination, and addressed the effects of mBGn addition (Col:mBG=2:1, 1:1 and 1:2) and its surface amination on the physicochemical and mechanical properties of the hydrogels. The amination of mBGn was shown to enable chemical bonding with collagen molecules. As a result, the nanocomposite hydrogels exhibited a significantly improved physicochemical and mechanical stability. The hydrolytic and enzymatic degradation of the Col–mBGn hydrogels were slowed down due to the incorporation of mBGn and its surface amination. The mechanical properties of the hydrogels, specifically the resistance to loading as well as the stiffness, significantly increased with the addition of mBGn and its aminated form, as assessed by a dynamic mechanical analysis. Mesenchymal stem cells cultivated within the Col–mBGn hydrogels were highly viable, with enhanced cytoskeletal extensions, due to the addition of surface aminated mBGn. While the Col hydrogel showed extensive shrinkage (down to ∼20% of initial size) during a few days of culture, the shrinkage of the mBGn-added hydrogel was substantially reduced, and the aminated mBGn-added hydrogel had no observable shrinkage over 21days. Results demonstrated the effective roles of aminated mBGn in significantly improving the physicochemical and mechanical properties of Col hydrogel, which are ultimately favorable for applications in stem cell culture for bone tissue engineering.

Multilevel Hierarchically Ordered Artificial Biomineral

Living organisms are known for creating complex organic-inorganic hybrid materials such as bone, teeth, and shells, which possess outstanding functions as compared to their simple mineral forms. This has inspired many attempts to mimic such structures, but has yielded few practical advances. In this study, a multilevel hierarchically ordered artificial biomineral (a composite of hydroxyapatite and gelatine) with favorable nanomechanical properties is reported. A typical optimized HAp/gelatin hybrid material in the perpendicular direction of the HAp c-axis has a modulus of 25.91 + 1.78 GPa and hardness of 0.90 + 0.10 GPa, which well matches that of human cortical bone (modulus 24.3 + 1.4 GPa, hardness 0.69 + 0.05 GPa). The bottom-up crystal constructions (from nano- to micro- to macroscale) of this material are achieved through a hard template approach by the phase transformation from DCP to HAp. The structural biomimetic material shows another way to mimic the complex hierarchical designs of sclerous tissues which have potential value for application in hard tissue engineering.

Synthesis, characterization and cytocompatibility of spherical bioactive glass nanoparticles for potential hard tissue engineering applications

Nanotechnology offers a new strategy to develop novel bioactive materials, given that nano-scaled biomaterials exhibit an enhanced biocompatibility and bioactivity. In this work, we developed a method for the synthesis of spherical bioactive glass nanoparticles (BGNP) aimed at producing biomaterials for potential use in the repair of hard tissues. The BGNP were prepared using the sol–gel process based on the reaction of alkoxides and other precursors in aqueous media for obtaining the oxide-ternary system with the stoichiometric proportion of 60% SiO2, 36% CaO and 4% P2O5. The system was extensively characterized by Fourier transform infrared, x-ray diffraction and scanning electron microscope/energy-dispersive x-ray spectroscopy with regard to chemical composition, crystallinity and morphology. Moreover, the results suggested the BGNP to be highly bioactive, which was confirmed by the formation of a hydroxyapatite biomimetic layer on the material surfaces upon immersion in simulated body fluid solution. In addition, the bioactivity response toward the developed BGNPs was assessed by direct contact of osteoblast cells using resazurin and alkaline phosphatase assays. The new BGNP have presented a significant increase in the osteoblast in vitro cytocompatibility behavior as compared to similar micro-sized bioactive glass particles. Such improvement in the overall bioactive behavior of BGNP was attributed to the much higher surface area causing enhanced interactions at the cell–nanomaterial interfaces. Hence, based on the results, the BGNP produced are the biomaterials to be potentially utilized in hard tissue engineering applications.