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.     

Microstructure,mechanical property and corrosion behavior of interpenetrating (HA + β-TCP)/MgCa composite fabricated by suction casting

The novel interpenetrating (HA + β-TCP)/MgCa composites were fabricated by infiltrating MgCa alloy into porous HA + β-TCP using suction casting technique. The microstructure, mechanical properties and corrosion behaviors of the composites have been evaluated by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical testing, electrochemical and immersion tests. It was shown that the composites had compact structure and the interfacial bonding between MgCa alloy and HA + β-TCP scaffolds was very well. The ultimate compressive strength of the composites was about 500–1000 fold higher than that of the original porous scaffolds, and it still retained quarter-half of the strength of the bulk MgCa alloy. The electrochemical and immersion tests indicated that the corrosion resistance of the composites was better than that of the MgCa matrix alloy, and the corrosion products of the composite surface were mainly Mg(OH)₂, HA and Ca₃(PO₄)₂. Meanwhile, the mechanical and corrosive properties of the (HA + β-TCP)/MgCa composites were adjustable by the choice of HA content.     

Selective laser sintering of β-TCP/nano-58S composite scaffolds with improved mechanical properties

Nano-sized 58S bioactive glass (nano-58S) as the dispersed phase was added to β-tricalcium phosphate (β-TCP) to reinforce the mechanical properties, and then the β-TCP/nano-58S composite scaffolds were prepared via selective laser sintering (SLS). The effects of nano-58S on microstructure, mechanical properties, bioactivity, and biocompatibility of the composite scaffolds were evaluated. The results showed that nano-58S was homogeneously dispersed in the β-TCP matrix and the mechanical properties were gradually improved when the amount of nano-58S was no more than a certain value (15 wt.%). However, exceeding this value, nano-58S became the continuous phase and exhibited the brittleness of bioactive glass. Accordingly, the mechanical properties gradually decreased. The maximum fracture toughness and compressive strength were 1.347 ± 0.025 MPa · m^1/2 and 18.2 ± 0.62 MPa, respectively. In vitro tests in the simulated body fluid (SBF) demonstrated that the apatite-like layer formed faster on the composite scaffolds than on the scaffold without nano-58S, indicating that the nano-58S glass could enhance the bioactivity of the composite scaffolds. The MG-63 cells culture experiment proved that nano-58S glass could further facilitate the growth of human osteoblastic cells.     

In vitro cell-biological performance and structural characterization of selective laser sintered and plasma surface functionalized polycaprolactone scaffolds for bone regeneration

In the present study a structural characterization and in vitro cell-biological evaluation was performed on polycaprolactone (PCL) scaffolds that were produced by the additive manufacturing technique selective laser sintering (SLS), followed by a plasma-based surface modification technique, either non-thermal oxygen plasma or double protein coating, to functionalize the PCL scaffold surfaces. In the first part of this study pore morphology by means of 2D optical microscopy, surface chemistry by means of hydrophilicity measurement and X-ray photoelectron spectroscopy, strut surface roughness by means of 3D micro-computed tomography (CT) imaging and scaffold mechanical properties by means of compression testing were evaluated before and after the surface modifications. The results showed that both surface modifications increased the PCL scaffold hydrophilicity without altering the morphological and mechanical properties. In the second part of this study the in vitro cell proliferation and differentiation of human osteoprogenitor cells, over 14 days of culture in osteogenic and growth medium were investigated. The O₂ plasma modification gave rise to a significant lower in vitro cell proliferation compared to the untreated and double protein coated scaffolds. Furthermore the double protein coating increased in vitro cell metabolic activity and cell differentiation compared to the untreated and O₂ plasma PCL scaffolds when OM was used.     

Preparation of porous scaffold from hydroxyapatite powders

Hydroxyapatite (HA) powder was prepared by wet chemical method. The hydroxyapatite phase was stable up to 1250 °C without decomposition to beta-tricalcium phosphate. Interconnected porous hydroxyapatite scaffold resembling trabecular bone structure was developed from polymeric replica sponge method. The prepared scaffold has 60 vol.% porosity having a major fraction of ~ 50–125 μm pore diameter. The pore content, pore morphology, pore interconnectivity of scaffold and their compressive strength were dependent on the solid loading and binder content. In-vitro bioactivity and bioresorbability confirmed the feasibility of the developed scaffolds.     

Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering

Calcium silicate possessed excellent biocompatibility, bioactivity and degradability, while the high brittleness limited its application in load-bearing sites. Hydroxyapatite whiskers ranging from 0 to 30 wt.% were incorporated into the calcium silicate matrix to improve the strength and fracture resistance. Porous scaffolds were fabricated by selective laser sintering. The effects of hydroxyapatite whiskers on the mechanical properties and toughening mechanisms were investigated. The results showed that the scaffolds had a uniform and continuous inner network with the pore size ranging between 0.5 mm and 0.8 mm. The mechanical properties were enhanced with increasing hydroxyapatite whiskers, reached a maximum at 20 wt.% (compressive strength: 27.28 MPa, compressive Young's modulus: 156.2 MPa, flexural strength: 15.64 MPa and fracture toughness: 1.43 MPa·m^1/2) and then decreased by addition of more hydroxyapatite whiskers. The improvement of mechanical properties was due to whisker pull-out, crack deflection and crack bridging. Moreover, the degradation rate decreased with the increase of hydroxyapatite whisker content. A layer of bone-like apatite was formed on the scaffold surfaces after being soaked in simulated body fluid. Human osteoblast-like MG-63 cells spread well on the scaffolds and proliferated with increasing culture time. These findings suggested that the calcium silicate scaffolds reinforced with hydroxyapatite whiskers showed great potential for bone regeneration and tissue engineering applications.     

In vitro study of hydroxyapatite/polycaprolactone (HA/PCL) nanocomposite synthesized by an in situ sol–gel process

Hydroxyapatite (HA) is the most substantial mineral constituent of a bone which has been extensively used in medicine as implantable materials, owing to its good biocompatibility, bioactivity high osteoconductive, and/or osteoinductive properties. Nevertheless, its mechanical property is not utmost appropriate for a bone substitution. Therefore, a composite consist of HA and a biodegradable polymer is usually prepared to generate an apt bone scaffold. In the present work polycaprolactone (PCL), a newly remarkable biocompatible and biodegradable polymer, was employed as a matrix and hydroxyapatite nanoparticles were used as a reinforcement element of the composite. HA/PCL nanocomposites were synthesized by a new in situ sol–gel process using calcium hydroxide and phosphoric acid precursors in the presence of Tetrahydrofuran (THF) as a solvent. Chemical and physical characteristics of the nanocomposite were studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared (FTIR) analyses. The results indicated that pure HA nanoparticles were well-incorporated and homogenously dispersed in the PCL matrix. It was found that the mechanical property of PCL was improved by addition of 20 wt.% HA nanoparticles. Furthermore, the biological property of nanocomposites was investigated under in vitro condition. For this purpose, HA/PCL scaffolds were prepared through a salt leaching process and immersed in a saturated simulated body fluid (SBF) after 3 and 7 days. It was found that a uniform layer of biomimetic HA could be deposited on the surface of HA/PCL scaffolds. Therefore, the prepared HA/PCL scaffolds showed good potential for bone tissue engineering and could be used for many clinical applications in orthopedic and maxillofacial surgery.     

Electrospun nanofibers composed of poly(ε-caprolactone) and polyethylenimine for tissue engineering applications

Poly(ε-caprolactone) (PCL) electrospun nanofibers have been reported as a scaffold for tissue engineering application. However, high hydrophobicity of PCL limits use of functional scaffold. In this study, PCL/polyethylenimine (PEI) blend electrospun nanofibers were prepared to overcome the limitation of PCL ones because the PEI as a cationic polymer can increase cell adhesion and can improve the electrospinnability of PCL. The structure, mechanical properties and biological activity of the PCL/PEI electrospun nanofibers were studied. The diameters of the PCL/PEI nanofibers ranged from 150.4 ± 33 to 220.4 ± 32 nm. The PCL/PEI nanofibers showed suitable mechanical properties with adequate porosity and increased hydrophilic behavior. The cell adhesion and cell proliferation of PCL nanofibers were increased by blending with PEI due to the hydrophilic properties of PEI.     

Synthesis of poly(ε-caprolactone)/hydroxyapatite nanocomposites using in-situ co-precipitation

Hybrid poly(ε-caprolactone) (PCL)/hydroxyapatite(HA) nanocomposites with various HA contents (0, 10, 20, 30 wt.%) were synthesized using an in-situ co-precipitation method. All nanocomposites prepared contained elongated HA nanocrystals dispersed uniformly in the PCL matrix without severe agglomeration. The tensile strength decreased from 13.5 ± 0.4 to 10.2 ± 0.3 MPa with increasing the HA content from 0 to 30 wt.%, whereas the elastic modulus increased from 85 ± 4.2 to 183 ± 6.6 MPa. In addition, the ALP activity was increased remarkably due to the presence of bioactive HA nanocrystals within the composites. The nanocomposite containing 30 wt.% HA showed a higher elastic modulus and ALP activity than the conventional PCL/HA composite with an initial HA content of 30 wt.%. This was attributed to the nanoscale hybridization of the HA nanocrystals without significant agglomeration.     

Electrospun poly(ε-caprolactone)/Ca-deficient hydroxyapatite nanohybrids: Microstructure,mechanical properties and cell response by murine embryonic stem cells

Nanohybrid scaffolds mimicking extracellular matrix are promising experimental models to study stem cell behaviour, in terms of adhesion and proliferation. In the present study, the structural characterization of a novel electrospun nanohybrid and the analysis of cell response by a highly sensitive cell type, embryonic stem (ES) cells, are investigated. Ca-deficient hydroxyapatite nanocrystals (d-HAp) were synthesized by precipitation. Fibrous PCL/d-HAp nanohybrids were obtained by electrospinning, d-HAp content ranging between 2 and 55 wt.%. Electrospun mats showed a non-woven architecture, average fiber size was 1.5 ±0.5 μm, porosity 80–90%, and specific surface area 16 m² g^? 1. Up to 6.4 wt.% d-HAp content, the nanohybrids displayed comparable microstructural, mechanical and dynamo-mechanical properties. Murine ES cell response to neat PCL and to nanohybrid PCL/d-HAp (6.4 wt.%) mats was evaluated by analyzing morphological, metabolic and functional markers. Cells growing on either scaffold proliferated and maintained pluripotency markers at essentially the same rate as cells growing on standard tissue culture plates with no detectable signs of cytotoxicity, despite a lower cell adhesion at the beginning of culture. These results indicate that electrospun PCL scaffolds may provide adequate supports for murine ES cell proliferation in a pluripotent state, and that the presence of d-HAp within the mat does not interfere with their growth.     

Preparation of high strength macroporous hydroxyapatite scaffold

Hydroxyapatite (HAp) powder was prepared from CaNO₃·4H₂O and (NH₄)₂HPO₄ by wet-chemical method and has phase stable up to 1250 °C. High strength macroporous HAp–naphthalene (HN) and HAp–naphthalene–benzene (HNB) scaffolds were fabricated by adapting sintering method. The resulting HAp scaffolds have porosity about 60 vol.% with compressive strength of ~ 11 MPa and average pore diameter in the range of ~ 125 μm. The incorporation of benzene in HN scaffold reduces the strength whereas enhanced both the porosity and pore size distribution. XRD, FTIR, SEM and mercury porosimeter techniques were used to study the phase purity, morphology, pore size and pore size distribution of scaffold. The study compared the effect of concentration of naphthalene on strength, porosity and pore size distribution on both HN and HNB scaffold. In-vitro bioactivity studies on HN and HNB scaffolds show the nucleation of spherical carbonated apatite particles on the surface in SBF solution.     

Co-electrospun blends of PU and PEG as potential biocompatible scaffolds for small-diameter vascular tissue engineering

A small-diameter vascular graft (inner diameter 4 mm) was fabricated from polyurethane (PU) and poly(ethylene glycol) (PEG) solutions by blend electrospinning technology. The fiber diameter decreased from 1023 ± 185 nm to 394 ± 106 nm with the increasing content of PEG in electrospinning solutions. The hybrid PU/PEG scaffolds showed randomly nanofibrous morphology, high porosity and well-interconnected porous structure. The hydrophilicity of these scaffolds had been improved significantly with the increasing contents of PEG. The mechanical properties of electrospun hybrid PU/PEG scaffolds were obviously different from that of PU scaffold, which was caused by plasticizing or hardening effect imparted by PEG composition. Under hydrated state, the hybrid PU/PEG scaffolds demonstrated low mechanical performance due to the hydrophilic property of materials. Compared with dry PU/PEG scaffolds with the same content of PEG, the tensile strength and elastic modulus of hydrated PU/PEG scaffolds decreased significantly, while the elongation at break increased. The hybrid PU/PEG scaffolds demonstrated a lower possibility of thrombi formation than blank PU scaffold in platelet adhesion test. The hemolysis assay illustrated that all scaffolds could act as blood contacting materials. To investigate further in vitro cytocompatibility, HUVECs were seeded on the scaffolds and cultured over 14 days. The cells could attach and proliferate well on the hybrid scaffolds than blank PU scaffold, and form a cell monolayer fully covering on the PU/PEG (80/20) hybrid scaffold surface. The results demonstrated that the electrospun hybrid PU/PEG tubular scaffolds possessed the special capacity with excellent hemocompatibility while simultaneously supporting extensive endothelialization with the 20 and 30% content of PEG in hybrid scaffolds.     

Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering

Surface properties of scaffolds such as hydrophilicity and the presence of functional groups on the surface of scaffolds play a key role in cell adhesion, proliferation and migration. Different modification methods for hydrophilicity improvement and introduction of functional groups on the surface of scaffolds have been carried out on synthetic biodegradable polymers, for tissue engineering applications. In this study, alkaline hydrolysis of poly (ε-caprolactone) (PCL) nanofibrous scaffolds was carried out for different time periods (1 h, 4 h and 12 h) to increase the hydrophilicity of the scaffolds. The formation of reactive groups resulting from alkaline hydrolysis provides opportunities for further surface functionalization of PCL nanofibrous scaffolds. Matrigel was attached covalently on the surface of an optimized 4 h hydrolyzed PCL nanofibrous scaffolds and additionally the fabrication of blended PCL/matrigel nanofibrous scaffolds was carried out. Chemical and mechanical characterization of nanofibrous scaffolds were evaluated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle, scanning electron microscopy (SEM) and tensile measurement. In vitro cell adhesion and proliferation study was carried out after seeding nerve precursor cells (NPCs) on different scaffolds. Results of cell proliferation assay and SEM studies showed that the covalently functionalized PCL/matrigel nanofibrous scaffolds promote the proliferation and neurite outgrowth of NPCs compared to PCL and hydrolyzed PCL nanofibrous scaffolds, providing suitable substrates for nerve tissue engineering.     

Osteogenic effect of hyaluronic acid sodium salt in the pores of a hydroxyapatite scaffold

According to previous reports, a large volume of bone marrow cells (1 × 10⁷ cells/ml) is required for bone regeneration in the pores of a scaffold in vivo. We theorized that immersion of a porous hydroxyapatite (HA) scaffold in hyaluronic acid solution would facilitate bone formation in the scaffold at 1 × 10⁶ cells/ml density of bone marrow cells. The cells were respectively seeded into pores of the cylindrical HA scaffolds with a hollow center after immersion in hyaluronic acid solution or in culture medium. The scaffolds were implanted in the dorsal subcutis of rats for 4 weeks. Thereafter, serially sectioned paraffin specimens were made and observed histologically. Bone formation was observed in many pores of HA scaffold by immersion in hyaluronic acid solution. However, there were no or less pores with new bone formation in the scaffold by immersion in culture medium. The cells were cultured with and without hyaluronic acid in vitro. There was no significant difference in bone formation in vitro with and without hyaluronic acid. The results of this study suggest that hyaluronic acid binds to the cells on the wall of three-dimensional structure and effectively promotes new bone formation.     

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.     

The effect of processing parameters and solid concentration on the mechanical and microstructural properties of freeze-casted macroporous hydroxyapatite scaffolds

The design and fabrication of macroporous hydroxyapatite scaffolds, which could overcome current bone tissue engineering limitations, have been considered in recent years. In the current study, controlled unidirectional freeze-casting at different cooling rates was investigated. In the first step, different slurries with initial hydroxyapatite concentrations of 7–37.5 vol.% were prepared. In the next step, different cooling rates from 2 to 14 °C/min were applied to synthesize the porous scaffold. Additionally, a sintering temperature of 1350 °C was chosen as an optimum temperature. Finally, the phase composition (by XRD), microstructure (by SEM), mechanical characteristics, and the porosity of sintered samples were assessed. The porosity of the sintered samples was in a range of 45–87% and the compressive strengths varied from 0.4 MPa to 60 MPa. The mechanical strength of the scaffolds increased as a function of initial concentration, cooling rate, and sintering temperature. With regards to mechanical strength and pore size, the samples with the initial concentration and the cooling rate of 15 vol.% and 5 °C/min, respectively, showed better results.     

Microstructure,mechanical property,bio-corrosion and cytotoxicity evaluations of Mg/HA composites

Mg/HA (10 wt.%, 20 wt.% and 30 wt.%) composites were prepared by pure magnesium and hydroxyapatite (HA) powders using powder metallurgy (PM) method. The microstructure, mechanical property, corrosion and cytotoxicity of these Mg/HA composites were studied, with the bulk pure magnesium as control. The results showed that the main constitutional phases of Mg/HA composites were simply α-Mg and HA. The HA particulates distributed uniformly in Mg matrix for Mg/10HA composite, and few HA clustering occasionally spread over the Mg/20HA composite, whereas severe agglomeration of HA particulates could be seen for Mg/30HA composite. The yield tensile strength of Mg/10HA composite increased compared with that of the as-extruded bulk pure magnesium, yet the yield tensile strength, ultimate tensile strength and ductility of Mg/HA composites decreased with the further increase of HA content. The corrosion rate of Mg/HA composites increased with the increment of HA content. The cytotoxicity tests indicated that Mg/10HA extract showed no toxicity to L-929 cells, whereas Mg/20HA and Mg/30HA composite extracts induced significantly reduced cell viability.     

Effect of ZrO2 addition on the mechanical properties of porous TiO2 bone scaffolds

This study aimed at the investigation of the effect of zirconium dioxide (ZrO₂) addition on the mechanical properties of titanium dioxide (TiO₂) bone scaffolds. The highly biocompatible TiO₂ has been identified as a promising material for bone scaffolds, whereas the more bioinert ZrO₂ is known for its excellent mechanical properties. Ultra-porous TiO₂ scaffolds (> 89% porosity) were produced using polymer sponge replication with 0–40 wt.% of the TiO₂ raw material substituted with ZrO₂. Microstructure, chemical composition, and pore architectural features of the prepared ceramic foams were characterised and related to their mechanical strength. Addition of 1 wt.% of ZrO₂ led to 16% increase in the mean compressive strength without significant changes in the pore architectural parameters of TiO₂ scaffolds. Further ZrO₂ additions resulted in reduction of compressive strength in comparison to containing no ZrO₂. The appearance of zirconium titanate (ZrTiO₄) phase was found to hinder the densification of the ceramic material during sintering resulting in poor intergranular connections and thus significantly reducing the compressive strength of the highly porous ceramic foam scaffolds.     

Novel,simple and reproducible method for preparation of composite hierarchal porous structure scaffolds

Demand to develop a simple and adaptable method for preparation the hierarchical porous scaffolds for bone tissue regeneration is ever increasing. This study presents a novel and reproducible method for preparing the scaffolds with pores structure spanning from nano, micro to macro scale. A macroporous Sr-Hardystonite (Sr–Ca₂ZnSi₂O₇, Sr–HT) scaffold with the average pore size of ~ 1200 μm and porosity of ~ 95% was prepared using polymer sponge method. The struts of the scaffold were coated with a viscous paste consisted of salt (NaCl) particles and polycaprolactone (PCL) to provide a layer with thickness of ~ 300–800 μm. A hierarchical porous scaffold was obtained with macro, micro and nanopores in the range of 400–900 μm, 1–120 μm and 40–290 nm, after salt leaching process. These scales could be easily adjusted based on the starting foam physical characteristics, salt particle size, viscosity of the paste and salt/PCL weight ratio.     

Fabrication of porous titanium scaffold with controlled porous structure and net-shape using magnesium as spacer

This paper reports a new approach to fabricating biocompatible porous titanium with controlled pore structure and net-shape. The method is based on using sacrificial Mg particles as space holders to produce compacts that are mechanically stable and machinable. Using magnesium granules and Ti powder, Ti/Mg compacts with transverse rupture strength (~ 85 MPa) sufficient for machining were fabricated by warm compaction, and a complex-shape Ti scaffold was eventually produced by removal of Mg granules from the net-shape compact. The pores with the average size of 132–262 μm were well distributed and interconnected. Due to anisotropy and alignment of the pores the compressive strength varied with the direction of compression. In the case of pores aligned with the direction of compression, the compressive strength values (59–280 MPa) high enough for applications in load bearing implants were achieved. To verify the possibility of controlled net-shape, conventional machining process was performed on Ti/Mg compact. Compact with screw shape and porous Ti scaffold with hemispherical cup shape were fabricated by the results. Finally, it was demonstrated by cell tests using MC3T3-E1 cell line that the porous Ti scaffolds fabricated by this technique are biocompatible.