Development of nano-sized hydroxyapatite reinforced composites for tissue engineering scaffolds

Nano-sized hydroxyapatite (nanoHA) reinforced composites, mimicking natural bone, were produced. Examination by transmission electron microscopy revealed that the nanoHA particles had a rod-like morphology, 20–30 nm in width and 50–80 nm in length. The phase composition of hydroxyapatite was confirmed by X-ray diffraction. The nanoHA particles were incorporated into poly-2-hydroxyethylmethacrylate (PHEMA)/polycaprolactone (PCL) matrix to make new nanocomposites: nanoHA-PHEMA/PCL. Porous nanocomposite scaffolds were then produced using a porogen leaching method. The interconnectivity of the porous structure of the scaffolds was revealed by non-destructive X-ray microtomography. Porosity of 84% was achieved and pore sizes were approximately around 300–400 μm. An in vitro study found that the nanocomposites were bioactive as indicated by the formation of a bone-like apatite layer after immersion in simulated body fluid. Furthermore, the nanocomposites were able to support the growth and proliferation of primary human osteoblast (HOB) cells. HOB cells developed a well organized actin cytoskeletal protein on the nanocomposite surface. The results demonstrate the potential of the nanocomposite scaffolds for tissue engineering applications for bone repair.     

Polymer-bioceramic composites for tissue engineering scaffolds

Designing tissue engineering scaffolds with the required mechanical properties and favourable microstructure to promote cell attachment, growth and new tissue formation is one of the key challenges facing the tissue engineering field. An important class of scaffolds for bone tissue engineering is based on bioceramics and bioactive glasses, including: hydroxyapatite, bioactive glass (e.g. Bioglass^®), alumina, TiO₂ and calcium phosphates. The primary disadvantage of these materials is their low resistance to fracture under loads and their high brittleness. These drawbacks are exacerbated by the fact that optimal scaffolds must be highly porous (>90% porosity). Several approaches are being explored to enhance the structural integrity, fracture strength and toughness of bioceramic scaffolds. This paper reviews recent proposed approaches based on developing bioactive composites by introducing polymer coatings or by forming interpenetrating polymer-bioceramic microstructures which mimic the composite structure of bone. Several systems are analysed and scaffold fabrication processes, microstructure development and mechanical properties are discussed. The analysis of the literature suggests that the scaffolds reviewed here might represent the optimal solution and be the scaffolds of choice for bone regeneration strategies.     

Electrospun chitosan/hydroxyapatite nanofibers for bone tissue engineering

Chitosan (CS) nanofibers were prepared by an electrospinning technique and then treated with simulated body fluid (SBF) to encourage hydroxyapatite (HA) formation on their surface. The CS/HA nanofibers were subjected to scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy, and X-ray diffraction (XRD) to confirm HA formation as well as determine the morphology of the nanofibrous scaffolds. The SEM image indicated that the distribution of HA on the CS nanofibers was homogeneous. The results from EDS and XRD indicated that HA was formed on the nanofibrous surfaces after 6-day incubation in the SBF. The calcium/phosphorus ratio of deposited HA was close to that of natural bone. To determine biocompatibility, the CS/HA scaffolds were applied to the culture of rat osteosarcoma cell lines (UMR-106). The cell densities on the CS/HA nanofibers were higher than those on the CS nanofibers, the CS/HA film, and the CS film, indicating that cell proliferation on CS/HA nanofibers was enhanced. Moreover, the early osteogenic differentiation on CS/HA was also more significant, due to the differences in chemical composition and the surface area of CS/HA nanofibers. The biocompatibility and the cell affinity were enhanced using the CS/HA nanofibers. This indicates that electrospun CS/HA scaffolds would be a potential material in bone tissue engineering.     

Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.     

Tuning polycaprolactone-carbon nanotube composites for bone tissue engineering scaffolds

This report describes the mechanical, thermal and biological characterisation of a solid free form microfabricated carbon nanotube-polycaprolactone composite, in which both the quantity of nanotubes in the matrix as well as the scaffold design were varied in order to tune the mechanical properties of the material. The creep and stress relaxation behaviour of the composite material was analysed to identify an optimal composition for bone tissue engineering. Moreover, the morphology and viability of osteoblast-like cells (MG63) on composite scaffolds were analysed using scanning electron microscopy and MTT assays. Our data demonstrate that by changing the ratio of CNT to PCL, the elastic modulus of the nanocomposite can be varied between 10 and 75 MPa. In this range, the geometry of the scaffold can be used to finely tune its stiffness. However our PCL-CNT nanocomposites were able to sustain osteoblast proliferation and modulate cell morphology. Thus we show the potential of custom designed CNT nanocomposites for bone tissue engineering.     

Preparation and characterization of polycaprolactone-chitosan composites for tissue engineering applications

Highly porous scaffold plays an important role in bone tissue engineering, which becomes a promising alternative approach for bone repair since its emergence. The objective of this work was to blend poly (є-caprolactone) (PCL) with chitosan (CS) for the purpose of preparation of porous scaffold. A simple unique method was employed under room-temperature condition to blend the two components together without separation of two phases. The reaction leads to formation of sponge-like porous 5, 10, 15 and 20 wt% CS composites. XRD, IR and SEM were used to determine components and morphology of the composites. DSC studies indicated that the miscibility of the two components. And pore volume fractures of composites were determined by a simple method in which a pycnometer was used. The results show that CS is successfully commingled into PCL matrix, and adding CS into PCL will not damage the crystalline structure of PCL. The composite shows no signs of phase separation and presents a unique porous structure under SEM observation. The porosity of composite increased with the increase of the content of CS in the composite. The highest porosity reached to 92% when CS content increased to 20 wt%. The mechanism of formation of this unique porous structure is also discussed.     

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.     

Biodegradable polylactide/hydroxyapatite nanocomposite foam scaffolds for bone tissue engineering applications

Supercritical carbon dioxide processing of poly-L-lactide (PLLA)/hydroxyapatite (nHA) nanocomposites was investigated as a means to prepare foams suitable as scaffolds in bone tissue engineering applications. For given foaming parameters, addition of nHA to the PLLA gave reduced cell sizes and improved homogeneity in the size distribution, but did not significantly affect the degree of crystallinity, which remained of the order of 50 wt% in all the foams. The compressive modulus and strength were primarily influenced by the porosity and there was no significant reinforcement of the matrix by the nHA. The mechanical properties of the foams were nevertheless comparable with those of trabecular bone, and by adjusting the saturation pressure and depressurization rate it was possible to generate porosities of about 85 %, an interconnected morphology and cell diameters in the range 200-400 μm from PLLA containing 4.17 vol% nHA, satisfying established geometrical requirements for bone replacement scaffolds.     

骨组织工程用多孔HA生物材料的制备

用PVB、NH4HCO3和(NH4)2CO3粒子作造孔剂,制备了骨组织工程用多孔HA生物材料.讨论了烧结工艺和造孔剂含量等对材料结构的影响.研究表明,较佳的烧结工艺为1200℃烧结4h,烧结后样品主要是HA相.造孔剂PVB、(NH4)2CO3、NH4HCO3含量分别为10vol%、15vol%和20vol%时,多孔HA陶瓷拥有大于100μm和5～50 μm的贯通孔,具有较好的孔连通性与孔结构,有利于细胞和组织的生长以及营养输送;其最大孔隙率为50.3%,抗压强度为6.33MPa.     

Design of tunable protein-releasing nanoapatite/hydrogel scaffolds for hard tissue engineering

Hierarchically structured porous scaffolds based on nanocrystalline carbonated hydroxyapatite reinforced hydrogels (Gellan or Agarose) have been tested as protein release matrices while evaluation their in vitro biocompatibility. The shaping method used develops under mild conditions thus allowing the incorporation of labile substances. The Bovine Serum Albumin (BSA), employed as a model protein, has been included by using two drug-inclusion strategies: during the scaffolds preparation (in situ process) or by injection of an aqueous protein solution within (ex situ process). The release studies showed a more controlled BSA delivery when the protein was incorporated during the scaffold preparation when compared to that where the protein has been loaded in a second step (ex situ process). The release patterns can also be tailored as a function of the scaffold composition (ceramic/polysaccharide ratio and nature) as well as the drying technology employed. Biocompatibility studies demonstrated that these scaffolds, regardless of the composition, allow the culture of osteoblasts on and around the material, thus supporting the potential use of these biomaterials for bone tissue engineering.     

Preparation of hydroxyapatite spheres with an internal cavity as a scaffold for hard tissue regeneration

Microparticulates are currently regarded as a useful matrix for the delivery of bioactive molecules and tissue cells. Herein, hydroxyapatite (HA) spherical microparticulates with an internal cavity were produced using an oil-in-water emulsion technique. The HA slurry in the organic solvent was formulated into spherical particles in a water bath containing a surfactant. Rapid evaporation of the solvent helped create a cavity within the microparticulates. The microparticulates were heat-treated at 1,200 degrees C to produce bioactive HA particles with a mean size of approximately 363 microm. Osteoblastic cells were observed to spread and grow favorably over the surface as well as within the cavity of the microparticulates. The currently developed HA microparticulates with an internal cavity are considered to be useful as a scaffolding matrix for bone tissue engineering and direct filling skeletal defects.     

Synthesis of organic derived hydroxyapatite scaffold from pig bone waste for tissue engineering applications

Micro porous hydroxyapatite (HAp) had drawn great attention in the field of tissue engineering due to its numerous applications such as tissue regeneration, dental, drug delivery, and adsorption and desorption of substances etc. The chemical synthesis of HAp is often faced with the high cost of starting materials and often lacks the presence of beneficial ions which can promote biological reactions. This paper examined a novel application of pig bone waste for the synthesis of HAp via heat treatment between 600 and 1000 °C. Thus synthesized powder was characterized by X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FT-IR), Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM), Energy dispersive X-ray analysis (EDX) and Transmission electron microscopy (TEM). XRD results revealed the main characteristic peaks of single phase HAp powder, while the presence of various functional groups such as PO₄^3?, CO₃^2? and OH^? corresponding to HAp were observed by FT-IR analysis. The elemental composition of as-synthesized HAp powder as observed by EDX showed the presence of Ca and P in addition to some beneficial metals such as Na, K, Mg and Si which plays vital roles in biological applications. SEM and TEM observation confirmed the microscopic sctructure of the as-synthesized HAp to be rod-like morphology with 38–52 nm in length. Porous HAp scaffold up to 65% porosity could be prepared using ammonium bicarbonate as pore forming agent. Therefore, bio-waste such as pig bones can be utilized in the synthesis of porous hydroxyapatite scaffold which can serve as an alternative for the conventional chemical method.     

Mechanical performance of in vitro degraded polylactic acid/hydroxyapatite composites

Journal of Materials Science - Polylactic acid (PLA)-based products can be found in a wide range of industrial applications due to their favorable performances. Unfortunately, neat PLA also...     

Porous scaffolds of polycaprolactone reinforced with in situ generated hydroxyapatite for bone tissue engineering

Polycaprolactone/hydroxyapatite (PCL/HA) composites were prepared by in situ generation of HA in the polymer solution starting from the precursors calcium nitrate tetrahydrate and ammonium dihydrogen phosphate via sol–gel process. Highly interconnected porosity was achieved by means of the salt-leaching technique using a mixture of sodium chloride and sodium bicarbonate as porogens. Structure and morphology of the PCL/HA composites were investigated by scanning electron microscopy, and mechanical properties were determined by means of tensile and compression tests. The possibility to employ the developed composites as scaffolds for bone tissue regeneration was assessed by cytotoxicity test of the PCL/HA composites extracts and cell adhesion and proliferation in vitro studies.     

Poly(lactide-co-glycolide)/hydroxyapatite nanofibrous scaffolds fabricated by electrospinning for bone tissue engineering

Poly(lactide-co-glycolide) (PLGA) nanofibrous composite scaffolds having nano-hydroxyapatite particles (HAp) in the fibers were prepared by electrospinning of PLGA and HAp with an average diameter of 266.6 ± 7.3 nm. Microscopy and spectroscopy characterizations confirmed integration of the crystalline HAp in the scaffolds. Agglomerates gradually appeared and increased on the fiber surface along with increase of the HAp concentration. In vitro mineralization in a 5 × simulated body fluid (SBF) revealed that the PLGA/HAp nanofibrous scaffolds had a stronger biomineralization ability than the control PLGA scaffolds. Biological performance of the nanofibrous scaffolds of the control PLGA and PLGA with 5 wt% HAp (PLGA/5HAp) was assessed by in vitro culture of neonatal mouse calvaria-derived MC3T3-E1 osteoblasts. Both types of the scaffolds could support cell proliferation and showed sharp increase of viability until 7 days, but the cells cultured on the PLGA/5HAp nanofibers showed a more spreading morphology. Despite the similar level of the cell viability and cell number at each time interval, the alkaline phosphatase secretion was significantly enhanced on the PLGA/5HAp scaffolds, indicating the higher bioactivity of the as-prepared nano-HAp and the success of the present method for preparing biomimetic scaffold for bone regeneration.     

Zinc-doped hydroxyapatite and poly(propylene fumarate) nanocomposite scaffold for bone tissue engineering

Hydroxyapatite (HA) is a bioceramic material that shares similar crystal and chemical structures with inorganic components of the bone. However, HA lacks osteoinductive activity and has a brittle nature, making it challenging to apply for direct load-bearing bone applications. In this study, we used a wet chemical method to synthesize zinc-doped hydroxyapatite powders with different Zn/(Zn+Ca) molar ratios of 0, 0.025, 0.05, and 0.1. The corresponding Zn-HA was designated as HA, Zn2.5-HA, Zn5-HA, and Zn10-HA. The Zn-HA powders at 30 wt% were used to fabricate poly(propylene fumarate) (PPF)-based nanocomposite scaffolds (HA/PPF, Zn2.5-HA/PPF, Zn5-HA/PPF, and Zn10-HA/PPF). The physical properties of obtained scaffolds were examined by scanning electron microscopy, energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Live/dead cell viability assay showed that these scaffolds were biocompatible and supported excellent adhesion of MC3T3-E1 preosteoblast cells. Additionally, the proliferation of cells was detected at 1, 4, and 7 days on these scaffolds. Alkaline phosphatase (ALP) activity measurement and alizarin red staining showed good osteogenic differentiation and matrix mineralization for MC3T3-E1 cells growing on these scaffolds. Taken together, the results here indicate that Zn5-HA/PPF nanocomposite scaffolds are promising scaffold material for bone tissue engineering.

Graphical abstract

     

In situ hydroxyapatite crystallization for the formation of hydroxyapatite/polymer composites

In an attempt to synthesize hydroxyapatite (HAP)/polymer composites, HAP crystallization was investigated in solution in the presence of ionic synthetic polymers. The side groups of the polymers used include carboxylate, dihydrogen phosphate, sulfate, and primary amine. Spontaneous HAP precipitation and amorphous-crystalline transformation occurring in both the presence or absence of ionic polymers were studied by measuring the solution turbidity change and titrating the released protons, respectively. The rates of HAP nucleation and growth were determined from an induction period before onset of crystallization and the subsequent propagation of HAP crystallites. The added anionic and cationic polymers, were found to suppress the crystallization in a concentration-dependent manner. An exception was a concentrated poly(acrylic acid), which was precipitated by calcium ion binding to accelerate the nucleation and the growth of HAP crystallites. These results suggest that a molecular interaction is operative between the ionic polymer chains and the growing HAP crystallites. Infrared spectroscopy and X-ray diffraction analysis revealed that the HAP precipitated in the presence of ionic polymers incorporated the polymer chains in the HAP microcrystalline aggregates. Based on these findings, HAP/poly(acrylic acid) composites were prepared through in situ HAP crystallization in the presence of poly(acrylic acid). Tensile testing of the composites and electron microscopic observation of their fractured surfaces revealed that the composite prepared through this wet process was superior to that obtained by simple physical mixing with respect to the wet state mechanical properties and homogeneity in mixing. This revised version was published online in November 2006 with corrections to the Cover Date.     

Mechanical properties and in vitro behavior of nanofiber-hydrogel composites for tissue engineering applications

Hydrogel-based biomaterial systems have great potential for tissue reconstruction by serving as temporary scaffolds and cell delivery vehicles for tissue engineering (TE). Hydrogels have poor mechanical properties and their rapid degradation limits the development and application of hydrogels in TE. In this study, nanofiber reinforced composite hydrogels were fabricated by incorporating electrospun poly(ε-caprolactone) (PCL)/gelatin 'blend' or 'coaxial' nanofibers into gelatin hydrogels. The morphological, mechanical, swelling and biodegradation properties of the nanocomposite hydrogels were evaluated and the results indicated that the moduli and compressive strengths of the nanofiber reinforced hydrogels were remarkably higher than those of pure gelatin hydrogels. By increasing the amount of incorporated nanofibers into the hydrogel, the Young's modulus of the composite hydrogels increased from 3.29 ± 1.02 kPa to 20.30 ± 1.79 kPa, while the strain at break decreased from 66.0 ± 1.1% to 52.0 ± 3.0%. Compared to composite hydrogels with coaxial nanofibers, those with blend nanofibers showed higher compressive strength and strain at break, but with lower modulus and energy dissipation properties. Biocompatibility evaluations of the nanofiber reinforced hydrogels were carried out using bone marrow mesenchymal stem cells (BM-MSCs) by cell proliferation assay and immunostaining analysis. The nanocomposite hydrogel with 25 mg ml(-1) PCL/gelatin 'blend' nanofibers (PGB25) was found to enhance cell proliferation, indicating that the 'nanocomposite hydrogels' might provide the necessary mechanical support and could be promising cell delivery systems for tissue regeneration.     

Electrospun composites of PHBV,silk fibroin and nano-hydroxyapatite for bone tissue engineering

Electrospinning of fibrous scaffolds containing nano-hydroxyapatite (nHAp) embedded in a matrix of functional biomacromolecules offers an attractive route to mimicking the natural bone tissue architecture. Functional fibrous substrates will support cell attachment, proliferation and differentiation, while the role of HAp is to induce cells to secrete extracellular matrix (ECM) for mineralization to form bone. Electrospinning of biomaterials composed of polyhydroxybutyrate-co-(3-hydroxyvalerate) with 2% valerate fraction (PHBV), nano-hydroxyapatite (nHAp), and Bombyx mori silk fibroin essence (SF), Mw = 90KDa, has been achieved for nHAp and SF solution concentrations of 2 (w/vol) % each and 5 (w/vol) % each. The structure and properties of the nanocomposite fibrous membranes were investigated by means of Scanning Electron Microscopy in combination with Energy Dispersive X-Ray Analysis (SEM/EDX), Fourier Transformed Infrared Spectroscopy (FT-IR), uniaxial tensile and compressive mechanical testing, degradation tests and in vitro bioactivity tests. SEM images showed smooth, uniform and continuous fibre deposition with no bead formation, and fibre diameters of between 10 and 15 μm. EDX and FT-IR confirmed the presence of nHAp and SF. After one month in deionised water, tests showed less than 2% weight loss with the samples retaining their fibrous morphology, confirming that this material biodegrades slowly. After 28 days of immersion in Simulated Body Fluid (SBF) an apatite layer was visible on the surface of the fibres, proving their bioactivity. Preliminary in vitro biological assessment showed that after 1 and 3 days in culture, cells were attached to the fibres, retaining their morphology while presenting a flattened appearance and elongated shape on the surface of fibres. Young's modulus was found to increase from 0.7 kPa (± 0.33 kPa) for electrospun samples of PHBV only to 1.4 kPa (± 0.54 kPa) for samples with 2 (w/vol) % each of nHAp and SF. Samples prepared with 5 (w/vol) % each of nHAp and SF did not show a similar improvement.     

Fabrication and characterization of titanium-matrix composite with 20 vol% hydroxyapatite for use as heavy load-bearing hard tissue replacement

Titanium-matrix composite with 20 vol% HA ceramic was fabricated by hot pressing technique and the microstructure of the composite was studied by transmission electron microscope (TEM). The mechanical and biological properties of the composite were investigated by mechanical and in vivo studies. The experimental results by TEM observation show the bonding state of Ti/HA interface in Ti-20 vol% HA composite with the relative density of 97.86% is good, however, there exists an interfacial transition zone between Ti and HA. In Ti matrix of the composite and pure Ti metal, an interesting substructure comprised of screw dislocations with Burgers vectors b of 1/3 〈 11ˉ20〉 was found. Screw dislocations are straight and regularly distributed, and cross slip can be observed. The subgrain boundaries consist of dislocation network walls with equidistant dislocation lines in the same direction. Elastic modulus and Vicker's hardness of Ti-20 vol%HA composite are 102.6 GPa and 3.41 GPa respectively. Owing to the existence of 20 vol% HA ceramic, bending strength and fracture toughness of the composite decrease sharply to 170.1 MPa and 3.57 MPa ⋅ m^1/2 respectively, which are only about 17.5 and 12% of those of pure Ti metal. In vivo studies indicate Ti-20 vol% HA composite has good biocompatibility, and even better osteointegration ability than pure titanium, especially in the early stage after the implantation. In conclusion, Ti-20 vol% HA composite is suitable for heavy load-bearing hard tissue replacement from the point of view of both mechanical properties and biocompatibility.