Novel biomimetic hydroxyapatite/alginate nanocomposite fibrous scaffolds for bone tissue regeneration

Hydroxyapatite/alginate nanocomposite fibrous scaffolds were fabricated via electrospinning and a novel in situ synthesis of hydroxyapatite (HAp) that mimics mineralized collagen fibrils in bone tissue. Poorly crystalline HAp nanocrystals, as confirmed by X-ray diffractometer peak approximately at 2θ = 32° and Fourier transform infrared spectroscopy spectrum with double split bands of PO₄(v ₄) at 564 and 602 cm^?1, were induced to nucleate and grow at the [–COO^?]–Ca²⁺–[–COO^?] linkage sites on electrospun alginate nanofibers impregnated with PO₄ ^3? ions. This novel process resulted in a uniform deposition of HAp nanocrystals on the nanofibers, overcoming the severe agglomeration of HAp nanoparticles processed by the conventional mechanical blending/electrospinning method. Preliminary in vitro cell study showed that rat calvarial osteoblasts attached more stably on the surface of the HAp/alginate scaffolds than on the pure alginate scaffold. In general, the osteoblasts were stretched and elongated into a spindle-shape on the HAp/alginate scaffolds, whereas the cells had a round-shaped morphology on the alginate scaffold. The unique nanofibrous topography combined with the hybridization of HAp and alginate can be advantageous in bone tissue regenerative medicine applications.     

Biodegradable polyurethane composite scaffolds containing Bioglass® for bone tissue engineering

Five types of solid and porous polyurethane composites containing 5–20 wt.% of Bioglass® inclusions were synthesized. Porous structures were fabricated by polymer coagulation combined with the salt-particle leaching method. In-vitro bioactivity tests in simulated body fluid (SBF) were carried out and the marker of bioactivity, e.g. formation of surface hydroxyapatite or calcium phosphate layers upon immersion in SBF, was investigated. The chemical and physical properties of the solid and porous composites before and after immersion in SBF were evaluated using different techniques: Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Dynamic Mechanical Analysis (DMA) and Thermogravimetric Analysis (TGA). Moreover the surface structure and microstructure of the composites was characterised by Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM), respectively. Mercury intrusion porosimetry, SEM and microtomography (μCT) were used to determine pore size distribution and porosity. The fabricated foams exhibited porosity >70% with open pores of 100–400 μm in size and pore walls containing numerous micropores of <10 μm. This pore structure satisfies the requirements for bone tissue engineering applications. The effects of Bioglass® addition on microstructure, mechanical properties and bioactivity of polyurethane scaffolds were evaluated. It was found that composite foams showed a higher storage modulus than neat polyurethane foams. The high bioactivity of composite scaffolds was confirmed by the rapid formation of hydroxyapatite on the foam surfaces upon immersion in SBF.     

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.     

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.     

Electrospun bioactive nanocomposite scaffolds of polycaprolactone and nanohydroxyapatite for bone tissue engineering

Nanocomposite scaffolds based on nanofibrous poly(epsilon-caprolactone) (PCL) and nanohydroxyapatite (nanoHA) with different compositions (wt%) were prepared by electrostatic co-spinning to mimic the nano-features of the natural extracellular matrix (ECM). NanoHA was found to be well dispersed in polymers up to the addition of 20 wt%, after ultrasonication. The composite scaffolds were characterized for structure and morphology using XRD, EDX, SEM, and DSC. The scaffolds have a porous nanofibrous morphology with fibers (majority) having diameters in the range of 450-650 nm, depending on composition, and interconnected pore structures. SEM, EDX, and XRD analyses have confirmed the presence of nanoHA in the fibers. As the nanoHA content in the fibers increases, the surface of fibers becomes rougher. The mechanical (tensile) property measurement of the electrospun composites reveals that as the nanoHA content increases, the ultimate strength increases from 1.68 MPa for pure PCL to 2.17, 2.65, 3.91, and 5.49 MPa for PCL/nanoHA composites with the addition of 5, 10, 15, and 20 wt% nanoHA, respectively. Similarly the tensile modulus also increases gradually from 6.12 MPa to 21.05 MPa with the increase of nanoHA content in the PCL/nanoHA fibers, revealing an increase in stiffness of the fibers due to the presence of HA. DSC analysis reveals that as nanoHA in the composite scaffolds increases, the melting point slightly increases due to the good dispersion and interface bonding between PCL and nanoHA.     

Carbon nanofibers produced from modified electrospun PAN/hydroxyapatite precursors as scaffolds for bone tissue engineering

In the current study we have proposed a method to obtain a carbon/HAp bioactive nanofibrous scaffold. The modified carbon nanofibrous nonwoven' fabrics were obtained by the use of electrospinning and subsequent stabilization and carbonization processes. The modified with HAp powder nanofibrous PAN nonwovens were thermally stabilized using a multi-stage process in the temperature ranging from 100 °C to 300 °C in an oxidative environment and then carbonized at 1000 °C in argon atmosphere. The changes of properties of composite precursor membranes taking place during stabilization and carbonization processes were investigated using the methods of: DSC, TGA, FTIR, SEM, EDX, WAXD and mechanical tests. Bioactivity was determined by assessing the formation of crystalline apatite on the surface of membranes upon immersion in Simulated Body Fluid (SBF). The FTIR, SEM and WAXD investigation clearly prove that hydroxyapatite added to the electrospinning solution was present also in composites nanofibrous nonwovens after stabilization and carbonization process. It was found that due to HAp addition: the significant decrease of fibers average diameter occurs and that the average pore size for modified membranes is smaller than for the unmodified one. On the other hand it was shown that the ceramic additive protects fibers from mass reduction during the stabilization treatment. Finally a drastic increase of mineralization activity of nCF/HAp scaffolds as compared to their nCF counterparts has been proved.     

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.     

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.     

Three-dimensional printing of polycaprolactone/hydroxyapatite bone tissue engineering scaffolds mechanical properties and biological behavior

Amniotic membrane (AM) is a biological tissue that surrounds the fetus in the mother’s womb. It has pluripotent cells, immune modulators, collagen, cytokines with anti-fibrotic and anti-inflammatory effect, matrix proteins, and growth factors. In spite of the biological characteristics, some results have been released in preventing the adhesion on traumatized surfaces. Application of the AM as a scaffold is limited due to its low biomechanical resistance and rapid biodegradation. Therefore, for using the AM during surgery, its modification by different methods such as cross-linking of the membrane collagen is necessary, because the cross-linking is an effective way to reduce the rate of biodegradation of the biological materials. In addition, their cross-linking is likely an efficient way to increase the tensile properties of the material, so that they can be easily handled or sutured. In this regard, various methods related to cross-linking of the AM subsuming the composite materials, physical cross-linking, and chemical cross-linking with the glutraldehyde, carbodiimide, genipin, aluminum sulfate, etc. are reviewed along with its advantages and disadvantages in the current work.

     

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.     

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

     

Novel forsterite/polycaprolactone nanocomposite scaffold for tissue engineering applications

Novel highly porous nanocomposite scaffolds consisting of polycaprolactone (PCL) and forsterite nanopowder were prepared by a solvent-casting/particle-leaching method. In addition, the effects of forsterite nanopowder contents on the structure of the scaffolds were investigated to provide an appropriate composite for bone regenerative medicine. Results showed that the scaffolds exhibited high porosity (up to 92%) with open pores of 100-300 μm average diameters. This porosity increased with decreasing forsterite nanopowder content. In addition, the pore walls contained numerous micropores. Microstructure studies showed that the pores were well distributed throughout the structures. Furthermore, the bioactive forsterite nanoparticles were homogenously distributed within the PCL matrix of the scaffolds, which contained up to 30 wt.% forsterite nanopowder. This porous structure with micropores provides the properties required for bone tissue engineering applications.     

Alginate based scaffolds for bone tissue engineering

The design and production of scaffolds for bone tissue regeneration is yet unable to completely reproduce the native bone properties. In the present study new alginate microparticle and microfiber aggregated scaffolds were produced to be applied in this area of regenerative medicine.The scaffolds' mechanical properties were characterized by thermo mechanical assays. Their morphological characteristics were evaluated by isothermal nitrogen adsorption and scanning electron microscopy. The density of both types of scaffolds was determined by helium pycnometry and mercury intrusion porosimetry. Furthermore, scaffolds' cytotoxic profiles were evaluated in vitro by seeding human osteoblast cells in their presence.The results obtained showed that scaffolds have good mechanical and morphological properties compatible with their application as bone substitutes. Moreover, scaffold's biocompatibility was confirmed by the observation of cell adhesion and proliferation after 5 days of being seeded in their presence and by non-radioactive assays.     

Synthesis and characterization of hydroxyapatite/chitosan nanocomposite materials for medical engineering applications

Incorporation of hydroxyapatite (HA) with organic polymer in favor of composites would be used in biomaterial engineering. According to prior researches, because of its chemical similarity to natural bone and dental, this product could improve bioactivity and bone bonding ability. In this research, nano-hydroxyapatite/chitosan composite material was prepared via in situ Hybridization route. The surface chemical characterization on the nanocomposite was evaluated by Fourier transformed infrared (FTIR) and X-ray diffraction (XRD). Surface topography, roughness and morphology of the samples were observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The characterization results confirmed homogeneity, interaction and integration between the HA and chitosan matrix. It was indicated that composite samples consist of homogeneous aggregations around 40–100 nm, in which many HA nanocrystals align along the chitosan molecules. HA grain gradually decreased in size when amount of chitosan increased from 0 to 6 g into 100 cc solution. It can be seen that by increasing chitosan, the aggregation of nanoparticles enhance and subsequently, improve the expected compatibility among HA filler and chitosan matrix. Furthermore, the mechanical compressive testing indicated that the synthesized composites have acceptable mechanical behavior for tissue substitution. The mechanistic of the biodegradable nanocomposite systems, their preparation and characterization for medical usage are strongly discussed.     

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.     

一种新型仿生骨组织工程支架的制备与表征

利用改性生物玻璃粉体和胶原、透明质酸钠、磷酸丝氨酸等天然生物分子复合制备仿生型三维多孔骨组织工程支架材料,利用体外模拟实验结合SEM、FTIR、XRD 等测试方法对材料的显微结构、生物矿化性能进行了综合研究,研究表明该材料具有良好的孔隙结构,在模拟生理溶液(SBF)中反应24h即可在支架表面形成碳酸羟基磷灰石(HCA).     

壳聚糖支架在组织工程中的应用

综述了壳聚糖作为细胞生长载体在软骨组织工程，骨组织工程和皮肤组织工程等方面的应用进展，表明壳聚糖有望成为优异的组织工程支架材料。     

Biomimetic design of a bacterial cellulose/hydroxyapatite nanocomposite for bone healing applications

This study describes the design and synthesis of bacterial cellulose/hydroxyapatite nanocomposites for bone healing applications using a biomimetic approach. Bacterial cellulose (BC) with various surface morphologies (pellicles and tubes) was negatively charged by the adsorption of carboxymethyl cellulose (CMC) to initiate nucleation of calcium-deficient hydroxyapatite (cdHAp). The cdHAp was grown in vitro via dynamic simulated body fluid (SBF) treatments over a one week period. Characterization of the mineralized samples was done with X-ray Photoelectron Spectroscopy (XPS) and Field Emission Scanning Electron Microscopy (FESEM) with Energy Dispersive Spectroscopy (EDS). The amount of cdHAp observed varied among different samples. XPS demonstrated that the atomic presence of calcium and phosphorus ranged from 0.44 at.% to 7.71 at.% Ca and 0.27 at.% to 11.18 at.% P. The Ca/P overall ratio ranged from 1.22 to 1.92. FESEM images showed that the cdHAp crystal size increased with increasing nanocellulose fibril density. To determine the viability of the scaffolds in vitro, the morphology and differentiation of osteoprogenitor cells was analyzed using fluorescence microscopy and alkaline phosphatase gene expression. The presence of cdHAp crystals on BC surfaces resulted in increased cell attachment.     

Synthesis, neutralization and blocking procedures of organic/inorganic hybrid scaffolds for bone tissue engineering applications

Bioactive glasses (BaG) can bind to human bone tissues and have been used in many biomedical applications for the last 30 years. However they usually are weak and brittle. On the other hand, composites that combine polymers and BaG are of particular interest, since they often show an excellent balance between stiffness and toughness. Bioactive glass-poly(vinyl alcohol) foams to be used in tissue engineering applications were previously developed by our group, using the sol-gel route. Since bioactive glass-polymer composite derived from the sol-gel process cannot be submitted to thermal treatments at high temperatures (above 400 degrees C), they usually have unreacted species that can cause cytotoxicity. This work reports a technique for stabilizing the sol-gel derived bioactive glass/poly(vinyl alcohol) hybrids by using glutaraldehyde (GA), NH(4)OH solutions and a blocking solution containing bovine serum albumin. PVA/BaG/GA hybrids were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM/EDX) analyses. Moreover, MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) biocompatibility and cytotoxicity assays were also conducted. The hybrids exhibited pore size varying from 80 to 820 mum. After treatments, no major changes in the pore structure were observed and high levels of cell viability were obtained.