Natural origin hydroxyapatite scaffold as potential bone tissue engineering substitute

Fish scales derived natural hydroxyapatite (FS-HAp) scaffolds were prepared through solvent casting technique, which could mimic the structure of cortical and cancellous bone tissues of body system. The hydroxyapatite (HAp) biomaterial was synthesized by thermal decomposition of chemically treated fish scales. Fabricated scaffolds were characterized through morphological analysis, volumetric shrinkage, mechanical tests, and in vitro, in vivo biological studies. The projected scaffolds successfully mimic the cancellous/cortical bone system in terms of structure, porosity, mechanical strength, and exhibit excellent bioactive behavior. The FS-HAp scaffolds manifest good mechanical behaviors with Vickers Hardness (HV) of ~0.78 GPa, 0.52 GPa compressive stress, 190 MPa tensile stress and ~35% porosity on sintering at 1200 °C. In vitro and in vivo studies suggest these nontoxic HAp scaffolds graft with osteoconductive support, facilitating new cell growth on the developed scaffold surface. The graded grafts have a great potential for application as traumatized tissue augmentation substitute, and ideal for load-bearing bone applications.     

Bioactivity evaluation of novel nanocomposite scaffolds for bone tissue engineering: The impact of hydroxyapatite

The objective of this study was to prepare scaffolds based on cellulose-graft-polyacrylamide composed of different contents of nano-hydroxyapatite (n-HAp). To this end, polyacrylamide was grafted onto cellulose in the presence of n-HAp through free radical polymerization. Then, the scaffolds of the dispersed grafted polymer nanocomposite powder were fabricated by the freeze-drying method. The grafted polymer nanocomposite scaffolds were tested and characterized using tensile test instrument, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD) analysis. Finally, bioactivity and apatite formation on the surface after immersion in a simulated body fluid (SBF) were determined by XRD and SEM analysis. According to the results, as the n-HAp content in the scaffold structure increased, the porosity, elastic modulus and compressive strength were increased. In addition, apatite was deposited very well on the interconnected irregular pores on the surface of the scaffolds after incubation in SBF, while the size of precipitated apatite was reduced by increasing the soaking time. The results indicated that the prepared grafted polymer nanocomposite scaffolds have a great potential as biocompatible materials for use in bone tissue engineering.     

Synthesis of ultralong hydroxyapatite micro/nanoribbons and their application as reinforcement in collagen scaffolds for bone regeneration

Ultralong hydroxyapatite (HAp) micro/nanoribbons were successfully synthesized by a simple hydrothermal method without using any organic solvents and templates. The ultralong HAp micro/nanoribbons were up to several hundred micrometers in length and 100–400?nm in width. The growth process and mechanism of this micro/nanoribbons were also analyzed in this study. Moreover, the ultralong HAp micro/nanoribbons were used as reinforcement in collagen scaffolds and the HAp/collagen composite scaffolds were fabricated by freeze-drying process without cross-linking. The morphological results demonstrated homogeneous interconnected porous structure in 20?wt% and 35?wt% HAp reinforced scaffolds. The compressive modulus of the 35?wt% HAp/collagen composite was about 6 times that of the pure collagen scaffold. The ultralong HAp reinforced collagen scaffold possesses a porous structure, good flexibility as well as elasticity, and thus it is promising for used as bone repair material.     

Evaluation of a redox-initiated in situ hydrogel as vitreous substitute

Currently there is no material clinically available as a long-term vitreous substitute. In this study, an in-situ gelation system based on α-poly(ethylene glycol) methacrylate (α-PEG-MA) and a redox-initiated radical polymerization/crosslinking reaction was evaluated for this purpose. Ammonium persulfate (APS) and N,N,N′,N′-tetramethyl ethylene diamine (TMEDA) were used as initiators. The gelation time, rheological properties, reaction kinetics and swelling profiles were studied in detail and the system with 10 wt% of α-PEG-MA and 8 mM APS/TMEDA was chosen as the optimal material for in vivo studies. Using the rabbit as the animal model, we showed that the system did form a space-filling and transparent gel in the vitreous cavity, and the inflammation response could be controlled to an acceptable level.     

Development and in vivo response of hydroxyapatite/whitlockite from chicken bones as bone substitute using a chitosan membrane for guided bone regeneration

Biomaterials that meet the requirements to stimulate bone tissue formation play a vital role in orthopedics and dentistry. In this work, chitosan and a biphasic, non-cytotoxic material hydroxyapatite/whitlockite were obtained from natural sources, which are available as organic waste. The osteogenic activity was assessed using a rabbit model animal with a chitosan barrier membrane in combination with a bone-filling graft substitute composed of hydroxyapatite/whitlockite. FT-IR results showed the typical absorption bands of the chitosan and hydroxyapatite. Moreover, the X-ray diffraction pattern revealed a typical hexagonal phase of hydroxyapatite and rhombohedral structures related to whitlockite. Masson's trichrome stain showed an early formation of extracellular matrix mineralized, in accord with the surface morphology of a cortical mature bond observed by Scanning Electron Microscopy. The immunocytochemistry results showed a significant increase of positive immunoreactive cells to osteonectin in the treated defects in comparison with the control defects 6 and 8 weeks postoperatively. Overall, the results confirm that the use of this low-cost and versatile biomaterial as a barrier membrane and a bone substitute graft are useful for bone tissue engineering.     

Development of bone scaffold using Puntius conchonius fish scale derived hydroxyapatite: Physico-mechanical and bioactivity evaluations

Naturally derived Hydroxyapatite (HAp) from fish scale is finding wide applications in the development of bone scaffold to promote bone regeneration. But porous HAp scaffold is fragile in nature making it unsuitable for bone repair or replacement applications. Thus, it is essential to improve the mechanical property of HAp scaffolds while retaining the interconnected porous structure for tissue ingrowth in vivo. In this study solvent casting particulate leaching technique is used to develop novel Puntius conchonius fish scale derived HAp bone scaffold by varying the wt.% of the HAp from 60 to 80% in PMMA matrix. Physico-chemical, mechanical, structural and bioactive properties of the developed scaffolds are investigated. The obtained results indicate that HAp-PMMA scaffold at 70?wt % HAp loading shows optimal properties with 7.26?±?0.45?MPa compressive strength, 75?±?0.8% porosity, 8.0?±?0.68% degradation and 190?±?11% water absorption. The obtained results of the scaffold can meet the physiological demands to guide bone regeneration. Moreover, in vitro bioactivity analysis also confirms the formation of bone like apatite in the scaffold surface after 28 days of SBF immersion. Thus, the developed scaffold has the potential to be effectively used in bone tissue engineering applications.     

PLGA/nHA hybrid nanofiber scaffold as a nanocargo carrier of insulin for accelerating bone tissue regeneration

The development of tissue engineering in the field of orthopedic surgery is booming. Two fields of research in particular have emerged: approaches for tailoring the surface properties of implantable materials with osteoinductive factors as well as evaluation of the response of osteogenic cells to these fabricated implanted materials (hybrid material). In the present study, we chemically grafted insulin onto the surface of hydroxyapatite nanorods (nHA). The insulin-grafted nHAs (nHA-I) were dispersed into poly(lactide-co-glycolide) (PLGA) polymer solution, which was electrospun to prepare PLGA/nHA-I composite nanofiber scaffolds. The morphology of the electrospun nanofiber scaffolds was assessed by field emission scanning electron microscopy (FESEM). After extensive characterization of the PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectrometry (EDS), and transmission electron microscopy (TEM), the PLGA/nHA-I and PLGA/nHA (used as control) composite nanofiber scaffolds were subjected to cell studies. The results obtained from cell adhesion, alizarin red staining, and Von Kossa assay suggested that the PLGA/nHA-I composite nanofiber scaffold has enhanced osteoblastic cell growth, as more cells were proliferated and differentiated. The fact that insulin enhanced osteoblastic cell proliferation will open new possibilities for the development of artificial scaffolds for bone tissue regeneration.     

Mechanochemical synthesis and cold sintering of mussel shell-derived hydroxyapatite nano-powders for bone tissue regeneration

According to the circular economy principles, processing routes aiming at reducing the natural resources consumption and the energy demand can be addressed as ‘green’. In this framework, mussel shells, a natural feedstock of calcium carbonate, were successfully transformed into nano-crystalline hydroxyapatite by mechanochemical synthesis at room temperature after mixing with a phosphoric acid solution. The as-synthesized powder was then consolidated up to 82 % relative density by cold sintering (600 MPa, 200 °C). The materials were fully investigated by physical, chemical and thermal characterization techniques. Cold-sintered samples were also subjected to biaxial flexural strength test, showing a flexural resistance of 23 MPa. Cell viability assessment revealed that cold sintered hydroxyapatite derived from mussel shells promotes faster adhesion and spreading of human bone marrow-derived mesenchymal stem cells, in comparison to a commercial hydroxyapatite sintered at 1050 °C. Therefore, cold-sintered mussel shells-derived hydroxyapatite can be a promising future candidate scaffold for bone tissue regeneration.     

A comprehensive review on the preparation and application of calcium hydroxyapatite: A special focus on atomic doping methods for bone tissue engineering

Hydroxyapatite (HAP) has been considered to be one of the most preferred scaffold materials among many in the last decade for the bone tissue engineering. Be it prosthetic implants, scaffolds or artificial bone cement, hydroxyapatite has received highest attraction among all due to its chemical and physical properties similar to that of human bone. Although it can be used in the bone tissue engineering as the original composition; for enhancing its different properties relevant to in vivo applications, the calcium in HAP may also be replaced by other atomic dopants depending on usage. Here, we review various HAP coating agents and methods, their merits and demerits. We also review various HAP doping materials, including both cationic as well as anionic materials. We discuss the effects and usage of substitution of hydroxyapatite and their subsequent usage in both bone tissue engineering and maxillofacial surgeries. We consider various research articles published in recent times to accomplish detailed discussion on the subject.     

Influence of magnesium and silver ions on rheological properties of hydroxyapatite/chitosan/calcium sulphate based bone cements

Rheology of bioceramic bone cements is usually described as properties of ceramic slurries, neglecting the self-setting character of these materials. In our studies calcium sulphate based bone cements with Ag⁺, Mg²⁺ and Mg²⁺/CO₃^2- modified hydroxyapatite were investigated. Despite of expectations, it has been proven that the presence of magnesium ions significantly influence the rheological properties of cement pastes. Changes in rheological properties were connected with (I) chemical interactions between Mg²⁺ and sulphate ions (II) chemical interaction between Mg²⁺ and chitosan. These effects were not observed for silver additive. Most of the developed calcium sulphate based pastes, except material containing MgHA and chitosan, have been categorized as thick pastes applicable with the spatula. It has been found that the chitosan present around and at the calcium sulphate grains acted as a lubricant and prolong the period of quasi-constant viscosity of the pastes.     

Adsorption capacity of bone char for removing fluoride from water solution. Role of hydroxyapatite content,adsorption mechanism and competing anions

The adsorption of fluoride from water on bone char (BC) was investigated in this work, and the fluoride adsorption capacity of BC was compared to that of hydroxyapatite (HAP). The adsorption capacity of BC and HAP drastically increased while decreasing the pH from 7.0 to 5.0. Furthermore, the fluoride adsorption on BC was due to its HAP content and was not considerably affected by the presence of the anions Cl⁻, HCO₃⁻, CO₃²⁻, SO₄²⁻, NO₃⁻ and NO₂⁻. The mechanism of fluoride adsorption on BC was attributed to electrostatic interactions between surface charge of BC and fluoride ions in solution.     

Polymer assisted isolation of hydroxyapatite from Thunnus obesus bone

The combination of micro and nanostructured hydroxyapatite (HAp) was isolated from Thunnus obesus bone via thermal calcination method in the presence of polymers such as poly ethylene glycol (PEG), poly (ethylene glycol)-block-poly (propylene glycol)-block-poly (ethylene glycol) (PEG-PPG-PEG) and poly vinyl alcohol (PVA). The thermal stability, crystalline phase, chemical composition and morphology of the derived HAp were characterized by thermal gravimetric analysis, X-ray diffraction analysis, Fourier transform infrared spectroscopy and field emission scanning electron microscopy analysis. The physicochemical characteristic examination revealed that derived HAp was coherent with standard HAp data. Moreover, FE-SEM depicted significant difference in the crystal size of HAp derived with thermal calcination, with and without added polymers. The crystallinity of HAp isolated in the presence of polymer was lower than that obtained in the absence of polymers. The biocompatibility of the derived HAp crystals was checked with MC3T3-E1 osteoblastic cells by MTT assay and Hoechst-33342 staining. The biocompatibility of HAp derived by polymer assisted thermal calcination method revealed that it is less toxic as compared to HAp derived in the absence of polymer. As an inference, polymer assisted thermal calcination derived HAp is good in terms of the presence of combined micro and nanostructured HAp and its low toxicity will bring about new orthopaedic applications.     

Sintering behavior and mechanical properties of hydroxyapatite ceramics prepared from Nile Tilapia (Oreochromis niloticus) bone and commercial powder for biomedical applications

In this work, Nile tilapia (Oreochromis niloticus) bone was calcined at 800 °C for 5 h in an air atmosphere to obtain hydroxyapatite powder (FB powder). The elemental composition, phase structure, and morphology of the FB powder were investigated and compared with commercial hydroxyapatite powder (SM powder). The FB-powder exhibited 1.01 at. % of Mg while the SM-powder showed Mg in ppm-level. Carbonate groups were detected in the two powders. Both HAp and β-tricalcium phosphate (β-TCP) structures were found in the FB powder, but the SM powder exhibit only the HAp phase. Irregular-shaped particles were observed in the FB powder. After the two HAp powders were sintered at 1200 °C and 1250 °C for 2 h (FB-1200, FB-1250, SM-1200, and SM-1250), the β-TCP intensity peaks of the FB-ceramic samples significantly increased with increasing sintering temperature. The highest relative density, well-packed grains, and β-TCP stabilization by Mg at the Ca5 site of the FB-1250 structure were the dominant factors governing the highest mechanical properties. Although high density was observed in the SM-1200 sample, Vickers hardness of the SM-1200 sample is lower than the FB-1250 sample. This may be attributed to the partial decomposition of HAp into β-TCP, α-tricalcium phosphate (α-TCP), and Ca₁₀(PO₄)₆O phases. In addition, the increase of grain size was the main factor that governs the increasing compressive strength and Young's modulus instead of density and phase decomposition of the SM-ceramic samples.     

Superoxide Dismutase as a Novel Macromolecular Nitric Oxide Carrier: Preparation and Characterization

Nitric oxide (NO) is an important molecule that exerts multiple functions in biological systems. Because of the short-lived nature of NO, S-nitrosothiols (RSNOs) are believed to act as stable NO carriers. Recently, sulfhydryl (SH) containing macromolecules have been shown to be promising NO carriers. In the present study, we aimed to synthesize and characterize a potential NO carrier based on bovine Cu,Zn-superoxide dismutase (bSOD). To prepare S-nitrosated bSOD, the protein was incubated with S-nitrosoglutathione (GSNO) under varied experimental conditions. The results show that significant S-nitrosation of bSOD occurred only at high temperature (50 °C) for prolonged incubation time (>2 h). S-nitrosation efficiency increased with reaction time and reached a plateau at ~4 h. The maximum amount of NO loaded was determined to be about 0.6 mol SNO/mol protein (~30% loading efficiency). The enzymatic activity of bSOD, however, decreased with reaction time. Our data further indicate that NO functionality can only be measured in the presence of extremely high concentrations of Hg²⁺ or when the protein was denatured by guanidine. Moreover, mildly acidic pH was shown to favor S-nitrosation of bSOD. A model based on unfolding and refolding of bSOD during preparation was proposed to possibly explain our observation.     

Preparation and evaluation of thermo-reversible copolymer hydrogels containing chitosan and hyaluronic acid as injectable cell carriers

Poly(N-isopropylacrylamide) end-capped with a carboxyl group (PNIPAM-COOH) was grafted to chitosan for synthesizing thermo-reversible chitosan-g-poly(N-isopropylacrylamide) (CPN), which was further grafted with hyaluronic acid (HA) to form hyaluronic acid-g-CPN (HA-CPN). PNIPAM-COOH, CPN and HA-CPN formed injectable free-flowing aqueous solutions and exhibited reversible sol-to-gel phase transition (above 5% polymer concentration) at 30 °C. Chemical properties and temperature-dependent physical properties of the polymer hydrogels, such as rheological behavior, phase transition kinetics, and water content were characterized in detail. The mechanical stiffness of hydrogels increased with the presence of chitosan in the copolymer, but decreased after conjugation with HA. Chitosan and HA grafting also endowed higher water content and resistance to volume contraction during phase change of the copolymer solution. In vitro cell culture experiments with chondrocytes and meniscus cells in HA-CPN hydrogel showed beneficial effects on the cell phenotypic morphology, proliferation, and differentiation. Progressive tissue formation was demonstrated by monotonic increases in extracellular matrix contents and mechanical properties.     

In situ grown fibrous composites of poly(dl-lactide) and hydroxyapatite as potential tissue engineering scaffolds

In situ grown hydroxyapatite (HA) within electrospun poly(dl-lactide) (PDLLA) fibers were initially investigated as potential tissue engineering scaffolds with respect to the mechanical performances, biomineralization capability, degradation behaviors, cell growth and differentiation profiles. The tensile strength and Young’s moduli of in situ grown composites (IGC) were 8.2 ± 1.1 and 63.5 ± 5.6 MPa, respectively, which were significantly higher than those of blend electrospun composites (BEC) with 25.2% of HA inoculation. The interactions between HA and matrix polymers were approved by the red-shifts of CO stretching and OH- stretching modes and the increases in glass transition temperatures of fibrous composites. The localization of apatite phase on the fiber surface improved the biomineralization capability and enhanced the morphological stability of the fibers and fibrous mats even when the degradation of matrix polymers was detected. The cell viability and alkaline phosphatase levels were significantly higher for composites IGC, indicating favorable scaffolds for cell proliferation and osteogenic differentiation.     

Mechanical and histological evaluation of a plasma sprayed hydroxyapatite coating on a titanium bond coat

To improve the biological stability of hydroxyapatite-coated implants, the use of an appropriate commercial pure titanium bond coat (CP-Ti) is an effective method for significant improvement in the interface bonding and stress reduction of the plasma-sprayed HA coating and Ti-alloy system. The purpose of this study was to determine the effect of plasma-sprayed CP-Ti on the in vivo properties of the HA coating on Ti6Al4V. According to the experimental results, samples with an HA coating on the CP-Ti (Ti-HA coating) display a lower and ineffective residual stress than samples without the bond coat. The CP-Ti provides a very rough surface and results in a higher adhesive strength between the HA and CP-Ti. In the in vivo test, after the intramedullary implantation, the histological observation showed the apposition of the new bone tissue directly onto the HA coating. The shear strength between the bone and the Ti-HA coating was higher than that without CP-Ti after 12 weeks of implantation (5.15 vs. 1.41 MPa). The failure mode analysis showed the failure mainly occurring at the surrounding bone, with some failure at the HA/CP-Ti interface. The excellent bio-durability and mechanical properties of the HA coating in this study contributed to the reduction of the compressive residual stress in the HA coating and to the enhancement of interface adhesive strength by introducing the commercial pure titanium bond coat.     

Preparation of electrospun nanofibers of carbon nanotube/polycaprolactone nanocomposite

Multiwalled carbon nanotube/polycaprolactone nanocomposites (MWNT/PCL) were prepared by in situ polymerization, whereby functionalized MWNTs (F-MWNTs) and unfunctionalized MWNTs (P-MWNTs) were used as reinforcing materials. The F-MWNTs were functionalized by Friedel-Crafts acylation, which introduced the aromatic amine (COC₆H₄-NH₂) groups on the side wall. The F-MWNTs were chemically bonded with the PCL chains in the F-MWNT/PCL, as indicated by the appearance of the amide II group in the FT-IR spectrum. The TGA thermograms showed that the F-MWNT/PCL had better thermal stability than PCL and P-MWNT/PCL. The PCL and the nanocomposite nanofibers were prepared by an electrospinning technique. The nanocomposites that contain more than 2 wt% of MWNTs were not able to be electrospun. The bead of the F-MWNT/PCL nanofiber was formed less than that of the P-MWNT/PCL. The nanocomposite nanofibers showed a relatively broader diameter than the pure PCL nanofibers. The MWNTs were embedded within the nanofibers and were well oriented along the axes of the electrospun nanofibers, as confirmed by transmission electron microscopy.     

Novel graphite/TiO2 electrochemical cells as a safe electric energy storage system

A graphite/TiO₂ full cell has been developed as a new safety energy storage system using a highly safety process. The crystal structures of the anatase TiO₂ electrode have been investigated with respect to the performance of the electrodes. Due to the large anion intercalation into the graphite positive electrode, the possible charging potential can be raised to around 5.3 V against the Li/Li⁺ electrode, which is a higher charging voltage than lithium-ion batteries (maximum voltage is around 4.3 V vs. Li/Li⁺). In situ XRD measurements have been carried out on both the cathode and anode electrodes of the graphite/TiO₂ cell during the charge process to elucidate the intercalation mechanism.     

Influence of strontium and niobium on the physical and biological performance of hydroxyapatite as a bioactive coating on implant materials

The coating of hydroxyapatite (HAP) on the surface of bio-inert metallic implants to augment their bioactivity is in use for the last two decades. Substitution of various materials in HAP further improves the functionality of these coatings. We demonstrate coating of Ti6Al4V alloy sheets with strontium and niobium reinforced HAP using microwave (MW) irradiation technique. Physical characterization revealed, uniform semicrystalline hydroxyapatite coating with enhanced surface roughness and microhardness. The increased surface roughness was accompanied by higher wettability and more protein adsorption. Electrochemical corrosion assessment showed a dramatic increase in corrosion potential and a noticeable decline in corrosion current density suggesting an enhanced anticorrosive behaviour. These implants also exhibited improved hemocompatibility and bacteriostatic properties. Cell viability and confocal microscopy studies of the coated samples showed enhanced cell attachment on the surface. We propose microwave irradiation as a fast and hassle-free alternative for one-pot synthesis and deposition of ionic substituted HAP on metallic implants.