Manufacturing methods, properties, and potential applications in bone tissue regeneration of hydroxyapatite-chitosan biocomposites: A review.

Hydroxyapatite (HA) and chitosan (CS) biopolymer are the major materials investigated for biomedical purposes. Both of these components play an important role in the orthopedic field as bone substitutes or drug release systems. Used separately, the hydroxyapatite is quite fragile, while CS mechanical strength is very weak. Therefore, a combination of HA and CS polymer is used, which provides excellent mechanical performance with high biocompatibility and biomimetic capacity. Moreover, the porous structure and reactivity of the hydroxyapatite-chitosan (HA-CS) composite allow their application not only as a bone repair but also as a drug delivery system providing controlled drug release directly to the bone site. These features make biomimetic HA-CS composite a subject of interest for many researchers. Through this review, we provide the important recent achievements in the development of HA-CS composites, focusing on manufacturing techniques, conventional and novel three-dimensional bioprinting technology, and physicochemical and biological properties. The drug delivery properties and the most relevant biomedical applications of the HA-CS composite scaffolds are also presented. Finally, alternative approaches are proposed to develop HA composites with the aim to improve their physicochemical, mechanical, and biological properties.

New method of synthesis and in vitro studies of a porous biomaterial.

Biomaterials for bone reconstruction represent a widely studied area. In this paper, a new method of synthesis of a porous glass-ceramic obtained by thermal treatment is presented. The prepared biomaterial was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and induced couple plasma-optical emission spectroscopy (ICP-OES), mercury porosimetry and by the Archimedes method. In vitro evaluations in a simulated body fluid (SBF) and in contact with SaOS2 human osteoblasts were also carried out. The porous glass-ceramic is composed of a total porous network of 60% suitable for body fluid and cell infiltration, with pore sizes varying from 60 nm to 143 µm. The presence of two crystalline phases decreases the kinetic of bioactivity compared to an amorphous biomaterial (bioactive glass). A hydroxyapatite layer appears from 15 days of immersion on the surface and inside the pores, showing a biodegradation and a bioactivity in four steps. Cytotoxicity assessments present an increase of the cellular viability after 72 h proving the non-cytotoxic effect of the glass-ceramic. Thus, the results of these different studies indicate that the porous biomaterial may have a potential application for the bone regeneration. This paper also presents the novelty of this method. It is a rapid synthesis which combines simplicity and low cost. This represents an advantage for an eventual industrialization.

3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

Effect of novel curcumin-encapsulated chitosan-bioglass drug on bone and skin repair after gamma radiation: experimental study on a Wistar rat model.

Radiation therapy contributes to a significant increase in bone osteoporosis and skin loss. Various natural health products might be beneficial to reduce bone and skin alterations. Curcumin (CUR) medicines derived from natural plants have played an important role in health care. This study aims at synthesizing and evaluating the performance therapy of CUR-encapsulated bioglass-chitosan (CUR-BG-CH). In vitro, the antioxidant assay was evaluated by using 1,1-diphenyl-2-picrylhydrazyl free-radical (DPPH) scavenging and the nitroblue tetrazolium reduction. The CUR-BG-CH antimicrobial effects were tested in liquid media. In vivo, after rat (60) Co γ-radiation, the tissue wound-healing process was studied by grafting CUR and CUR-BG-CH in femoral condyle and dorsal skin rat tissue. The antioxidant studies indicated that CUR-BG-CH quenches free radicals more efficiently than unmodified CUR and had effective DPPH (91%) and superoxide anion (51%) radical scavenging activities. The CUR-BG-CH biomaterial exhibited an important antimicrobial activity against Staphylococcus aureus. The histomorphometric parameters showed amelioration in CUR-BG-CH-treated rats. An improved mechanical property was noticed (33.16 ± 5.0 HV) when compared with that of unmodified CUR group (23.15 ± 4.9 HV). A significant decrease in tumour necrosis factor-α cytokine production was noted in the CUR-BG-CH rats (90 pg/ml) as compared with that of unmodified CUR group (240 pg/ml). The total amount of hydroxyproline was significantly enhanced (33.5%) in CUR-BG-CH group as compared with that of control. Our findings suggested that CUR-BG-CH might have promising potential applications for wound healing.

Detoxification of rats subjected to nickel chloride by a biomaterial-based carbonated orthophosphate.

Recently, the therapeutic approaches of the detoxification against the metals (nickel) in the body are the use of biomaterials such as carbonated hydroxyapatite. The aim of this study is therefore to analyze the physiological and physicochemical parameters of strain white rats "Wistar" receiving nickel chloride and to study the protective associative of apatite against adverse effects of this metal, and this in comparison with control rats. Our results showed that the nickel induced in rats an oxidative stress objectified by elevated levels of thiobarbituric acid-reactive substances and conjugated dienes associated with inhibition of the activity of the antioxidant defense system such as glutathione peroxidase, superoxide dismutase and catalase in the liver, kidney, spleen and erythrocyte. Disorders balances of ferric, phosphocalcic, a renal failure and a liver toxicity were observed in rats exposed to nickel. As well as a significant increase in the rate of nickel in the bones and microcytic anemia was revealed. However, the implantation of carbonated hydroxyapatite in capsule form protects rats intoxicated by the nickel against the toxic effects of this metal by lowering the levels of markers of lipid peroxidation and improving the activities of defense enzymes. Our implantation technique is effective to correct ferric balance and phosphocalcic equilibrium, to protect liver and kidney function, to reduce the rate of bone nickel and to correct anemia. They clearly explain the beneficial and protective of our biomaterial which aims the detoxification of rats receiving nickel by substituting cationic (Ca(2+) by Ni(2+)) and anionic (OH(-) by Cl(-)) confirmed by physicochemical characterization like the IR spectroscopy and X-ray diffraction. These techniques have shown on the one hand a duplication of OH(-) bands (IR) and on the other hand the increase of the volume of the apatite cell after these substitutions (X-ray diffraction).

Biological therapy of strontium-substituted bioglass for soft tissue wound-healing: responses to oxidative stress in ovariectomised rats.

New synthetic biomaterials are constantly being developed for wound repair and regeneration. Bioactive glasses (BG) containing strontium have shown successful applications in tissue engineering account of their biocompatibility and the positive biological effects after implantation. This study aimed to assess whether BG-Sr was accepted by the host tissue and to characterize oxidative stress biomarker and antioxidant enzyme profiles during muscle and skin healing. Wistar rats were divided into five groups (six animals per group): the group (I) was used as negative control (T), after ovariectomy, groups II, III, IV and V were used respectively as positive control (OVX), implanted tissue with BG (OVX-BG), BG-Sr (OVX-BG-Sr) and presented empty defects (OVX-NI). Soft tissues surrounding biomaterials were used to estimate superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and malondialdehyde (MDA) concentration. Our results show that 60 days after operation, treatment of rats with BG-Sr significantly increased MDA concentration and caused an increase of SOD, CAT and GPx activities in both skin and muscular tissues. BG-Sr revealed maturation of myotubes followed a normal appearance of muscle regenerated with high density and mature capillary vessels. High wound recovery with complete re-epithelialization and regeneration of skin was observed. The results demonstrate that the protective action against reactive oxygen species (ROS) was clearly observed in soft tissue surrounding BG-Sr. Moreover, the potential use of BG-Sr rapidly restores the wound skin and muscle structural and functional properties. The BG advantages such as ion release might make BG-Sr an effective biomaterial choice for antioxidative activity.

Apatite forming ability and cytocompatibility of pure and Zn-doped bioactive glasses.

The use of bone grafts permits the filling of a bone defect without risk of virus transmission. In this work, pure bioactive glass (46S6) and zinc-doped bioactive glass (46S6Zn10) with 0.1 wt% zinc are used to elaborate highly bioactive materials by melting and rapid quenching. Cylinders of both types of glasses were soaked in a simulated body fluid (SBF) solution with the aim of determining the effect of zinc addition as a trace element on the chemical reactivity and bioactivity of glass. Several physico-chemical characterization methods such as x-ray diffraction, Fourier transform infrared spectroscopy and nuclear magnetic resonance methods, with particular focus on the latter, were chosen to investigate the fine structural behaviour of pure and Zn-doped bioactive glasses as a function of the soaking time of immersion in SBF. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) was used to measure the concentrations of Ca and P ions in the SBF solution after different durations of immersion. The effect of the investigated samples on the proliferation rate of human osteoblast cells was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and tested on two different sizes of pure and zinc-doped glasses in powder form, with particle sizes that ranged between 40 to 63 µm and 500 to 600 µm. The obtained results showed the delay release of ions by Zn-doped glass (46S6Zn10) and the slower CaP deposition. Cytotoxicity and cell viability were affected by the particle size of the glass. The release rate of ions was found to influence the cell viability.

Aluminosilicates and biphasic HA-TCP composites: studies of properties for bony filling.

Aluminosilicate materials synthesized at room temperature present good mechanical properties. Hydroxyapatite, tricalcium phosphate or both offer a high biocompatibility in the biomedical field. In this work, we focused on the composites resulting from associations of these materials. The best compromise between porosity and biomechanical properties versus different parameters was determined. The in vitro behaviour of compounds in contact with the simulated body fluid (SBF) was studied and in vivo experiments in a rabbit's thighbones were carried out. The inductively coupled plasma-optical emission spectroscopy (ICP-OES) method permitted us to study the eventual release of Al from composites to SBF and to evaluate the chemical stability of composites characterized by the succession of SiO(4) and AlO(4) tetrahedra. The kinetic biomineralization, the bioconsolidation and biological studies were made. The results obtained show the chemical stability of composites. In the bone-implant interface, the intimate links reveal the high quality of the biointegration and the bioconsolidation between composites and bony matrix. Histological studies confirm good bony bonding and highlight the total absence of inflammation or fibrous tissues.

Comparison of the bony remodelling of two synthetic biomaterials: aragonite 55% and aragonite 55% with active substance.

In this work, the in vivo behaviour of pure aragonite and vectabone, which is an association of aragonite and an active substance such as gentamicin, was studied to highlight the kinetic resorption of these two biomaterials with 55% of porosity destined for the filling or replacement of bony defects. The synthesis conditions and parameters we used permit us to obtain a biomaterial without a sintering stage. These conditions allow introducing of active substances at the first stage of the elaboration. In this work, the gentamycin antibiotic was associated with calcium carbonate (aragonite 55% with gentamycin) to deliver this active substance on the surgical site for local treatment. The tricalcium phosphate biomaterial was used as the control because of its high biocompatibility. The bony remodelling of these three biomaterials was studied by in vivo experiments. This study was ensured with neutron activation analysis (NAA). The resorption kinetic was elaborated and comparisons of the remodelling biomaterials CaCO(3) 55% and CaCO(3) 55% with gentamicin (vectabone) and tricalcium phosphate were carried out. The obtained results show that, 6 months after implantation, the mineral composition of vectabone and tricalcium phosphate becomes close to that of young bone. Twelve months after implantation, it becomes similar to that of mature bone.

Nuclear methods to characterize biomaterials.

Techniques using X-rays are often used to study biomaterials fields. However, when one is interested by quantitative and very sensitive measurements, it is valuable to develop nuclear instruments and methods, in addition and complement with others. Fast neutron activation is appropriate for non-destructive analysis. Thermal neutron activation can evaluate trace elements as a reference. Proton-induced X-rays emission is applied to cartography of heavy elements. If necessary, proton-induced gamma-rays emission and charged particles scattering are suitable for evaluation and cartography of light elements. Radioactivated nuclei and labelled molecules can tag element transfers and biofunctionality. In our work, these methods are related to biomaterials field.

Dihydrotestosterone prevents glucocorticoid-negative effects on fetal rat metatarsal bone in vitro.

The effects of dihydrotestosterone (DHT) on glucocorticoid-pretreated fetal rat long bone were studied in an in vitro culture system. First, dose-response curves of corticosterone, hydrocortisone, and dexamethasone were studied at several concentrations. Then, hydrocortisone (H) at 10(-5) M was selected for the second part of the study, as it slackened rudiment mineralization (104 +/- 16% of the initial dark zone vs. 141 +/- 9% in control bones), as well as its lengthening (140 +/- 4% of the harvesting day length vs. 160 +/- 1% in control bones), by both inhibition of cell proliferation and stimulation of resorption. On the contrary, in H-pretreated metatarsal bones, DHT (10(-7) M) partly limited slackening of mineralization (124 +/- 5%) and lengthening (153 +/- 2%). Moreover, a control-like cell proliferation was re-established and resorption holes were filled in. Thus, in this study, DHT partly limited hydrocortisone-induced impairment of fetal rat metatarsal bone development.

Determination of metallic ion transfer from an implanted prosthesis by the PIXE method.

Prostheses can release some metallic elements to the surrounding tissues, particularly when they are not covered with a biomaterial layer and when an unsealing process happens. We try to measure major and trace elements in these tissues with an experimentally sensitive method. Proton-induced X-ray emission is used to detect about 10 elements in tissue. Tissues are calcinated and deposited in a thin layer before irradiation. Results are obtained in a standard and samples from three patients. We observe contamination by Ti, Cr, Ni, and Zn in the tissues. Correlations are to be studied between these atomic transfers and prosthesis in the patient.

Study of implanted biomaterial functionality by diphosphonate molecules labeled with radioactive 99mTc.

An implanted biomaterial can be transformed into young bone after some months, but it has not necessary reached full biofunctionality. Mineral concentration kinetics and crystal-structure studies, still being carried out in our group, are completed here by biofunctionality determinations. A natural coral is implanted in vivo at the cortical level of the femoral diaphyoff++ in rabbits. Diphosphonates molecules labeled with radioactive 99mTc are then injected in rabbits and the fixation of the radioactivity is analyzed in several sites for 8 mo after the implantation. Nuclear instruments and methods are used for the measurements. Four successive cycles of osseous remodeling are determined before reaching a biofunctional phase.

Resorption kinetics of osseous substitute: natural coral and synthetic hydroxyapatite.

Coral and hydroxyapatite may be used as substitution biomaterials for bone grafts. In this work, we extracted the implants from the femora to study the kinetics of elementary mineral transformation of the osseous substitutes. The use of physical analysis methods such as PIXE (particle-induced X-ray emission) shows that coral and hydroxyapatite, after their implantation in vivo, reach a mineral composition comparable with that of bone. For the first time we have measured the concentration of mineral elements, at different time intervals after implantation, along a cross-section. The distribution according to mineral elements (Ca, P, Sr, Zn, Fe) in the implant, in the receiver site and also at the interface, showed that the kinetics of coral resorption was faster than that of hydroxyapatite; in the same way, the osseous attack was not global but, rather, centripetal.

Diffusion of mineral elements evaluated by PIXE at the bone-coral interface.

Substituting the tissue of human organs with biomaterials is problematic. However, its importance and relevance justify all the efforts made. An interdisciplinary approach is required. We report on our study of a product for bone substitution. Coral is a natural product, the interest of which we have already demonstrated in our previous work. Following sterilization, natural coral was implanted in sheep femurs. We regularly extracted the implants from the femurs to study the kinetics of elemental mineral transformation of the bone substitutes. For the first time ever, and thanks to the PIXE method (particles induced X-ray emission), we were able to measure the concentration of mineral elements at different time intervals after implantation over a whole cross-section. We found a discontinuity of the mineral elements (Ca, P, Sr, Zn, Fe) at the interface between the implant and the receiver. This shows that the osseous attack is not global but, on the contrary, centripetal. Moreover, the fit of the concentration time course indicates that the kinetics of ossification are different for each atomic element and characterize a distinct biological phenomenon. Our analyses confirm the biocompatibility and the ossification of the implanted coral.

Quantitative study of the coral transformations 'in vivo' by several physical analytical methods.

Coral has been used for the last ten years as bone substitution in the body because of its mechanic and osteoconductor properties. Our primary studies have shown, for the first time, the quantitative behaviour of the atomic components.