Marine Collagen/Apatite Composite Scaffolds Envisaging Hard Tissue Applications.

The high prevalence of bone defects has become a worldwide problem. Despite the significant amount of research on the subject, the available therapeutic solutions lack efficiency. Autografts, the most commonly used approaches to treat bone defects, have limitations such as donor site morbidity, pain and lack of donor site. Marine resources emerge as an attractive alternative to extract bioactive compounds for further use in bone tissue-engineering approaches. On one hand they can be isolated from by-products, at low cost, creating value from products that are considered waste for the fish transformation industry. One the other hand, religious constraints will be avoided. We isolated two marine origin materials, collagen from shark skin (Prionace glauca) and calcium phosphates from the teeth of two different shark species (Prionace glauca and Isurus oxyrinchus), and further proposed to mix them to produce 3D composite structures for hard tissue applications. Two crosslinking agents, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/N-Hydroxysuccinimide (EDC/NHS) and hexamethylene diisocyanate (HMDI), were tested to enhance the scaffolds' properties, with EDC/NHS resulting in better properties. The characterization of the structures showed that the developed composites could support attachment and proliferation of osteoblast-like cells. A promising scaffold for the engineering of bone tissue is thus proposed, based on a strategy of marine by-products valorisation.

In vivo evaluation of shark teeth-derived bioapatites.

OBJECTIVE: The present work proposes the shark teeth as a new source of bioapatites for bone filler applications in maxillary sinus elevation, periodontal regeneration or implants placement. This abundant fishing by-product provides an improved hydroxyapatite (HA) with fluorine contributions. The in vivo evaluation of osteointegration and bone mineral density levels promoted by these marine bioapatites was the main objective. MATERIALS AND METHODS: Marine bioapatite granules of two sizes (1 mm, <20 µm) were obtained and characterized (XRD, SEM, ICP-OES) to determine morphology and composition. In vivo evaluation was performed, after bioapatites implantation in critical defects of parietal bone of 25 rats, for 3 weeks. Commercial synthetic HA/ßTCP (60/40%) material and unfilled defects were used as controls. Radiology, micro-CT, histology and quantification of bone mineral density are presented. RESULTS: These marine bioapatites presented a globular porous morphology. A biphasic composition ~70% apatitic (HA, apatite-CaF, fluorapatite) and ~30% non-apatitic phase (whitlockite, tricalcium bis(orthophosphate)), with contributions of F (1.0 ± 0.5%wt), Na (0.9 ± 0.2%wt) and Mg (0.65 ± 0.04%wt) was confirmed. After implantation period, higher osteointegration of 1-mm marine bioapatites than commercial synthetic granules was observed, together with bone formation from the defect surroundings but also at central area (potential osteoinductive properties). New bone cells penetrated inside pores and inter-granular cavities. Higher bone mineral density, in both 1-mm and <20-µm granules, than on commercial synthetic graft was determined, being significant in 1-mm bioapatites (a P < 0.05). CONCLUSION: Shark teeth bioapatites were successfully validated as new functionally efficient bone filler in rat model, promoting significantly increased bone mineral density than synthetic control.

Isolation and preparation of nanoscale bioapatites from natural sources: a review.

This review summarizes recent work on isolating and preparing bioapatites from various natural sources. First, the physicochemical properties of bioapatites are described and discussed. Then, a general summary of natural (animals and vegetals) sources for bioapatite production from various environments (terrestrial and water) is made. Special attention is paid to describing individual methods for acquiring bioapatite from biogenic sources, e.g., direct isolation of bioapatite, or indirect biomimetic synthesis, with the aid of naturally derived biomolecules or biomembranes. The results of a comprehensive physicochemical characterization and a biological evaluation (in vitro and in vivo) for bioapatites and their applications in clinical practice are presented. Finally, future perspectives are summarized and discussed.

Fossils as candidate material for orthopedic applications.

Ceramic powders from fossil deposits were thoroughly characterized from the material point of view and sintered to produce massive components. The raw material, a mixture of apatite minerals, feldspars, and quartz, seems ideally suitable to obtain a biologically compatible glass ceramic. Preliminary in vitro tests of proliferation and adhesion of MG63 human osteoblast-like cell line on a selected sample are encouraging. Results are correlated with sintering conditions and phase composition: the fossil can be sintered to almost full density at temperatures as low as 900 °C and seems to quickly promote cell activation with respect to hydroxylapatite.

Apatite formation in the reaction-setting mixture of Ca(OH)2--KH2PO4 system.

Development of a new calcium phosphate cement (CPC) system as an alternative to that commonly used, basically consisting of tetracalcium phosphate and dicalcium phosphate self-setting mixtures, could be of interest in achieving special properties of the product. Powder mixtures of Ca(OH)(2) and KH(2)PO(4) were studied to assess their potential for the precipitation of apatite-like phase with the use of potassium phosphate salt solution as the cement liquid. X-ray diffraction and infrared (IR) spectroscopy studies and pH and setting time measurements were performed. The set cement was revealed to consist of a low crystalline carbonate-substituted apatite-like phase. The setting time of the cement was about 5 min. Its dissolution in distilled water led to an increase in solution pH to about 11.5, the pH slowly decreasing to 10.2 at day 10. The results showed the cement to be of an increased carbonate substitution ability compared to the tetracalcium phosphate-dicalcium phosphate anhydrous cement.

Influence of synthesis and sintering parameters on the characteristics of carbonate apatite.

A new method to synthesise carbonate-substituted hydroxyapatite (CHA) powder has been set up introducing a CO(2) flux, as a source of carbonate, in the HA synthesis process based on the neutralisation reaction. The reactants are abundant and inexpensive. The yield is good compared to other CHA powder synthesis. The reaction may be performed at low temperature and without pH control and does not produce any by-products. The influence of the synthesis parameters (temperature, H(3)PO(4) solution dropping rate, i.e. reaction time, CO(2) flux, ageing time) has been tested to optimise the process conditions in order to obtain the highest carbonation degree and favour the B-type CHA precipitation with respect to A-type one. The prepared powder (5.8wt% of total carbonate with an A/B ratio of 0.78) was thermally treated at various temperatures in the range 500-1400 degrees C in different atmospheres (air, wet and dry carbon dioxide). The thermal treatments were performed with a double aim, to eliminate selectively the carbonate groups in A-position maintaining the B-type substitution, and to evaluate the thermal stability of the CHA and the total loss of carbonate as a function of temperature. The thermal treatment at 900 degrees C in wet CO(2) gave the best result in terms of a high carbonate residue and a low A/B ratio. We also investigate the use of different techniques (inductively coupled plasma, TGA, Fourier transformed infrared spectroscopy, X-ray diffraction) for characterising CHA and calculating sensitivity and accuracy in the quantification of carbonate ions for each molecular site.

Biological activities of sustained polymyxin B release from calcium phosphate biomaterial prepared by dynamic compaction: an in vitro study.

Calcium phosphate ceramics (CaP) have recently been proposed as a potential matrix for a bioactive drug delivery system (DDS) in which the effect in situ of a released therapeutic agent is favored by the biocompatibility, osteoconductivity, and bioresorption of the ceramic material. Polymyxin B (PMB) is a polypeptidic antibiotic which undergoes thermodamage above 60 degrees C. The dynamic compaction method was developed to consolidate the drug load on CaP powder without external heating. Two projectile velocities (50 and 25 m/s) were used here to achieve powder consolidation. Among the different techniques used to associate therapeutic agents with CaP, wet adsorption was performed before the dynamic compaction process. The PMB release profile was measured by a capillary electrophoresis technique, CaP crystallography was studied by x-ray diffraction, and CaP physicochemical analysis was performed by infrared spectroscopy. The biological activities of PMB-loaded compacted CaP were determined by the effect of the antibiotic and monocyte/macrophage degradation on compact surfaces. PMB release began after 2-3 days of incubation for blocks compacted at 25 m/s velocity and on day 5 for those compacted at 50 m/s velocity. A discrepancy was noted between the amounts of PMB released (0.5-2.1 mg) and the amounts initially compacted (2-8 mg) with CaP powder. The biological activities (antibacterial activity and inhibited lipopolysaccharide effects on monocyte/macrophage CaP degradation) of PMB released from compacted calcium-deficient apatite were unaltered. Thus, dynamic compaction allows PMB to be used with CaP ceramics without any loss in its integrity and biological effects.

Fibro-osseous lesion with calcified spherules (cementifying fibromalike lesion) of the tibia.

The histologic and ultrastructural features of a tibial lesion strongly resembling cementifying fibroma of the jaws are described. Histologically the lesion consisted of fibrous tissue containing small calcified spherules. In some areas acellular trabeculae or islets with lobulated margins of a cementlike substance occurred. Spherules and small calcified bodies were composed of apatite crystal depositions along the collagen fibers and were often in close contact with membrane-surrounded cytoplasmic processes resembling matrix vesicles. In fibrous tissue fibroblasts, myofibroblasts, and undifferentiated cells with numerous cytoplasmic processes and intracytoplasmic microfibrils were present. The character of the cellular component of this lesion and the presence of calcified spherules arranged in a manner rarely observed in fibrous dysplasia indicate a histogenic relationship between the two lesions.

Electron imaging and diffraction study of individual crystals of bone, mineralized tendon and synthetic carbonate apatite.

A transmission electron microscope study of carbonate apatite crystals isolated from bone and mineralizing tendon, as well as those produced synthetically under approximated-physiological conditions, shows that they are thin irregular shaped plates. Electron diffraction patterns of individual crystals confirm that the large developed crystal face is (100), and that the longest dimensions of the biogenic crystals are aligned with the crystallographic c axes. As the latter are also aligned with collagen fibril axes, the observations provide additional information on the tissue organization itself. The marked similarity between the biologic and synthetic crystals suggests that the biological environment in which the crystals form may not be primarily responsible for controlling their shape.

The nature of bone carbonate.

Models of the bone salt and its synthetic analogues have been strenuously, and sometimes emotionally debated since the late nineteenth century. The main protagonist in the drama is the ubiquitous CO3=ion whose role has never been clearly understood. Initially regarded as an essential part of the calcium phosphate crystal complex, it came to be dubiously designated as a separate phase CaCO3, as an adsorbed ion, or even as a mere contaminant. More recent studies provide evidence that the original impression may be more nearly correct. Of particular interest in defining the role of CO3= in bone are the reactions involved in the formation of CO3-apatite under conditions approximating the physiological. These observations suggest that the synthesis of bone mineral involves hydrolysis of an initial acidic calcium phosphate precipitate to octacalcium phosphate, which is then converted to octacalcium phosphate carbonate (OCPC) by virtue of the replacement of PO4 identical to (HPO4=) by CO3=. OCPC satisfies many criteria for a satisfactory definition of the nature of the bone mineral. It can explain its solubility behavior and the intrinsic relationship between PO4 identical to (HPO4=) and CO3=, the normal variations in bone composition, the sequence of events in bone mineral maturation, and the loss of CO3= under normal and pathological conditions.