2.1. Thermogravimetry (TG)

Thermogravimetry (TG) is an experimental technique suitable for monitoring physical and chemical changes in the sample exposed to heating. By using this method the mass of a sample is measured as a function of sample temperature or time and presented in TG curves. Since the sample reacts with the surrounding atmosphere, there are many different effects which may result in mass change (loss or even in gain of mass) of the sample. These effects include evaporation of volatile constituents, drying, adsorption and desorption of gases, moisture and other volatile substances, loss of water of crystallization, oxidation of metals, oxidative decomposition of organic substances, uptake or loss of water in a humidity controlled experiments, thermal decomposition in an inert atmosphere, and heterogeneous chemical reaction [9], [10], [11], [12], [13].

Using thermogravimetric analysis (TGA), the influence of hot pressing on the properties, crystallization kinetics and decomposition of HAp/PLLA composite biomaterial was analyzed [14]. In the study by Azavedo et al. [15], TGA was applied to determine the real HAp content present in the polycaprolactone/hydroxyapatite (PCL/HAp) composites, obtained after processing by two different methods. Sivakumar et al. [16] used TGA to characterize calcium carbonate skeleton of the corals, to confirm HAp formation and also to optimize the processing parameters of converting this skeleton into HAp granules. It was found, using TGA, that faster heating rate and larger sample sizes produced higher decomposition temperatures of ultrahigh-molecular-weight polyethylene (UHMWPE) [17]. The results of this analysis are important to provide solutions for the current problems associated with clinical applications of polymeric biomaterials. The results of XRD and thermal analysis (TGA/DTA) in the characterization of β-TCP suspension indicate that it is possible to obtain β-TCP stable suspensions by controlling the properties of used dispersing agents and specific operations [18]. In the paper by Voicu et al. [19], thermal analysis (TGA and DTA) was used to estimate the kinetics of the hydration process in binding systems based on white mineral trioxide aggregate (WMTA) and partially stabilized cements (PSC) WMTA/PSCs as materials for dental applications. Iliescu et al. [20] performed the preparation and determined irinotecan incorporation efficiency in montmorillonite (Mt) and sodium alginate (AL) beads as drug carriers, both by UV–Vis spectroscopy and thermal analysis. Onishi et al. [21] concluded that thermogravimetric analysis in combination with mass spectrometry (TGA-MS) represents an appropriate method for the identification of origin and for monitoring the ageing trends of skeletal remains in a forensic context. Also, the effects of storage conditions and preparation methods in maintaining the integrity of bone, as a form of evidence for forensic examiners, have been examined by TG analysis [22]. TG analysis could be an excellent method for detailed analysis of human femur bone, as a complex substance [23]. In order to improve the biocompatibility of chitosan (Ch) and poly-d,l-lactide-co-glycolide (PLGA), Ignjatović et al. [24] combined them with compounds that exhibit complementary properties. The authors used TGA in analysis of nanoparticulate form of hydroxyapatite (HAp) coated with two different polymeric systems, Ch and a Ch-PLGA polymer blend. It was confirmed that the detected changes in the TGA curve of HAp/Ch-PLGA, for the most part, came from the thermal decomposition of Ch and PLGA [24]. An example of TGA on line MS (gas analysis) of HAp, HAp/Ch and HAp/Ch-PLGA is given in Fig. 2.

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Fig. 2. Thermal analysis: TGA-on line MS (gas analysis) of HAp (a), HAp/Ch (b) and HAp/Ch-PLGA (c) (Reproduced with the permission of the Elsevier) [24].

2.2. Differential thermal analysis (DTA) and differential scanning calorimetry (DSC)

Differential thermal analysis (DTA) and differential scanning calorimetry (DSC) are similar and comparable thermal techniques most widely used in the investigation of the mechanisms of thermal processes. Thermal behavior provides indirect information on the examined structure and a wealth of information about materials. The design principle is similar in both methods; the tested sample is compared with an inert reference sample that cannot undergo phase changes in measured temperature range. The sample temperature becomes lower in endothermic, or higher in exothermic processes, to a reference sample temperature [9], [10], [11], [12], [13]. DSC is based on the measurement of the heat flow in the samples upon heating and recording of possible structural modifications and phase changes that occur in the sample depending on the temperature. These changes can be monitored by DSC devices quantitatively on two principles, isothermal and adiabatic. The obtained DSC curve also allows the quantitative determination of the energy flow as a function of temperature or time (ΔH, enthalpy changes). The softening temperature, crystallization temperature, melting point, and a range of more complex changes, are also measured by the DSC [12], [13].

DSC measurements were used to study the changes in thermal behavior and crystallinity of many different materials. Ignjatović et al. [14] have shown that hot processing is a necessary step in obtaining HAp/PLLA composite blocks with mechanical properties similar to those of the bones. In the paper by Ignjatović et al. [25] biphasic calcium phosphate/poly-d,l-lactide-co-glycolide (BCP/DLPLG) composite biomaterial, with and without biostimulative agents, in the form suitable for reconstruction of bone defects was examined. Prior to in vivo examination, material was characterized by DSC method and the desired material properties in the terms of structure and composition were confirmed by this method. Effects of gamma irradiation on the structure and physicochemical properties of HAp/PLLA composite were discussed in the study by Suljovrujić et al. [26]. In the study by Ignjatović et al. [27] DTA, TGA and DSC methods have confirmed that CP particles were coated with DLPLG polymer in two kinds of CP/DLPLG composite biomaterials. Affatato et al. [28] used DSC to determine the crystallinity of vitamin E-stabilized crosslinked acetabular cups based on polyethylene, in comparison with those of conventional standard and crosslinked polyethylene acetabular cups. A significant impact of the pH adjustment on the apatite formation, the morphology and phase purity of the ceramic samples in aqueous sol-gel chemistry process, was studied by thermoanalytical methods (TGA/DTA), among the others [29]. The authors used DSC method to confirm that the rate and the heat released during the polymerization reaction of acrylic bone cements could be changed by adding TCP [30]. In the domain of biomaterials engineering and reconstructive medicine, human and animal bone grafts are excellent biomaterials due to their natural origin, if they are used in the optimal conditions that support their osteoconductivity and osteoinductivity. TGA/DSC measurements are used in highlighting the best characteristics of the heat treated human compact bone samples, in order to obtain bone graft products so called allografts [31]. The physicochemical characterization of lyophilized bovine bone grafts by TG analysis, confirmed that this is a product of excellent biocompatibility and characteristics similar to natural bone and to other bone grafts that are widely used [32].

The composite HAp/Ch-PLGA particles loaded with 17β-hydroxy-17α-picolyl-androst-5-en-3β-yl-acetate, a chemotherapeutic derivative of androstane with a selective anticancer activity against lung cancer cells, were examined by Ignjatović et al. [33]. The drug loading process and the prediction of the loading capacity of poorly water-soluble drugs were analyzed by thermogravimetric and differential thermal analysis coupled with mass spectrometry [33]. An example of DTA curves for HAp, Ch, PLGA and Androstane (A), and A-HAp/Ch-PLGA is given in Fig. 3.

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Fig. 3. Differential thermal analysis (DTA) curves for (a) HAp, Ch, PLGA and (b) Androstane (A) and A-HAp/Ch-PLGA (Reproduced with the permission of the Elsevier) [33].

2.3. X-ray diffraction (XRD) analysis

Diffraction methods like X-ray diffraction, neutron diffraction, electron diffraction, etc., are the most important methods for the structural characterization of biomaterials in a crystalline form. XRD method is based on X-ray diffraction on the lattice, depending on the scattering of X-rays by the electron density of atoms and molecules. The diffraction maxima are achieved when the scattered rays of the crystal planes are in phase, which obeys Bragg's law. This law can be used to determine the type and parameters of the simple crystal lattice as well as parameters of linear thermal expansion of tested biomaterials. In the case of complex crystal structures it is necessary to take into account the geometrical structure factor. This factor modifies intensity of the diffraction maxima, so it is possible to determine the distribution of electron density in the crystal. In a complex biopolymer structures heavier atoms are united with biopolymers so the spatial distribution of electron density for many biomaterials and biopolymers was determined [2], [3].

In the studies by Kandić et al. [34] and Veselinović et al. [35] XRD was used to investigate the qualitative and quantitative composition, crystallite size of phases, and the degree of crystallinity of biocomposite materials, since these properties have a great influence on the quality of composites and their application in the bone tissue repair. Effects of hot pressing on the structural changes that occurred in the composite biomaterial were studied by wide angle X-ray structural (WAXS) analysis. Obtained results indicated a possible decrease in the degree of crystallinity with a hot pressing time increase [14]. Results from characterization of composite biomaterials performed by WAXS showed that the application of composite for healing of infra bone defects in patients caused high level of osseous regeneration [36]. XRD was used to show that the bioactivity of nano-sized HAp can be improved by incorporating of some ions, such as Co2 +, into the HAp lattice [35], [37]. In the study by Ahmadi et al. [38]synthesis of silicon and magnesium co-doped fluorapatite (Si-Mg-FA) was performed by the high-energy ball milling method and obtained materials were analyzed by using XRD method. The XRD patterns showed that the diffraction peaks of starting materials disappeared with increasing the milling time and that the new peaks, associated with the apatite phase, appeared [38]. Kashkarova et al. [39] have shown that HAp produced from the egg-shell of birds could be used in medical purposes, for improvement of the adhesion properties of dental cement pastes, by using XRD analysis. Also, an alternative method for dating osseous remains, with important legal implications, could be the use of both XRD and biochemical analysis [40].

An example of XRD spectra of HAp/Ch-PLGA, HAp/Ch, HAp, Ch and PLGA is given in Fig. 4.

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Fig. 4. The XRD spectra of HAp/Ch-PLGA, HAp/Ch, HAp, Ch and PLGA biomaterials (Reproduced with the permission of the Elsevier) [73].

3.1. Particle size distribution (PSD)

The particle size distribution and uniformity are characteristics responsible for the properties of disperse materials. There is the interest in controlling and measuring of particle size distribution, considering that this value influence the content uniformity, dissolution and absorption rates of active ingredients in the pharmaceutical products. This value is an important indicator of quality and performance in other areas like nanotechnology, proteins, cosmetics, polymers, soils, abrasives, fertilizers and many others. The laser light diffraction on the particles is precise and simple method for testing size distribution in the disperse systems. In order to obtain reliable results it is necessary to understand and take into account important factors like physical and chemical properties of the material, instrument, measurement methodology and verification of results [43].

The effect of particle size on the kinetics of reaction and on the final micro- and nanostructural features of CP cement was studied by Ginebra et al. [44]. Khin et al. [41] analyzed the effects of particle size and particle surface coating on the cellular uptake of fluorescent polystyrene nanoparticles and PLGA nanoparticles coated with polyvinyl alcohol (PVA) or vitamin ETPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate). It has been shown that the cellular uptake is notably improved by surface modification. An example of PSD of HAp, HAp/Ch and HAp/Ch-PLGA is given in Fig. 5.

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Fig. 5. PSD of HAp, HAp/Ch and HAp/Ch-PLGA (Reproduced with the permission of the Elsevier) [73].

3.2. Dynamic light scattering (DLS)

Dynamic light scattering (DLS), also known as photon correlation spectroscopy(PCS), is probably the most commonly used technique for characterizing of nanomaterial-bioconjugates. This is one of the most popular light scattering modalities that are simple, noninvasive, nondestructive and relatively cheap to apply. Compared to the other methods, it has short experiment duration (in minutes) and lower apparatus costs with more reproducible results. DLS exploits the scattering of radiation through its interaction with a sample. Fluctuations in the scattered light intensity are used to determine the particle diffusion coefficient, related to hydrodynamic radius. This method is used in physicochemical characterization of nanomaterials, in determining the hydrodynamic size, shape, structure, aggregation state and biomolecular conformation of monodisperse samples. It is useful for analyzing the samples in a wide range of concentrations (∼ 108–1012 particles/mL) and detecting small amounts of higher molecular weight species. DLS techniques also provide information about flexibility of particles and offer insight in the nature of interactions between particles and their environment [45]. There are, however, several disadvantages of DLS, such as difficulty in correlating size fractions with a particular composition when certain amount of aggregates is present and also dust particles interfering with the scattering intensity [46]. DLS is also known to have a poor peak resolution and can only resolve particle populations (within the same sample) if they differ in size by at least a factor of 3 [47]. Atomic force microscopy (AFM) has advantages over DLS for characterizing size distributions of polystyrene nanoparticles in the size range of 20–100 nm for non-monodispersed solutions [48].

3.3. Small-angle X-ray scattering (SAXS)

In the last few years, small-angle X-ray scattering (SAXS) was used to examine crystalline nanostructure in a range of materials. SAXS represents a non-destructive method that provides information of important characteristics of polymers, proteins and biomaterials. The size, size distribution, shape, orientation distributions and structure of a variety of polymers and nanomaterial-bioconjugate systems in solution was evaluated by SAXS [49]. This is an established method for investigating internal low-resolution molecular structure of biological systems in the absence of single-crystals or structure of disordered and partially ordered systems. Modern small-angle scattering by X-rays also provides overall three-dimensional structures of biological macromolecules [50]. There are significant future perspectives for SAXS and its potential impact on structural molecular biology [51], [52].

SAXS was used to determine the changes of bone crystallite size and shape during an experimental heating treatment by Hiller et al. [53]. In the paper by Fratzl et al. [54] recent developments of this method for application in bone research are reviewed. These authors also used this nondestructive technique to obtain important information about the size, habit, typical shape and arrangement of mineral crystals in bone [55]. Microfocus SAXS has the ability of probing textural differences in a sample at a micron scale resolution and to detect such local changes in texture. It also has a great potential in determining crystal characteristics in healthy and diseased bone samples [56].

4.1. Zeta potential (ZP) and surface charge

Zeta (ζ) potential and surface charge represent physical properties which are exhibited by any particle in suspension and emulsion that can be optimized using this value. The knowledge of these properties is also important in reducing the time needed to produce trial formulations and in prediction of long-term stability. The choice of the method for measuring a particle's ζ-potential is dependent on the nature of both particle and suspension formulation, but also on the size and concentration of particles [64].

Smeets et al. [65] found that negative ζ-potential of biphasic calcium composite (BCC) material is favorable for bone regeneration and osseointegration of dental implants. In the study by Swanand et al. [66] the effect of ζ-potential was identified as an important factor in protein adsorption and cellular uptake of nanoparticles. ζ-Potential measurements by Botelho et al. [67] confirmed that silicon incorporation into HAp induced a decrease in the net surface charge and isoelectric point of HAp. Hallab et al. [68] showed that surface alloying for optimum adherence may be possible since there is a charge and electrical potential-dependent adhesion maxima of the cells. Hunt et al. [69] examined polymer biocompatibility and showed that the net surface charge can influence the early phase of acute inflammatory response to an implanted material. Gessner et al. [70] determined physicochemical parameters important for the future controlled design of the colloidal drug carriers and biocompatible surfaces of other devices that come into contact with proteins (e.g. microparticles and implants). The findings in the study by Chunabi et al. [71]concerning the particle size and surface charge are useful in the rational design of drug nanocarriers with maximized therapeutic efficacy and predictable in vivo properties. In the study by Ignjatović et al. [72] ζ-potential of nanoparticles with cholecalciferol (D3) and nanoparticulate carriers composed of HAp and PLGA indicated their stability in suspended state and possibility for their therapeutic application by injection. The ζ-potential analysis was performed to study the HAp particles coated with chitosan (HAp/Ch) and chitosan-poly-d,l-lactide-co-glycolide (HA/Ch-PLGA) polymer blend prior to further examination of those particles as an organ-targeting system, in the study by Ignjatović et al. [73].

4.2. Contact angle (CA) analysis

Contact angle (CA) analysis is one of the most sensitive and inexpensive surface analysis techniques for the characterization of surface wettability of biomaterials. It is a relatively simple method for measuring the surface tension of solid samples that should be clean enough and do not swell or dissolve in the test liquid. There is no need for special sample preparation, and this technique is capable of measuring ~ 3–20 Å deep. However, there are certain concerns, including operator error, surface roughness, surface heterogeneity, contaminated fluids, sample geometry, misinterpretation of results that can influence the accuracy of the overall result [74]. The contact angle (q) is the angle of the liquid at the interface relative to the plane of the model surface, and it can be measured by five common techniques: the static or sessile drop method, the Wilhelmy balance technique, the captive air bubble method, the capillary rise method and the tilting plate method developed by Adam and Jessop [74], [75], [76].

The Wilhelmy balance technique is an indirect force method, ideal for more accurate contact angle values that reflect the property of the entire sample. An alternative to this method is the captive air bubble method, with the advantage of ensuring that the surface is in contact with a saturated atmosphere. This method was used in the determination of wetting characteristics of commercial ultrafiltration (UF) membranes [77]. In the paper by Lander et al. [78] it has been shown that the Wilhelmy balance and the tilting plate method yield very similar values of contact angle hysteresis. There is operator subjectivity and difficulty in performing the most commonly used sessile drop method, so it is not recommended [78]. Krishnan et al. [79] proposed Wilhelmy-balance tensiometry as the gold standard in comparison to tilting-plate goniometry and captive-drop goniometry. Under certain conditions, from contact-angle measurements, the adhesion of number of different cell types, including bacteria, granulocytes, and erythrocytes can be determined [80].

Biomaterials are becoming increasingly popular in the medical, biomedical, optometric, dental, and pharmaceutical applications, since they are coming into close contact with living cells and biological fluids. In the article by Menzies and Jones [81] the impact of contact angle on the biocompatibility of tissue engineered substrates, blood-contacting devices, dental implants, intraocular lenses and contact lens materials, is reviewed. Bumgardner et al. [82] evaluated the contact angle and showed that there is an increased protein adsorption and osteoblast precursor cell attachment on the chitosan-coated Ti, so they proposed chitosan to be a beneficial tool in enhancing osseointegration of implant devices.

4.3. Energy dispersive X-ray (EDX) and wavelength dispersive X-ray (WDX) spectroscopy

Energy dispersive X-ray (EDX) spectroscopy is a surface analysis method that provides semi-quantitative chemical analysis of the sample. This method relies on the radiation of samples with high energy electrons and observation of emitted X-rays resulting from the de-excitation of core electron holes. The emitted X-ray energies will generally be different from element to element considering that each element has a unique atomic structure. In the obtained spectra there could be some overlapping of peaks from different elements, but other peaks or limited knowledge of the sample history allow a detection of many different elements, at least theoretically. There is an important use of this method in TEM or SEM electron microscopes where it provides elemental analysis combined with high resolution images of surface topography in nanometer area. Two techniques, energy dispersive X-ray (EDX) and wavelength dispersive X-ray (WDX), each with some advantages and disadvantages, are used [3]. EDX technique is more frequently used, especially in automated analysis, thanks to its speed and whole spectrum acquisition. Some limitations are, for example, relatively poor spectral resolution and high detection limits. WDX analysis is a geometry based technique and, contrary to the EDX analysis, the whole range of wavelengths has to be scanned. Using this method, it is possible to achieve a good spectral resolution and lower detection limits by changing the accelerating voltage, specimen current, and spectral background settings. According to previous research, WDX is the most suitable choice to measure trace element distribution between phases, for multi-element quantification and for samples containing peak-overlapping elements [83]. This technique has better detection limit for low-Z elements in contrast to the EDX [84]. It was shown by Weinbruch et al. [85] that semi-quantitative WDX analysis is feasible even for irregularly shaped particles down to 0.8 mm or equivalent diameter, but the main limitation is the relatively long analysis time required. In order to address this limitation both EDX and WDX systems were combined, so the advantages of each method were used [86]. EDX technique was used for characterization of composite biomaterials based on BCP and DLPLG [87]. This method has also a significant role in the examination of the effect of bone sites and sex to mineral and matrix content and composition [88].

Scanning electron microscopy (SEM) is often used coupled with an EDX detector system, especially in composite biomaterials, with high sensitivity [89], [90]. Farzadi et al. [91] used SEM-EDX analysis to confirm the even distribution and influence of Mg incorporated in the stoichiometric HAp powders on the lattice parameters. This is also an important method in forensic science, commonly used for distinguishing dental and/or osseous samples from other morphologically similar materials [92].

EDX analysis could have a great application in testing of bone substitutebiomaterials since the changes in elemental content are very important for predicting and assessing the behavior of biomaterials in living system. Stojanović et al. [93] analyzed the changes in the Ca/P ration of commonly used bone substitute biomaterial (Bio-Oss®) in simulated in vitro conditions, by incubating the material in standard cell culture media, at 37 °C. After 3 days of incubation, extract was discarded and Bio-Oss® particles were further analyzed using SEM equipped with EDX. We showed that calcium and phosphorus ions concentration on the surface of particles simultaneously increases after incubation of Bio-Oss® in DMEM, and new layer consisting mainly of apatite or hydroxyapatite is formed. This observation is of great importance for prediction of Bio-OSS® particles' behavior in living system [93]. An example of combined SEM-EDX application is given in Fig. 6.

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Fig. 6. The EDX spectra with related SEM images of CP/PLGA biomaterial before (a) and after (b) incubation in DMEM.

4.4. X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS), also called electron spectroscopy for chemical analysis (ESCA), is widely used ultrahigh-vacuum (UHV) surface analysis technique. There is a need for adequate characterization of the biomaterial surface, because of its responsibility for the success or failure of a biomaterial device. The principle of XPS is based on the absorption of X-rays by atoms in the sample that leads to the ejection of core and valence electrons (photoelectrons). These photoelectrons have energies that are unique for each element and sensitive to their chemical states. The peak intensity in the photoelectron spectrum is a direct measure of the elemental concentration and provides information on chemical bonding as well. Except for the hydrogen and helium, qualitative and quantitative information on all other elements can be obtained by the characteristic binding energies of the electrons. This is a relatively non-destructive, quite mature method, increasingly used in different areas of biology, cell analysis, bacteria analysis and tissue analysis as well as bioengineering [94]. This extremely sensitive surface technique is in expanding use in the field of biomolecules' characterization like proteins, peptides, lipids, mucins, enzymes and DNA thanks to the new instrumentation, technique development and enhanced data analysis [95].

Sabbatini and Zambonin [96] examined the surface properties with strong influence on biological response and long-term performances of polymeric biomaterials. They emphasized that XPS has been far more widely employed than other surface techniques in analysis of chemical composition, structure, in determining the presence of contamination, segregation of elements and molecules, microdomain distribution, functional group orientation and molecular mobility at the top layer of a biomaterial. The results of XPS obtained in the study by Kasemo and Lausmaa [97] are valuable for basic research concerning implant-tissue interactions as well as production control and implant standardization. Yoshida et al. [98] used XPS to provide the direct evidence of chemical bonding and also to enable the quantification of functional groups of the polyalkenoic acids that are bonded to calcium in HAp. This technique can be used in the development of new biomaterials that are more durable and reliable in the fields of both restorative dentistry and orthopedics. In the study by Xu and Khor [99] chemical composition of the silica(SiO2) and silicate-based biomaterials the replace possibility of silicate substitution was determined by XPS. The results of this study indicated that silicon can be partially substituted into HAp and that enhance dissolution due to silicon substitution and calcium phosphate phase content may contribute to the higher cell proliferation compared with pure HAp, suggesting a good biocompatibility of examined biomaterials. Casaletto et al. [100] used XPS qualitative and quantitative analyses to show that good stoichiometry of the HAp coatings were obtained by the specific preparation procedures. In the study by Maachou et al. [101] chitosan (Ch), HAp and microparticles phosphorylated Ch and Ch/HAp hybrids, were analyzed by XPS before and after immersion in biological fluids.

4.5. Vibrational spectroscopy

Vibrational spectroscopy is one of the most important spectroscopic method that covers infrared, near-infrared and Raman spectroscopies. It is based on the measurement of vibrational energy levels which are associated with the chemical bonds in the sample. So, the obtained spectra could be used as a fingerprint for identification of unknown compounds or to assess the close intermolecular interactions of specific groups. The spectra obtained by scattering and absorption of infrared light are used to provide vibrational data from solid state or gas phase samples. Vibrational spectroscopy has a lot of perspectives for the study of biomaterials, especially for the characterization of different biomaterials based on CP and HAp. Infrared and Raman, vibrational spectroscopic techniques are of particular significance in providing the information about the molecular composition, structure and interactions within a sample [91]. CP based biomaterials, especially apatites, represent a complex structures of major interest. Their structural examination is mainly performed by diffraction methods. In quantitative determination of a very limited amount of material vibrational spectroscopic techniques may be used. Biological tissues or materials are also characterized by using microscopic techniques that allow accurate mapping of specific mineral characteristics [102].