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Raman Spectroscopy of Natural Bone andSynthetic ApatitesAther Farooq Khan a , Muhammad Awais a , Abdul Samad Khan a ,Sobia Tabassum a , Aqif Anwar Chaudhry a & Ihtesham Ur Rehman ba Interdisciplinary Research Centre in Biomedical Materials, COMSATSInstitute of Information Technology, Lahore, Pakistanb Department of Materials Science and Engineering, Koroto ResearchInstitute, University of Sheffield, Sheffield, UKAccepted author version posted online: 10 Jan 2013.

To cite this article: Ather Farooq Khan , Muhammad Awais , Abdul Samad Khan , SobiaTabassum , Aqif Anwar Chaudhry & Ihtesham Ur Rehman (2013) Raman Spectroscopy ofNatural Bone and Synthetic Apatites, Applied Spectroscopy Reviews, 48:4, 329-355, DOI:10.1080/05704928.2012.721107

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Applied Spectroscopy Reviews, 48:329–355, 2013Copyright © Taylor & Francis Group, LLCISSN: 0570-4928 print / 1520-569X onlineDOI: 10.1080/05704928.2012.721107

Raman Spectroscopy of Natural Bone andSynthetic Apatites

ATHER FAROOQ KHAN,1 MUHAMMAD AWAIS,1 ABDULSAMAD KHAN,1 SOBIA TABASSUM,1 AQIF ANWARCHAUDHRY,1 AND IHTESHAM UR REHMAN2

1Interdisciplinary Research Centre in Biomedical Materials, COMSATS Instituteof Information Technology, Lahore, Pakistan2Department of Materials Science and Engineering, Koroto Research Institute,University of Sheffield, Sheffield, UK

Abstract: Raman spectroscopy of natural bones and hydroxyapatites is described. Inaddition, how Raman spectroscopy has proved crucial in providing baseline data forthe modification of synthetic apatite powders that are routinely used now as bone re-placement materials is explained. It is important to understand the chemical structuralproperties of natural bone. Bone consists of two primary components: an inorganicor mineral phase, which is mainly a carbonated form of a nanoscale crystalline cal-cium phosphate, closely resembling hydroxyapatite, and an organic phase, which iscomposed largely of type I collagen fibers. Other constituents of bone tissue includewater and organic molecules such as glycosaminoglycans, glycoproteins, lipids, andpeptides. Ions such as sodium, magnesium, fluoride, and citrate are also present, aswell as hydrogenophosphate. Hence, the mineral phase in bone may be characterizedessentially as nonstoichiometric substituted apatite. Such a distinction is important inthe development of synthetic calcium phosphates for application as skeletal implants.An understanding of bone function and its interfacial relationship to an implant clearlydepends on the associated structure and composition. Therefore, it is essential to fullyunderstand the chemical composition of bone, and Raman spectroscopy is an excellenttechnique for such an analysis.

Keywords Raman spectroscopy, bone, calcium phosphates, hydroxyapatite

Bone

Bone is a naturally occurring composite comprised mainly of collagen matrix and biologicalapatite (bone mineral), mainly hydroxyapatite (HA), as a reinforcement. Other organicconstituents include noncollagenous proteins, lipids, vascular elements, and cells in additionto water. Collagen acts as a structural framework in which plate-like tiny crystals of HA areembedded to strengthen the bone (1). It contains trace ions, including carbonate, citrate,sodium, magnesium, fluoride, chloride, and potassium. The major role of biological apatiteis to provide toughness and rigidity to the bone, whereas collagen provides tensile strength

Address correspondence to Dr. Ihtesham ur Rehman, Reader in Biomedical Materials, Depart-ment of Materials Science and Engineering, Koroto Research Institute, The University of Sheffield,North Campus, Broad Lane, Sheffield S3 7HQ, United Kingdom. E-mail: i.u.rehman@sheffield.ac.uk

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and flexibility. The minerals bind to collagen through noncollagenous proteins, whichare approximately 3–5% of the bone and active sites for biomineralization and cellularattachment. Bone can be considered as an assemblage of hierarchical structural unitselegantly designed on many scales, macro to nano, to meet multiple functions. Bone isstructurally weak and unorganized initially; however, the primary bone remodels to becomelamellar bone within a few days. The matured lamellar bone can be distinguished into twotypes, a spongy bone and a compact bone at the macrostructural level, and radically variesin density. Spongy bones and compact bones are organized with multilevel pores, nano tomacro, to establish multiple functions, including transportation of nutrients, oxygen, andbody fluids (2).

Raman Spectroscopy of Bone

Raman spectroscopy is a nondestructive technique used to distinguish between differentphases present in a material. It has been used to determine the chemical properties ofbiological materials, such as bone, with a spatial resolution reaching the micrometer level.Although Fourier transform infrared (FTIR) and Raman spectroscopic investigations pro-vide almost equivalent chemical information of the composition of bone tissue, the mainadvantage of Raman spectroscopy is that it can be applied to fresh tissues. In addition,it offers superior resolution on the order of 0.6–1 μm compared to FTIR’s resolution of5–10 μm. The acquisition time for Raman spectra is one to two orders higher than thosetypically used in infrared spectroscopy due to a small portion of scattered light (3). Ramanspectroscopy is nonresponsive to water, which allows analysis of hydrated samples, whereasFTIR is more sensitive toward water. Moreover, Raman can simultaneously measure theorganic and mineral phases of the material; however, there are some limitations of Ramanspectroscopy. To date, only the major components of bone such as matrix collagen, mineralphosphate, and carbonate have been observed spectroscopically. The Raman spectroscopyof bone lipids and phospholipids has also been reported, but generally they are inaccessiblebecause they are removed during sample preparation (4, 5). There are some contributionsfrom the noncollagenous proteins in the Raman spectrum (6); nonetheless, it is very difficultto separate them from the whole spectrum of the bone, because they are structurally verysimilar to collagen and are less abundant.

In this article, firstly, the Raman spectral data of natural bone, including human corticaland spongy bone, and cortical and trabecular bone of mice and their comparison as well asthe Raman spectra of stressed bone are summarized. In the second part, we describe Ramanspectral studies of synthetic bone substitutes, including HA, substituted HA, tricalciumphosphate (TCP), and substituted TCPs.

Specimen Preparation

Raman spectroscopy is a highly specific and versatile technique and requires minimal orno sample preparation. Fresh, hydrated, intact bone is preferred for analysis. Subtraction ofreference spectra can be used to remove the minor contribution from the embedded media(7). In many studies, bone specimens are fixed to avoid bacterial growth and degradationof the specimen before collection of the data. Yeni et al. (7) and Bachmann et al. (8)studied the effect of ethanol and glycerol with different fixating media like eponate, glycolmethacrylate, polymethylmethacrylate (PMMA), Technovit, Araldite, and LR White onthe Raman spectral properties of bone. A comparison between nonembedded control andglycerol-fixed and ethanol-fixed bone samples revealed that fixation has a significant effect

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Raman Spectroscopy of Natural Bone 331

on crystallinity and mineralization but no effect on carbonation. Glycerol-fixed sampleshad a significantly greater PO4

−3: amide I ratio. The results showed that ethanol fixationis relatively better than glycerol fixation. The effect of embedded media on the Ramanspectrum may be attributed to the interaction of the different functional groups between thebone matrix and embedding medium.

Specimen Fluorescence

The major problem associated with Raman spectroscopy is bone florescence when using agreen laser (most commonly used in Raman studies). It results in background noise severalorders of magnitude higher than Raman peaks. Several protocols have been developed todeproteinate the bone specimen to obtain refined peaks (9, 10). However, there is concernthat any deproteination procedure may affect the chemical and structural properties of theparticular parts of the bone that remain present after processing (11). With recent advances,the problem of tissue florescence can now be minimized by using shorter wavelengths forexcitation sources and therefore deproteination is not necessary (12).

Typical Raman Band Assignments of Bone

The Raman spectra of bone exhibit a strong molecular character associated with the internalmodes of the PO4

−3 tetrahedral (9). The vibrational normal modes of the free tetrahedronthat consists of Td point group symmetry [which were well established (10)] increase ν1,v2, v3, and v4 frequencies. The v1 frequency is associated with the symmetric stretching ofthe phosphate to oxygen (P-O) bonds, called A1 symmetry. The v2 frequency arises fromthe doubly degenerate O-P-O bending modes, which involve E symmetry, whereas thev3 frequency corresponds to the triply degenerate T2 mode (asymmetric P-O stretching),and the v4 frequency is mainly due to the triply degenerate T2 modes of O-P-O bendingcharacter (11). The characteristic spectral peaks of bone are tabulated in Table 1. The bandsin the range 957–962 cm−1 and 422–454 cm−1 correspond to v1 stretching of the P-O bandand v2 bending of the O-P-O in PO4

−3, respectively; 1006–1055 cm−1 is attributed to v3

Table 1Typical Raman band assignments for bone (137)

Peak position (cm−1) Assignment

422–454 PO4−3 v2

568–617 PO4−3 v4

815–921 C-C stretching957–962 PO4

−3 v1

1003–1005 HPO4−3 v3

1006–1055 PO4−3 v3

1065–1071 CO3−2

1243–1269 Amide III1447–1452 CH2 wag1595–1720 Amide I2840–2986 CH2 stretching3572–3575 OH stretching

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Figure 1. Comparison of human and sheep whole and deproteinated bone (16).

PO4−3; and 568–617 cm−1 is assigned to v4 bending PO4

−3. The band associated withcollagen appears at 1243–1269 cm−1 for amide III, 1447–1452 cm−1 for CH2 wag, and1595–1720 cm−1 for amide I (12–14).

Raman Spectroscopy of Human Cortical Bone

Cortical or dense bone occupies 80% of the whole bone. Cortical bone at the primary levelmainly consists of two phases; that is, an inorganic phase, which essentially consists ofcalcium phosphate crystals, and an organic phase, which largely entails type I collagenfibers (15). There are many techniques available to assess the actual structure of bone, suchas X-ray diffraction and electron microscopy.

A comparative study between human and sheep, using whole and deproteinated bones,revealed no significant differences according to Raman spectroscopic studies, as shown inFigure 1 (16). A deproteination procedure was performed using the protocol described in theliterature (14). The peak values in the Raman spectra of both complete and deproteinatedbone are shown in Table 2. (13, 14). The scattering intensity of some peaks decreasedfor the deproteinated samples, especially in the regions 1443 and 2930 cm−1, which are

Table 2Peak positions for both complete and deproteinated bones obtained using Raman spec-

troscopy (13, 14, 138)

Peak positions (cm−1)

Human bone (whole) Human bone (deproteinated) Assignment

422 422 PO43− (bend)

583 583 PO43− (bend)

960 960 (P O) stretch, phosphate1270 1070 (C O) stretch, carbonate1450 (C H) bend1660 Amide I band

2924 (O H) stretch2940 (C H) stretch

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Raman Spectroscopy of Natural Bone 333

mainly attributed to the organic part of the tissue (CH2 wag, amide I, and CH2 stretching).The peaks at 950 cm−1 and other positions remained unaffected during the deproteinationbecause they are associated with the mineral part of the bone tissue.

Raman Spectroscopy of Cortical Bones of Different Ages

Bones of vertebrates remain under a continuous remodeling process during which old anddamaged tissues are replaced with new tissues. Ager et al. (17) studied the effect of agingon the mechanical strength of human cortical bone. They used ultraviolet (UV) excitationin order to eliminate the background florescence and collect Raman spectra. Freshly frozencadaveric cortical bone samples from cadavers of different ages (age 34 to 85) were obtained.Excitation from a deep UV source resulted in a well-defined enhanced Raman spectra ofthe organic matrix; in particular, three organic bands were obtained, including amide I(1656–1626 cm−1), CH2 wag (1454–1461 cm−1), and amide III (1245–1260 cm−1). Largepeak shifts were observed for the amide I peak, especially for the case of elderly bones. Inaddition, there was a significant difference in intensity with age (Figure 2). The CH2 wagand amide III bands did not show much peak difference.

The amide bands, particularly amide I and amide III, are good indicators of proteinconformation due to their role in cross-linking and bonding, and at the molecular level,it has been suggested that aging can contribute to changes in the cross-linking chemistryof bone (18). The changes in the nature of the cross-linking also affect the biomechanicalproperties of the bone. The observed enhanced signal of the amide I band may resultfrom nonreducible pyridinoline (Pyr) cross-linked cement; it has been suggested that withage, the reducible dihydroxy-lysinonorleucine (DHLNL) matures intro Pyr cross-linkingcement (19). Similar findings were observed by Paschalis et al. (20), who studied age-related

Figure 2. UV Raman spectra of human humerus bone from donors of three different ages. Therelative height of the amide I feature compared to the CH2 wag was greater in the sample from theolder donor, and there is a positional shift of this band to higher energies with aging (17) (Color figureavailable online.)

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Figure 3. Raman spectra of trabecular bone tissue (22).

changes in the amide band of bovine bone and associated this change with an increase inthe nonreducible Pyr cross-link.

Composition and Structure of Trabecular Bone Tissue

Spongy or trabecular bone mainly consists of a network of trabecula of different thicknessesand shapes with an arrangement along the directions in which mechanical pressure isexerted. The metabolic activity of spongy bone is much greater than that of cortical bone,which results in a more intense process of organization and composition. An array ofcharacterization techniques have been used to examine the structure of spongy bone;however, Raman spectroscopy is better due to its higher spatial resolution, insensitivity towater, and lack of a florescence effect (21). In Raman spectra of spongy bone, the organicand mineral phases are clearly separated. The inorganic part is mainly dominated by v1

PO4−3 at 961 cm−1, whereas v2 and v4 vibrations were detected at 430 and 587 cm−1,

respectively. Moreover, v3 vibration of PO4−3 is overlapped by a carbonate B-type peak

appearing at 1070–1075 cm−1 (Figure 3).The peak for amide III of the organic part ranges from 1200 to 1320 cm−1, whereas

for amide I the peaks appeared at 1595–1700 cm−1. Similarly, peaks at 1400–1470 cm−1

and 2800–3100 cm−1 corresponded to the bending and stretching modes of C-H groups,respectively (Table 3) (22).

Comparison of Raman Spectra of Cortical and Trabecular Bone

Cortical and trabecular bone are diverse at the macroscopic level, as described earlier;however, at the microscopic level their structures are very similar, because they are mainlycomposed of mineralized collagen. There are a number of reports on the comparison of thecortical and trabecular bones. For example, Gong et al. (23) compared the composition ofmammals by ashing and found that cortical bone has a higher organic fraction. Goodyearet al. (24) compared the properties of cortical and trabecular bones. The peaks in both

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Raman Spectroscopy of Natural Bone 335

Table 3Peak position and assignments in Raman spectra of spongy bone tissue (22)

Peak position (cm−1) Assignment

422 PO43− (bend)

583 PO43− (bend)

960 (P O) stretch, phosphate1270 (C O) stretch, carbonate1450 (C H) bend1660 Amide I band2940 (C H) asymmetric stretch

spectra appeared at similar positions. The bending vibrations, v2 and v4, for the phosphateappeared at 438 and 589 cm−1, respectively. The peak due to the superimposition of carbonand phosphate—that is, v3—appeared at 1070 cm−1. The high-frequency peak of amide IIIappeared at 1260 cm−1 and for amide I at 1960 cm−1. However, the intensities of the peakswere different. The mineral-to-matrix ratio in cortical bone was greater than in trabecularbone. Similarly, the carbonate-to-phosphate ratio was greater for cortical bone (Figure 4).

High Pressure Raman Spectroscopy of a Stressed Bone

The mechanical properties of bone are mainly due to organic and mineral parts of the boneand depend on their interaction. A number of studies have reported Raman spectroscopicinvestigations of the changes in bone’s ultrastructure and crystal lattice changes associatedwith mechanical stress (25, 26). For stress-related studies the PO4

−3 v1 symmetric stretch(atmospheric pressure peak intensity 958 cm−1) and B-type carbonate (CO3

−2) v1 symmetricstretch (1070 cm−1) were observed. The amide I vibration (1655 cm−1), methylene (CH2)wag (1464 cm−1), and CH stretch (2937 cm−1) were noticed for bone matrix bands. Bonesof different animals, such as rat and mouse, have been studied and it was reported that the

Figure 4. Raman spectra of cortical and trabecular bone (24) (Color figure available online.)

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Figure 5. Phosphate v1 and B-type carbonate v1 symmetric stretches for (a) powdered and depro-teinated rat bone, (b) powdered rat bone, and (c) powdered mouse bone under low and high pressure(25) (Color figure available online.)

spectra for phosphate were similar to each other, which confirmed that the observed effectswere not dependent on the species (25). Peak shifts for all specimens were found toward thehigher frequencies for both phosphate and carbonate peaks due to an increase in pressure.The observed peaks were symmetric and those at lower frequencies were found to bebroader than those at higher frequencies. This effect was more prominent in deproteinatedrat bone, where a low-frequency shoulder can be clearly seen at higher pressure (25). Acomparison was also made between bones at atmospheric pressure before and after loading,which revealed no permanent changes. Therefore, the spectral changes with pressure arereversible. In the collagen matrix the peaks of both CH2 wag and CH stretch shifted tohigher frequencies and the bands were much more dependent on pressure than mineralbands are (25). However, the amide I band shifted to lower frequencies, and according tothe preliminary conclusion of de Carmejane et al. (25), the amide I (and possibly CH2 wag)was shielded from the effect of bulk water and the internal hydrogen bonding was strongerand more stable. Amide I is in contact with a very thin layer of internal water, which doesnot respond to pressure in the same way as bulk water and therefore responds differentlyto compression from the other bone components (Figure 5).

Synthetic Replacement of Natural Bone

HydroxyapatiteThere are several calcium phosphates available as bone substitute materials (27),

such as dicalcium phosphate (DCP) (28–30), amorphous calcium phosphate (ACP) (31,32), tricalcium phosphate (TCP) (28, 29, 33–34), octacalcium phosphate (35), calcium-deficient hydroxyapatite (36, 38), substituted hydroxyapatite (39, 40), fluorapatite (41, 42),chlorapatite (41, 43, 44), and hydroxyapatite (45–51). Hydroxyapatite [Ca10(PO4)6(OH)2]is commonly used as a bone substitute because its chemical structure is very similar to

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Raman Spectroscopy of Natural Bone 337

the mineral component of bone (52). Thermodynamically it is the most stable calciumphosphate (53). HA follows an osteoconductive (54, 55) pathway that causes bone togrow along the material without any intercellular response from the hard tissue (56). HA isproduced using several techniques, including sol-gel (57, 58), wet precipitation (51, 59–61),liquid mix (62, 63), mechanical alloying (64, 65), aerosol (66, 67), and hydrothermal(68–71) routes.

The Raman spectra of HA over its whole optical frequency range mainly consist offour scattering bands, which can be associated with four frequencies (v1, v2, v3, and v4) ofthe PO4 group (5, 72–81), as in the case of bone. The bands in the range between 1020 and1095 cm−1 (especially a medium peak at 1046 cm−1 and weak peaks at 1030, 1054, and1076 cm−1) are assigned to the triply degenerate asymmetric stretching mode (ν3) of thePO4 group (P-O bond) (61, 72, 74, 75, 77). A very strong peak in the region 960–963 cm−1

is assigned to the symmetric stretching mode (v1) of the PO4−3 tetrahedron (72, 73, 78,

80–82). The peaks ranging from 570 to 625 cm−1 (especially a medium peak at 594 cm−1

and weak peaks at 582, 610, and 620 cm−1) correspond to the triply degenerate bendingmode (ν4) of the PO4 group (O-P-O bond), in addition the shoulder peak at 571 cm−1, arerelated to factor group components of v4 (72, 73, 78, 80–82). Similarly, a medium peak at447 cm−1 and a weak peak at 433 cm−1 are attributed to the doubly degenerate bendingmode (ν2) of the PO4 group (O-P-O bond) (61, 72, 74, 75, 77). In addition, several peaksdetected in the range from 50 to 320 cm−1 are the translational modes of Ca2+ and PO4

3−

sublattices and rotational modes of the PO43− group (78).

Comparison of Hydroxyapatite with Natural BoneThe chemical and structural properties of HA were thought to be similar to natural bone

(83). Rehman et al. (16) utilized Raman spectroscopy to compare the properties of bonewith that of synthetic HA. Both human whole and deproteinated bones were employed inRehman et al.’s (16) study. The Raman spectra of synthetic HA were found to be differentfrom that of whole natural bone (due to the presence of the proteinous part in the spectrumof whole bone); however, no significant difference was observed for the deproteinatedhuman bone (Figure 6) (16, 84).

Figure 6. Comparison of synthetic HA (84) with human bone (16) (Color figure available online.)

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Table 4Raman band positions and assignments of different peaks

Peak position (cm−1) Assignment

431m, 449w ν2 bending O-P-O, doubly degenerate581w, 592m, 608w ν4 bending O-P-O, doubly degenerate664vw ν4 C-O-A730vw ν4 C-O-B963vs ν1 symmetric stretching P-O1048m, 1078w ν3 asymmetric stretching P-O, triply degenerate1069 ν1 C-O-B1114 ν1 C-O-A

s = strong; m = medium; vs = very strong; w = weak; vw = very weak; b = broad; sh = shoulder.

Substituted HydroxyapatitesCarbonate-Substituted Hydroxyapatite. In bone mineral, carbonate (CO3

−2) is themost abundant substituent, with 2.3–8 wt%, and the amount is dependent upon the age ofthe individual (85). CO3

−2 ions play an important role in bone metabolism (86). Reportsin the literature (87) have elucidated that CO3

−2 ions have a strong impact on the proper-ties of HA: (1) they decrease crystallinity, (2) reduce crystal growth, and (3) increase thesolubility of HA in the acidic environment. These properties result in improved clinicaltreatment effects in bone repair. Carbonated apatites can be classified into three differentclasses depending upon their position in the crystal lattice: AB-type, where CO3

−2 oc-cupies both PO4

3− and OH− sites; A-type, where its ions occupy the OH- sites (placedin the channels); and B-type, where CO3

−2 ions occupy the PO4−3 sites. On the ba-

sis of charge neutrality, the chemical formula of carbonated hydroxyapatite (CHA) isCa10−x/2[(PO4)6-x(CO3)x][(OH)2–2y(CO3)y], where x and y are the number of CO3

−2 ionssubstituting PO4

−3 and OH−, respectively (88). Bone mineral apatite mainly consists ofB-type carbonate and mixed AB type, whereas in older bones the A-type substituted apatiteis more common (89). The Raman band positions and assignments of different peaks arelisted in Table 4.

A series of B-type carbonate-substituted HA was prepared by Awonusi et al. for Ramanstudies of CHA (90). The Raman spectra of CHA containing different concentrationsof carbonate (0.03–7.9%) are shown in Figure 7. Significant changes occurred in the900–1100 cm−1 region. The band at 1070 cm−1 resulted due to a carbonate v1 peak, andthe intensity of the peak was largely dependent on the amount of carbonate ions present inthe inorganic matrix of bone. The phosphate v3 peak at 1076 cm−1 maintained a constantarea and height; however, it was enveloped by the carbonate v1 peak when the carbonateconcentration exceeded 3% (Figure 10).

Fluorine-Substituted Hydroxyapatite. Fluorine-substituted hydroxyapatite (FHA)contains both hydroxyl and fluoride in its structure, whereas in fluorapatite, the hydroxylgroup is completely replaced by fluoride ions. This occurs naturally in both tooth and boneand helps to reduce susceptibility to degradation as well as increases biocompatibility invitro and in vivo (91). The incorporation of fluorine in HA also causes changes in the phys-iochemical properties of HA by changing the solubility, biocompatibility, and resorptionrate of a graft in a living body. Fluoride has been used in the treatment of osteoporosis toincrease bone mineral density (92). The general formula for FHA is Ca10(PO4)6(OH)2–2xF2x

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Raman Spectroscopy of Natural Bone 339

Figure 7. Raman spectra of a synthetic carbonated apatite (2.2% carbonate). Insets show expandedsections of spectra of samples with carbonate contents of (a) 0.03% (NIST standard; standard hy-droxyapatite powder [SRM 2910] obtained from the National Institute of Standards and Technology[NIST], Gaithersburg, MD), (b) 1.1%, (c) 2.0%, (d) 4.7%, (e) 6.9%, and (f) 7.9% (90).

with varying fluoride contents (x = 1 fluoroapatite, 0 < x < 1 for FHA) (91). FHA is gen-erally synthesized using CaF2 and hydroxyapatite by using different weight percentages ofCaF2 (93) using a pH recycling method (94). In Figure 8, the Raman spectra of HA arecompared to those of FHA (95). The dominant peak in the spectra belongs to phosphatevibrations. The v3 vibrations of the phosphate group of HA, FHA, and FA are shown inFigure 8. The Raman spectra of HA and FHA did not show any remarkable difference with

Figure 8. FT-Raman spectra v3 vibrations of HA, FHA, and FA (96).

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Figure 9. Raman spectra of HA and FA in the region of the v1 vibration (95) (Color figure availableonline.)

fluoride substitution ranging from 5 to 95%, which shows that the phosphate environmentof FHA is very similar to HA. However, in the case of FA the splitting pattern at v3 wasvery different from HA or FHA. The blue shift of the v1 peak was also observed in FAcompared with HA. This blue shift of v1 was mainly attributed to the volume reduction ofthe unit cell (95, 96) (Figure 9).

Manganese-Substituted Hydroxyapatite. HA has a significant ability to exchange Ca+2

ions with similar metallic additives that modify its crystalline structure and physiochem-ical and biological properties; manganese (Mn) is one such additive. Mn is important,from a biological point of view, for the synthesis of compounds responsible for cartilageformation, namely, mucopolysaccharide. A deficiency of Mn may cause lower activity ofthe osteoblasts, which may result in delayed osteogenesis processes (97). Incorporation ofMn2+ in HA lattice influences the adhesion of bone cells to implant materials, their spread,and viability. The Mn-HA can be synthesized using different techniques like wet chemicaland coprecipitation methods (98, 99). The degree of Mn2+ substitution in HA depends onthe amount of manganese and the temperature of calcination. Mn-HA powders doped with0.1 to 1 wt% of Mn are stable up to 800◦C and begin to show the presence of MnO4

−3 whensintered above 800◦C (Figure 10). When the Mn content in HA is 5 wt% it decomposes at800◦C into α-TCP, β-TCP, and Mn3O4 as a secondary phase. In addition, when sinteredat high temperature the presence of oxides of Mn results in a change in the color of thesample. This color change depends on the calcination temperature, including grey–green,grey, brown, or black (99). The Raman spectra of the samples calcined at 800◦C showed thepresence of symmetric vibrations of O-P-O bonds with an intense band at 960 cm−1. Thebands at ca. 435 cm−1 (v2), 590 cm−1 (v4), and 1075 cm−1 (v3) correspond to characteristicvibrations of PO4

−3 groups occurring in the HA structure (65). The bands that appear at ca.820 and 828 cm−1 in the Raman spectra of MnHA materials are due to v1 and v3 vibrations,respectively, of MnO4

−1 (100).

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Raman Spectroscopy of Natural Bone 341

Figure 10. Raman spectra of HA powders modified with (a) nonmodified material, (b) 0.1 wt%, (c)0.5 wt%, (d) 1.0 wt%, and (e) 5.0 wt% of Mn. Calcination temperature: 1250◦C (99).

Si-Substituted Hydroxyapatite. Silicon is an essential element for connective tissuesand healthy bones. Additionally, silicon-based biomaterials play an important role in osteo-integration of implants due to in vivo and in vitro biological responses (101). Hing et al.(102) highlighted the sensitivity of healing response to Si level in the femoral intercondylarnotch of New Zealand white rabbits and suggested that an optimal response can be obtainedwhen HA is substituted with 0.8 wt% Si by examining its effect on the activity of bothbone-forming and bone-resorbing cells. Similarly, some reports (103, 104) have claimedthat inclusion of Si in HA improves its in vivo activity and proves to be a better alternateto conventional HA materials for bone replacement applications; however, it is still notclear that why Si substitution positively influences the biological response in Si-substitutedcalcium phosphates, in particular, Si-substituted HA. Presently, several explanations canbe put forward, such as a change in surface chemistry or a change in surface topography(105). Si-HA has been synthesized utilizing many techniques, such as precipitation andmechanochemical methods using different Si contents (0–2 wt%) (106, 107). It has beenreported in a number of different analytical studies that Si substitutes the phosphate site inthe HA structure in the form of SiO4

−4 (108). It has also been suggested that the negativecharge of the SiO4

−4 ion substituting PO4−3 is balanced by the creation of hydroxide

vacancies (107) and thus leads to the general formula Ca10(PO4)6-x(SiO4)x(OH)2-x. It hasbeen reported that at silicon substitution greater than 2%, HA starts to destabilize andα-TCP is formed when heat-treated at 1100◦C or higher (109). In the Raman spectra ofSi-HA typical phosphate vibrations were observed. No special peak could be assigned tosilicate to differentiate it from the Raman spectra of HA; however, the intensities of thehydroxyl (OH) absorption band at 3571 cm−1 were slightly less than that of the pure phaseHA. These results indicated that the presence of silicon can result in breakdown of the OHgroup present in HA (Figure 11) (110).

Magnesium-Doped Hydroxyapatite. Magnesium is an important trace element, which,despite its low concentration (0.5–1.5 wt%), plays an important role in bone metabolism.It is particularly important in early stages of osteogenesis where it stimulates osteoblast

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Figure 11. Raman spectra of various Si-HA sintered at 1000◦C (110) (Color figure available online.)

proliferation (111) and its depletion results in bone loss and bone fragility (112). Manyresearchers around the world (40, 68, 112–117) are working on the preparation of Mg-containing hydroxyapatite (Mg-HA) because it has shown bioactivity (113). Due to a largesize difference between Mg+2 and Ca+2 the replacement of calcium by magnesium islimited. This difference results in the strong distortion on the crystal lattice of HA andeventually reduces crystallinity. Thus, Mg-HA can be more easily converted to substi-tuted β-TCP (β-Ca3-xMgx(PO4)2). Mg-HA also increases biocompatibility and solubilityin physiological fluids (114). It was also reported that higher concentrations of Mg canbe observed at the surface of HA if it is cosubstituted with other ions like CO3

−2 (115,116). There are a few reports on the exact position of Mg in HA lattice; however, it isstill unclear whether it occupies one or both crystallographic positions of Ca. Accordingto Sprio et al. (117), Mg occupies the Ca(I) position, whereas Bigi et al. suggested the CaII (118) position. More recently, using density functional theory, it was suggested (119)that Mg+2 incorporation is more suitable at the Ca(II) position than at the Ca(I) position.However, this is still an open question. Raman spectroscopy proves to be more useful thanother techniques to investigate the efficiency of the introduction of Mg+2 in the structureas well as the presence of other phases. Diallo-Garcia et al. (120) synthesized magnesium-substituted hydroxyapatite with the formula Ca10-xMgx(PO4)6(OH)2 with x ranging from0.25 to 2 using a precipitation method (Table 5).

Diallo-Garcia et al. (120) used Raman spectroscopy to investigate the efficiency of theintroduction of Mg+2 in the structure as well as the presence of other phases. The classicalpeak at 960 cm−1 for the v1 vibration mode of PO4

3− was present in all samples containingdifferent quantities of Mg+2. Peaks at 959 and 975 cm−1 for the whitlockite phase wereobserved in an Mg1.54 HA sample when heat-treated at 1173◦C. Broadening of the bandat 960 cm−1 was also observed for all samples except for Mg1.54 HA (Figure 12). Thereason for the broadening may be associated with the appearance of a new vibration bandcentered at 950 cm−1, which could be due to the substitution of Mg in the HA lattice ordecomposition of Mg-HA into magnesium-substituted β-TCP (Ca3-zMgz(HPO4)x(PO4)2-x/3

(78). However, the difference between the values of v4 and v2 (12 cm−1) was more consistentwith HA (130–150 cm−1) than β-TCP (55 cm−1), which indicates that peak broadeningmay be due to cationic disorder (117).

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Raman Spectroscopy of Natural Bone 343

Table 5Sample labeling of nonsubstituted HA (Ca-HA) and Mg-HA (Ca10-xMgx(PO4)6(OH)2)samples and OCP sample, sample compositions, expressed as Ca/P, Mg/P, and (Ca +

Mg)/P ratio calculated from ICP analysis

Sample name x Mg/P Ca/P Mg+Ca/P

Ca-HA-2 0.00 1.67 (1.67) 1.67 (1.67)Mg0.5-HA-2 0.50Mg1-HA-1 1.00 0.077 (0.082) 1.55 (1.50) 1.68 (1.67)Mg1.5-HA-2 1.50 0.185 (0.250) 1.44 (1.42) 1.62 (1.67)Mg1.54-HA 1.54 0.197 (0.257) 1.38 (1.41) 1.59 (1.67)Mg2-HA 2.00 0.249 (0.333) 1.36 (1.33) 1.61 (1.67)

Nominal expected compositions are reported in brackets.

Calcium-Deficient HABone mineral is essentially calcium-deficient HA (CDHA) in which Ca/P is 1.5. The

chemical properties and composition of CDHA are very similar to TCP, whereas in terms ofstructure it is related to HA. Several general formulae, all with six phosphate ions per unitcell and one or two variables, have been proposed for CDHA with a Ca/P mole ratio varyingfrom 8/6 to 10/6 (Table 6). The efficiency of CDHA in the induction of precipitation isgreater compared to HA (121). Similarly, the solubility of CDHA in water is much higherthan HA. In addition, CDHA has higher surface area compared to HA and TCP and ithas more reproducible seeding efficacy (122). Due to these significant advantages, CDHAhas been utilized for bone substitution, implant coating, drug delivery, and so forth (123).There are characteristic peaks in the Raman spectra at 964 and 1046 cm−1 of P-O stretchingfrequency and at 433 and 594 cm−1 of O-P-O bending frequency due to PO4

−3 of HA(124). The absence of a florescence band around 690–770 cm−1 (125), which arises fromthe hexagonal structure of stoichiometric HA, is clear evidence of perturbed CDHA due

Figure 12. Raman spectra of Ca-HA-2, Mg0.5-HA-2, Mg1-HA-1, Mg1.5-HA-2, Mg2-HA-2, Mg1.54-HA, and Mg1.54-HA further calcined at 1173 K (120).

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Table 6Proposed chemical formulas of CDHA from the literature

No Formula Conditions Reference

1 Ca10-x(HPO4)2x(PO4)6–2x(OH)2 0 ≤ x ≤ 2 (139)2 Ca10-x(HPO4)x(PO4)6-x(OH)2-x 0 ≤ x ≤ 2 (140)3 Ca10-x-y(HPO4)x(PO4)6-x(OH)2-x-2y 0 ≤ x ≤ 2, y ≤ 1 − x/2 (141)4 Ca10-x(HPO4)x(PO4)6-x(OH)2-x Ca/P = 1.5–1.67, 0 ≤ x ≤ 1 (142)5 Ca9-x(HPO4)1+2x(PO4)5–2x(OH)H2O Ca/P = 1.4–1.5, 0 ≤ x ≤ 1 (143)6 Ca10-x(HPO4)x(PO4)6-x(OH)2-x(H2O)x 0 ≤ x ≤ 1 (144)7 Ca9+z(PO4)5+y+z(HPO4)1-y-z(OH)1-y+z y + z ≤ 1 (145)8 Ca10–1/2x-1/2y(PO4)6-x(HPO4)x(OH)2-y 0 ≤ y ≤ 2; 0 ≤ x (123)

to incorporation of HPO42− (126). Acid phosphate bands were also reported at 878 and

1004 cm−1 (123).

Tricalcium PhosphateCalcium phosphate is extensively investigated due to its biocompatible and osteo-

conductive applications in bone grafting and as a coating material for metallic prostheses.Calcium orthophosphates are exhibited in the forms of HA as already discussed, amorphouscalcium phosphate, dicalcium phosphate dihydrate, octacalcium phosphate, and tricalciumphosphate.

Tricalcium phosphate exists in two phases, β-Ca3(PO4)2 and α-Ca3(PO4). A charac-teristic feature of TCP is the molar ratio of calcium and phosphate (Ca/P 1.5). The β formof TCP is thermodynamically stable and bioactive at room temperature, and its range ofstability is between 25◦C to about 1120◦C, whereas α-TCP is thermodynamically stablebetween 1140 and 1470◦C (127); below 1120◦C α-TCP is metastable. Ruan et al. (128)reported that α-TCP has a self-setting ability; this property is required for biomedical ma-terials of ear ossicle and dentals. The dissolution rate of TCP in biological fluid is fasterthan hydroxyapatite and between two phases β-TCP is dissolved to a greater degree. TCPis commonly used as a temporary support for regenerated bone and as a bone cement (129).

In order to characterize and distinguish TCP from other phases of calcium phosphate,there is a need for comprehensive data on the vibrational spectra of different phases ofcalcium phosphate. The Raman spectra of TCP have been reported in the literature (130).

Jillavenkatesa and Condrate (139) reported the Raman spectra of β- and α-TCP. Theymeasured Raman spectra of TCP powders using an ISA U-1000 double monochromatorRaman spectrometer. The Raman band of TiO2 observed at 142 cm−1 was used to calibratethe Raman spectrometer. Highly pure commercially available powder of β-TCP was usedfor this study and α-TCP was synthesized in their lab by a solid-state reaction of dicalciumphosphate and hydroxyapatite as shown in Eq. (1).

2CaHPO4 + Ca10(PO4)6(OH)2 → 4Ca3(PO4) + 2H2O (1)

The Raman spectra of β-TCP showed the most prominent bands at 948 and 970 cm−l.Both bands correspond to symmetric P-O stretching (v1) vibration of the phosphate ion.There are three distinct bands at 954, 964, and 976 cm−1 in α-TCP, corresponding to v1

vibrations. The band at 954 cm−1 has a weak shoulder at 964 cm−1. The isolated phosphateions have characteristic bending vibrations at 420 cm−l corresponding to doubly degenerate

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Raman Spectroscopy of Natural Bone 345

Table 7Frequencies (cm−1) of the Raman peaks of α-TCP and β-TCP related to PO4

−3 internalmodes

Peak positions (cm−1)

α-TCP β-TCP Assignment

954 (with a weak shoulderat 964)

948 v1, symmetric stretching ofP O

976 970567 (band splits into 563,

577, 593, 610, and 620)567 (band splits into no

distinct peak)v4, triply degenerate

fundamental vibrationalmode, bending modes ofO-P-O

420 (splits into 421 and451)

420 (splits into very weakbands at 419, 438, 458,

and 497)

v2 bending mode of PO4−3

v2 and 567 cm−l corresponding to triply degenerate v4 fundamental vibrational modes. Inthe Raman spectra of β-TCP, a v2 band of phosphate ions appeared at 420 cm−l, whichfurther split into very weak bands at 419, 438, 458, and 497 cm−1. α-TCP phosphate ionshave a very distinct band at 420 splitting subsequently into 421 and 451 cm−1 and the bandat 567 cm−1 is further divided into 563, 577, 593, 610, and 620 cm−l (Table 7).

Ruan et al. (128) reported structure and phase transitions of α-TCP between 212 and1264◦C. α-TCP is orthorhombic above 212◦C and monoclinic at room temperature. TheRaman spectra showed four sharp peaks for PO4

−3 (symmetrical expansion vibration) at952, 964, 973, and 984 cm−1 at 23◦C and at 230◦C and there was a single intense peak of967 cm−1, which indicates higher symmetry of α-TCP above 212◦C.

Comparison of TCP and HAIn 1997, de Aza et al. (132) reported a comparative study of the Raman spectra of

polycrystalline Ca10(PO4)6(OH)2 (HA) and β-Ca3(PO4)2 (β-TCP). Though the Ramanactivity of the OH−1 group of HA would apparently be sufficient to establish a differencebetween HA and TCP, the detection of OH−1 stretching vibration at 3576 cm−1 is not easyto observe in samples with a low degree of crystallinity. Therefore, other aspects of theRaman spectra of HA and β-TCP need to be explored in order to recognize these two typesof calcium phosphate.

The Raman spectra of HA and β-TCP have shown similar internal modes of PO4−3;

however, there are several characteristic features of the Raman spectra that make themdifferent. In β-TCP and HA the internal PO4

−3 bands are present at 20 cm−1 above thecorresponding free PO4

−3 with similar frequency values. This may be due to a reduction inthe interatomic distances in both crystals. Compared to HA, the internal bands of β-TCPextend over a wider frequency range. For HA a single peak appears at 961 cm−1 for v1

modes (A + E1 + E2), whereas for β-TCP there is a main peak at 971 cm−1, a wide peak at946–949 cm−1, and a shoulder at 961 cm−1. β-TCP has a wider frequency range for v2 andv4 type modes; therefore, bands related to them appeared close together compared to thewell-spaced bands in HA. A greater numbers of peaks were detected in the Raman spectra

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Table 8Raman peaks (cm−1) of polycrystalline β-TCP and HA for PO4

−3 internal modes (76)

v1 (cm−1) v2 (cm−1) v3 (cm−1) v4 (cm−1)

TCP HA TCP HA TCP HA TCP HA

946 405 1005 547949 430 1016 555961 439 1029 578

962 447 1031 579970 452 1033 588 588

460 1038 591475 1043 594483 1046 599

1048 6071059 1054 611 61074 615

1077 6241084 6311091

of β-TCP compared to HA, as shown in Table 8. The greater number of peaks appearingin β-TCP is due to the presence of three nonequivalent PO4

−3 tetrahedral structures. ForHA there are one, two, three, and four peaks for modes v1, v2, v3, and v4, respectively, asshown in Table 8. In HA the peak appearing at 615 cm−1 (v4) is due to OH−1 rotationalmodes (Figure 13).

Figure 13. Comparison between the Raman spectra of (a) HA and (b) β-TCP in the frequencyregions corresponding to the internal PO4

−3 modes. The intensity scale is the same for both spectra(133).

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Raman Spectroscopy of Natural Bone 347

Table 9Peak positions, relative areas v1 symmetric stretching Raman band for pure β-TCP,

Ca10Na(PO4)7, and Ca10K(PO4)7

Pure β-TCP Ca10Na(PO4)7 Ca10K(PO4)7

Peak position % Peak position % Peak position %(cm−1) Area (cm−1) Area (cm−1) Area

946 7.1 950 17.6 944 17.9949 7.1 956 36.5 957 36.3959 21.4 971 42.9 968 45.8962 21.4970 42.7

In contrast, in the v1 region β-TCP exhibits two peaks with one shoulder and threemajor broad peaks in the v2 region. The v3 modes display the largest spreading region withthree peaks and the v4 modes have six major peaks.

Substituted TCPThe biological activities and biodegradation of synthetic calcium phosphate materials

are improved by a range of ionic substitutions such as CaNaPO4, Ca2KNa(PO4)2, andCa10Na(PO4)7 (133). These substitutions can be monovalent, divalent, or trivalent. In1988, Oralkov et al. (146) reported that K+- and/or Na+-substituted β-TCP for the firsttime. K-, Li-, and Na-substituted β-TCP have better thermal stabilities (134) and theirmechanical strength is improved by Mg (135). Moreover, Mg increases the transition phasetemperature of the two phases (β and α) of TCP.

For analysis of TCP and its ionic substituents, infrared absorption and Raman scattering(vibrational spectroscopy) are practical tools. Quillard et al. (136) used the symmetricvibration v1, a prominent mode in Raman spectra, to detect the influence of potassium and/orsodium substitution in β-TCP. In β-TCP and substituted β-TCP, the different environmentsaround phosphate cause small frequency shifts. The Raman spectra for pure and substitutedβ-TCP were recorded (136) and the v1 vibration range was 920–1000 cm−1 for β-TCPsamples. For pure TCP samples, there are five bands 946, 949, 959, 962, and 970 cm−1.The relative areas are in the ratios of 7.1, 7.1, 21.4, 21.4, and 42.7%, corresponding to P(1),P′(1), P(2), P′(2), and P(3), respectively. In substituted samples of β-TCP with sodium orpotassium, three new bands were formed and bands related to pure β-TCP decreased as thepercentage of substitution increased. For fully Na- or K-substituted β-TCP, there are threebands. Their peak positions and percentage areas are shown in Table 9. The most stablefrequency related to the PO4

−3 group appeared at 970 cm−1 for pure β-TCP, 971 cm−1 forNa, and 968 cm−1 for K-substituted β-TCP.

Summary

The quality of the structural and compositional details that can be revealed by Ramanspectroscopy makes it a very attractive technique for the analysis of natural bone tissuesand synthetic analogue materials (apatite powders). To analyze neat (no significant samplepreparation) materials, bone, or apatite powders, Raman spectroscopy offers an attractivechoice for characterization without the need to reduce the particle size or dilute with

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348 A. F. Khan et al.

KBr, allowing analysis of biomaterials extracted directly from physiological conditions.Quantitative analysis is also possible to confirm the presence of more carbonate moiety inthe natural bone, compared to commercial hydroxyapatite and carbonate-substituted apatitepowders.

Raman spectroscopy is a powerful technique used to characterize both natural andsynthetic materials and provides a complete picture of the chemical and structural charac-terization of natural bone and its replacement materials.

Spectral information on synthetic apatite showed a number of peaks similar to thoseof human and sheep bone. Nevertheless, the spectra indicate that there are a number ofdifferences between synthetic hydroxyapatite and bone. The most obvious difference isthe presence of a hydroxyl stretch on the hydroxyapatite powders and the absence of ahydroxyl stretch on the bone spectra. In addition, a greater amount of carbonate is presentin bone compared to synthetic hydroxyapatite, the two proofs of which are that the peakarea is much larger in bone and the phosphate : carbonate ion ratio is larger in synthetichydroxyapatite.

Raman spectroscopy provides baseline data about the chemical and structural propertiesof the inorganic and organic matrices of the bone, which is crucial in mimicking theproperties of bone and introducing the same amount of ionic substitution within the latticestructure of apatite powders. This makes Raman spectroscopy an ideal technique that can beadded to the armory of biomaterials scientists who are interested in research on bioceramics.

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