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The influence of transition metal ions on collagen mineralization
Materials Science and Engineering C 33 (2013) 2399–2406
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
The influence of transition metal ions on collagen mineralization Mingfen Jia a, Yuanping Hong a, Shuyuan Duan a, Yongjun Liu b, Bo Yuan b, Fengzhi Jiang a, b,⁎ a b
Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming, 650091, PR China Advanced Analysis and Measurement Center, Yunnan University, Kunming, 650091, PR China
a r t i c l e
i n f o
Article history: Received 5 June 2012 Received in revised form 11 January 2013 Accepted 4 February 2013 Available online 10 February 2013 Keywords: Transition metal ions Collagen Hydroxyapatite Mineralization Collagen sponge
a b s t r a c t The ions in body fluid play an important role in bone formation besides being a synthesizing material. Transition metal ions Co 2+, Ni 2+, Zn2+, Fe 3+, Mn2+, Cu 2+, Cd 2+ and Hg2+ doped hydroxyapatite (HAP)/collagen composites were synthesized successfully in the presence of collagen traces at mild acidic pH for the first time. However, the amount of doped Hg2+ and Cd2+ was relatively low. Meanwhile, through soaking the collagen sponge as a template in simulated body fluid (SBF) which contains different transition metal ions (Mn2+, Cu 2+, Ni 2+, Co 2+, Cd 2+, Hg 2+), bone-like HAP/collagen composites were synthesized. Hg 2+ had a certain inhibitory effect on the formation of HAP crystals on the surface of the collagen sponge while Co2+ can promote the formation of HAP on the collagen sponge. For both HAP/collagen composites and HAP/collagen sponge, it was found that transition metal ions Mn2+ had a significant effect on the morphology of HAP particles and could induce to form floc-like HAP particle aggregates. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Natural bone is a kind of hydroxyapatite (HAP)/collagen composite which is in a complex hierarchical structure [1,2]. It has attracted considerable attention for its unique structure and mechanical properties. In fact, bone formation is a biological process in which deposition of inorganic salts was induced by a template of collagen network formed through self-assembly. But knowledge of the detailed construction process of bone in vivo still remains at an early stage. The development of biocompatible materials for hard tissue repair and replacement has been studied for a long time [3]. Among these materials, HAP/collagen composite is of great interest if it is synthesized by biomimetic strategy inspired by natural bone [4–9]. And it is promising in clinical application because of its compositional and partly structural analogy to natural bone [10–14]. Meanwhile, the ions in body fluid play an important role in bone formation besides being a synthesizing material. As it is well known, Ca2+ and some trace element ions (Co2+, Ni2+, Zn2+, Fe3+, Mn2+, Cu2+) are important for normal growth and metabolism of bone tissues [15–17]. Recent researches have shown that cobalt ions directly inhibit osteoblast function [18] and Cr3+, Co2+, Ni2+can be incorporated into the hydroxyapatite crystal during mineralization and affect the crystal lattice parameters as well as the size of the crystal [19]. Study of the influence of special elements on collagen selfassembly and mineralization is helpful for preventing and treating bone disease caused by the special elements, as well as for developing new bone repairing materials with excellent property of promoting ⁎ Corresponding author at: Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming, 650091, PR China. Tel./fax: +86 871 5036200. E-mail address: [email protected] (F. Jiang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.02.008
bone formation, biocompatibility, bioactivity and biodegradability. Recently, calcium phosphate/collagen biocomposites were prepared in order to potentially enhance osteoconductivity as new bone substitutes that resemble tissue more closely than current available materials [20]. However, reports concerning the influence of the general traces on the collagen mineralization were still very limited so far. In this study, we used two methods to prepare HAP/collagen composites and tried to find out the influence of the traces on collagen mineralization from both methods. HAP/collagen composites were synthesized in solutions containing transition metal ions Co2+, Ni2+, Zn2+, Fe3+, Mn2+, Cu2+, Cd2+ and Hg2+ at mild acidic pH. Meanwhile, through soaking the collagen sponge as a template in simulated body fluid (SBF) which contains different transition mental ions (Mn2+, Cu2+, Ni2+, Co2+, Cd2+, Hg2+), bone-like HAP/collagen composites were synthesized. In order to find out obvious influence of the various traces on collagen mineralization in a short experiment time, we used extremely high ion concentrations. Although the results could not reflect the situation in vivo, we still may find out some clues for the influence of different traces on collagen mineralization. The prepared composites were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) to investigate the effect of the transition metal ions on the shape and doping ratio of the deposited HAP.
2. Materials and methods 2.1. Materials Type I collagen solution was purchased from Hangzhou Biological Technology CO LTD. CaCl2, Na2HPO4·12H2O, NiCl2·6H2O, CoCl2·6H2O,
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FeCl3·6H2O, CuCl2·2H2O, CdCl2, HgCl2, ZnSO4·7H2O, MnSO4·H2O,HCl and NaOH were of reagent grade and ultra-pure water was used as solvent. The collagen sponge (from Beijing Yierkang Bioengineering Development Center) used in the experiment was a sponge-like tissue engineering scaffold and its molecular precursor is the type I collagen. 2.2. Preparation of transition metal ions containing SBF
Table 2 Composition of SBF contains different transition metal ions. Serial number
Components
Concentration of transition mental ions (mM)
1 2 3 4 5 6 7
SBF + CuCl2 SBF + CoCl2 SBF + NiCl2 SBF + MnSO4 SBF + CdCl2 SBF + HgCl2 SBF
[Cu2+] = 0.5 [Co2+]= 0.5 [Ni2+]= 0.5 [Mn2+] = 0.5 [Cd2+] = 0.5 [Hg2+]= 0.5 —
An SBF solution [21] with ion concentrations close to those of human blood plasma [22] (Table 1) was prepared by dissolving NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2, and Na2SO4 in ultra-pure water and the solution was divided into seven shares. To investigate the effects of metal ions on the mineralization of collagen, the SBF was supplemented with 0.5 mM of Ni2+, Co2+, Mn2+, Cu2+, Cd2+, or Hg2+ respectively except the seventh share. The solution was buffered at pH 7.4 with 1 M tris (Hydroxymethyl) aminomethane and 1 M HCl at 36.5 °C, and the ionic concentration of the solution was shown in Table 2. The solution was filtered using a Filtropur syringe filter (Sarstedt, Germany).
shaker (SKY-100 shaker incubator) at 36.5 °C and 80 rpm. After 5 days, collagen sponges were taken out and gently rinsed with ultrapure water, and freeze-dried. The mineralization time for collagen sponges was chosen as 5 days. The mineralization growth on sponges was not homogeneous. Within the beginning 3 days, no precipitate was observed. When the time was last to day 4, white precipitate appeared and another 24 h was run for complete mineralization.
2.3. Preparation of mineralized collagen composites
2.5. Materials characterization
Solutions of HCl (0.5 ml 1 M), transition metal solutions (10 ml 0.5 mM) and type I collagen solution (0.1 ml 5 mg/ml) were gradually added into CaCl2 solutions (5 ml 0.2 M) at 36.5 °C while stirring and then standing for 10 min after mixing. The transition metal solutions were one of NiCl2·6H2O, CoCl2·6H2O, FeCl3·6H2O, CuCl2·2H2O, CdCl2, HgCl2, ZnSO4·7H2O and MnSO4·H2O. While stirring, Na2HPO4·12H2O solutions (3 ml 0.2 M) were added slowly into the above mixture solution and calcium phosphate started to co-precipitate with collagen. Then NaOH solutions (0.9 ml 1 M) were added slowly into the reaction system and a lot of white precipitate appeared. The pH of the solution was then adjusted to 5.2 by dropwise adding HCl solution (1 M) or NaOH (1 M). The molar ratio of Ca/P = 1.67 used in this experiment was equal to the stoichiometric ratio of HAP molecule Ca10(PO4)6(OH)2. The supersaturated solutions were placed in shaker incubator (SKY-100 shaker incubator) and maintained at 36.5 °C for 24 h with the rotation speed of 100 rpm. Then the precipitates were collected by centrifugation at a speed of 5000 rpm and suspended in deionized water to remove the salts. The centrifugation and suspension cycle were repeated 4 times. Then the collected precipitates were washed with ethanol. Finally, the sample of a mechanically poor hydrogel was dried in vacuum at 60 °C for 12 h to get powder. In the preparation procedure, the precipitate formed at the beginning of the process and the least incubating time was determined according to the situation of collagen gelification. Collagen fibrils stabilized over 4–5 h while the solution was standing. In our experiment, a longer time of 24 h was chosen since the solution was in the shaker incubator.
Morphology and microstructure of the collagen mineralization composite and mineralized collagen sponge were examined by SEM (FEI QUANTA200) observation at 30 kV. To prepare the test sample, the synthesized powders were ultrasonically dispersed in ethanol to form dilute suspensions which were dropped on silicon slice. After the samples were dried, they were covered with a fine gold layer for SEM measurement. An energy-dispersive X-ray (EDX) analyzer attached to the SEM was used to determine the elemental composition of HAP/collagen composites. For the mineralized collagen sponge, a fine gold layer was applied before SEM measurement. The crystallinity of the prepared materials was investigated by X-ray powder diffractometry. The XRD pattern was recorded on a Rigaku D/max-3B diffractometer using the monochromatized X-ray beam from graphite filtered CuKα radiation at 40 kV and 30 mA. The 2θ range was from 10° to 60° at a scanning speed of 10°/min. In order to analyze the composite and the combination of the inorganic and organic phase in the prepared materials, FTIR spectroscopy (Thermo Nicolet Avatar 360) measurements were taken. The spectra were obtained using KBr pellets over the wave number range of 1800–400 cm −1 with a resolution of 2 cm −1.
2.4. Preparation of mineralized collagen sponge Collagen sponge was cut into small sheets of 1 cm× 1 cm× 0.2 cm in size and then the sheets were numbered (the number is as same as the serial number of Table 2) and immersed in 15 ml SBF or SBF containing transition metal ions for 5 days in the constant temperature breeding
Table 1 The comparison of ionic concentration between blood plasma and SBF. Species
Blood plasma SBF
Concentration (mM) Na+
K+
Mg2+
Ca2+
Cl−
HCO3−
HPO42−
SO42−
142.0 142.0
5.0 5.0
1.5 1.5
2.5 2.5
103.0 147.8
27.0 4.2
1.0 1.0
0.5 0.5
3. Results and discussion 3.1. Influence of transition metal ions on the morphology of mineralized collagen composite particles HAP/collagen composite is usually synthesized at alkaline condition. Formation of crystalline hydroxyapatite was described by idealized chemical reaction of 10Ca 2+ + 6PO43− + 2OH− → Ca10(PO4)6(OH)2. It is favorable for the nucleation of hydroxyapatite crystal at alkaline condition [23]. However, it was reported that hydroxyapatite crystal was also synthesized successfully and was stable at acidic pH [24]. In our study, in order to avoid the transition metal ions from forming precipitation at alkaline condition, HAP/collagen composites were synthesized in solutions containing transition metal ions Co2+, Ni2+, Zn2+, Fe3+, Mn2+, Cu2+, Cd 2+ and Hg2+ at mild acidic pH. Fig. 1 showed the SEM micrographs of synthesized HAP/collagen composite powders obtained from the solutions containing: (a) no transition metal ion, (b) Co2+, (c) Ni2+, (d) Zn2+, (e) Fe 3+, (f) Mn2+, (g) Cu2+, (h) Cd2+, and (i) Hg2+ at mild acidic pH. The insets of Fig. 1 were SEM images at higher magnification of the corresponding materials. Although we called the prepared material as “collagen/HAP composites”, it could be thought as composite of HAP powder
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Fig 1. SEM images at 1000 × magnification and 10,000 × magnification (insets) of the synthesized HAP/collagen composites in solution containing (a) no transition metal ion;(b) Co 2+;(c) Ni 2 +;(d) Zn 2 +;(e) Fe 3+;(f) Mn2 +;(g) Cu2 +;(h) Cd2 +;and (i) Hg2 +.
containing trace collage since in our preparation system the molar ratio of collagen was low. When the composite was synthesized in the solution without transition metal ions (Fig. 1a), a lot of irregular particles aggregated to form big pores. Meanwhile, the irregular particles were in a porous structure as shown in the inset of Fig. 1a. The prepared HAP/collagen composites revealed no fibrillar nature of collagen since the molar ratio of collagen in the composites was low. The shapes of synthesized HAP/collagen composite powders obtained from the solutions containing (b) Co2+, (c) Ni2+, (d) Zn2+, (g) Cu2+, (h) Cd2+, (i) Hg2+ were quite similar to that of in Fig. 1a with a porous structure. It suggested no significant influence of transition metal ions Co2+, Ni2+, Zn2+, Cu2+, Cd2+, and Hg2+ on the shape of HAP/collagen composite particles. Meanwhile, as can be observed in Fig. 1e, solution containing Fe3+ led to the attainment of much smaller porous particles with respect to those in solution without added transition metals. However, in the solution containing Mn2+, most HAP deposits were in a loose flocculus-like structure although few flaky crystals can be observed (Fig. 1f). SEM images of untreated collagen sponge and mineralization of collagen sponge in SBF containing different transition mental ions were shown in Fig. 2. Fig. 2a is the SEM image of the collagen sponge before being immerged. No inorganic salt deposition was observed on the collagen sponge substrate as shown in Fig. 2a. Compared to the mineralized collagen sponge in SBF shown in Fig. 2b, Cu 2+ and Ni 2+ have no significant effect on the morphology of HAP particles and similar deposited HAP particles were observed as shown in
Fig. 2d and e. However, the size of HAP particles formed in SBF containing Co 2+ (Fig. 2f), Cd 2+ (Fig. 2g) and Hg 2+ (Fig. 2h) was smaller than that in SBF (Fig. 2b) and the coverage ratio of HAP on collagen sponge was different from that in SBF. To quantify the coverage of HAP on collagen sponge, we printed the SEM images and cut the paper into covered and uncovered parts, then weighted the two parts to give the coverage ratio. The coverage ratio equals to the ratio of weight of covered part to the total weight of covered and uncovered parts. Coverage ratios of HAP on collagen sponge for SBF, SBF + Co 2+, SBF + Cd 2+ and SBF + Hg 2+ were 32.3%, 52.4%, 33.8% and 24.3%, respectively. That is, Co 2+ in the SBF significantly increased the coverage of HAP on the surface of collagen sponge (Fig. 2f) and Hg 2+ decreased the coverage (Fig. 2h) while Cd 2+ maintained the similar coverage (Fig. 2g). Interestingly, we can clearly see that the morphology of the HAP particles in SBF containing Mn 2+ (Fig. 2c) was totally different from that in SBF and a lot of floc aggregates formed on the surface. Meanwhile, Mn2+ in the SBF leads a significant increased coverage ratio (67.4%) for HAP on the surface of collagen sponge (Fig. 2c). Similar floc aggregates were also observed in the HAP/collagen composite in solution containing Mn2+ as shown in the inset of Fig. 1f. The difference of Figs. 2c and 1f was due to the fact that the deposition time was quite different. The deposition time was 24 h for Fig. 1f whereas deposition time was 5 days for Fig. 2c. For the studied transition metal ions, Mn2+ could significantly change the shapes of the HAP particles deposited on the collagen sponge while Co2+, Cd2+ and Hg2+ may change
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Fig. 2. The SEM images of untreated collagen sponge and mineralization of collagen sponge in SBF and SBF containing different transition mental ions (a) untreated collagen sponge; (b) SBF; (c) SBF + Mn2+; (d) SBF + Cu2+; (e) SBF + Ni2+; (f) SBF + Co2+; (g) SBF + Cd2+; and (h) SBF + Hg2+.
the size of the HAP particles on the collagen surface. Meanwhile, the coverage of HAP can be increased by Mn2+ and Co2+ or decreased by Hg2+. In order to compare the morphology of hydroxyapatite particles found in HAP/collagen sponge, the different particle sizes and coverage percentage from SBF containing different metal ions were listed in Table 3. 3.2. Doping the collagen mineralized composite with transition metal ions In order to check the doping transition metals in the collagen mineralization composite, EDX analysis was performed. The EDX spectra of
the synthesized HAP/collagen composites powders in mild acidic pH solutions containing (a) Co2+, (b) Ni2+, (c) Zn2+, (d) Fe3+, (e) Mn2+, (f) Cu2+, (g) Cd2+ and (h) Hg2+ were shown in Fig. 3. From Fig. 3, we can clearly see that the peaks of Ca and P were pretty high for all synthesized composites since these two elements were the major content in the composite. Meanwhile, the appearance of C and O demonstrated that HAP in the prepared composite was carbonate substitutive HAP. Meanwhile, the peaks of the transition metals were very low since their concentrations in solutions were all as low as 0.5 mM. Taking Mn2+ for example, its mass concentration was 27.5 μg/ml, which was much higher than that in human blood plasma of 0.83 μg/100 ml. Similarly, the concentrations of other transition metal ions in the solutions
M. Jia et al. / Materials Science and Engineering C 33 (2013) 2399–2406 Table 3 Comparing of hydroxyapatite morphology found in HAP/collagen sponge. Species
HAP HAP HAP HAP HAP HAP HAP
(SBF) (SBF + Mn2+) (SBF + Cu2+) (SBF + Ni2+) (SBF + Co2+) (SBF + Cd2+) (SBF + Hg2+)
The hydroxyapatite morphology Particle size (μm)
Coverage percentage (%)
1.1–1.6 0.07–0.15 0.4–1.5 0.5–1.5 0.1–0.9 0.1–0.3 0.2–0.9
32.3 67.40 35.18 32.34 52.4 33.8 24.3
were much higher than that in human blood plasma. If all of Mn2+ in the solution were doped into the composite, the mass ratio of Mn to the synthesized HAP/collagen composite was about 2.75‰. Similarly, if doping of other transition metal elements was successful, the mass ratios were all higher than 2.75‰ since their mole masses were greater than
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that of Mn. The detection limit of the EDX equipment was higher than 1‰. Compared to Ca and P, the peaks of Zn, Mn, and Cu were too low to see as shown in Fig. 3. But the doped weight rations of the transition metals were measured and recorded by EDX. The EDX spectra demonstrated the presence of Co, Ni, Zn, Fe, Mn and Cu in the composite constitution. Therefore, it was believed that these elements had been successfully doped into the synthesized HAP/collagen composites. However, no elements other than Ca, P, C and O were detectable in Fig. 2g and h. This means that the amounts of Cd and Hg in the synthesized HAP/collagen composites were very little and lower than the EDX sensibility limit of 1‰. 3.3. XRD analysis of the HAP/collagen composites powder and mineralized collagen sponge XRD patterns of synthesized HAP/collagen composites were shown in Fig. 4. The XRD patterns of all prepared HAP/collagen composites in solutions containing different transition metal ions at mild
Fig. 3. The EDX spectra of synthesized HAP/collagen composites in solutions containing (a) Co2+; (b) Ni2+; (c) Zn2+; (d) Fe3+; (e) Mn2+; (f) Cu2+; (g) Cd2+; and (h) Hg2+.
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M. Jia et al. / Materials Science and Engineering C 33 (2013) 2399–2406 Table 4 Lattice parameters of standard HAP and HAP in HAP/Collagen composites. Species
Standard HAP HAP/collagen HAP/collagen (Co2+) HAP/collagen (Ni2+) HAP/collagen (Zn2+) HAP/collagen (Fe3+) HAP/collagen (Mn2+) HAP/collagen (Cu2+) HAP/collagen (Cd2+) HAP/collagen (Hg2+)
Fig. 4. The XRD patterns of HAP/collagen composites prepared in the solutions containing (a) no transition metal ion; (b) Co2+; (c) Ni2+; (d) Zn2+; (e) Fe3+; (f) Mn2+; (g) Cu2+; (h) Cd2+; and (i) Hg2+.
Lattice parameters a
b
c
9.424 9.4004 9.3849 9.5897 9.4336 9.4561 9.4706 9.4986 9.3910 9.4491
9.424 9.4004 9.3849 9.5897 9.4336 9.4561 9.4706 9.4986 9.3910 9.4491
6.879 6.8782 6.8869 6.8576 6.8566 6.8551 6.9256 6.9541 6.9548 6.8918
equation of Dhkl = kλ∕βhklcosθ [26]. All of the average crystal sizes of the prepared HAP particles in the HAP/collagen composites were calculated by XRD analysis software. The results demonstrated that all of the crystal sizes were in the range of 10–20 nm. In different metal ions containing solution, the crystal sizes of HAP in composite were as follows: 15 nm (no transition metal ion), 18 nm (Cu 2+), 17 nm (Cd 2+), 15 nm (Hg 2+), 16 nm (Ni 2+), 14 nm (Co 2+), 15 nm (Fe 3+), 15 nm (Zn 2+) and 16 nm (Mn 2+). The crystallographic structure of the mineralized collagen sponge was also studied with X-ray diffraction. Fig. 5 showed the XRD patterns of mineralized collagen sponge and commercially purchased collagen sponge. Only an extensive broadening peak the 2θ range of 10–30° was observed for the untreated collagen sponge. Several diffraction peaks (211, 222, 322) were clearly seen in the mineralized collagen sponge and the positions of main diffraction peaks correspond well to the expected Bragg peaks for hydroxyapatite (ICDD PDF No.9-432). This indicated that the HAP crystal was formed on the surface of collagen sponge and the transition metal ions did not affect the crystal of the HAP. Compared with the standard HAP, the peaks of the inorganic phase in the mineralized collagen sponge become wider and several peaks overlapped together. This indicated that the crystallinity of inorganic phase was very poor and crystallite size was tiny, which closely resemble the natural bone tissue [25]. All of the average crystal sizes of the HAP on the collagen sponge were calculated by XRD analysis software. The results demonstrated that all of the crystal sizes were in the range of 10–15 nm. In SBF containing different metal ions, the crystal sizes of HAP on the collagen sponge were as follows: 10.4 nm (SBF); 13 nm (SBF + Mn 2 +); 15 nm (SBF + Cu 2+); 14 nm (SBF + Ni 2+); 13 nm (SBF + Co 2+); 14 nm (SBF + Cd 2+); and 12 nm (SBF + Hg 2 +). 3.4. IR analysis of the HAP/collagen composites
acidic pH correspond to that of standard HAP. No peaks other than HAP were detected. This confirmed that the inorganic phase in the synthesized HAP/collagen composites was calcium phosphate apatite. In order to find out the influence of the doped metal ions on the HAP crystal structure, lattice parameters of the composites were listed in Table 4. From Table 4, we can find that compared to the HAP standard sample, the lattice parameters of HAP in all the doped HAP/collagen composite changed while that of HAP in undoped composite was very similar. This means that doped transition metal ions Co 2+, Ni 2+, Zn 2+, Fe 3+, Mn 2+, Cu 2+, Cd 2+ and Hg 2+ have gotten into the HAP crystals and changed the crystal structure of HAP in collagen mineralization composites. Furthermore, metal ions of Cd 2+ and Hg 2+ were believed to be doped into the composites although the amount was lower than the EDX sensibility limit of 1‰. As can be seen in Fig. 4, the extensive broadening and overlapping of the peaks of the synthesized HAP/collagen were similar to that of natural bone [25], which indicated that the crystal grains of HAP were extremely fine. The average crystallite size of prepared HAP particles in the HAP/collagen composite can be estimated by Scherrer
In order to investigate the content of the prepared composites, FTIR spectroscopy measurements of commercially purchased HAP powder and synthesized HAP/collagen composites were taken (Fig. 6). For purchased HAP powder shown in Fig. 6a, the absorption bands at 1087, 1046, 962, 601 and 571 cm −1 were due to PO43−, and those at 1419 and 875 cm −1 were due to CO32− existing in the apatite [27]. According to the literature report, FTIR spectrum of untreated collagen fibrils showed the typical amide bands in protein: 1660 cm −1 was ascribed to amide I band; 1550 cm −1 to amide II and 1240 cm −1 to amide III [26,28]. For the synthesized HAP/collagen composites (Fig. 6b–j), we found that the amide I band shifted from 1660 cm −1 to 1640 cm −1 and the peaks of amide II and amide III disappeared because of the formation of the bonding between Ca ions and C_O bonds. The presence of CO32− in the structure of composites was believed to arise from the air during the apatite formation [29,7]. The presence of CO32− bands indicated that the HAP crystals formed here were carbonate-substituted hydroxyapatite, which was the same as natural bone apatite.
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Fig. 5. XRD patterns of (a) untreated collagen sponge and collagen sponge soaked in the (b) SBF; (c) SBF+Mn2+; (d) SBF+Cu2+; (e) SBF+Ni2+; (f) SBF+Co2+; (g) SBF+Cd2+; and (h)SBF+Hg2+.
FTIR analysis of mineralized collagen sponge was also carried out to illustrate the formation mechanism of HAP and the chemical interaction of HAP and collagen sponge. The IR spectra of the untreated collagen sponge and mineralized collagen sponge were shown in Fig. 7. For the untreated collagen sponge shown in Fig. 7a, the absorption bands at 1660 cm−1, 1550 cm−1 and 1240 cm−1 were due to the amide groups of the collagen sponge [30]. Compared with Fig. 7a, it is easily seen that the characteristic absorption bands of 1058 cm−1 and 876 cm−1 appeared in Fig. 7b–h, which correspond to PO43 − and CO32 −, respectively. This demonstrated that mineralized collagen sponge was HAP/collagen composition, and the deposition on the collagen sponge was HAP crystals which were carbonate-substituted apatite [31], and the composition was similar to apatite in natural bone.
4. Conclusions Trace elements in body fluid play a very important role in the person's life activity. HAP/collagen composites were synthesized in the presence of collagen in very low concentration and HAP/collagen sponge was prepared by soaking the collagen sponge in SBF. For both HAP/collagen composites and HAP/collagen sponge, it was found that transition metal ions Mn 2+ had a significant effect on the morphology of HAP particles and could induce to form floc-like HAP particles.
Fig. 6. The IR spectra of (a) the purchased HAP and HAP/collagen composites prepared in the solutions containing (b) no transition metal ion; (c) Co2+; (d) Ni2+; (e) Zn2+; (f) Fe3+; (g) Mn2+; (h) Cu2+; (i) Cd2+; and (j) Hg2+.
The essential beneficial trace element ions of Co 2+, Ni 2+, Zn 2+, Fe 3+, Mn 2+ and Cu 2+ were easily doped into HAP/collagen composites while the amount of doped harmful elements of Cd 2+and Hg 2+ was relatively low.
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Acknowledgment This work is supported by the National Natural Science Foundation of China (No. 20763009) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
References
Fig. 7. The IR spectra of (a) untreated collagen sponge and collagen sponge soaked in the (b) SBF; (c) SBF + Mn2+; (d) SBF + Cu2+; (e) SBF + Ni2+; (f) SBF + Co2+; (g) SBF + Cd2+; and (h) SBF + Hg2+.
Meanwhile, from the HAP/collagen sponge, we can conclude that HAP particle size got smaller in SBF containing Co 2+, Cd 2+and Hg 2+. Meanwhile, the coverage of HAP could be increased significantly by Co 2+ or decreased by Hg 2+.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
The influence of transition metal ions on collagen mineralization Mingfen Jia a, Yuanping Hong a, Shuyuan Duan a, Yongjun Liu b, Bo Yuan b, Fengzhi Jiang a, b,⁎ a b
Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming, 650091, PR China Advanced Analysis and Measurement Center, Yunnan University, Kunming, 650091, PR China
a r t i c l e
i n f o
Article history: Received 5 June 2012 Received in revised form 11 January 2013 Accepted 4 February 2013 Available online 10 February 2013 Keywords: Transition metal ions Collagen Hydroxyapatite Mineralization Collagen sponge
a b s t r a c t The ions in body fluid play an important role in bone formation besides being a synthesizing material. Transition metal ions Co 2+, Ni 2+, Zn2+, Fe 3+, Mn2+, Cu 2+, Cd 2+ and Hg2+ doped hydroxyapatite (HAP)/collagen composites were synthesized successfully in the presence of collagen traces at mild acidic pH for the first time. However, the amount of doped Hg2+ and Cd2+ was relatively low. Meanwhile, through soaking the collagen sponge as a template in simulated body fluid (SBF) which contains different transition metal ions (Mn2+, Cu 2+, Ni 2+, Co 2+, Cd 2+, Hg 2+), bone-like HAP/collagen composites were synthesized. Hg 2+ had a certain inhibitory effect on the formation of HAP crystals on the surface of the collagen sponge while Co2+ can promote the formation of HAP on the collagen sponge. For both HAP/collagen composites and HAP/collagen sponge, it was found that transition metal ions Mn2+ had a significant effect on the morphology of HAP particles and could induce to form floc-like HAP particle aggregates. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Natural bone is a kind of hydroxyapatite (HAP)/collagen composite which is in a complex hierarchical structure [1,2]. It has attracted considerable attention for its unique structure and mechanical properties. In fact, bone formation is a biological process in which deposition of inorganic salts was induced by a template of collagen network formed through self-assembly. But knowledge of the detailed construction process of bone in vivo still remains at an early stage. The development of biocompatible materials for hard tissue repair and replacement has been studied for a long time [3]. Among these materials, HAP/collagen composite is of great interest if it is synthesized by biomimetic strategy inspired by natural bone [4–9]. And it is promising in clinical application because of its compositional and partly structural analogy to natural bone [10–14]. Meanwhile, the ions in body fluid play an important role in bone formation besides being a synthesizing material. As it is well known, Ca2+ and some trace element ions (Co2+, Ni2+, Zn2+, Fe3+, Mn2+, Cu2+) are important for normal growth and metabolism of bone tissues [15–17]. Recent researches have shown that cobalt ions directly inhibit osteoblast function [18] and Cr3+, Co2+, Ni2+can be incorporated into the hydroxyapatite crystal during mineralization and affect the crystal lattice parameters as well as the size of the crystal [19]. Study of the influence of special elements on collagen selfassembly and mineralization is helpful for preventing and treating bone disease caused by the special elements, as well as for developing new bone repairing materials with excellent property of promoting ⁎ Corresponding author at: Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming, 650091, PR China. Tel./fax: +86 871 5036200. E-mail address: [email protected] (F. Jiang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.02.008
bone formation, biocompatibility, bioactivity and biodegradability. Recently, calcium phosphate/collagen biocomposites were prepared in order to potentially enhance osteoconductivity as new bone substitutes that resemble tissue more closely than current available materials [20]. However, reports concerning the influence of the general traces on the collagen mineralization were still very limited so far. In this study, we used two methods to prepare HAP/collagen composites and tried to find out the influence of the traces on collagen mineralization from both methods. HAP/collagen composites were synthesized in solutions containing transition metal ions Co2+, Ni2+, Zn2+, Fe3+, Mn2+, Cu2+, Cd2+ and Hg2+ at mild acidic pH. Meanwhile, through soaking the collagen sponge as a template in simulated body fluid (SBF) which contains different transition mental ions (Mn2+, Cu2+, Ni2+, Co2+, Cd2+, Hg2+), bone-like HAP/collagen composites were synthesized. In order to find out obvious influence of the various traces on collagen mineralization in a short experiment time, we used extremely high ion concentrations. Although the results could not reflect the situation in vivo, we still may find out some clues for the influence of different traces on collagen mineralization. The prepared composites were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) to investigate the effect of the transition metal ions on the shape and doping ratio of the deposited HAP.
2. Materials and methods 2.1. Materials Type I collagen solution was purchased from Hangzhou Biological Technology CO LTD. CaCl2, Na2HPO4·12H2O, NiCl2·6H2O, CoCl2·6H2O,
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FeCl3·6H2O, CuCl2·2H2O, CdCl2, HgCl2, ZnSO4·7H2O, MnSO4·H2O,HCl and NaOH were of reagent grade and ultra-pure water was used as solvent. The collagen sponge (from Beijing Yierkang Bioengineering Development Center) used in the experiment was a sponge-like tissue engineering scaffold and its molecular precursor is the type I collagen. 2.2. Preparation of transition metal ions containing SBF
Table 2 Composition of SBF contains different transition metal ions. Serial number
Components
Concentration of transition mental ions (mM)
1 2 3 4 5 6 7
SBF + CuCl2 SBF + CoCl2 SBF + NiCl2 SBF + MnSO4 SBF + CdCl2 SBF + HgCl2 SBF
[Cu2+] = 0.5 [Co2+]= 0.5 [Ni2+]= 0.5 [Mn2+] = 0.5 [Cd2+] = 0.5 [Hg2+]= 0.5 —
An SBF solution [21] with ion concentrations close to those of human blood plasma [22] (Table 1) was prepared by dissolving NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2, and Na2SO4 in ultra-pure water and the solution was divided into seven shares. To investigate the effects of metal ions on the mineralization of collagen, the SBF was supplemented with 0.5 mM of Ni2+, Co2+, Mn2+, Cu2+, Cd2+, or Hg2+ respectively except the seventh share. The solution was buffered at pH 7.4 with 1 M tris (Hydroxymethyl) aminomethane and 1 M HCl at 36.5 °C, and the ionic concentration of the solution was shown in Table 2. The solution was filtered using a Filtropur syringe filter (Sarstedt, Germany).
shaker (SKY-100 shaker incubator) at 36.5 °C and 80 rpm. After 5 days, collagen sponges were taken out and gently rinsed with ultrapure water, and freeze-dried. The mineralization time for collagen sponges was chosen as 5 days. The mineralization growth on sponges was not homogeneous. Within the beginning 3 days, no precipitate was observed. When the time was last to day 4, white precipitate appeared and another 24 h was run for complete mineralization.
2.3. Preparation of mineralized collagen composites
2.5. Materials characterization
Solutions of HCl (0.5 ml 1 M), transition metal solutions (10 ml 0.5 mM) and type I collagen solution (0.1 ml 5 mg/ml) were gradually added into CaCl2 solutions (5 ml 0.2 M) at 36.5 °C while stirring and then standing for 10 min after mixing. The transition metal solutions were one of NiCl2·6H2O, CoCl2·6H2O, FeCl3·6H2O, CuCl2·2H2O, CdCl2, HgCl2, ZnSO4·7H2O and MnSO4·H2O. While stirring, Na2HPO4·12H2O solutions (3 ml 0.2 M) were added slowly into the above mixture solution and calcium phosphate started to co-precipitate with collagen. Then NaOH solutions (0.9 ml 1 M) were added slowly into the reaction system and a lot of white precipitate appeared. The pH of the solution was then adjusted to 5.2 by dropwise adding HCl solution (1 M) or NaOH (1 M). The molar ratio of Ca/P = 1.67 used in this experiment was equal to the stoichiometric ratio of HAP molecule Ca10(PO4)6(OH)2. The supersaturated solutions were placed in shaker incubator (SKY-100 shaker incubator) and maintained at 36.5 °C for 24 h with the rotation speed of 100 rpm. Then the precipitates were collected by centrifugation at a speed of 5000 rpm and suspended in deionized water to remove the salts. The centrifugation and suspension cycle were repeated 4 times. Then the collected precipitates were washed with ethanol. Finally, the sample of a mechanically poor hydrogel was dried in vacuum at 60 °C for 12 h to get powder. In the preparation procedure, the precipitate formed at the beginning of the process and the least incubating time was determined according to the situation of collagen gelification. Collagen fibrils stabilized over 4–5 h while the solution was standing. In our experiment, a longer time of 24 h was chosen since the solution was in the shaker incubator.
Morphology and microstructure of the collagen mineralization composite and mineralized collagen sponge were examined by SEM (FEI QUANTA200) observation at 30 kV. To prepare the test sample, the synthesized powders were ultrasonically dispersed in ethanol to form dilute suspensions which were dropped on silicon slice. After the samples were dried, they were covered with a fine gold layer for SEM measurement. An energy-dispersive X-ray (EDX) analyzer attached to the SEM was used to determine the elemental composition of HAP/collagen composites. For the mineralized collagen sponge, a fine gold layer was applied before SEM measurement. The crystallinity of the prepared materials was investigated by X-ray powder diffractometry. The XRD pattern was recorded on a Rigaku D/max-3B diffractometer using the monochromatized X-ray beam from graphite filtered CuKα radiation at 40 kV and 30 mA. The 2θ range was from 10° to 60° at a scanning speed of 10°/min. In order to analyze the composite and the combination of the inorganic and organic phase in the prepared materials, FTIR spectroscopy (Thermo Nicolet Avatar 360) measurements were taken. The spectra were obtained using KBr pellets over the wave number range of 1800–400 cm −1 with a resolution of 2 cm −1.
2.4. Preparation of mineralized collagen sponge Collagen sponge was cut into small sheets of 1 cm× 1 cm× 0.2 cm in size and then the sheets were numbered (the number is as same as the serial number of Table 2) and immersed in 15 ml SBF or SBF containing transition metal ions for 5 days in the constant temperature breeding
Table 1 The comparison of ionic concentration between blood plasma and SBF. Species
Blood plasma SBF
Concentration (mM) Na+
K+
Mg2+
Ca2+
Cl−
HCO3−
HPO42−
SO42−
142.0 142.0
5.0 5.0
1.5 1.5
2.5 2.5
103.0 147.8
27.0 4.2
1.0 1.0
0.5 0.5
3. Results and discussion 3.1. Influence of transition metal ions on the morphology of mineralized collagen composite particles HAP/collagen composite is usually synthesized at alkaline condition. Formation of crystalline hydroxyapatite was described by idealized chemical reaction of 10Ca 2+ + 6PO43− + 2OH− → Ca10(PO4)6(OH)2. It is favorable for the nucleation of hydroxyapatite crystal at alkaline condition [23]. However, it was reported that hydroxyapatite crystal was also synthesized successfully and was stable at acidic pH [24]. In our study, in order to avoid the transition metal ions from forming precipitation at alkaline condition, HAP/collagen composites were synthesized in solutions containing transition metal ions Co2+, Ni2+, Zn2+, Fe3+, Mn2+, Cu2+, Cd 2+ and Hg2+ at mild acidic pH. Fig. 1 showed the SEM micrographs of synthesized HAP/collagen composite powders obtained from the solutions containing: (a) no transition metal ion, (b) Co2+, (c) Ni2+, (d) Zn2+, (e) Fe 3+, (f) Mn2+, (g) Cu2+, (h) Cd2+, and (i) Hg2+ at mild acidic pH. The insets of Fig. 1 were SEM images at higher magnification of the corresponding materials. Although we called the prepared material as “collagen/HAP composites”, it could be thought as composite of HAP powder
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Fig 1. SEM images at 1000 × magnification and 10,000 × magnification (insets) of the synthesized HAP/collagen composites in solution containing (a) no transition metal ion;(b) Co 2+;(c) Ni 2 +;(d) Zn 2 +;(e) Fe 3+;(f) Mn2 +;(g) Cu2 +;(h) Cd2 +;and (i) Hg2 +.
containing trace collage since in our preparation system the molar ratio of collagen was low. When the composite was synthesized in the solution without transition metal ions (Fig. 1a), a lot of irregular particles aggregated to form big pores. Meanwhile, the irregular particles were in a porous structure as shown in the inset of Fig. 1a. The prepared HAP/collagen composites revealed no fibrillar nature of collagen since the molar ratio of collagen in the composites was low. The shapes of synthesized HAP/collagen composite powders obtained from the solutions containing (b) Co2+, (c) Ni2+, (d) Zn2+, (g) Cu2+, (h) Cd2+, (i) Hg2+ were quite similar to that of in Fig. 1a with a porous structure. It suggested no significant influence of transition metal ions Co2+, Ni2+, Zn2+, Cu2+, Cd2+, and Hg2+ on the shape of HAP/collagen composite particles. Meanwhile, as can be observed in Fig. 1e, solution containing Fe3+ led to the attainment of much smaller porous particles with respect to those in solution without added transition metals. However, in the solution containing Mn2+, most HAP deposits were in a loose flocculus-like structure although few flaky crystals can be observed (Fig. 1f). SEM images of untreated collagen sponge and mineralization of collagen sponge in SBF containing different transition mental ions were shown in Fig. 2. Fig. 2a is the SEM image of the collagen sponge before being immerged. No inorganic salt deposition was observed on the collagen sponge substrate as shown in Fig. 2a. Compared to the mineralized collagen sponge in SBF shown in Fig. 2b, Cu 2+ and Ni 2+ have no significant effect on the morphology of HAP particles and similar deposited HAP particles were observed as shown in
Fig. 2d and e. However, the size of HAP particles formed in SBF containing Co 2+ (Fig. 2f), Cd 2+ (Fig. 2g) and Hg 2+ (Fig. 2h) was smaller than that in SBF (Fig. 2b) and the coverage ratio of HAP on collagen sponge was different from that in SBF. To quantify the coverage of HAP on collagen sponge, we printed the SEM images and cut the paper into covered and uncovered parts, then weighted the two parts to give the coverage ratio. The coverage ratio equals to the ratio of weight of covered part to the total weight of covered and uncovered parts. Coverage ratios of HAP on collagen sponge for SBF, SBF + Co 2+, SBF + Cd 2+ and SBF + Hg 2+ were 32.3%, 52.4%, 33.8% and 24.3%, respectively. That is, Co 2+ in the SBF significantly increased the coverage of HAP on the surface of collagen sponge (Fig. 2f) and Hg 2+ decreased the coverage (Fig. 2h) while Cd 2+ maintained the similar coverage (Fig. 2g). Interestingly, we can clearly see that the morphology of the HAP particles in SBF containing Mn 2+ (Fig. 2c) was totally different from that in SBF and a lot of floc aggregates formed on the surface. Meanwhile, Mn2+ in the SBF leads a significant increased coverage ratio (67.4%) for HAP on the surface of collagen sponge (Fig. 2c). Similar floc aggregates were also observed in the HAP/collagen composite in solution containing Mn2+ as shown in the inset of Fig. 1f. The difference of Figs. 2c and 1f was due to the fact that the deposition time was quite different. The deposition time was 24 h for Fig. 1f whereas deposition time was 5 days for Fig. 2c. For the studied transition metal ions, Mn2+ could significantly change the shapes of the HAP particles deposited on the collagen sponge while Co2+, Cd2+ and Hg2+ may change
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Fig. 2. The SEM images of untreated collagen sponge and mineralization of collagen sponge in SBF and SBF containing different transition mental ions (a) untreated collagen sponge; (b) SBF; (c) SBF + Mn2+; (d) SBF + Cu2+; (e) SBF + Ni2+; (f) SBF + Co2+; (g) SBF + Cd2+; and (h) SBF + Hg2+.
the size of the HAP particles on the collagen surface. Meanwhile, the coverage of HAP can be increased by Mn2+ and Co2+ or decreased by Hg2+. In order to compare the morphology of hydroxyapatite particles found in HAP/collagen sponge, the different particle sizes and coverage percentage from SBF containing different metal ions were listed in Table 3. 3.2. Doping the collagen mineralized composite with transition metal ions In order to check the doping transition metals in the collagen mineralization composite, EDX analysis was performed. The EDX spectra of
the synthesized HAP/collagen composites powders in mild acidic pH solutions containing (a) Co2+, (b) Ni2+, (c) Zn2+, (d) Fe3+, (e) Mn2+, (f) Cu2+, (g) Cd2+ and (h) Hg2+ were shown in Fig. 3. From Fig. 3, we can clearly see that the peaks of Ca and P were pretty high for all synthesized composites since these two elements were the major content in the composite. Meanwhile, the appearance of C and O demonstrated that HAP in the prepared composite was carbonate substitutive HAP. Meanwhile, the peaks of the transition metals were very low since their concentrations in solutions were all as low as 0.5 mM. Taking Mn2+ for example, its mass concentration was 27.5 μg/ml, which was much higher than that in human blood plasma of 0.83 μg/100 ml. Similarly, the concentrations of other transition metal ions in the solutions
M. Jia et al. / Materials Science and Engineering C 33 (2013) 2399–2406 Table 3 Comparing of hydroxyapatite morphology found in HAP/collagen sponge. Species
HAP HAP HAP HAP HAP HAP HAP
(SBF) (SBF + Mn2+) (SBF + Cu2+) (SBF + Ni2+) (SBF + Co2+) (SBF + Cd2+) (SBF + Hg2+)
The hydroxyapatite morphology Particle size (μm)
Coverage percentage (%)
1.1–1.6 0.07–0.15 0.4–1.5 0.5–1.5 0.1–0.9 0.1–0.3 0.2–0.9
32.3 67.40 35.18 32.34 52.4 33.8 24.3
were much higher than that in human blood plasma. If all of Mn2+ in the solution were doped into the composite, the mass ratio of Mn to the synthesized HAP/collagen composite was about 2.75‰. Similarly, if doping of other transition metal elements was successful, the mass ratios were all higher than 2.75‰ since their mole masses were greater than
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that of Mn. The detection limit of the EDX equipment was higher than 1‰. Compared to Ca and P, the peaks of Zn, Mn, and Cu were too low to see as shown in Fig. 3. But the doped weight rations of the transition metals were measured and recorded by EDX. The EDX spectra demonstrated the presence of Co, Ni, Zn, Fe, Mn and Cu in the composite constitution. Therefore, it was believed that these elements had been successfully doped into the synthesized HAP/collagen composites. However, no elements other than Ca, P, C and O were detectable in Fig. 2g and h. This means that the amounts of Cd and Hg in the synthesized HAP/collagen composites were very little and lower than the EDX sensibility limit of 1‰. 3.3. XRD analysis of the HAP/collagen composites powder and mineralized collagen sponge XRD patterns of synthesized HAP/collagen composites were shown in Fig. 4. The XRD patterns of all prepared HAP/collagen composites in solutions containing different transition metal ions at mild
Fig. 3. The EDX spectra of synthesized HAP/collagen composites in solutions containing (a) Co2+; (b) Ni2+; (c) Zn2+; (d) Fe3+; (e) Mn2+; (f) Cu2+; (g) Cd2+; and (h) Hg2+.
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M. Jia et al. / Materials Science and Engineering C 33 (2013) 2399–2406 Table 4 Lattice parameters of standard HAP and HAP in HAP/Collagen composites. Species
Standard HAP HAP/collagen HAP/collagen (Co2+) HAP/collagen (Ni2+) HAP/collagen (Zn2+) HAP/collagen (Fe3+) HAP/collagen (Mn2+) HAP/collagen (Cu2+) HAP/collagen (Cd2+) HAP/collagen (Hg2+)
Fig. 4. The XRD patterns of HAP/collagen composites prepared in the solutions containing (a) no transition metal ion; (b) Co2+; (c) Ni2+; (d) Zn2+; (e) Fe3+; (f) Mn2+; (g) Cu2+; (h) Cd2+; and (i) Hg2+.
Lattice parameters a
b
c
9.424 9.4004 9.3849 9.5897 9.4336 9.4561 9.4706 9.4986 9.3910 9.4491
9.424 9.4004 9.3849 9.5897 9.4336 9.4561 9.4706 9.4986 9.3910 9.4491
6.879 6.8782 6.8869 6.8576 6.8566 6.8551 6.9256 6.9541 6.9548 6.8918
equation of Dhkl = kλ∕βhklcosθ [26]. All of the average crystal sizes of the prepared HAP particles in the HAP/collagen composites were calculated by XRD analysis software. The results demonstrated that all of the crystal sizes were in the range of 10–20 nm. In different metal ions containing solution, the crystal sizes of HAP in composite were as follows: 15 nm (no transition metal ion), 18 nm (Cu 2+), 17 nm (Cd 2+), 15 nm (Hg 2+), 16 nm (Ni 2+), 14 nm (Co 2+), 15 nm (Fe 3+), 15 nm (Zn 2+) and 16 nm (Mn 2+). The crystallographic structure of the mineralized collagen sponge was also studied with X-ray diffraction. Fig. 5 showed the XRD patterns of mineralized collagen sponge and commercially purchased collagen sponge. Only an extensive broadening peak the 2θ range of 10–30° was observed for the untreated collagen sponge. Several diffraction peaks (211, 222, 322) were clearly seen in the mineralized collagen sponge and the positions of main diffraction peaks correspond well to the expected Bragg peaks for hydroxyapatite (ICDD PDF No.9-432). This indicated that the HAP crystal was formed on the surface of collagen sponge and the transition metal ions did not affect the crystal of the HAP. Compared with the standard HAP, the peaks of the inorganic phase in the mineralized collagen sponge become wider and several peaks overlapped together. This indicated that the crystallinity of inorganic phase was very poor and crystallite size was tiny, which closely resemble the natural bone tissue [25]. All of the average crystal sizes of the HAP on the collagen sponge were calculated by XRD analysis software. The results demonstrated that all of the crystal sizes were in the range of 10–15 nm. In SBF containing different metal ions, the crystal sizes of HAP on the collagen sponge were as follows: 10.4 nm (SBF); 13 nm (SBF + Mn 2 +); 15 nm (SBF + Cu 2+); 14 nm (SBF + Ni 2+); 13 nm (SBF + Co 2+); 14 nm (SBF + Cd 2+); and 12 nm (SBF + Hg 2 +). 3.4. IR analysis of the HAP/collagen composites
acidic pH correspond to that of standard HAP. No peaks other than HAP were detected. This confirmed that the inorganic phase in the synthesized HAP/collagen composites was calcium phosphate apatite. In order to find out the influence of the doped metal ions on the HAP crystal structure, lattice parameters of the composites were listed in Table 4. From Table 4, we can find that compared to the HAP standard sample, the lattice parameters of HAP in all the doped HAP/collagen composite changed while that of HAP in undoped composite was very similar. This means that doped transition metal ions Co 2+, Ni 2+, Zn 2+, Fe 3+, Mn 2+, Cu 2+, Cd 2+ and Hg 2+ have gotten into the HAP crystals and changed the crystal structure of HAP in collagen mineralization composites. Furthermore, metal ions of Cd 2+ and Hg 2+ were believed to be doped into the composites although the amount was lower than the EDX sensibility limit of 1‰. As can be seen in Fig. 4, the extensive broadening and overlapping of the peaks of the synthesized HAP/collagen were similar to that of natural bone [25], which indicated that the crystal grains of HAP were extremely fine. The average crystallite size of prepared HAP particles in the HAP/collagen composite can be estimated by Scherrer
In order to investigate the content of the prepared composites, FTIR spectroscopy measurements of commercially purchased HAP powder and synthesized HAP/collagen composites were taken (Fig. 6). For purchased HAP powder shown in Fig. 6a, the absorption bands at 1087, 1046, 962, 601 and 571 cm −1 were due to PO43−, and those at 1419 and 875 cm −1 were due to CO32− existing in the apatite [27]. According to the literature report, FTIR spectrum of untreated collagen fibrils showed the typical amide bands in protein: 1660 cm −1 was ascribed to amide I band; 1550 cm −1 to amide II and 1240 cm −1 to amide III [26,28]. For the synthesized HAP/collagen composites (Fig. 6b–j), we found that the amide I band shifted from 1660 cm −1 to 1640 cm −1 and the peaks of amide II and amide III disappeared because of the formation of the bonding between Ca ions and C_O bonds. The presence of CO32− in the structure of composites was believed to arise from the air during the apatite formation [29,7]. The presence of CO32− bands indicated that the HAP crystals formed here were carbonate-substituted hydroxyapatite, which was the same as natural bone apatite.
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Fig. 5. XRD patterns of (a) untreated collagen sponge and collagen sponge soaked in the (b) SBF; (c) SBF+Mn2+; (d) SBF+Cu2+; (e) SBF+Ni2+; (f) SBF+Co2+; (g) SBF+Cd2+; and (h)SBF+Hg2+.
FTIR analysis of mineralized collagen sponge was also carried out to illustrate the formation mechanism of HAP and the chemical interaction of HAP and collagen sponge. The IR spectra of the untreated collagen sponge and mineralized collagen sponge were shown in Fig. 7. For the untreated collagen sponge shown in Fig. 7a, the absorption bands at 1660 cm−1, 1550 cm−1 and 1240 cm−1 were due to the amide groups of the collagen sponge [30]. Compared with Fig. 7a, it is easily seen that the characteristic absorption bands of 1058 cm−1 and 876 cm−1 appeared in Fig. 7b–h, which correspond to PO43 − and CO32 −, respectively. This demonstrated that mineralized collagen sponge was HAP/collagen composition, and the deposition on the collagen sponge was HAP crystals which were carbonate-substituted apatite [31], and the composition was similar to apatite in natural bone.
4. Conclusions Trace elements in body fluid play a very important role in the person's life activity. HAP/collagen composites were synthesized in the presence of collagen in very low concentration and HAP/collagen sponge was prepared by soaking the collagen sponge in SBF. For both HAP/collagen composites and HAP/collagen sponge, it was found that transition metal ions Mn 2+ had a significant effect on the morphology of HAP particles and could induce to form floc-like HAP particles.
Fig. 6. The IR spectra of (a) the purchased HAP and HAP/collagen composites prepared in the solutions containing (b) no transition metal ion; (c) Co2+; (d) Ni2+; (e) Zn2+; (f) Fe3+; (g) Mn2+; (h) Cu2+; (i) Cd2+; and (j) Hg2+.
The essential beneficial trace element ions of Co 2+, Ni 2+, Zn 2+, Fe 3+, Mn 2+ and Cu 2+ were easily doped into HAP/collagen composites while the amount of doped harmful elements of Cd 2+and Hg 2+ was relatively low.
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Acknowledgment This work is supported by the National Natural Science Foundation of China (No. 20763009) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
References
Fig. 7. The IR spectra of (a) untreated collagen sponge and collagen sponge soaked in the (b) SBF; (c) SBF + Mn2+; (d) SBF + Cu2+; (e) SBF + Ni2+; (f) SBF + Co2+; (g) SBF + Cd2+; and (h) SBF + Hg2+.
Meanwhile, from the HAP/collagen sponge, we can conclude that HAP particle size got smaller in SBF containing Co 2+, Cd 2+and Hg 2+. Meanwhile, the coverage of HAP could be increased significantly by Co 2+ or decreased by Hg 2+.
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