J. Mater. Sci. Technol. ›› 2019, Vol. 35 ›› Issue (9): 1894-1905.DOI: 10.1016/j.jmst.2019.05.010
• Orginal Article • Previous Articles Next Articles
Mingli Lin, Huanhuan Liu, Jingjing Deng, Ran An, Minjuan Shen, Yanqiu Li, Xu Zhang*()
Received:
2019-03-11
Revised:
2019-04-07
Accepted:
2019-04-24
Online:
2019-09-20
Published:
2019-07-26
Contact:
Zhang Xu
About author:
1 These authors contributed equally to this work.
Mingli Lin, Huanhuan Liu, Jingjing Deng, Ran An, Minjuan Shen, Yanqiu Li, Xu Zhang. Carboxymethyl chitosan as a polyampholyte mediating intrafibrillar mineralization of collagen via collagen/ACP self-assembly[J]. J. Mater. Sci. Technol., 2019, 35(9): 1894-1905.
Fig. 1. Transmittance and zeta potential of CMC solutions at a pH of 1 to 9: (a) transmittance of CMC solution at pH 3 to pH 4 (i.e., showing the greatest precipitation and lowest concentration of CMC); (b) net charge of CMC solution almost zero at pH 3.5, which was consistent with the result measured based on transmittance. Thus, the IP of the CMC solution was approximately pH 3.5. Above the IP, the CMC chains presented negative charges, whereas below the IP, they showed positive charges.
Fig. 2. TEM images of ACP nanoparticle aggregates in solutions of nanocomplexes of CMC/ACP at 2h after preparation: (a) low-magnification image of electron-dense aggregates of CaP nanoparticles at pH 2 and the corresponding SAED pattern indicating the amorphous phase. The corresponding EDX showing that the calcium content was lower than the phosphorus content; (b) high-magnification image of the area in a showing discernible nanoparticles in the aggregates (inset, white circles); (c) low-magnification image of aggregates of CaP nanoparticles at pH 7 showing the amorphous phase (inset, SAED pattern). The corresponding EDX showing that the phosphorus content was lower than the calcium content; (d) high-magnification image of the area in (c) also clearly showing discernible nanoparticles in and around the aggregates (red arrows or white circles in the inset).
Fig. 3. TEM images of synchronous self-assembly/mineralization of collagen (SSM) starting from pH 2:(a) image of the uranyl acetate-stained mineralization of collagen via SSM after 2h (pH approximately 2.5) showing the spindle-shaped CaP nanoparticles with lengths of 40 nm oriented along a certain axis;(b) image of unstained collagen fibrils with pre-assembly and post-assembly parts (indicated by the red dashed line) after 12 h of mineralization; (c) high-magnification image of the area marked in (b) showing the internal and external ACP nanoparticles associated with the collagen fibrils; (d) images of uranyl acetate-stained, completely self-assembled collagen fibrils after 12h of mineralization clearly showing alternating gaps and overlap regions with the nanoparticles mostly located at the gap regions; (e) high-magnification image of area marked in (d) showing the typical spindle-shaped ACP nanoparticles located in the gap region extending to the overlap region (black dashed square); (f) high-magnification image of area marked in (d) showing the ACP nanoparticles non-selectively covering both the gap and overlap regions (red arrows); (g) high-magnification image of area marked in € showing that the typical spindle-shaped ACP nanoparticles were aggregates of smaller nanoparticles (red arrows and white circle).
Fig. 4. TEM images of unstained synchronous self-assembly/mineralization of collagen (SSM) starting from pH 2: (a) mineralized collagen fibrils showing a corn-like arrangement of particles lined up along the long axis of the fibril; (b) high-magnification image of area marked in (a) showing more CaP nanoparticles highly organized in the gap regions shown in Fig. 2(d); (c) high-magnification image of area marked in (a) showing the collagen fibrils (open arrows) containing fewer CaP nanoparticles (red arrows); (d) heavily intrafibrillar mineralization of collagen fibrils; (e) high-magnification image showing the collagen fibrils filled with needle-like HAP crystals along the long axis (red double-headed arrow) and the disordered needle-like HAP crystals outside the fibrils (red arrow). The corresponding SAED pattern indicates the HAP crystal structure with ring characteristics; (f) high-magnification image showing the collagen fibrils with poor intrafibrillar mineralization (open arrows).
Fig. 5. ATR-FTIR spectra for pure collagen (a) and after SSM for 2 h (b), 2 d (c) and 4 d (d). ATR-FTIR spectra shows obvious characteristic peaks of amide I, amide II and amide III of pure collagen (a), indicating the organic phase of collagen. The functional group corresponding to the V3PO4 stretching vibration from 1021 to 1030 cm-1 (b-d).
Fig. 6. Elemental maps of the SSM for 4 d: (a) TEM image; (b-d) elemental maps of oxygen (O), calcium (Ca) and phosphorus (P). The elemental maps show that Ca and P were well distributed inside the collagen fibrils.
Fig. 7. Kinetic characterization and representative TEM images (taken at 120 min and 1800 min) of self-assembly and mineralization of collagen in the presence of CMC or CMC/ACP. The points that the pH of samples were close to IP were indicated by brown: (a) turbidity-time curve showing that the turbidity increase with dialysis time; (b, c) TEM images of collagen mixed with CMC/ACP (SSM model); (d, e) TEM images of pure collagen; (f, g) TEM images of collagen mixed with CMC.
Fig. 8. TEM images of unstained mineralization following self-assembly of collagen (MFS): (a) low-magnification image showing aggregates of ACP nanoparticles irregularly covering self-assembled collagen fibrils after 2 h of mineralization; (b) high-magnification image of area marked in (a) showing the aggregates of ACP nanoparticles outside (open arrows) the collagen fibrils and less elongated nanoparticles inside the fibrils (red arrows); (c) low-magnification image of collagen fibrils after 2 d of mineralization; (d) high-magnification image of area marked in (c) showing the aggregates of ACP nanoparticles homogeneously covering the collagen fibrils (open arrows) and dense elongated nanoparticles with a corn-like pattern inside the collagen fibrils (red arrows), and a number of nanoparticles are fused and extended (black arrows); (e) low-magnification image of collagen fibrils after 4 d of mineralization; (f) high-magnification image of area marked in (e) showing the collagen fibrils filled with dense needle-like crystals along the long axis (red arrows). Corresponding SAED pattern indicates the HAP crystal structure rather than an amorphous phase.
Fig. 9. Schematic diagram comparing SSM and MFS models. In the MFS model, collagen molecules first self-assemble into collagen fibrils as a mineralization template. After that, the ACP nanoparticles infiltrate the collagen fibrils via gap zones where apatite crystals nucleate and grow. In the SSM model, the ACP nanoparticles first mix and interact with the collagen microfibrils. With further collagen self-assembly, ACP nanoparticles are trapped and localized in the gap zones. The ACP nanoparticles are converted in situ into HAP crystals to accomplish the intrafibrillar mineralization of collagen.
Fig. 10. Schematic diagram showing possible process of in vivo collagen mineralization. The procollagen molecules are secreted by osteoblasts and assembled into microfibrils extracellularly, and the matrix vesicles containing ACP nanoparticles can be released from osteoblasts. The in vivo mineralization of collagen can be accomplished via the MFS model only (Route II) or initially via the SSM model and subsequently via the MFS model (Route I).
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