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Biomimetic mineralization of collagen fibrils induced by amine-terminated PAMAM dendrimers—PAMAM dendrimers for remineralization ab
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c
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a
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Kunneng Liang , Yuan Gao , Jianshu Li , Ying Liao , Shimeng Xiao , Xuedong Zhou
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Jiyao Li a
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China b
Department of Operative Dentistry and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China c
Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, China Accepted author version posted online: 03 Jul 2015.
Click for updates To cite this article: Kunneng Liang, Yuan Gao, Jianshu Li, Ying Liao, Shimeng Xiao, Xuedong Zhou & Jiyao Li (2015): Biomimetic mineralization of collagen fibrils induced by amine-terminated PAMAM dendrimers—PAMAM dendrimers for remineralization, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2015.1068606 To link to this article: http://dx.doi.org/10.1080/09205063.2015.1068606
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Publisher: Taylor & Francis Journal: Journal of Biomaterials Science, Polymer Edition DOI: http://dx.doi.org/10.1080/09205063.2015.1068606
Full paper Biomimetic mineralization of collagen fibrils induced by
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amine-terminated PAMAM dendrimers—PAMAM dendrimers for remineralization Kunneng Liang a, b, Yuan Gao a, b, Jianshu Li c, Ying Liao a, b, Shimeng Xiao a, Xuedong Zhou a, b, Jiyao Li a, b, *. a. State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China b. Department of Operative Dentistry and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China c. Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, China
* Corresponding author.
Full postal address: Department of Operative Dentistry and Endodontics. West China Hospital of 1
Stomatology. NO.14, Unit 3, Renmin Nan Road, Chengdu city. Sichuan province, China, 610041 Telephone number：+86-28-85501439 Fax numbers: +86-28-85582167 E-mail address: [email protected]
Abstract: Objective: Achieving biomimetic mineralization of collagen fibrils by mimicking
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the role of non-collagenous proteins (NCPs) with biomimetic analogues is of great interest in the fields of material science and stomatology. Amine-terminated PAMAM dendrimer (PAMAM-NH2), which possesses a highly ordered architecture and many calcium coordination sites, may be a desirable template for simulating NCPs to induce mineralization of collagen fibrils.
In this study, we focused on the ability of
PAMAM-NH2 to mineralize collagen fibrils. Design: Type I collagen fibrils were reconstituted over 400-mesh formvar-and-carbon-coated gold grids and treated with a third generation PAMAM-NH2 (G3-PAMAM-NH2) solution. The treated collagen fibrils were immersed in artificial saliva for different lengths of time. The morphologies of the mineralized reconstituted type I collagen fibrils were characterized by transmission electron microscopy (TEM). Results: No obvious mineralized collagen fibrils were detected in the control group. On the contrary, collagen fibrils were heavily mineralized in the experimental group. Most importantly, intrafibrillar mineralization was achieved within the 2
reconstituted type I collagen fibrils. Conclusions: In this study, we successfully induced biomimetic mineralization within type I collagen fibrils using G3-PAMAM-NH2. This strategy may serve as a potential therapeutic technique for restoring completely demineralized collagenous mineralized tissues (CMTs).
1. Introduction Mineralized type I collagen fibrils are the basic structural and functional units of
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collagenous mineralized tissues (CMTs) such as dentin and bone.[1] These CMTs possess complex morphologies and superior biomechanical properties, which are attributed to their elaborate arrangements of hydroxyapatite (HA).[2] Biomineralization is a multistep process involving secretion of extracellular matrix and controlled deposition of minerals in an organized fashion. Although some scientists posit that collagen fibrils can independently act as nucleators to induce apatite deposition,[3] it is generally accepted that collagen fibrils alone are insufficient to induce mineralization. [4] Non-collagenous proteins (NCPs) play a critical role in biomineralization.[5-7] Studies have demonstrated that mutations in genes that code for NCPs result in abnormal mineralization in dentin and bone.[8, 9] NCPs have been reported to inhibit mineralization when present in solution and induce mineralization when present in a bound state,[10] a phenomenon which is attributed to the two different functional motifs that are present in NCPs: a sequestration functional motif on the N-terminal fragments and a templating functional motif on the C-terminal fragments.[11] 3
In nature, biominerals are believed to be formed by an amorphous precursor pathway that is mediated by NCPs: the initial stage is the sequestration of calcium and phosphate ions from solutions for their conversions into amorphous calcium phosphate (ACP) nano-precursors, followed by the templating of mineral nucleation at specific sites of a collagen substrate.[12，13] The amorphous precursor pathway leads to highly ordered intrafibrillar mineralization at the nanoscale, which is very important for the biomechanical properties of CMTs.[14, 15] However, it is difficult
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to extract native NCPs and the therapeutic use of native NCPs is not yet economically viable, thus scientists have resorted to searching for biomimetic analogues that can play similar roles to NCPs in the biomineralization process.[11] In recent years, biomimetic mineralization of collagen fibrils has become a focus of research. Many research groups have attempted to mineralize collagen fibrils using different biomimetic analogue systems. Some scientists believe that the templating analogues are not necessary and that collagen itself could be a template. Thus, they have only employed sequestration analogues such as polyaspartic acid.[16-21] Other researchers believe that both sequestration and templating analogues are necessary and they have used dual-analogue biomimetic systems such as polyacrylic acid (PAA)/polyvinylphosphonic acid (PVPA) and polyacrylic acid (PAA)/sodium trimetaphosphate (STMP).[2, 11, 22] These biomimetic systems achieved a high degree of intrafibrillar mineralization. However, they are not all available for application in clinical dentistry at present. It is necessary to develop new biomaterials that can induce biomimetic mineralization of collagen fibrils. 4
Poly (amido amine) (PAMAM) dendrimer, which has many reactive functional groups, a well-defined size and a controlled spatial structure, is often referred to as ‘artificial protein’.[23] It has been reported that PAMAM dendrimers possess the same sequestration and templating functions as NCPs. When present in solution, PAMAM dendrimers can inhibit mineralization and stabilize ACP nano-precursors to prevent phase transformation.[24, 25] When bound at a surface, PAMAM dendrimers can act as nucleation templates to induce remineralization.[26] These characteristics
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of PAMAM dendrimer make it suitable for mimicking natural NCPs. In our previous studies, we successfully remineralized demineralized dentin with PAMAM dendrimers.[27, 28] However, we only remineralized partially demineralized dentin and intrafibrillar remineralization was not observed due to the limitations in our detection system. In some clinical situations (i.e., hybrid layers created by etch-and-rinse adhesive systems), dentin surfaces are completely demineralized, which results in the exposure of type I collagen fibrils. Remineralizing collagen fibrils following the amorphous precursor pathway is different from traditional remineralization: it requires both sequestration and templating functional analogues. It has been reported that a portion of bound PAMAM dendrimers will be released into solution if their binding capacity to a tooth is not strong enough.[29] Therefore, collagen mineralization may be achieved by an amorphous precursor pathway mediated by PAMAM dendrimers due to their sequestration and templating functions. In this study, we attempted to mineralize reconstituted type I collagen fibrils with 5
third generation PAMAM-NH2 (G3-PAMAM-NH2). First, the type I collagen fibrils were reconstituted and immobilized with G3-PAMAM-NH2. Then, to test whether the bound G3-PAMAM-NH2 molecules would be released into solution, we analyzed their ultraviolet-visible spectra (UV-visible spectra). Finally, transmission electron microscopy (TEM) was employed to characterize the morphologies of the mineralized reconstituted type I collagen fibrils. Selected area electron diffraction (SAED) was performed to identify the mineral phase at different periods. The hypothesis was that
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G3-PAMAM-NH2 could induce biomimetic mineralization on reconstituted type I collagen fibrils.
2. Materials and methods 2.1. Self-assembly and crosslinking of collagen fibrils Lyophilized type I collagen powder derived from bovine skin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.1 M acetic acid (pH 3.0) at 4 °C. A 100 μL of type I collagen fibril stock solution (0.15 mg/mL) was reconstituted over 400-mesh formvar-and-carbon-coated gold TEM grids by neutralizing the collagen stock solution with ammonia vapor in a humidity chamber for 3 h. The neutralized collagen solution was incubated at 37 °C for 5 days to ensure that it transformed into a gel. To stabilize the structures of the reconstituted collagen fibrils, a cross-linking procedure was performed as follows: first, a solution containing 0.3 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.06 M N-hydroxysuccinimide (NHS) was prepared. Then, the pH of the EDC/NHS solution 6
was adjusted to 5.7 with 2-morpholinoethane sulphonic (MES) powder. Last, the collagen grids were treated with 100 μL of the EDC/NHS solution for 3 h, washed with deionized water and air-dried.
2.2 UV-visible spectra
TEM grids coated with and without cross-linked collagen fibrils were treated
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with 50 μL of G3-PAMAM-NH2 solution (1000 ppm) for 2 h. In addition, a TEM grid coated with cross-linked collagen fibrils was treated with 50 μL of deionized water for 2 h. All groups were washed with deionized water, air-dried and then immersed in 1 mL of deionized water. After 6 h, the TEM grids were removed from the deionized water and the absorbance of the remaining deionized water was measured at a wavelength of 280 nm using an ultraviolet spectrophotometer (UV-1800, MPD Instrument Company, Shanghai, China) to examine whether there were free PAMAM dendrimers in the remaining deionized water. A G3-PAMAM-NH2 solution (100 ppm) was used as a positive control.
2.3 Collagen mineralization TEM grids coated with cross-linked collagen fibrils were treated with 50 μL of G3-PAMAM-NH2 solution (1000 ppm) or 50 μL of deionized water (the control) for 2 h, washed with deionized water, air-dried and then immersed in 5 mL of artificial saliva solution (pH 7.0) containing 1.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 1.0 mM NaN3 and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 7
(HEPES) at 37 °C.[30] The TEM grids were removed from the artificial saliva after 6, 24 and 48 hours, washed with deionized water, air-dried and characterized by TEM (200 kV, Tecnai GF20S-TWIN, FEI, USA). SAED of the mineralized collagen fibrils was performed to identify the mineral phase. For each group, 5 TEM grids were analyzed.
3. Results and discussion
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3.1 UV-visible spectra UV-visible spectra of the four tested solutions are displayed in Figure (1). The standard spectrum of the G3-PAMAM-NH2 solution (100 ppm) showed an obvious characteristic peak at approximately 280 nm. After storing the collagen that was treated with deionized water for 6 h, the spectrum of the remaining deionized water exhibited a smooth curve, which indicated that there were hardly any free G3-PAMAM-NH2 molecules. After storing the TEM grid that was treated with 1000 ppm of G3-PAMAM-NH2 for 6 h, a similar smooth curve was observed revealing that G3-PAMAM-NH2 could scarcely bind to the TEM grid. On the contrary, after storing the collagen that was treated with 1000 ppm of G3-PAMAM-NH2 for 6 h, the spectrum of the remaining deionized water displayed a weak characteristic peak at 280 nm, which suggested the presence of a small number of free PAMAM macromolecules in the solution. Collagen fibrils have a size-exclusion feature,[31] that prevents molecules larger than 40 kDa from penetrating into their internal water compartments while 8
allowing molecules smaller than 6 kDa to freely diffuse into their water compartments. In other words, molecules with a molecular weight (MW) between 6 - 40 kDa can be retained within collagen fibrils. The molecular weight of G3-PAMAM-NH2 is 6909 Da, which is just within this range. In addition, a previous study has demonstrated that carboxyl-terminated PAMAM (PAMAM-COOH) can enter into collagen fibrils,[32] and the structure of PAMAM-NH2 is similar to PAMAM-COOH. Therefore, G3-PAMAM-NH2 may penetrate into collagen fibrils and be retained within them. On
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the other hand, there are many charged sites on collagen surfaces，[33] enabling G3-PAMAM-NH2 molecules to potentially bind to collagen surfaces via electrostatic interactions [34] because their many charged functional groups (positively charged amine groups and negatively charged amide groups).
In summary, we speculated
that when collagen fibrils were exposed to a large number of G3-PAMAM-NH2 molecules, some molecules were retained within the collagen fibrils due to their size-exclusion feature, while other molecules may have bound to the collagen surfaces via weaker electrostatic interactions. When the collagen fibrils coated with G3-PAMAM-NH2 were immersed in solution, a small portion of the bound molecules could be released into the solution because their binding strength to the collagen fibrils was not strong enough.
3.2 Collagen mineralization The presence of remnant phosphoproteins that remain bound to collagen fibrils after dentin demineralization also has the potential to induce remineralization, which 9
may lead to confusion when interpreting the experimental results.[11] To avoid the effect of remnant phosphoproteins, we chose a pure type I collagen fibril model to simulate completely demineralized CMTs. Biomimetic mineralization is different from traditional mineralization, as it can induce intrafibrillar mineralization within collagen fibrils, which is very important for the biomechanical properties of mineralized dentin. In this study, we employed TEM to observe the morphologies of the mineralized, reconstituted type I collagen fibrils
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and explored whether G3-PAMAM-NH2 could induce intrafibrillar mineralization within them. As can be seen in Figure 2 (A), after mineralization for 6 h, collagen fibril immobilized with G3-PAMAM-NH2 was slightly darkly stained by amorphous minerals. Electron-dense mineral nano-precursors (10–50 nm in diameter) (red arrows) aggregated along the periphery of the collagen fibril. These nano-precursors exhibited fluidity by coalescing to produce continuous phases, which revealed their amorphous nature (Figure 2, B). The nano-precursors were arranged within the collagen fibrils in an ordered manner with a roughly 70 nm periodicity (Figure 2, C) (red arrows showed the periodicity), which is in accordance with a previous report that the characteristic periodicity of the native collagen assembly is approximately 67 nm.[1] In the control group (Figure 2, E), without the sequestration function imparted by free G3-PAMAM-NH2 molecules, the nano-precursors aggregated much faster. Cluster larger than 100 nm in diameter was commonly observed (red arrow). The collagen fibril could not easily be detected because it was not darkly stained by the 10
nano-precursors (Figure 2, D). The electronic diffraction pattern with a 004 reflection (Figure 2, F) revealed that the nano-precursors in the control group had begun to crystallize, while the electronic diffraction pattern of the experimental group had only a broad diffraction ring (Figure 2, B, C) (inset) indicating that the mineralized phase was still amorphous nature. These results confirmed that G3-PAMAM-NH2 could stabilize ACP nano-precursors and reduced the speed of their transformation into crystal.
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After mineralization for 24 h, the mineralized collagen fibrils immobilized with G3-PAMAM-NH2 revealed arrays of ribbon-like mineral particles deposited within them with a roughly 70 nm periodicity (Figure 3, A) (red arrows showed the periodicity). These deposited minerals were closely connected to the collagen fibrils. A typical HA diffraction pattern with distinct (002), (211) and (004) reflections (Figure 3, A) (inset) confirmed that the ACP nano-precursors had been transformed from an amorphous state into apatite. In the control group, the mineral crystals aggregated further. Crystal clusters larger than 200 nm in diameter were observed (Figure 3, B). After mineralization for 48 h, the collagen fibrils immobilized with G3-PAMAM-NH2 were heavily mineralized (Figure 4, A). Obvious mineralized collagen fibrils could be observed due to intrafibrillar mineral deposits even at low magnification. Branching of the collagen fibrils (black arrow) and extrafibrillar mineral deposits (red arrow) were frequently observed. At high magnification (Figure 4, B, C, D), the collagen fibrils were further darkly stained by minerals, which 11
obscured their banding periodicity. In fact, it was difficult to observe the banding characteristics of the completely mineralized collagen fibrils. In the control group, at low magnification (Figure 4, E), a large number of crystals (200 nm-500 nm in diameter) were randomly oriented and evenly distributed throughout the grid. The collagen fibrils were difficult to observe. At high magnification (Figure 4, F), the collagen fibril was slightly stained by a few minerals, which made them more easily observed than after 6 h of mineralization (Figure 2, D). However, it seemed that the
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minerals were only deposited on the collagen surfaces, as the collagen fibrils were not heavily mineralized such as in the experimental group. These results suggested that the collagen surfaces may have served as nucleation templates, but the nucleation appeared to be weak. The size-exclusion feature of collagen fibrils limits the dimension of ACP nano-precursors that can penetrate them. Molecules larger than 40 kDa are completely excluded from collagen fibrils, which may explain why relatively larger ACP nano-precursors (larger than 100 nm in diameter) were incapable of participating in intrafibrillar mineralization. [11] G3-PAMAM-NH2 possesses many amine groups on its external surface and a large number of amide groups in its internal cavity. It can attract calcium ions by calcium complexation through either its amine or amide groups to form temporary a “PAMAM-Ca2+” complex,[26] which can decrease the amount of calcium ions in artificial saliva to prevent calcium and phosphate ions from aggregating too quickly. Based on the above results, we posited that G3-PAMAM-NH2 is capable of stabilizing metastable ACP nanoprecursors to form 12
relatively smaller ACP nanoprecursors (10 nm - 50 nm in diameter), which could diffuse into the water compartments within the collagen fibrils. Comparatively, in the control group, the crystals that were directly formed by calcium and phosphate ions in the solution were too large (larger than 100 nm in diameter) to infiltrate into collagen fibrils, which resulted in failure of intrafibrillar mineralization. Previous studies and the results of this research indicate that G3-PAMAM-NH2 molecules may enter into collagen fibrils due to the size-exclusion feature of collagen
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fibrils and may bind to the collagen surfaces via electrostatic interactions.[31-34] Furthermore, free and bound PAMAM molecules possessed different sequestration and templating functions. In summary, we proposed a possible mechanism behind G3-PAMAM-NH2-coating-induced mineralization of collagen fibrils (Figure 5). First, when collagen fibrils were exposed to G3-PAMAM-NH2 solutions, some PAMAM molecules entered into them and were retained within the intrafibrillar spaces (67 nm intervals) due to the size-exclusion feature. At the same time, other PAMAM molecules bound to the surfaces of the collagen fibrils via electrostatic interactions. After being immersed in artificial saliva, some of the PAMAM molecules that were bound to the surfaces were released into solution. Then, with the help of the free PAMAM molecules, smaller ACP nano-particles penetrated into the collagen fibrils and the bound PAMAM molecules acted as templates to attract the ACP nano-particles through calcium complexation. Finally, as the ACP nano-particles within the collagen fibrils slowly transformed into HA, the collagen fibrils were completely mineralized. 13
In addition to PAMAM-NH2, other types of PAMAM dendrimers have been used for the biomimetic mineralization of teeth such as PAMAM-COOH, PAMAM-COOH-alendronate (ALN) conjugate (ALN-PAMAM-COOH), and polyhydroxy-terminated PAMAM (PAMAM-OH) dendrimers.[27, 29, 32] However, there are some limitations to using these PAMAM dendrimers such as the high cost of PAMAM-COOH, the relatively weak remineralization ability of PAMAM-OH and the extremely difficult synthesis condition of ALN-PAMAM-COOH. Compared with
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these PAMAM dendrimers, the only limitation to using PAMAM-NH2 is that its biocompatibility is slightly poor. However, a previous study has demonstrated that there was nearly no cytotoxicity to oral cells when the concentration of PAMAM-NH2 was lower than 1250 ug/mL. [28] Therefore, the concentration of PAMAM-NH2 in this study (1000 ug/mL) was safe and could potentially be used in clinical dentistry. Dentin demineralization is a common pathological state, which is related to dentin caries, dentin hypersensitivity and other dentin diseases. Remineralization of demineralized dentin is a non-invasive therapeutic method and is preferred to traditional invasive substitutes.[35] In recent years, various biomaterials have been employed for remineralizing demineralized dentin such as fluoride, bioactive glasses, amorphous calcium phosphate (ACP)-releasing resins, and dopamine.[36-40] However, all of the above are only suitable for remineralizing partially demineralized dentin, which relies on the epitaxial deposition of calcium and phosphate ions over remnant crystallites.[41] In some clinical situations, such as the hybrid layers that are created by etch-and-rinse adhesive systems and in the superficial portion of 14
caries-affected dentin lesions that are left behind after minimally invasive caries removal, dentin is completely demineralized. With regard to completely demineralized dentin, traditional remineralization agents are ineffective, as there are no remnant crystallites in the collagen fibrils. It is a difficult problem to remineralize completely demineralized dentin in clinical dentistry. Biomimetic remineralization, which is based on a non-classical particle-based crystallization concept and does not rely on remnant crystal seeds, may solve this clinical dental puzzle. This study has
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demonstrated that G3-PAMAM-NH2 can mineralize type I collagen fibrils, which is similar to the special clinical conditions mentioned above. Hence, PAMAM dendrimers may be used to remineralize completely demineralized dentin. In previous studies, PAMAM-COOH has been demonstrated to possess the abilities to remineralize dentin and enamel.[32，42] PAMAM-NH2 has a similar structure to PAMAM-COOH, which suggests similar abilities of remineralization. We have confirmed that PAMAM-NH2 can remineralize demineralized dentin. In future experiments, we will explore the remineralization ability of PAMAM-NH2 on enamel. Our hope is that G3-PAMAM-NH2 could be used in other dental clinical treatments, such as in the treatments of enamel caries and root lesions. [43, 44]
4. Conclusions In this study, we used G3-PAMAM-NH2 dendrimers to induce the mineralization of type I collagen fibrils. Our results suggested that G3-PAMAM-NH2 could simulate both the sequestration and templating functions of natural NCPs in biomineralization. 15
Intrafibrillar mineralization within collagen fibrils was achieved. G3-PAMAM-NH2 has great potential for the mineralization of type I collagen fibrils and may be a promising restorative biomaterial for other biomineralized tissues.
Acknowledgements Financial support from the National Natural Science Foundation of China (Grant Nos. 81170958, 81400508 and 51073102), Specialized Research Fund for the
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Doctoral Program of Higher Education of China (Grant No. 20100181110056 and 20130181120125) and Sichuan Province Science and Technology support program (Grant No. 2011SZ0130) is gratefully acknowledged. We also thank Xiao Yang, Xin Xu, Lei Cheng and Libang He for their guidance in this study.
Author contributions This study was accomplished through the cooperation of two independent departments at Sichuan University: the State Key Laboratory of Oral Diseases, and the Department of Biomedical Polymers and Artificial Organs. Prof. Xuedong Zhou and Prof. Jiyao Li are from the State Key Laboratory of Oral Diseases and they were in charge of the design and planning of the entirety of the study. Kunneng Liang, Yuan Gao, Ying Liao and Shimeng Xiao are also from this lab and their work included sample preparation and the mineralization experiment. Because the TEM tests were conducted in this lab, they were also in charge of analyzing these results. Finally, Liang wrote the article. 16
Prof. Jianshu Li is from the Department of Biomedical Polymers and Artificial Organs. He was responsible for the synthesis of the PAMAM dendrimers and the tests of UV-visible spectra.
Keywords: Amine-terminated PAMAM dendrimer, Biomaterial, Biomimetic mineralization, Collagen fibrils, Intrafibrillar mineralization.
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Figure 1. UV-visible spectra of a 100 ppm of G3-PAMAM-NH2 solution, the remaining deionized water after storing collagen that was treated with deionized water, the remaining deionized water after storing collagen that was treated with 1000 ppm of G3-PAMAM-NH2 solution and the remaining deionized water after storing a TEM grid that was treated with 1000 ppm of G3-PAMAM-NH2 for 6 h.
23
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Figure 2. TEM images of the mineralized collagen fibrils immobilized with (A, B, C) and without (D, E, F) G3-PAMAM-NH2 after being immersed in artificial saliva for 6 h. (B) is the high magnification photo of coalesced nano-precursors in (A) (arrows). Electron diffraction of the mineralized collagen fibrils (insets) showed their mineral phase. (F) is the electron diffraction of (E).
24
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Figure 3. TEM images of the mineralized collagen fibrils immobilized with (A) and without (B) G3-PAMAM-NH2 after being immersed in artificial saliva for 24 h. Electron diffraction of the mineralized collagen fibrils (insets) showed the mineral phase.
25
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Figure 4. TEM images of the mineralized collagen fibrils immobilized with (A, B, C, D) and without (E, F) G3-PAMAM-NH2 after being immersed in artificial saliva for 48 h. (B, C, D, F) are high magnification photos.
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Figure 5. Schematic demonstration of the mechanism of biomimetic mineralization of collagen fibrils induced by G3-PAMAM-NH2. The rectangle represents a collagen fibril. The green cylinders within the rectangle represent microfibrils. The orange balls, yellow balls and purple balls represent Ca2+ ions, PO43- ions and ACP nano-particles, respectively. In the control group, without the help of free PAMAM, large ACP nano-particles could not enter the collagen fibrils. In the experimental group, free PAMAM molecules stabilized the ACP nano-particles to form smaller ACPs, which could penetrate into collagen fibrils and combine with the bound PAMAM.
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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Biomimetic mineralization of collagen fibrils induced by amine-terminated PAMAM dendrimers—PAMAM dendrimers for remineralization ab
ab
c
ab
a
ab
Kunneng Liang , Yuan Gao , Jianshu Li , Ying Liao , Shimeng Xiao , Xuedong Zhou
&
ab
Jiyao Li a
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China b
Department of Operative Dentistry and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China c
Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, China Accepted author version posted online: 03 Jul 2015.
Click for updates To cite this article: Kunneng Liang, Yuan Gao, Jianshu Li, Ying Liao, Shimeng Xiao, Xuedong Zhou & Jiyao Li (2015): Biomimetic mineralization of collagen fibrils induced by amine-terminated PAMAM dendrimers—PAMAM dendrimers for remineralization, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2015.1068606 To link to this article: http://dx.doi.org/10.1080/09205063.2015.1068606
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Publisher: Taylor & Francis Journal: Journal of Biomaterials Science, Polymer Edition DOI: http://dx.doi.org/10.1080/09205063.2015.1068606
Full paper Biomimetic mineralization of collagen fibrils induced by
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amine-terminated PAMAM dendrimers—PAMAM dendrimers for remineralization Kunneng Liang a, b, Yuan Gao a, b, Jianshu Li c, Ying Liao a, b, Shimeng Xiao a, Xuedong Zhou a, b, Jiyao Li a, b, *. a. State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China b. Department of Operative Dentistry and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China c. Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, China
* Corresponding author.
Full postal address: Department of Operative Dentistry and Endodontics. West China Hospital of 1
Stomatology. NO.14, Unit 3, Renmin Nan Road, Chengdu city. Sichuan province, China, 610041 Telephone number：+86-28-85501439 Fax numbers: +86-28-85582167 E-mail address: [email protected]
Abstract: Objective: Achieving biomimetic mineralization of collagen fibrils by mimicking
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the role of non-collagenous proteins (NCPs) with biomimetic analogues is of great interest in the fields of material science and stomatology. Amine-terminated PAMAM dendrimer (PAMAM-NH2), which possesses a highly ordered architecture and many calcium coordination sites, may be a desirable template for simulating NCPs to induce mineralization of collagen fibrils.
In this study, we focused on the ability of
PAMAM-NH2 to mineralize collagen fibrils. Design: Type I collagen fibrils were reconstituted over 400-mesh formvar-and-carbon-coated gold grids and treated with a third generation PAMAM-NH2 (G3-PAMAM-NH2) solution. The treated collagen fibrils were immersed in artificial saliva for different lengths of time. The morphologies of the mineralized reconstituted type I collagen fibrils were characterized by transmission electron microscopy (TEM). Results: No obvious mineralized collagen fibrils were detected in the control group. On the contrary, collagen fibrils were heavily mineralized in the experimental group. Most importantly, intrafibrillar mineralization was achieved within the 2
reconstituted type I collagen fibrils. Conclusions: In this study, we successfully induced biomimetic mineralization within type I collagen fibrils using G3-PAMAM-NH2. This strategy may serve as a potential therapeutic technique for restoring completely demineralized collagenous mineralized tissues (CMTs).
1. Introduction Mineralized type I collagen fibrils are the basic structural and functional units of
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collagenous mineralized tissues (CMTs) such as dentin and bone.[1] These CMTs possess complex morphologies and superior biomechanical properties, which are attributed to their elaborate arrangements of hydroxyapatite (HA).[2] Biomineralization is a multistep process involving secretion of extracellular matrix and controlled deposition of minerals in an organized fashion. Although some scientists posit that collagen fibrils can independently act as nucleators to induce apatite deposition,[3] it is generally accepted that collagen fibrils alone are insufficient to induce mineralization. [4] Non-collagenous proteins (NCPs) play a critical role in biomineralization.[5-7] Studies have demonstrated that mutations in genes that code for NCPs result in abnormal mineralization in dentin and bone.[8, 9] NCPs have been reported to inhibit mineralization when present in solution and induce mineralization when present in a bound state,[10] a phenomenon which is attributed to the two different functional motifs that are present in NCPs: a sequestration functional motif on the N-terminal fragments and a templating functional motif on the C-terminal fragments.[11] 3
In nature, biominerals are believed to be formed by an amorphous precursor pathway that is mediated by NCPs: the initial stage is the sequestration of calcium and phosphate ions from solutions for their conversions into amorphous calcium phosphate (ACP) nano-precursors, followed by the templating of mineral nucleation at specific sites of a collagen substrate.[12，13] The amorphous precursor pathway leads to highly ordered intrafibrillar mineralization at the nanoscale, which is very important for the biomechanical properties of CMTs.[14, 15] However, it is difficult
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to extract native NCPs and the therapeutic use of native NCPs is not yet economically viable, thus scientists have resorted to searching for biomimetic analogues that can play similar roles to NCPs in the biomineralization process.[11] In recent years, biomimetic mineralization of collagen fibrils has become a focus of research. Many research groups have attempted to mineralize collagen fibrils using different biomimetic analogue systems. Some scientists believe that the templating analogues are not necessary and that collagen itself could be a template. Thus, they have only employed sequestration analogues such as polyaspartic acid.[16-21] Other researchers believe that both sequestration and templating analogues are necessary and they have used dual-analogue biomimetic systems such as polyacrylic acid (PAA)/polyvinylphosphonic acid (PVPA) and polyacrylic acid (PAA)/sodium trimetaphosphate (STMP).[2, 11, 22] These biomimetic systems achieved a high degree of intrafibrillar mineralization. However, they are not all available for application in clinical dentistry at present. It is necessary to develop new biomaterials that can induce biomimetic mineralization of collagen fibrils. 4
Poly (amido amine) (PAMAM) dendrimer, which has many reactive functional groups, a well-defined size and a controlled spatial structure, is often referred to as ‘artificial protein’.[23] It has been reported that PAMAM dendrimers possess the same sequestration and templating functions as NCPs. When present in solution, PAMAM dendrimers can inhibit mineralization and stabilize ACP nano-precursors to prevent phase transformation.[24, 25] When bound at a surface, PAMAM dendrimers can act as nucleation templates to induce remineralization.[26] These characteristics
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of PAMAM dendrimer make it suitable for mimicking natural NCPs. In our previous studies, we successfully remineralized demineralized dentin with PAMAM dendrimers.[27, 28] However, we only remineralized partially demineralized dentin and intrafibrillar remineralization was not observed due to the limitations in our detection system. In some clinical situations (i.e., hybrid layers created by etch-and-rinse adhesive systems), dentin surfaces are completely demineralized, which results in the exposure of type I collagen fibrils. Remineralizing collagen fibrils following the amorphous precursor pathway is different from traditional remineralization: it requires both sequestration and templating functional analogues. It has been reported that a portion of bound PAMAM dendrimers will be released into solution if their binding capacity to a tooth is not strong enough.[29] Therefore, collagen mineralization may be achieved by an amorphous precursor pathway mediated by PAMAM dendrimers due to their sequestration and templating functions. In this study, we attempted to mineralize reconstituted type I collagen fibrils with 5
third generation PAMAM-NH2 (G3-PAMAM-NH2). First, the type I collagen fibrils were reconstituted and immobilized with G3-PAMAM-NH2. Then, to test whether the bound G3-PAMAM-NH2 molecules would be released into solution, we analyzed their ultraviolet-visible spectra (UV-visible spectra). Finally, transmission electron microscopy (TEM) was employed to characterize the morphologies of the mineralized reconstituted type I collagen fibrils. Selected area electron diffraction (SAED) was performed to identify the mineral phase at different periods. The hypothesis was that
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G3-PAMAM-NH2 could induce biomimetic mineralization on reconstituted type I collagen fibrils.
2. Materials and methods 2.1. Self-assembly and crosslinking of collagen fibrils Lyophilized type I collagen powder derived from bovine skin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.1 M acetic acid (pH 3.0) at 4 °C. A 100 μL of type I collagen fibril stock solution (0.15 mg/mL) was reconstituted over 400-mesh formvar-and-carbon-coated gold TEM grids by neutralizing the collagen stock solution with ammonia vapor in a humidity chamber for 3 h. The neutralized collagen solution was incubated at 37 °C for 5 days to ensure that it transformed into a gel. To stabilize the structures of the reconstituted collagen fibrils, a cross-linking procedure was performed as follows: first, a solution containing 0.3 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.06 M N-hydroxysuccinimide (NHS) was prepared. Then, the pH of the EDC/NHS solution 6
was adjusted to 5.7 with 2-morpholinoethane sulphonic (MES) powder. Last, the collagen grids were treated with 100 μL of the EDC/NHS solution for 3 h, washed with deionized water and air-dried.
2.2 UV-visible spectra
TEM grids coated with and without cross-linked collagen fibrils were treated
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with 50 μL of G3-PAMAM-NH2 solution (1000 ppm) for 2 h. In addition, a TEM grid coated with cross-linked collagen fibrils was treated with 50 μL of deionized water for 2 h. All groups were washed with deionized water, air-dried and then immersed in 1 mL of deionized water. After 6 h, the TEM grids were removed from the deionized water and the absorbance of the remaining deionized water was measured at a wavelength of 280 nm using an ultraviolet spectrophotometer (UV-1800, MPD Instrument Company, Shanghai, China) to examine whether there were free PAMAM dendrimers in the remaining deionized water. A G3-PAMAM-NH2 solution (100 ppm) was used as a positive control.
2.3 Collagen mineralization TEM grids coated with cross-linked collagen fibrils were treated with 50 μL of G3-PAMAM-NH2 solution (1000 ppm) or 50 μL of deionized water (the control) for 2 h, washed with deionized water, air-dried and then immersed in 5 mL of artificial saliva solution (pH 7.0) containing 1.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 1.0 mM NaN3 and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 7
(HEPES) at 37 °C.[30] The TEM grids were removed from the artificial saliva after 6, 24 and 48 hours, washed with deionized water, air-dried and characterized by TEM (200 kV, Tecnai GF20S-TWIN, FEI, USA). SAED of the mineralized collagen fibrils was performed to identify the mineral phase. For each group, 5 TEM grids were analyzed.
3. Results and discussion
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3.1 UV-visible spectra UV-visible spectra of the four tested solutions are displayed in Figure (1). The standard spectrum of the G3-PAMAM-NH2 solution (100 ppm) showed an obvious characteristic peak at approximately 280 nm. After storing the collagen that was treated with deionized water for 6 h, the spectrum of the remaining deionized water exhibited a smooth curve, which indicated that there were hardly any free G3-PAMAM-NH2 molecules. After storing the TEM grid that was treated with 1000 ppm of G3-PAMAM-NH2 for 6 h, a similar smooth curve was observed revealing that G3-PAMAM-NH2 could scarcely bind to the TEM grid. On the contrary, after storing the collagen that was treated with 1000 ppm of G3-PAMAM-NH2 for 6 h, the spectrum of the remaining deionized water displayed a weak characteristic peak at 280 nm, which suggested the presence of a small number of free PAMAM macromolecules in the solution. Collagen fibrils have a size-exclusion feature,[31] that prevents molecules larger than 40 kDa from penetrating into their internal water compartments while 8
allowing molecules smaller than 6 kDa to freely diffuse into their water compartments. In other words, molecules with a molecular weight (MW) between 6 - 40 kDa can be retained within collagen fibrils. The molecular weight of G3-PAMAM-NH2 is 6909 Da, which is just within this range. In addition, a previous study has demonstrated that carboxyl-terminated PAMAM (PAMAM-COOH) can enter into collagen fibrils,[32] and the structure of PAMAM-NH2 is similar to PAMAM-COOH. Therefore, G3-PAMAM-NH2 may penetrate into collagen fibrils and be retained within them. On
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the other hand, there are many charged sites on collagen surfaces，[33] enabling G3-PAMAM-NH2 molecules to potentially bind to collagen surfaces via electrostatic interactions [34] because their many charged functional groups (positively charged amine groups and negatively charged amide groups).
In summary, we speculated
that when collagen fibrils were exposed to a large number of G3-PAMAM-NH2 molecules, some molecules were retained within the collagen fibrils due to their size-exclusion feature, while other molecules may have bound to the collagen surfaces via weaker electrostatic interactions. When the collagen fibrils coated with G3-PAMAM-NH2 were immersed in solution, a small portion of the bound molecules could be released into the solution because their binding strength to the collagen fibrils was not strong enough.
3.2 Collagen mineralization The presence of remnant phosphoproteins that remain bound to collagen fibrils after dentin demineralization also has the potential to induce remineralization, which 9
may lead to confusion when interpreting the experimental results.[11] To avoid the effect of remnant phosphoproteins, we chose a pure type I collagen fibril model to simulate completely demineralized CMTs. Biomimetic mineralization is different from traditional mineralization, as it can induce intrafibrillar mineralization within collagen fibrils, which is very important for the biomechanical properties of mineralized dentin. In this study, we employed TEM to observe the morphologies of the mineralized, reconstituted type I collagen fibrils
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and explored whether G3-PAMAM-NH2 could induce intrafibrillar mineralization within them. As can be seen in Figure 2 (A), after mineralization for 6 h, collagen fibril immobilized with G3-PAMAM-NH2 was slightly darkly stained by amorphous minerals. Electron-dense mineral nano-precursors (10–50 nm in diameter) (red arrows) aggregated along the periphery of the collagen fibril. These nano-precursors exhibited fluidity by coalescing to produce continuous phases, which revealed their amorphous nature (Figure 2, B). The nano-precursors were arranged within the collagen fibrils in an ordered manner with a roughly 70 nm periodicity (Figure 2, C) (red arrows showed the periodicity), which is in accordance with a previous report that the characteristic periodicity of the native collagen assembly is approximately 67 nm.[1] In the control group (Figure 2, E), without the sequestration function imparted by free G3-PAMAM-NH2 molecules, the nano-precursors aggregated much faster. Cluster larger than 100 nm in diameter was commonly observed (red arrow). The collagen fibril could not easily be detected because it was not darkly stained by the 10
nano-precursors (Figure 2, D). The electronic diffraction pattern with a 004 reflection (Figure 2, F) revealed that the nano-precursors in the control group had begun to crystallize, while the electronic diffraction pattern of the experimental group had only a broad diffraction ring (Figure 2, B, C) (inset) indicating that the mineralized phase was still amorphous nature. These results confirmed that G3-PAMAM-NH2 could stabilize ACP nano-precursors and reduced the speed of their transformation into crystal.
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After mineralization for 24 h, the mineralized collagen fibrils immobilized with G3-PAMAM-NH2 revealed arrays of ribbon-like mineral particles deposited within them with a roughly 70 nm periodicity (Figure 3, A) (red arrows showed the periodicity). These deposited minerals were closely connected to the collagen fibrils. A typical HA diffraction pattern with distinct (002), (211) and (004) reflections (Figure 3, A) (inset) confirmed that the ACP nano-precursors had been transformed from an amorphous state into apatite. In the control group, the mineral crystals aggregated further. Crystal clusters larger than 200 nm in diameter were observed (Figure 3, B). After mineralization for 48 h, the collagen fibrils immobilized with G3-PAMAM-NH2 were heavily mineralized (Figure 4, A). Obvious mineralized collagen fibrils could be observed due to intrafibrillar mineral deposits even at low magnification. Branching of the collagen fibrils (black arrow) and extrafibrillar mineral deposits (red arrow) were frequently observed. At high magnification (Figure 4, B, C, D), the collagen fibrils were further darkly stained by minerals, which 11
obscured their banding periodicity. In fact, it was difficult to observe the banding characteristics of the completely mineralized collagen fibrils. In the control group, at low magnification (Figure 4, E), a large number of crystals (200 nm-500 nm in diameter) were randomly oriented and evenly distributed throughout the grid. The collagen fibrils were difficult to observe. At high magnification (Figure 4, F), the collagen fibril was slightly stained by a few minerals, which made them more easily observed than after 6 h of mineralization (Figure 2, D). However, it seemed that the
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minerals were only deposited on the collagen surfaces, as the collagen fibrils were not heavily mineralized such as in the experimental group. These results suggested that the collagen surfaces may have served as nucleation templates, but the nucleation appeared to be weak. The size-exclusion feature of collagen fibrils limits the dimension of ACP nano-precursors that can penetrate them. Molecules larger than 40 kDa are completely excluded from collagen fibrils, which may explain why relatively larger ACP nano-precursors (larger than 100 nm in diameter) were incapable of participating in intrafibrillar mineralization. [11] G3-PAMAM-NH2 possesses many amine groups on its external surface and a large number of amide groups in its internal cavity. It can attract calcium ions by calcium complexation through either its amine or amide groups to form temporary a “PAMAM-Ca2+” complex,[26] which can decrease the amount of calcium ions in artificial saliva to prevent calcium and phosphate ions from aggregating too quickly. Based on the above results, we posited that G3-PAMAM-NH2 is capable of stabilizing metastable ACP nanoprecursors to form 12
relatively smaller ACP nanoprecursors (10 nm - 50 nm in diameter), which could diffuse into the water compartments within the collagen fibrils. Comparatively, in the control group, the crystals that were directly formed by calcium and phosphate ions in the solution were too large (larger than 100 nm in diameter) to infiltrate into collagen fibrils, which resulted in failure of intrafibrillar mineralization. Previous studies and the results of this research indicate that G3-PAMAM-NH2 molecules may enter into collagen fibrils due to the size-exclusion feature of collagen
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fibrils and may bind to the collagen surfaces via electrostatic interactions.[31-34] Furthermore, free and bound PAMAM molecules possessed different sequestration and templating functions. In summary, we proposed a possible mechanism behind G3-PAMAM-NH2-coating-induced mineralization of collagen fibrils (Figure 5). First, when collagen fibrils were exposed to G3-PAMAM-NH2 solutions, some PAMAM molecules entered into them and were retained within the intrafibrillar spaces (67 nm intervals) due to the size-exclusion feature. At the same time, other PAMAM molecules bound to the surfaces of the collagen fibrils via electrostatic interactions. After being immersed in artificial saliva, some of the PAMAM molecules that were bound to the surfaces were released into solution. Then, with the help of the free PAMAM molecules, smaller ACP nano-particles penetrated into the collagen fibrils and the bound PAMAM molecules acted as templates to attract the ACP nano-particles through calcium complexation. Finally, as the ACP nano-particles within the collagen fibrils slowly transformed into HA, the collagen fibrils were completely mineralized. 13
In addition to PAMAM-NH2, other types of PAMAM dendrimers have been used for the biomimetic mineralization of teeth such as PAMAM-COOH, PAMAM-COOH-alendronate (ALN) conjugate (ALN-PAMAM-COOH), and polyhydroxy-terminated PAMAM (PAMAM-OH) dendrimers.[27, 29, 32] However, there are some limitations to using these PAMAM dendrimers such as the high cost of PAMAM-COOH, the relatively weak remineralization ability of PAMAM-OH and the extremely difficult synthesis condition of ALN-PAMAM-COOH. Compared with
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these PAMAM dendrimers, the only limitation to using PAMAM-NH2 is that its biocompatibility is slightly poor. However, a previous study has demonstrated that there was nearly no cytotoxicity to oral cells when the concentration of PAMAM-NH2 was lower than 1250 ug/mL. [28] Therefore, the concentration of PAMAM-NH2 in this study (1000 ug/mL) was safe and could potentially be used in clinical dentistry. Dentin demineralization is a common pathological state, which is related to dentin caries, dentin hypersensitivity and other dentin diseases. Remineralization of demineralized dentin is a non-invasive therapeutic method and is preferred to traditional invasive substitutes.[35] In recent years, various biomaterials have been employed for remineralizing demineralized dentin such as fluoride, bioactive glasses, amorphous calcium phosphate (ACP)-releasing resins, and dopamine.[36-40] However, all of the above are only suitable for remineralizing partially demineralized dentin, which relies on the epitaxial deposition of calcium and phosphate ions over remnant crystallites.[41] In some clinical situations, such as the hybrid layers that are created by etch-and-rinse adhesive systems and in the superficial portion of 14
caries-affected dentin lesions that are left behind after minimally invasive caries removal, dentin is completely demineralized. With regard to completely demineralized dentin, traditional remineralization agents are ineffective, as there are no remnant crystallites in the collagen fibrils. It is a difficult problem to remineralize completely demineralized dentin in clinical dentistry. Biomimetic remineralization, which is based on a non-classical particle-based crystallization concept and does not rely on remnant crystal seeds, may solve this clinical dental puzzle. This study has
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demonstrated that G3-PAMAM-NH2 can mineralize type I collagen fibrils, which is similar to the special clinical conditions mentioned above. Hence, PAMAM dendrimers may be used to remineralize completely demineralized dentin. In previous studies, PAMAM-COOH has been demonstrated to possess the abilities to remineralize dentin and enamel.[32，42] PAMAM-NH2 has a similar structure to PAMAM-COOH, which suggests similar abilities of remineralization. We have confirmed that PAMAM-NH2 can remineralize demineralized dentin. In future experiments, we will explore the remineralization ability of PAMAM-NH2 on enamel. Our hope is that G3-PAMAM-NH2 could be used in other dental clinical treatments, such as in the treatments of enamel caries and root lesions. [43, 44]
4. Conclusions In this study, we used G3-PAMAM-NH2 dendrimers to induce the mineralization of type I collagen fibrils. Our results suggested that G3-PAMAM-NH2 could simulate both the sequestration and templating functions of natural NCPs in biomineralization. 15
Intrafibrillar mineralization within collagen fibrils was achieved. G3-PAMAM-NH2 has great potential for the mineralization of type I collagen fibrils and may be a promising restorative biomaterial for other biomineralized tissues.
Acknowledgements Financial support from the National Natural Science Foundation of China (Grant Nos. 81170958, 81400508 and 51073102), Specialized Research Fund for the
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Doctoral Program of Higher Education of China (Grant No. 20100181110056 and 20130181120125) and Sichuan Province Science and Technology support program (Grant No. 2011SZ0130) is gratefully acknowledged. We also thank Xiao Yang, Xin Xu, Lei Cheng and Libang He for their guidance in this study.
Author contributions This study was accomplished through the cooperation of two independent departments at Sichuan University: the State Key Laboratory of Oral Diseases, and the Department of Biomedical Polymers and Artificial Organs. Prof. Xuedong Zhou and Prof. Jiyao Li are from the State Key Laboratory of Oral Diseases and they were in charge of the design and planning of the entirety of the study. Kunneng Liang, Yuan Gao, Ying Liao and Shimeng Xiao are also from this lab and their work included sample preparation and the mineralization experiment. Because the TEM tests were conducted in this lab, they were also in charge of analyzing these results. Finally, Liang wrote the article. 16
Prof. Jianshu Li is from the Department of Biomedical Polymers and Artificial Organs. He was responsible for the synthesis of the PAMAM dendrimers and the tests of UV-visible spectra.
Keywords: Amine-terminated PAMAM dendrimer, Biomaterial, Biomimetic mineralization, Collagen fibrils, Intrafibrillar mineralization.
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Figure 1. UV-visible spectra of a 100 ppm of G3-PAMAM-NH2 solution, the remaining deionized water after storing collagen that was treated with deionized water, the remaining deionized water after storing collagen that was treated with 1000 ppm of G3-PAMAM-NH2 solution and the remaining deionized water after storing a TEM grid that was treated with 1000 ppm of G3-PAMAM-NH2 for 6 h.
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Figure 2. TEM images of the mineralized collagen fibrils immobilized with (A, B, C) and without (D, E, F) G3-PAMAM-NH2 after being immersed in artificial saliva for 6 h. (B) is the high magnification photo of coalesced nano-precursors in (A) (arrows). Electron diffraction of the mineralized collagen fibrils (insets) showed their mineral phase. (F) is the electron diffraction of (E).
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Figure 3. TEM images of the mineralized collagen fibrils immobilized with (A) and without (B) G3-PAMAM-NH2 after being immersed in artificial saliva for 24 h. Electron diffraction of the mineralized collagen fibrils (insets) showed the mineral phase.
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Figure 4. TEM images of the mineralized collagen fibrils immobilized with (A, B, C, D) and without (E, F) G3-PAMAM-NH2 after being immersed in artificial saliva for 48 h. (B, C, D, F) are high magnification photos.
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Figure 5. Schematic demonstration of the mechanism of biomimetic mineralization of collagen fibrils induced by G3-PAMAM-NH2. The rectangle represents a collagen fibril. The green cylinders within the rectangle represent microfibrils. The orange balls, yellow balls and purple balls represent Ca2+ ions, PO43- ions and ACP nano-particles, respectively. In the control group, without the help of free PAMAM, large ACP nano-particles could not enter the collagen fibrils. In the experimental group, free PAMAM molecules stabilized the ACP nano-particles to form smaller ACPs, which could penetrate into collagen fibrils and combine with the bound PAMAM.
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