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Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo
Biomaterials xxx (2014) 1e8
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Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo Benedetto Marelli a, 1, Chiara E. Ghezzi a, 1, Yu Ling Zhang b, Isabelle Rouiller c, Jake E. Barralet b, d, **, Showan N. Nazhat a, * Department of Mining and Materials Engineering, Faculty of Engineering, McGill University, 3610 University St., Montr eal, QC, H3A 0C5, Canada Faculty of Dentistry, McGill University, 3640 University St., Montr eal, QC, H3A 0C5, Canada c Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, 3640 University St., Montreal, QC, H3A 0C5, Canada d Department of Surgery, Montreal General Hospital, McGill University, Montreal, Canada a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2014 Accepted 2 October 2014 Available online xxx
We demonstrate that intrafibrillar, homogenous collagen biomineralization can be achieved by controlling self-assembly under mildly alkaline conditions. Using dense collagen (DC) gels as an osteoid model, we modulated their fibrillogenesis environment to evaluate the effects of fibrillogenesis pH on the protein charge distribution and ultimately on biomineralization. Cationic and anionic dye staining and electron cryomicroscopy analyses established that fibrillogenesis under mildly alkaline conditions promotes the formation of electronegative charges within the protein (anionic DC gels). These charges are stable upon titration of the gel pH to physiological values. Subsequent exposure of anionic DC gels to simulated body fluid induced the intrafibrillar biomineralization of the gels, promoting a rapid, extensive formation of carbonated hydroxyapatite, and strongly impacting gel mechanical properties. The generality and significance of this approach has been addressed by implanting freshly made anionic DC gels in vivo, in a rat subcutaneous model. Subcutaneous implants showed an extensive, homogenous biomineralization as early as at day 7, indicating that anionic collagen gels rapidly self-mineralize upon contact with body fluids in a non-osseous implantation site. The control of collagen fibrillogenesis pH provides not only new interpretations to what has been called the collagen mineralization enigma by demonstrating that neat collagen can intrafibrilarly self-mineralize, but it will also set a new starting point for the use of DC gels in bone regenerative medicine, in addition as potential applications as mineralized tissue model or as slow-release delivery carriers. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Collagen Mineralization Hydroxyapatite Gel Bone In vivo
1. Introduction Bone is a biocomposite comprised of type I collagen nanofibrils reinforced with ~60 wt% nanocrystals of carbonated hydroxyapatite (CHA). Nucleation and growth of the inorganic crystalline phase occurs within collagen intrafibrillar and interfibrillar spaces of the osteoid (premineralized bone matrix) and it is thought to be regulated by anionic non-collagenous proteins (NCPs) through polyanionic domains. Collagen mineralization has been widely
* Corresponding author. ** Corresponding author. Faculty of Dentistry, McGill University, 3640 University al, QC, H3A 0C5, Canada. St., Montre E-mail addresses: [email protected] (J.E. Barralet), showan.nazhat@mcgill. ca (S.N. Nazhat). 1 Present address: Department of Biomedical Engineering, Tufts University, Medford, MA, 02155, United States.
investigated and has inspired several engineering efforts to mimic hard tissue formation for bone regeneration applications [1e4]. Biomimetic alternatives to replace bone autografts are being actively sought to overcome the limitations intrinsic in currently applied surgical procedures (e.g. extended waiting and operative times, risk of infection, and limited autologous bone supply) [5e10]. As notable efforts to replicate collagen biomineralization in vitro, negatively charged polymers (e.g. polyaspartic acid) have been added to calcium-phosphate (CaP) solution to achieve intrafibrillar mineralization of collagen fibrils via the formation of a polymerinduced liquid-precursor [1e3]. This process has also been applied with polyvinylphosphonic and polyacrylic acids on turkey tendon [2], regenerated collagen fibrils, or demineralized human dentin; yielding hierarchically organized apatite crystals in the mineralized collagen fibres [11e14]. Attributable to its binding affinity to CaP, fetuin has also been shown to induce intrafibrillar mineralization [15]. Fetuin's small dimension (<6 kDa) allows it to
http://dx.doi.org/10.1016/j.biomaterials.2014.10.008 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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diffuse in all the water compartments within the collagen fibrils [12,16e18]. In addition, a flowing solution of inorganic cations and tropocollagen forced through a nanoporous membrane into an anion-rich receiving solution has successfully led to the formation of mineralized nanofibres [19]. Despite this significant interest in the collagen mineralization process, these strategies have only been applied to non-medically relevant amounts of collagenous materials and are yet to be successfully transferred to tissue-like constructs. Indeed, the only reported strategies for bulk, three-dimensional (3D) mineralization of osteoid-like constructs within physiological boundaries relied on the addition of an amorphous inorganic phase (i.e. bioactive glass [20,21] and amorphous calcium carbonate [22]) or on the inclusion of NCP-mimicking anionic fibroin derived polypeptides [23] within nanofibrillar dense collagenous frameworks. Although successful, these strategies did not mimic the physiological intrafibrillar biomineralization process, responsible for the biomechanical properties of mineralized collagen [13,24]. In this study, we hypothesized that the simple fibrillogenesis of collagen in mildly alkaline environment may lead to the formation of a collagen richer in negative charges, a characteristic which has been previously reported to induce formation of CaP [25,26]. To test this hypothesis we investigated the mineralization of osteoidequivalent dense collagen (DC) gels formed in neutral (Nf) [27,28], slightly alkaline (ANf) and mildly alkaline (Af) environments by exposing them to simulated body fluid (SBF, pH 7.4) and by subcutaneous implantation. Plastic compression was used to fabricate DC gels with morphological, physical and mechanical properties similar to the mineralizing osteoid [28,29]. As made Nf, ANf and Af DC gels were investigated through cryo-transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM), anionic and cationic staining, transmittance Fourier Transform Infrared spectroscopy (t-FTIR), m-Raman and tensile testing to evaluate possible fibrillogenesis pH-dependent changes in the properties of collagen. The nano-to macro-scale mineralization of collagen fibrils was assessed with cryo-TEM, SEM, X-ray micro-computed tomography (microCT), thermogravimetric analysis (TGA), attenuated total reflectance-FTIR (ATR-FTIR) spectroscopy, m-Raman and X-ray diffraction (XRD). The effect of collagen biomineralization on construct mechanical properties was investigated through tensile testing. 2. Materials and methods
2.3. Mineralization of collagen gel sheets and rolls After densification, gels were incubated in phosphate buffered saline (PBS; pH 7.4) at 37 C for 120 min to equilibrate the pH of the collagen fibril to a physiological level prior to exposure to Kokubo's SBF [30]. The extent of mineralization within collagen gels prepared at increasing alkalinity was investigated as previously described [29]. Gels (n ¼ 5) were conditioned at 37 C using a standardized ratio of 15:1 (ml/mg) SBF:collagen and mineralization was investigated after 3, 6, and 12 h, and at days 1, 3, 7 and 14. The solution was replaced at two day intervals by fresh, sterile SBF (pH 7.4). 2.4. Cryo-transmission electron microscopy Cryo-TEM was performed according to a previously reported protocol [15], with some modifications. Collagen solution (1 ml; 1.5 mg/ml) at neutral and mildly alkaline pH was fibrillized on Cryo-TEM Au grids (R2/2 Quantifoil Au grids) for 15 min at 37 C and 100% RH. To investigate mineralization, the Au grids with fibrilized collagen on top were transferred to 5 ml of SBF solution at 37 C for 24 h. Samples were washed with MilliQ water for 1 min, stained with uranyl acetate (0.5%) in MilliQ water for 15 s, washed in MilliQ water for 1 min and manually blotted immediately before vitrification by plunge freezing in liquid ethane, and stored in liquid nitrogen until these grids were loaded onto the cryogenic sample holder Gatant 626. Samples were then imaged with a Tecnai F20 equipped with a field emission gun operating at 200 kV. Images were recorded under low dose conditions (~10 eÅ2) using a Gatan Ultrascan 4000 4k 4k CCD Camera System Model 895. 2.5. X-ray microtomography MicroCT was used to calculate the vol% of mineralized collagen in the gel at days 1, 3, 7 and 14 in SBF. MicroCT analysis was performed on freeze-dried specimens with a SkyScan 1172 (SkyScan, Kontich, Belgium) using a 360 flat-field corrected scan at 67 kV and 175 mA, with a step size of 0.4 , a resolution of 8 mM, a large camera pixel, no filter, and 10 random movements. Volumetric reconstruction (NRecon software, SkyScan) was generated with a beam hardening correction of 15, a ring artifact correction of 10 and an “auto” misalignment correction. A grayscale intensity range of 20e255 (8 bit images) was used in 2-D analysis (software CTAn, SkyScan) to remove background noise. A previously reported protocol was used to analyze mineralized samples [29]. 2.6. FTIR spectroscopy t-FTIR spectroscopy (Spotlight 400, PerkineElmer, USA) was used in point mode to characterize the collagen gels as made, and to investigate mineralization. A resolution of 2 cm1, an IR range of 4000 to 500 cm1, 64 scans per sample and an aperture of 100 were used. The kinetic of carbonated hydroxyapatite (CHA) formation was indicated through changes in the ratio of the absorbance of v3 PO3 4 to Amide I (1022 cm1/1661 cm1) while the crystallinity index of the CHA was measured as proposed by Termine and Posner [31]. In particular, the spectra were baseline corrected and normalized against the intensity of v3 PO3 peak at 4 1022 cm1. The crystallinity index of the v4 PO3 at 560e600 cm1 was then 4 1 1 calculated as the sum of the 562 cm and 600 cm peak heights divided by the height of the minimum between this doublet following the method of Weiner and Bar-Yosef [32].
2.1. Preparation of dense collagen gel sheets and rolls at different alkalinity
2.7. m-Raman analysis
Collagen gels were prepared by adding dilute acid solubilised rat-tail tendon type I collagen (2.05 mg/ml in 0.6% acetic acid; First Link Ltd., U.K.) to 10 times concentrated Dulbecco Modified Eagle Medium (10x DMEM; Sigma Aldrich, Canada) at a ratio of 4:1. The solution pH was adjusted to the desired value (Nf pH ¼ 7.4, ANf pH ¼ 8.2 and Af pH ¼ 9.0) by adding 5 M NaOH (Fisher Scientific, Canada) at the range of 1.7e2.0 vol%. Gels were prepared by casting the neutralized collagen solution in a cylindrical mold (Amold ¼ 2 cm2, Vsolution ¼ 2 ml) and incubating at 37 C for 25 min. Gel collagen fibrillar density was increased by applying the plastic compression method, as previously described [28]. Briefly, as cast highly-hydrated gels (0.2 wt%) were subjected to 1 kPa for 5 min to generate DC sheets (14.1 wt%). Gel collagen fibrillar density was measured by weighing the gels before and after freeze-drying (BenchTop K, VirTis, Canada). Gels (n ¼ 5) were frozen in liquid nitrogen for 3 min followed by freeze-drying for 6 h at 105 C and 13 mTorr. Cylindrically rolled DC gels were prepared by plastically compressing prism gels (n ¼ 5, Amold ¼ 18 43 mm2, Vsolution ¼ 4 ml) to obtain sheets (14.1 wt% collagen), which were rolled along their long axis to give cylinders of 1.5 mm in diameter and 20 mm in length.
m-Raman analysis was carried out on wet gels with an InVia Raman (Renishaw, USA) equipped with an NIR laser (785 cm1, 22 mW) and a Carl Zeiss optical microscope (50 objective with an aperture of 50). In order to reduce any potential collagen protein damage due to auto-fluorescence, a photobleaching pre-treatment of 2 min was used, thus reducing the intensity of the laser to 1%. A range of 1800 to 400 cm1, a resolution of 0.5 cm1, three accumulations per sample and an exposure time of 60 s were used. Cosmic rays were removed using the appropriate function in The Wire v.3.0 software (Renishaw). 2.8. X-ray diffraction XRD patterns of the freeze-dried gels were analyzed with a Bruker D8 Discover (Germany) from 6 to 60 2q at 40 kV and 20 mA. Two frames of 30 were recorded for 15 min and merged during data post processing. Sample cross-sections were analyzed with a pinhole size of 0.05 mM. The XRD spectra were analyzed with EVA software (Bruker) correcting the baseline (threshold ¼ 0.750). The phase composition was assigned by comparing the acquired spectra with peaks identified in the International Centre for Diffraction Data (ICDD) database.
2.2. Ionic dye binding
2.9. Thermogravimetric analysis
The capacity of collagen gels fibrilized at increasing alkalinity to bind to both anionic (acidic fuchsine, 0.3%) and cationic (methylene blue, 0.1%) dyes (SigmaeAldrich, Canada) was investigated. Nf and Af gels were immersed in the dye solutions at 37 C for 30 min and then rinsed in distilled deionized water. Gels were maintained in the dark to prevent dye photobleaching.
TGA was used to quantify mineralization within the gels at different time points in SBF. Gels (n ¼ 5) were carefully rinsed with distilled deionized water prior to freeze-drying. Specimens were then heated up to 700 C with a 10 C/min ramp function using a TA SDT Q600 TGA (TA Instruments, USA). The weight residue percent was used as the percentage of mineral in the gels.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
B. Marelli et al. / Biomaterials xxx (2014) 1e8 2.10. Mechanical analysis Tensile testing was carried out on cylindrically rolled DC sheets (14.1 wt% fibrillar density), prepared by plastically compressing prism gels (A ¼ 18 43 mM2, V ¼ 4 ml). Cylindrically shaped specimens (n ¼ 3) of approximately 1.5 mM in diameter and 21 mM in length (measured through microCT, as previously described [20]) were tested using an ElectroForce® Biodynamic Test Instrument 5160 (Bose Corp., USA) with a 15 N load cell. Customized grips were used in displacement control at a rate of 0.01 mm/s. Tests were conducted on as made gels (preconditioned in DMEM at 37 C for 30 min), and on gels allowed to mineralize for 7 or 14 days in SBF. Specimens were maintained hydrated by adding SBF in a drop wise manner during testing. From the resulting stressestrain curves, apparent modulus, ultimate tensile strength (UTS), and strain at UTS were measured as a function of conditioning time. 2.11. Subcutaneous implantation All animal experiments were performed according to the Guidelines of the Canadian Council for Animal Care and approved by the Animal Care Committee, McGill University. Three rats underwent dorsal implantation of Nf and Af gels. For subcutaneous implantation, dense collagen gels were fabricated with a rectangular shape and were then rolled along their long axis yielding Swiss-roll like, cylindrical constructs (∅ ¼ 3 mm, l ¼ 6 mm), as previously described [21]. A 3 cm skin incision was made over the spine extending posteriorly from the shoulders. One pocket was formed on each side of the incision to yield a total of 2 pockets, where Nf and Af gels were implanted for 7 days. Explants were analyzed by von Kossa Staining and with microCT, ATR-FTIR, and XRD. 2.12. Histological analysis Explants were washed in PBS, fixed in 10% neutral buffered formalin overnight, dehydrated through a series of graded ethanol, embedded in paraffin and cut in
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transverse sections of 5 mm-thickness. von Kossa staining was performed on the trimmed sections. 2.13. Statistical analysis All the quantitative data were compared with one-way ANOVA test with a Tukey means comparison implemented with software Origin Pro v.8 (OriginLab, USA).
3. Results and discussion 3.1. Effects of fibrillogenesis pH on collagen To test the hypothesis that fibrillogenesis pH controls mineral nucleation, dense collagen gels (collagen fibrillar density ¼ 14.1 wt %) obtained through plastic compression [28], were fabricated by increasing the alkalinity of tropocollagen self-assembly solution (Fig 1(a)). Fibrilized gels were then washed in physiological solution (i.e. phosphate buffer saline) prior to conditioning in SBF. A higher fibrillogenesis pH corresponded with an increased amount of charge within the collagen fibrils, as inferred from cationic and anionic dying of the gels (Fig. 1(b)) and increased electron microscopy staining (Fig. 2(a)). Banding in Af collagen fibrils was more evident than those in Nf, attributable to the higher uptake of the stain caused by the more charged nature of the fibrils formed in mildly alkaline conditions. The a priori alkaline fibrillogenesis of collagen was preferred to the exposure of an already assembled gel to a basic environment, to avoid protein denaturation [33,34]. In
Fig. 1. pH control of collagen gel fibrillogenesis. a) Densification of reconstituted highly-hydrated type I collagen gels, where fibrillogenesis occurred at neutral (Nf), slightly alkaline (ANf) and alkaline (Af) pH, obtained by application of 1 kPa compressive stress for 5 min. Dense collagen gels were then incubated in PBS (pH 7.4) at 37 C for 120 min to equilibrate the pH of the collagen fibril to physiological level. The mineralization of dense gels was then investigated in SBF for up to day 14. b) Ionic dye staining of collagen gels fibrilized at neutral and mildly alkaline conditions; i) Anionic dye staining. At pH 7.4, Nf collagen have a net positive charge and attract more anionic dye than Af collagen; ii) Cationic dye staining. Af collagen fibrils possess more negatively charged groups and attract more cationic dye than Nf collagen.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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III at 1661, 1550 cm1 and 1240 cm1, respectively) (Fig. S1(b)), confirming that the initial pH of the collagen solution didn't alter the protein structure [15,36e39]. However, the FTIR spectra revealed differences in the resonance between 1100 and 900 cm1 as the absorption of the infrared light increased (i.e. a decrease in the transmittance) in DC gels generated at a mildly alkaline environment, with the formation of a new local minimum at 1065 cm1, which may be attributed both to a different vibration of the v CeO moieties in the hydroxyproline [40] or to the formation of an insoluble and low crystalline PO3 4 salt (e.g.CaP) in the gels [41]. The lower transmittance between 1100 and 900 cm1 would then be caused by the superimposition of the amorphous CaP resonance to that of the collagen. m-Raman scatterings of the as made DC gels obtained at different fibrillogenesis pH were very similar, confirming the overall similarities in the protein structure (Fig. S1(c)). However, DC gels generated at mildly alkaline environment exhibited two shifts at 948 and 428 cm1, which can be attributed to the v4 of phosphate species present in low crystalline or amorphous CaP [42]. 3.2. Nano-to macro-scale mineralization, in vitro
Fig. 2. Effect of fibrillogenesis pH control on nano-to macro-scale mineralization of collagen, in vitro. a) Cryo-TEM nanographs of (i) Nf, (ii) Af as made, and (iii) Nf, (iv) Af after 24 h exposure to SBF. Banding of Af was more evident than in Nf due to the more charged nature of the fibrils that causes a higher uptake of heavy metal ions. Intrafibrillar mineralization was evident in Af but not in Nf after exposure to SBF, which masked the collagen D-banding structure. b) (i) Localized mineralization achieved at day 7 in SBF in Af gels embossed within a larger Nf gel. (ii) MicroCT 3D reconstruction demonstrating mineralized collagen in red, within non-mineralized regions in grey. c) Images of (i) Nf and (ii) Af roll gels at day 7 in SBF, respectively. e) MicroCT 3D reconstruction of (i) Nf and (ii) Af roll gels at day 7 in SBF, respectively. Af gels exhibited bulk and homogeneous mineralization (mineralized collagen phase highlighted in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
addition, NaOH was used to titrate the tropocollagen solutions due to the lower tendency of Naþ to denature collagen compared to other cations (e.g. Kþ, Ca2þ and Mg2þ) [34,35]. Morphological and chemical analyses demonstrated no effect of fibrillogenesis pH on the structure of as made gels (Fig. S1). SEM micrographs showed that procollagen fibrillization was not affected by the initial alkalinity of the solution, where the nanofibrils exhibited similar dimensions (∅ y 135 nm) and maintained the banding pattern, typical of non-denatured collagen (Fig. 2(a) and Fig. S1(a)) [33,34]. Furthermore, t-FTIR showed no fibrillogenesis pH-dependent modifications of the three main Amide vibrations (Amide I, II and
The in vitro mineralization of DC gels reconstituted at different fibrillogenesis pH was dramatically influenced by fibrillogenesis pH from the nano-to the macro-scale. At a nanoscopic level, Cryo-TEM analysis of Nf and Af gels exposed to SBF for 24 h depicted the masking of the typical collagen fibrils banding for Af samples only, which has been correlated with intrafibrillar mineralization (Fig. 2(a)) [15,43]. SEM analysis of Nf, ANf and Af gels at day 1 in SBF depicted the absence of mineralization in Nf and ANf gels, while mineral formation was only visible in the framework of Af gels (Fig. S2(a)). SEM micrographs of Af samples taken at days 3, 7 and 14 in SBF showed the continuous homogeneous growth of the crystals (Fig. S2(b)). In contrast, mineral formation in Nf and ANf gels was less homogeneous and extensive, visible only at days 7 and 14, as previously reported [29]. At a macroscopic scale, the effect of locally altering collagen fibrillogenesis pH was investigated by embossing letter-shaped Af gels (mM in size) within a larger Nf gel (cm in size) (Fig. 2(b) i). The hybrid AfeNf gel was then conditioned in SBF for 7 days. During exposure to SBF, the Af portion of the hybrid gel turned from palewhite to bright-white, indicating the formation of a new phase in the Af gels, which induced light scattering. MicroCT 3D reconstruction of the collagen gel at day 7 in SBF confirmed regional optical differences to be attributable to a denser phase formed within Af gels (highlighted in red, Fig. 2(b) ii). In addition, the 3D mineralization of Nf and Af gels was investigated by exposing cylindrically rolled DC gels (Ø ¼ 1.5 mM, l ¼ 20 mM) to SBF (Fig. 2(c)). At day 7 in SBF, Af cylindrical rolls self-sustained their own weight and appeared more white in colour, while Nf gels did not show any visible difference from those as made. MicroCT 3D reconstruction of Nf and Af cylindrical rolls at day 7 in SBF depicted the homogenous and bulk formation of a denser phase within Af gels (highlighted in red in Fig. 2(d) ii). 3.3. Characterization of the mineral phase t-FTIR, m-Raman and XRD analyses confirmed calciumphosphate nucleation and growth in collagen gels generated at mildly alkaline pH. Fig. 3(a) displays the change in the t-FTIR spectra of Af gels from t ¼ 0 to t ¼ 336 h (day 14) in SBF. Amide I peak remained unchanged at 1661 cm1 indicating no denaturation of the protein during the mineralization process [39], while Amide II decreased in intensity, indicating the nucleation of a mineral phase within collagen fibrils [6]. Additionally, the v3 PO34 and v4
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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Fig. 3. Characterization of Af collagen gel mineralization in SBF. a) t-FTIR spectroscopy showed no changes in the collagen triple helical structure during the mineralization process (Amide I peak at 1661 cm1). The v1, v2, v3 resonances of PO3 4 were visible in all spectra, suggesting the formation of CaP minerals. At day 1, typical apatite peaks (e.g. v3 vibration at 1033 cm1) were present which shifted to a lower wavenumber with time, revealing an increase in its crystallinity. The v2 CO2 3 vibration peak was present within 1 h in SBF and increased during the mineralization process, suggesting the presence of carbonated hydroxyapatite (CHA). b) The increase in the 1028 to 1661 cm1 peak ratio with time in SBF indicated an increase in CaP in the collagen framework. c) CaP crystallinity index increased after day 1 in SBF. d) m-Raman analysis depicted that collagen triple helical structure (peak at 1661 cm1) was not affected by the mineralization process. Phosphate species were formed as soon as after 3 h of exposure to SBF, suggesting the formation of CaP minerals. Mineral extent and crystallinity increased with time in SBF, as the Raman scattering of the phosphate species increased in intensity and became sharper for longer time points. e) XRD analysis showed diffractographs compatible with formation of bone-like hydroxyapatite within the collagenous matrix as soon as at day 1 upon exposure to SBF, as indicated by the peak around 25 and 31 2theta. Samples' crystallinity increased with time in SBF, as the peaks compatible with hydroxyapatite species increased in intensity and became sharper for longer time points.
PO3 absorbances at 1022 and 560e600 cm1, respectively, 4 increased as a function of conditioning time in SBF. The peaks and the shape of the PO3 4 resonances together with the presence and 1 temporal growth of the v2 CO2 suggested CHA 3 peak at 871 cm formation. Indeed, the formation of an apatitic phase was spectroscopically evident for Af gels within a 3 h exposure time to SBF (Fig. 3(b)) and v3 PO3 4 absorbance increased with time prior to stabilization at 168 h (day 7). Furthermore, the crystallinity index of CHA was not statistically different for the first two days in SBF (p > 0.05 between 3 and 48 h) (Fig. 3(c)), while there was an increase in the crystallinity of CHA after 72 h, which continued up to 168 h. The t-FTIR results were corroborated by m-Raman spectra (in Fig. 3(d)). The Raman shift of the collagen (Amide I and III at 1671 and 1243 cm1, respectively) remained unchanged during conditioning in SBF, while the shift of the v1 and of v4 PO3 4 indicated the formation and the growth of CHA. In particular, there was a tem1 poral shift in the v1 PO3 4 peak from 962 to 960 cm , an increase in its intensity when compared to the Amide I peak and a decrease in its half width, suggesting the formation and growth of CHA, with
increasing crystallinity at longer conditioning time in SBF [42]. XRD diffractographs of Af at various conditioning times in SBF confirmed apatite formation (Fig. 3(e)). The presence of two broad peaks at 7 and 25 , indicated the typically random collagen fibril organization in the as made gel [44]. By day 1 in SBF, HA formation was confirmed (ref. 009-0432), and its crystallinity increased with time, as indicated by the more intense and narrower peaks at 31 2theta. In contrast, the formation of low crystalline CHA in Af and ANf gels was detectable only at days 7 and 14 (data not shown), as previously reported [29]. TGA allowed for quantitation of mineral growth in DC gels through measurement of weight residue. Nf and ANf gels presented statistically similar thermogravimetric results (p > 0.05), with a linear increase in the weight residue reaching 13 dry weight% at day 14 in SBF (Fig. 4(a)). In contrast, as early as day 1 in SBF, mineralization in Af gels approached 10 wt%, which increased to 69 wt% at day 7 prior to stabilization at 76 wt% at day 14. Furthermore, microCT volumetric reconstruction analysis was implemented to calculate the volume percentage of mineralized collagen within the
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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Fig. 4. Characterization of mineralization extent, in vitro. a) TGA generated dry weight residue (mineral phase) increased with time in SBF for all gels. There was no statistically significant difference between mineralization extent in Nf and ANf gels (12 wt% at day 14). Af gels yielded 40 wt% mineral by day 3, and increased to 76 wt% at day 14. b) Mineralized collagen vol% calculated through microCT analysis. Increase in mineralization extent corresponded with an increase in mineralized collagen phase (95 vol% at day 14).
gel (Fig. 4(b)). Almost 27% of the collagen was mineralized in Af gels at day 1 in SBF, increasing to 50 and 82% at days 3 and 7, respectively, before reaching 95% at day 14. 3.4. Mechanical analysis The effects of fibrillogenesis pH and of conditioning time, at days 7 and 14, in SBF on the mechanical properties of Nf, ANf and Af gels were investigated through tensile testing. There were no statistically significant differences (p > 0.05) between the apparent modulus, UTS and strain at UTS values of cylindrically rolled as made gels of Nf, ANf and Af (Fig. 5(aec)) prior to mineralization. By day 14 in SBF, the values of these parameters remained statistically
insignificant (p > 0.05) between Nf and ANf and showed negligible changes. On the other hand, by day 14 in SBF, the apparent modulus and UTS values in Af gels were statistically higher (p < 0.05) when compared to those of Nf and ANf, which could be attributed to the enhanced mineral formation within the gels [45]. The strain at UTS of Af gels underwent a significant decrease (p < 0.05) with time in SBF. Representative stressestrain curves of gels conditioned up to day 14 in SBF displayed typical soft-tissuelike responses of Nf, ANf gels, with toe, linear and failure regions (Fig. 5(d)) [46]. In contrast, Af gels indicated a transition from softto-hard tissue-like response to tensile stress, exhibiting an increase in apparent modulus and UTS and a decrease in the strain at UTS.
Fig. 5. Mechanical properties of dense collagen gels as made, and at days 7 and 14 in SBF. a), b) and c) apparent modulus, UTS, and strain at UTS, respectively, as a function of conditioning time in SBF. d) Representative stressestrain curves of Nf, ANf and Af gels at day 14 in SBF. Higher mineralization extent in Af gels resulted in a transition from soft-tohard tissue-like response to tensile stress. There was a reduction in the toe region and an increase in the apparent modulus and UTS with time in SBF. This effect was particularly evident in Af gels. Nf and ANf gels registered no differences (p > 0.05) in apparent modulus with time in SBF, and a general reduction in UTS and strain at UTS, was measured. Af gels displayed a significant increase (p < 0.05) in apparent modulus at days 7 and 14 and in UTS at day 14 in SBF, attributable to the formation of a highly compact mineralized hybrid where both collagen fibrils and hydroxyapatite crystal bore the load. Strain at UTS decreased (p < 0.05) with increasing mineralization time. This effect was particularly evident in Af gels.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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Fig. 6. Effects of fibrillogenesis pH control on collagen mineralization, in vivo. a) Histological sections of (i) Nf and (ii) Af roll gels implanted subcutaneously for 7 days. Mineralized collagen was highlighted with von Kossa staining (black, brown in the images). b) MicroCT 3D reconstruction of (i) Nf and (ii) Af gels implanted subcutaneously for 7 days. Collagen is highlighted in grey while mineralized collagen is highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.5. In vivo mineralization The in vitro results were confirmed in vivo; in that subcutaneously implanted (i.e. in a non-osseous site thus avoiding the role of in vivo produced NCPs) Af gels extensively mineralized after 7 days. This is in contrast to Nf gels, which showed only minor evidences of mineralization (Fig. 6). von Kossa staining of histological sections indicated extensive mineral formation throughout the thickness of Af gels with no cell infiltration in the gel or cell-mediated remodelling (Fig. 6(a)). MicroCT 3D reconstruction confirmed that mineral formation was homogenously distributed throughout the thickness of Af with 69 vol% mineralization, compared to only 7 vol% in Nf gels (Fig. 6(b)). ATR-FTIR and XRD analyses confirmed the mineral phase formed within the explants to be CHA, in line with the in vitro data (Fig. S3(a and b), respectively).
CaP solution mineralized extrafibrilarly [47]. To investigate the effects of the alkaline environment on collagen fibrillogenesis, Nessler's reagent was used to evaluate the production of ammonia during fibrillogenesis. Interestingly, a detectable amount of ammonia was produced during the self-assembly of Af gels only, indicating the partial reaction of the amino groups present in the side chains of the collagen peptidic sequence during fibrillogenesis under mildly alkaline conditions, which may have resulted in a more charged collagen. In addition, the 3D, bulk in vivo mineralization of Af gels within 7 days upon subcutaneous implantation corroborates, in a nonosseous model, the predominant role of collagen in the nucleation and growth of bone apatite without the presence of bone inducing factors. Indeed, our findings support the recent questioning of the consensus in the literature on the need for Ca-rich NCPs for collagen mineralization to occur, in vivo [43].
3.6. Effects of fibrillogenesis pH on collagen mineralization 4. Conclusions Together these results suggest that fibrillogenesis pH has a dramatic impact on the ability of collagen fibrils to nucleate CHA, both in vitro, when exposed to SBF and in vivo, when implanted subcutaneously. The investigation of the in vitro mineralization of collagen gels reconstituted at mildly alkaline pH depicted a twostage mineralization process; intrafibrillar mineralization with the formation of a low crystalline apatite phase within the first day of exposure to SBF, followed by extrafibrillar CHA crystal formation, which grew in the absence of the mineral shape-regulating NCPs. Interestingly, exposing Nf gels to an alkaline solution for 30 min prior to conditioning in SBF did not impact mineralization, indicating that the alkaline self-assembly induced a distinct change in the properties of collagen. This difference in the mineralization of collagen gels exposed a priori or a posteriori to an alkaline solution underpins a different mineralization mechanism from that previously reported, where collagen membranes exposed to an alkaline
In summary, this work has demonstrated that intrafibrillar, bulk and homogeneous mineralization of dense collagen gels may be rapidly achieved in vitro and in vivo by simply adjusting the alkalinity of collagen during fibrillogenesis. This process provides a model to investigate mineralized tissues in vitro as well as a means to fabricate an immediately implantable osteoid-like tissue for bone regeneration. Acknowledgements The support of the Canadian Natural Sciences and Engineering bec Ministe re de l'Enseignement supe rieur, Research Council, Que de la Recherche, de la Science et de la Technologie, Canadian Institutes of Health Research and Canadian Foundation for Innovation are gratefully acknowledged. Funding for Benedetto Marelli is also
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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supported by the Werner Graupe Fellowship and McGill Engineering Doctoral Award. Funding for Chiara E. Ghezzi is also supported by Showan Nazhat's McGill Engineering Gerald Hatch Faculty Fellowship. Jake Barralet acknowledges the provision of a Canada Research Chair. Appendix A. Supplementary data
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Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo Benedetto Marelli a, 1, Chiara E. Ghezzi a, 1, Yu Ling Zhang b, Isabelle Rouiller c, Jake E. Barralet b, d, **, Showan N. Nazhat a, * Department of Mining and Materials Engineering, Faculty of Engineering, McGill University, 3610 University St., Montr eal, QC, H3A 0C5, Canada Faculty of Dentistry, McGill University, 3640 University St., Montr eal, QC, H3A 0C5, Canada c Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, 3640 University St., Montreal, QC, H3A 0C5, Canada d Department of Surgery, Montreal General Hospital, McGill University, Montreal, Canada a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2014 Accepted 2 October 2014 Available online xxx
We demonstrate that intrafibrillar, homogenous collagen biomineralization can be achieved by controlling self-assembly under mildly alkaline conditions. Using dense collagen (DC) gels as an osteoid model, we modulated their fibrillogenesis environment to evaluate the effects of fibrillogenesis pH on the protein charge distribution and ultimately on biomineralization. Cationic and anionic dye staining and electron cryomicroscopy analyses established that fibrillogenesis under mildly alkaline conditions promotes the formation of electronegative charges within the protein (anionic DC gels). These charges are stable upon titration of the gel pH to physiological values. Subsequent exposure of anionic DC gels to simulated body fluid induced the intrafibrillar biomineralization of the gels, promoting a rapid, extensive formation of carbonated hydroxyapatite, and strongly impacting gel mechanical properties. The generality and significance of this approach has been addressed by implanting freshly made anionic DC gels in vivo, in a rat subcutaneous model. Subcutaneous implants showed an extensive, homogenous biomineralization as early as at day 7, indicating that anionic collagen gels rapidly self-mineralize upon contact with body fluids in a non-osseous implantation site. The control of collagen fibrillogenesis pH provides not only new interpretations to what has been called the collagen mineralization enigma by demonstrating that neat collagen can intrafibrilarly self-mineralize, but it will also set a new starting point for the use of DC gels in bone regenerative medicine, in addition as potential applications as mineralized tissue model or as slow-release delivery carriers. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Collagen Mineralization Hydroxyapatite Gel Bone In vivo
1. Introduction Bone is a biocomposite comprised of type I collagen nanofibrils reinforced with ~60 wt% nanocrystals of carbonated hydroxyapatite (CHA). Nucleation and growth of the inorganic crystalline phase occurs within collagen intrafibrillar and interfibrillar spaces of the osteoid (premineralized bone matrix) and it is thought to be regulated by anionic non-collagenous proteins (NCPs) through polyanionic domains. Collagen mineralization has been widely
* Corresponding author. ** Corresponding author. Faculty of Dentistry, McGill University, 3640 University al, QC, H3A 0C5, Canada. St., Montre E-mail addresses: [email protected] (J.E. Barralet), showan.nazhat@mcgill. ca (S.N. Nazhat). 1 Present address: Department of Biomedical Engineering, Tufts University, Medford, MA, 02155, United States.
investigated and has inspired several engineering efforts to mimic hard tissue formation for bone regeneration applications [1e4]. Biomimetic alternatives to replace bone autografts are being actively sought to overcome the limitations intrinsic in currently applied surgical procedures (e.g. extended waiting and operative times, risk of infection, and limited autologous bone supply) [5e10]. As notable efforts to replicate collagen biomineralization in vitro, negatively charged polymers (e.g. polyaspartic acid) have been added to calcium-phosphate (CaP) solution to achieve intrafibrillar mineralization of collagen fibrils via the formation of a polymerinduced liquid-precursor [1e3]. This process has also been applied with polyvinylphosphonic and polyacrylic acids on turkey tendon [2], regenerated collagen fibrils, or demineralized human dentin; yielding hierarchically organized apatite crystals in the mineralized collagen fibres [11e14]. Attributable to its binding affinity to CaP, fetuin has also been shown to induce intrafibrillar mineralization [15]. Fetuin's small dimension (<6 kDa) allows it to
http://dx.doi.org/10.1016/j.biomaterials.2014.10.008 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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diffuse in all the water compartments within the collagen fibrils [12,16e18]. In addition, a flowing solution of inorganic cations and tropocollagen forced through a nanoporous membrane into an anion-rich receiving solution has successfully led to the formation of mineralized nanofibres [19]. Despite this significant interest in the collagen mineralization process, these strategies have only been applied to non-medically relevant amounts of collagenous materials and are yet to be successfully transferred to tissue-like constructs. Indeed, the only reported strategies for bulk, three-dimensional (3D) mineralization of osteoid-like constructs within physiological boundaries relied on the addition of an amorphous inorganic phase (i.e. bioactive glass [20,21] and amorphous calcium carbonate [22]) or on the inclusion of NCP-mimicking anionic fibroin derived polypeptides [23] within nanofibrillar dense collagenous frameworks. Although successful, these strategies did not mimic the physiological intrafibrillar biomineralization process, responsible for the biomechanical properties of mineralized collagen [13,24]. In this study, we hypothesized that the simple fibrillogenesis of collagen in mildly alkaline environment may lead to the formation of a collagen richer in negative charges, a characteristic which has been previously reported to induce formation of CaP [25,26]. To test this hypothesis we investigated the mineralization of osteoidequivalent dense collagen (DC) gels formed in neutral (Nf) [27,28], slightly alkaline (ANf) and mildly alkaline (Af) environments by exposing them to simulated body fluid (SBF, pH 7.4) and by subcutaneous implantation. Plastic compression was used to fabricate DC gels with morphological, physical and mechanical properties similar to the mineralizing osteoid [28,29]. As made Nf, ANf and Af DC gels were investigated through cryo-transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM), anionic and cationic staining, transmittance Fourier Transform Infrared spectroscopy (t-FTIR), m-Raman and tensile testing to evaluate possible fibrillogenesis pH-dependent changes in the properties of collagen. The nano-to macro-scale mineralization of collagen fibrils was assessed with cryo-TEM, SEM, X-ray micro-computed tomography (microCT), thermogravimetric analysis (TGA), attenuated total reflectance-FTIR (ATR-FTIR) spectroscopy, m-Raman and X-ray diffraction (XRD). The effect of collagen biomineralization on construct mechanical properties was investigated through tensile testing. 2. Materials and methods
2.3. Mineralization of collagen gel sheets and rolls After densification, gels were incubated in phosphate buffered saline (PBS; pH 7.4) at 37 C for 120 min to equilibrate the pH of the collagen fibril to a physiological level prior to exposure to Kokubo's SBF [30]. The extent of mineralization within collagen gels prepared at increasing alkalinity was investigated as previously described [29]. Gels (n ¼ 5) were conditioned at 37 C using a standardized ratio of 15:1 (ml/mg) SBF:collagen and mineralization was investigated after 3, 6, and 12 h, and at days 1, 3, 7 and 14. The solution was replaced at two day intervals by fresh, sterile SBF (pH 7.4). 2.4. Cryo-transmission electron microscopy Cryo-TEM was performed according to a previously reported protocol [15], with some modifications. Collagen solution (1 ml; 1.5 mg/ml) at neutral and mildly alkaline pH was fibrillized on Cryo-TEM Au grids (R2/2 Quantifoil Au grids) for 15 min at 37 C and 100% RH. To investigate mineralization, the Au grids with fibrilized collagen on top were transferred to 5 ml of SBF solution at 37 C for 24 h. Samples were washed with MilliQ water for 1 min, stained with uranyl acetate (0.5%) in MilliQ water for 15 s, washed in MilliQ water for 1 min and manually blotted immediately before vitrification by plunge freezing in liquid ethane, and stored in liquid nitrogen until these grids were loaded onto the cryogenic sample holder Gatant 626. Samples were then imaged with a Tecnai F20 equipped with a field emission gun operating at 200 kV. Images were recorded under low dose conditions (~10 eÅ2) using a Gatan Ultrascan 4000 4k 4k CCD Camera System Model 895. 2.5. X-ray microtomography MicroCT was used to calculate the vol% of mineralized collagen in the gel at days 1, 3, 7 and 14 in SBF. MicroCT analysis was performed on freeze-dried specimens with a SkyScan 1172 (SkyScan, Kontich, Belgium) using a 360 flat-field corrected scan at 67 kV and 175 mA, with a step size of 0.4 , a resolution of 8 mM, a large camera pixel, no filter, and 10 random movements. Volumetric reconstruction (NRecon software, SkyScan) was generated with a beam hardening correction of 15, a ring artifact correction of 10 and an “auto” misalignment correction. A grayscale intensity range of 20e255 (8 bit images) was used in 2-D analysis (software CTAn, SkyScan) to remove background noise. A previously reported protocol was used to analyze mineralized samples [29]. 2.6. FTIR spectroscopy t-FTIR spectroscopy (Spotlight 400, PerkineElmer, USA) was used in point mode to characterize the collagen gels as made, and to investigate mineralization. A resolution of 2 cm1, an IR range of 4000 to 500 cm1, 64 scans per sample and an aperture of 100 were used. The kinetic of carbonated hydroxyapatite (CHA) formation was indicated through changes in the ratio of the absorbance of v3 PO3 4 to Amide I (1022 cm1/1661 cm1) while the crystallinity index of the CHA was measured as proposed by Termine and Posner [31]. In particular, the spectra were baseline corrected and normalized against the intensity of v3 PO3 peak at 4 1022 cm1. The crystallinity index of the v4 PO3 at 560e600 cm1 was then 4 1 1 calculated as the sum of the 562 cm and 600 cm peak heights divided by the height of the minimum between this doublet following the method of Weiner and Bar-Yosef [32].
2.1. Preparation of dense collagen gel sheets and rolls at different alkalinity
2.7. m-Raman analysis
Collagen gels were prepared by adding dilute acid solubilised rat-tail tendon type I collagen (2.05 mg/ml in 0.6% acetic acid; First Link Ltd., U.K.) to 10 times concentrated Dulbecco Modified Eagle Medium (10x DMEM; Sigma Aldrich, Canada) at a ratio of 4:1. The solution pH was adjusted to the desired value (Nf pH ¼ 7.4, ANf pH ¼ 8.2 and Af pH ¼ 9.0) by adding 5 M NaOH (Fisher Scientific, Canada) at the range of 1.7e2.0 vol%. Gels were prepared by casting the neutralized collagen solution in a cylindrical mold (Amold ¼ 2 cm2, Vsolution ¼ 2 ml) and incubating at 37 C for 25 min. Gel collagen fibrillar density was increased by applying the plastic compression method, as previously described [28]. Briefly, as cast highly-hydrated gels (0.2 wt%) were subjected to 1 kPa for 5 min to generate DC sheets (14.1 wt%). Gel collagen fibrillar density was measured by weighing the gels before and after freeze-drying (BenchTop K, VirTis, Canada). Gels (n ¼ 5) were frozen in liquid nitrogen for 3 min followed by freeze-drying for 6 h at 105 C and 13 mTorr. Cylindrically rolled DC gels were prepared by plastically compressing prism gels (n ¼ 5, Amold ¼ 18 43 mm2, Vsolution ¼ 4 ml) to obtain sheets (14.1 wt% collagen), which were rolled along their long axis to give cylinders of 1.5 mm in diameter and 20 mm in length.
m-Raman analysis was carried out on wet gels with an InVia Raman (Renishaw, USA) equipped with an NIR laser (785 cm1, 22 mW) and a Carl Zeiss optical microscope (50 objective with an aperture of 50). In order to reduce any potential collagen protein damage due to auto-fluorescence, a photobleaching pre-treatment of 2 min was used, thus reducing the intensity of the laser to 1%. A range of 1800 to 400 cm1, a resolution of 0.5 cm1, three accumulations per sample and an exposure time of 60 s were used. Cosmic rays were removed using the appropriate function in The Wire v.3.0 software (Renishaw). 2.8. X-ray diffraction XRD patterns of the freeze-dried gels were analyzed with a Bruker D8 Discover (Germany) from 6 to 60 2q at 40 kV and 20 mA. Two frames of 30 were recorded for 15 min and merged during data post processing. Sample cross-sections were analyzed with a pinhole size of 0.05 mM. The XRD spectra were analyzed with EVA software (Bruker) correcting the baseline (threshold ¼ 0.750). The phase composition was assigned by comparing the acquired spectra with peaks identified in the International Centre for Diffraction Data (ICDD) database.
2.2. Ionic dye binding
2.9. Thermogravimetric analysis
The capacity of collagen gels fibrilized at increasing alkalinity to bind to both anionic (acidic fuchsine, 0.3%) and cationic (methylene blue, 0.1%) dyes (SigmaeAldrich, Canada) was investigated. Nf and Af gels were immersed in the dye solutions at 37 C for 30 min and then rinsed in distilled deionized water. Gels were maintained in the dark to prevent dye photobleaching.
TGA was used to quantify mineralization within the gels at different time points in SBF. Gels (n ¼ 5) were carefully rinsed with distilled deionized water prior to freeze-drying. Specimens were then heated up to 700 C with a 10 C/min ramp function using a TA SDT Q600 TGA (TA Instruments, USA). The weight residue percent was used as the percentage of mineral in the gels.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
B. Marelli et al. / Biomaterials xxx (2014) 1e8 2.10. Mechanical analysis Tensile testing was carried out on cylindrically rolled DC sheets (14.1 wt% fibrillar density), prepared by plastically compressing prism gels (A ¼ 18 43 mM2, V ¼ 4 ml). Cylindrically shaped specimens (n ¼ 3) of approximately 1.5 mM in diameter and 21 mM in length (measured through microCT, as previously described [20]) were tested using an ElectroForce® Biodynamic Test Instrument 5160 (Bose Corp., USA) with a 15 N load cell. Customized grips were used in displacement control at a rate of 0.01 mm/s. Tests were conducted on as made gels (preconditioned in DMEM at 37 C for 30 min), and on gels allowed to mineralize for 7 or 14 days in SBF. Specimens were maintained hydrated by adding SBF in a drop wise manner during testing. From the resulting stressestrain curves, apparent modulus, ultimate tensile strength (UTS), and strain at UTS were measured as a function of conditioning time. 2.11. Subcutaneous implantation All animal experiments were performed according to the Guidelines of the Canadian Council for Animal Care and approved by the Animal Care Committee, McGill University. Three rats underwent dorsal implantation of Nf and Af gels. For subcutaneous implantation, dense collagen gels were fabricated with a rectangular shape and were then rolled along their long axis yielding Swiss-roll like, cylindrical constructs (∅ ¼ 3 mm, l ¼ 6 mm), as previously described [21]. A 3 cm skin incision was made over the spine extending posteriorly from the shoulders. One pocket was formed on each side of the incision to yield a total of 2 pockets, where Nf and Af gels were implanted for 7 days. Explants were analyzed by von Kossa Staining and with microCT, ATR-FTIR, and XRD. 2.12. Histological analysis Explants were washed in PBS, fixed in 10% neutral buffered formalin overnight, dehydrated through a series of graded ethanol, embedded in paraffin and cut in
3
transverse sections of 5 mm-thickness. von Kossa staining was performed on the trimmed sections. 2.13. Statistical analysis All the quantitative data were compared with one-way ANOVA test with a Tukey means comparison implemented with software Origin Pro v.8 (OriginLab, USA).
3. Results and discussion 3.1. Effects of fibrillogenesis pH on collagen To test the hypothesis that fibrillogenesis pH controls mineral nucleation, dense collagen gels (collagen fibrillar density ¼ 14.1 wt %) obtained through plastic compression [28], were fabricated by increasing the alkalinity of tropocollagen self-assembly solution (Fig 1(a)). Fibrilized gels were then washed in physiological solution (i.e. phosphate buffer saline) prior to conditioning in SBF. A higher fibrillogenesis pH corresponded with an increased amount of charge within the collagen fibrils, as inferred from cationic and anionic dying of the gels (Fig. 1(b)) and increased electron microscopy staining (Fig. 2(a)). Banding in Af collagen fibrils was more evident than those in Nf, attributable to the higher uptake of the stain caused by the more charged nature of the fibrils formed in mildly alkaline conditions. The a priori alkaline fibrillogenesis of collagen was preferred to the exposure of an already assembled gel to a basic environment, to avoid protein denaturation [33,34]. In
Fig. 1. pH control of collagen gel fibrillogenesis. a) Densification of reconstituted highly-hydrated type I collagen gels, where fibrillogenesis occurred at neutral (Nf), slightly alkaline (ANf) and alkaline (Af) pH, obtained by application of 1 kPa compressive stress for 5 min. Dense collagen gels were then incubated in PBS (pH 7.4) at 37 C for 120 min to equilibrate the pH of the collagen fibril to physiological level. The mineralization of dense gels was then investigated in SBF for up to day 14. b) Ionic dye staining of collagen gels fibrilized at neutral and mildly alkaline conditions; i) Anionic dye staining. At pH 7.4, Nf collagen have a net positive charge and attract more anionic dye than Af collagen; ii) Cationic dye staining. Af collagen fibrils possess more negatively charged groups and attract more cationic dye than Nf collagen.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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III at 1661, 1550 cm1 and 1240 cm1, respectively) (Fig. S1(b)), confirming that the initial pH of the collagen solution didn't alter the protein structure [15,36e39]. However, the FTIR spectra revealed differences in the resonance between 1100 and 900 cm1 as the absorption of the infrared light increased (i.e. a decrease in the transmittance) in DC gels generated at a mildly alkaline environment, with the formation of a new local minimum at 1065 cm1, which may be attributed both to a different vibration of the v CeO moieties in the hydroxyproline [40] or to the formation of an insoluble and low crystalline PO3 4 salt (e.g.CaP) in the gels [41]. The lower transmittance between 1100 and 900 cm1 would then be caused by the superimposition of the amorphous CaP resonance to that of the collagen. m-Raman scatterings of the as made DC gels obtained at different fibrillogenesis pH were very similar, confirming the overall similarities in the protein structure (Fig. S1(c)). However, DC gels generated at mildly alkaline environment exhibited two shifts at 948 and 428 cm1, which can be attributed to the v4 of phosphate species present in low crystalline or amorphous CaP [42]. 3.2. Nano-to macro-scale mineralization, in vitro
Fig. 2. Effect of fibrillogenesis pH control on nano-to macro-scale mineralization of collagen, in vitro. a) Cryo-TEM nanographs of (i) Nf, (ii) Af as made, and (iii) Nf, (iv) Af after 24 h exposure to SBF. Banding of Af was more evident than in Nf due to the more charged nature of the fibrils that causes a higher uptake of heavy metal ions. Intrafibrillar mineralization was evident in Af but not in Nf after exposure to SBF, which masked the collagen D-banding structure. b) (i) Localized mineralization achieved at day 7 in SBF in Af gels embossed within a larger Nf gel. (ii) MicroCT 3D reconstruction demonstrating mineralized collagen in red, within non-mineralized regions in grey. c) Images of (i) Nf and (ii) Af roll gels at day 7 in SBF, respectively. e) MicroCT 3D reconstruction of (i) Nf and (ii) Af roll gels at day 7 in SBF, respectively. Af gels exhibited bulk and homogeneous mineralization (mineralized collagen phase highlighted in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
addition, NaOH was used to titrate the tropocollagen solutions due to the lower tendency of Naþ to denature collagen compared to other cations (e.g. Kþ, Ca2þ and Mg2þ) [34,35]. Morphological and chemical analyses demonstrated no effect of fibrillogenesis pH on the structure of as made gels (Fig. S1). SEM micrographs showed that procollagen fibrillization was not affected by the initial alkalinity of the solution, where the nanofibrils exhibited similar dimensions (∅ y 135 nm) and maintained the banding pattern, typical of non-denatured collagen (Fig. 2(a) and Fig. S1(a)) [33,34]. Furthermore, t-FTIR showed no fibrillogenesis pH-dependent modifications of the three main Amide vibrations (Amide I, II and
The in vitro mineralization of DC gels reconstituted at different fibrillogenesis pH was dramatically influenced by fibrillogenesis pH from the nano-to the macro-scale. At a nanoscopic level, Cryo-TEM analysis of Nf and Af gels exposed to SBF for 24 h depicted the masking of the typical collagen fibrils banding for Af samples only, which has been correlated with intrafibrillar mineralization (Fig. 2(a)) [15,43]. SEM analysis of Nf, ANf and Af gels at day 1 in SBF depicted the absence of mineralization in Nf and ANf gels, while mineral formation was only visible in the framework of Af gels (Fig. S2(a)). SEM micrographs of Af samples taken at days 3, 7 and 14 in SBF showed the continuous homogeneous growth of the crystals (Fig. S2(b)). In contrast, mineral formation in Nf and ANf gels was less homogeneous and extensive, visible only at days 7 and 14, as previously reported [29]. At a macroscopic scale, the effect of locally altering collagen fibrillogenesis pH was investigated by embossing letter-shaped Af gels (mM in size) within a larger Nf gel (cm in size) (Fig. 2(b) i). The hybrid AfeNf gel was then conditioned in SBF for 7 days. During exposure to SBF, the Af portion of the hybrid gel turned from palewhite to bright-white, indicating the formation of a new phase in the Af gels, which induced light scattering. MicroCT 3D reconstruction of the collagen gel at day 7 in SBF confirmed regional optical differences to be attributable to a denser phase formed within Af gels (highlighted in red, Fig. 2(b) ii). In addition, the 3D mineralization of Nf and Af gels was investigated by exposing cylindrically rolled DC gels (Ø ¼ 1.5 mM, l ¼ 20 mM) to SBF (Fig. 2(c)). At day 7 in SBF, Af cylindrical rolls self-sustained their own weight and appeared more white in colour, while Nf gels did not show any visible difference from those as made. MicroCT 3D reconstruction of Nf and Af cylindrical rolls at day 7 in SBF depicted the homogenous and bulk formation of a denser phase within Af gels (highlighted in red in Fig. 2(d) ii). 3.3. Characterization of the mineral phase t-FTIR, m-Raman and XRD analyses confirmed calciumphosphate nucleation and growth in collagen gels generated at mildly alkaline pH. Fig. 3(a) displays the change in the t-FTIR spectra of Af gels from t ¼ 0 to t ¼ 336 h (day 14) in SBF. Amide I peak remained unchanged at 1661 cm1 indicating no denaturation of the protein during the mineralization process [39], while Amide II decreased in intensity, indicating the nucleation of a mineral phase within collagen fibrils [6]. Additionally, the v3 PO34 and v4
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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Fig. 3. Characterization of Af collagen gel mineralization in SBF. a) t-FTIR spectroscopy showed no changes in the collagen triple helical structure during the mineralization process (Amide I peak at 1661 cm1). The v1, v2, v3 resonances of PO3 4 were visible in all spectra, suggesting the formation of CaP minerals. At day 1, typical apatite peaks (e.g. v3 vibration at 1033 cm1) were present which shifted to a lower wavenumber with time, revealing an increase in its crystallinity. The v2 CO2 3 vibration peak was present within 1 h in SBF and increased during the mineralization process, suggesting the presence of carbonated hydroxyapatite (CHA). b) The increase in the 1028 to 1661 cm1 peak ratio with time in SBF indicated an increase in CaP in the collagen framework. c) CaP crystallinity index increased after day 1 in SBF. d) m-Raman analysis depicted that collagen triple helical structure (peak at 1661 cm1) was not affected by the mineralization process. Phosphate species were formed as soon as after 3 h of exposure to SBF, suggesting the formation of CaP minerals. Mineral extent and crystallinity increased with time in SBF, as the Raman scattering of the phosphate species increased in intensity and became sharper for longer time points. e) XRD analysis showed diffractographs compatible with formation of bone-like hydroxyapatite within the collagenous matrix as soon as at day 1 upon exposure to SBF, as indicated by the peak around 25 and 31 2theta. Samples' crystallinity increased with time in SBF, as the peaks compatible with hydroxyapatite species increased in intensity and became sharper for longer time points.
PO3 absorbances at 1022 and 560e600 cm1, respectively, 4 increased as a function of conditioning time in SBF. The peaks and the shape of the PO3 4 resonances together with the presence and 1 temporal growth of the v2 CO2 suggested CHA 3 peak at 871 cm formation. Indeed, the formation of an apatitic phase was spectroscopically evident for Af gels within a 3 h exposure time to SBF (Fig. 3(b)) and v3 PO3 4 absorbance increased with time prior to stabilization at 168 h (day 7). Furthermore, the crystallinity index of CHA was not statistically different for the first two days in SBF (p > 0.05 between 3 and 48 h) (Fig. 3(c)), while there was an increase in the crystallinity of CHA after 72 h, which continued up to 168 h. The t-FTIR results were corroborated by m-Raman spectra (in Fig. 3(d)). The Raman shift of the collagen (Amide I and III at 1671 and 1243 cm1, respectively) remained unchanged during conditioning in SBF, while the shift of the v1 and of v4 PO3 4 indicated the formation and the growth of CHA. In particular, there was a tem1 poral shift in the v1 PO3 4 peak from 962 to 960 cm , an increase in its intensity when compared to the Amide I peak and a decrease in its half width, suggesting the formation and growth of CHA, with
increasing crystallinity at longer conditioning time in SBF [42]. XRD diffractographs of Af at various conditioning times in SBF confirmed apatite formation (Fig. 3(e)). The presence of two broad peaks at 7 and 25 , indicated the typically random collagen fibril organization in the as made gel [44]. By day 1 in SBF, HA formation was confirmed (ref. 009-0432), and its crystallinity increased with time, as indicated by the more intense and narrower peaks at 31 2theta. In contrast, the formation of low crystalline CHA in Af and ANf gels was detectable only at days 7 and 14 (data not shown), as previously reported [29]. TGA allowed for quantitation of mineral growth in DC gels through measurement of weight residue. Nf and ANf gels presented statistically similar thermogravimetric results (p > 0.05), with a linear increase in the weight residue reaching 13 dry weight% at day 14 in SBF (Fig. 4(a)). In contrast, as early as day 1 in SBF, mineralization in Af gels approached 10 wt%, which increased to 69 wt% at day 7 prior to stabilization at 76 wt% at day 14. Furthermore, microCT volumetric reconstruction analysis was implemented to calculate the volume percentage of mineralized collagen within the
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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Fig. 4. Characterization of mineralization extent, in vitro. a) TGA generated dry weight residue (mineral phase) increased with time in SBF for all gels. There was no statistically significant difference between mineralization extent in Nf and ANf gels (12 wt% at day 14). Af gels yielded 40 wt% mineral by day 3, and increased to 76 wt% at day 14. b) Mineralized collagen vol% calculated through microCT analysis. Increase in mineralization extent corresponded with an increase in mineralized collagen phase (95 vol% at day 14).
gel (Fig. 4(b)). Almost 27% of the collagen was mineralized in Af gels at day 1 in SBF, increasing to 50 and 82% at days 3 and 7, respectively, before reaching 95% at day 14. 3.4. Mechanical analysis The effects of fibrillogenesis pH and of conditioning time, at days 7 and 14, in SBF on the mechanical properties of Nf, ANf and Af gels were investigated through tensile testing. There were no statistically significant differences (p > 0.05) between the apparent modulus, UTS and strain at UTS values of cylindrically rolled as made gels of Nf, ANf and Af (Fig. 5(aec)) prior to mineralization. By day 14 in SBF, the values of these parameters remained statistically
insignificant (p > 0.05) between Nf and ANf and showed negligible changes. On the other hand, by day 14 in SBF, the apparent modulus and UTS values in Af gels were statistically higher (p < 0.05) when compared to those of Nf and ANf, which could be attributed to the enhanced mineral formation within the gels [45]. The strain at UTS of Af gels underwent a significant decrease (p < 0.05) with time in SBF. Representative stressestrain curves of gels conditioned up to day 14 in SBF displayed typical soft-tissuelike responses of Nf, ANf gels, with toe, linear and failure regions (Fig. 5(d)) [46]. In contrast, Af gels indicated a transition from softto-hard tissue-like response to tensile stress, exhibiting an increase in apparent modulus and UTS and a decrease in the strain at UTS.
Fig. 5. Mechanical properties of dense collagen gels as made, and at days 7 and 14 in SBF. a), b) and c) apparent modulus, UTS, and strain at UTS, respectively, as a function of conditioning time in SBF. d) Representative stressestrain curves of Nf, ANf and Af gels at day 14 in SBF. Higher mineralization extent in Af gels resulted in a transition from soft-tohard tissue-like response to tensile stress. There was a reduction in the toe region and an increase in the apparent modulus and UTS with time in SBF. This effect was particularly evident in Af gels. Nf and ANf gels registered no differences (p > 0.05) in apparent modulus with time in SBF, and a general reduction in UTS and strain at UTS, was measured. Af gels displayed a significant increase (p < 0.05) in apparent modulus at days 7 and 14 and in UTS at day 14 in SBF, attributable to the formation of a highly compact mineralized hybrid where both collagen fibrils and hydroxyapatite crystal bore the load. Strain at UTS decreased (p < 0.05) with increasing mineralization time. This effect was particularly evident in Af gels.
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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Fig. 6. Effects of fibrillogenesis pH control on collagen mineralization, in vivo. a) Histological sections of (i) Nf and (ii) Af roll gels implanted subcutaneously for 7 days. Mineralized collagen was highlighted with von Kossa staining (black, brown in the images). b) MicroCT 3D reconstruction of (i) Nf and (ii) Af gels implanted subcutaneously for 7 days. Collagen is highlighted in grey while mineralized collagen is highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.5. In vivo mineralization The in vitro results were confirmed in vivo; in that subcutaneously implanted (i.e. in a non-osseous site thus avoiding the role of in vivo produced NCPs) Af gels extensively mineralized after 7 days. This is in contrast to Nf gels, which showed only minor evidences of mineralization (Fig. 6). von Kossa staining of histological sections indicated extensive mineral formation throughout the thickness of Af gels with no cell infiltration in the gel or cell-mediated remodelling (Fig. 6(a)). MicroCT 3D reconstruction confirmed that mineral formation was homogenously distributed throughout the thickness of Af with 69 vol% mineralization, compared to only 7 vol% in Nf gels (Fig. 6(b)). ATR-FTIR and XRD analyses confirmed the mineral phase formed within the explants to be CHA, in line with the in vitro data (Fig. S3(a and b), respectively).
CaP solution mineralized extrafibrilarly [47]. To investigate the effects of the alkaline environment on collagen fibrillogenesis, Nessler's reagent was used to evaluate the production of ammonia during fibrillogenesis. Interestingly, a detectable amount of ammonia was produced during the self-assembly of Af gels only, indicating the partial reaction of the amino groups present in the side chains of the collagen peptidic sequence during fibrillogenesis under mildly alkaline conditions, which may have resulted in a more charged collagen. In addition, the 3D, bulk in vivo mineralization of Af gels within 7 days upon subcutaneous implantation corroborates, in a nonosseous model, the predominant role of collagen in the nucleation and growth of bone apatite without the presence of bone inducing factors. Indeed, our findings support the recent questioning of the consensus in the literature on the need for Ca-rich NCPs for collagen mineralization to occur, in vivo [43].
3.6. Effects of fibrillogenesis pH on collagen mineralization 4. Conclusions Together these results suggest that fibrillogenesis pH has a dramatic impact on the ability of collagen fibrils to nucleate CHA, both in vitro, when exposed to SBF and in vivo, when implanted subcutaneously. The investigation of the in vitro mineralization of collagen gels reconstituted at mildly alkaline pH depicted a twostage mineralization process; intrafibrillar mineralization with the formation of a low crystalline apatite phase within the first day of exposure to SBF, followed by extrafibrillar CHA crystal formation, which grew in the absence of the mineral shape-regulating NCPs. Interestingly, exposing Nf gels to an alkaline solution for 30 min prior to conditioning in SBF did not impact mineralization, indicating that the alkaline self-assembly induced a distinct change in the properties of collagen. This difference in the mineralization of collagen gels exposed a priori or a posteriori to an alkaline solution underpins a different mineralization mechanism from that previously reported, where collagen membranes exposed to an alkaline
In summary, this work has demonstrated that intrafibrillar, bulk and homogeneous mineralization of dense collagen gels may be rapidly achieved in vitro and in vivo by simply adjusting the alkalinity of collagen during fibrillogenesis. This process provides a model to investigate mineralized tissues in vitro as well as a means to fabricate an immediately implantable osteoid-like tissue for bone regeneration. Acknowledgements The support of the Canadian Natural Sciences and Engineering bec Ministe re de l'Enseignement supe rieur, Research Council, Que de la Recherche, de la Science et de la Technologie, Canadian Institutes of Health Research and Canadian Foundation for Innovation are gratefully acknowledged. Funding for Benedetto Marelli is also
Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008
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supported by the Werner Graupe Fellowship and McGill Engineering Doctoral Award. Funding for Chiara E. Ghezzi is also supported by Showan Nazhat's McGill Engineering Gerald Hatch Faculty Fellowship. Jake Barralet acknowledges the provision of a Canada Research Chair. Appendix A. Supplementary data
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Please cite this article in press as: Marelli B, et al., Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.008