Multiscale Analysis of Mineralized Collagen Combining X-ray

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Multiscale analysis of mineralized collagen: X-ray scattering and fluorescence with Raman spectroscopy under controlled mechanical, thermal and humidity environments Admir Masic, Roman Schuetz, Luca Bertinetti, Chenghao Li, Stefan Siegel, Till Hartmut Metzger, Wolfgang Wagermaier, and Peter Fratzl ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00676 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on December 31, 2016

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ACS Biomaterials Science & Engineering

Journal: special issue of ACS Biomat Sci Eng

Title: Multiscale analysis of mineralized collagen combining X-ray scattering and fluorescence with Raman spectroscopy under controlled mechanical, thermal and humidity environments Admir Masica, Roman Schuetzb, Luca Bertinettib, Chenghao Lib, Stefan Siegelb, Hartmut Metzgerb, Wolfgang Wagermaierb and Peter Fratzlb

a

MIT, Department of Civil and Environmental Engineering, 77 Massachusetts Avenue, 02139

Cambridge, USA b

Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Research Campus

Golm, 14424 Potsdam, Germany

Correspondence email: [email protected]

ACS Paragon Plus Environment

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KEYWORDS. Hierarchical structure, bone, tendon, Raman Spectroscopy, synchrotron x-ray scattering.

ABSTRACT. Biological materials, such as mineralized collagen, are structured over many length scales. This represents a challenge for quantitative characterization, in particular when complex specimen environments are required. This paper describes an approach based on synchrotron x-ray scattering and Raman spectroscopy to analyze the structure of biological materials from the molecular to the macroscopic range in controlled environments including humidity, temperature and mechanical load. This is achieved by a new set-up, installed at the microfocus beamline µSpot at the BESSY II synchrotron in Berlin, where a perforated mirror is placed into the x-ray beam to focus laser light into the specimen to excite a Raman signal. We show that this allows simultaneous micrometer-scale mapping of chemical groups in the organic matrix together with the size and orientation of mineral nanoparticles in mineralized collagen. The approach is especially suitable to study time-dependent modifications of materials, such as molecular changes during tensile deformation, dehydration or thermal denaturation.

1.

Introduction Biological materials, including mineralized (exo-)skeletal elements in mammals, arthropods or

sponges, cellulose-based plant tissues or protein-based fibers and sheets are often characterized by a multi-scalar structure1-3, which results from growth and remodeling and which is essential in providing the required physical properties, such as fracture resistance, for example. This poses a serious challenge for structural characterization. Indeed, biological materials are inhomogeneous at the micrometer scale and the nanoscale structure will vary throughout the material.


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Bone, for example, is a hierarchically structured material with remarkable mechanical performance, which can serve as a model for the development of biomimetic materials4-7. While the full hierarchical structure of bone is extremely complex and variable, its basic building block, the mineralized collagen fibril, is rather universal7. A great variety of structures results from the assembly of these basic blocks, thus allowing the adaptation of bones to their actual mechanical functions. Bone is assembled through different levels of hierarchy; starting with nanoscopic platelets of hydroxyapatite (HA) that are oriented and aligned within self-assembled collagen fibrils; the collagen fibrils are layered in parallel arrangement within lamellae; some of the lamellae are arranged concentrically around blood vessels to form osteons; finally the osteons are either packed densely into compact bone or construct a trabecular network of microporous bone, referred to as spongy or cancellous bone. In a more quantitative terms, bone is a composite material which consists of 33-43% apatite minerals, 32-44% organics, and 15-25% water8. Even though, water is a minor constituent, its significance should not be overlooked, because it contributes to the overall toughness of the biocomposite, acting like a plasticizer9. The collagen consists of –(Gly-X-Y)n- aminoacid sequences, where X and Y are frequently proline and hydroxyproline residues, allowing the protein to assemble into triple helical structures referred to as tropocollagen molecules. Recently, the mechanism of hydration-driven force generation in collagen has been explored10. This multi-scale experimental approach, which employed several in situ methods under controlled environmental conditions, allowed direct observation of inhomogeneous waterinduced conformational changes of the collagen molecule in both mineralized and mineral-free tendons.


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To image complex functional material structures such as bone, multi-scale approaches are required. In this endeavor, photon diffraction/scattering and spectroscopic methods present the major advantage that specimens can be characterized in an (almost) non-destructive way and in near-physiological conditions. In Figure 1, this is illustrated for the hierarchical structure of (mineralized) type I collagen, as in tendon or bone: molecular structure may be assessed by Raman scattering11-12, crystalline structures by X-ray diffraction (XRD)13-14, supramolecular arrangements by small-angle x-ray scattering (SAXS)15-16 and local chemical composition (at least in a qualitative way) by x-ray fluorescence imaging 17. SAXS can routinely be employed to map mineral particle characteristics in two-dimensional sections of bone16 or dentin18. All these techniques have been used separately in the past to study biological materials, such as tendon in their native hydrated state but also as a function of various parameters, such as external stress19-20 or air humidity 21. Here we first review the principles of SAXS/XRD as well as Raman spectroscopic scanning imaging of mineralized tissues and then discuss the potential of combining the two approaches. The combination of Raman spectroscopy and x-ray microdiffraction was pioneered by Christian Riekel22-24 at the European synchrotron source. Here, we are adding SAXS which is especially useful in the characterization of mineralized tissues.


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Figure 1: Schematic representation of the structure of (mineralized) collagen fibrils. Fibrils (F) contain triple-helical collagen molecules (M) and – if mineralized – also plate-like mineral particles (P)- The axial staggering period as well as the lateral molecular spacing in the fibrils can be measured by small-angle x-ray scattering (SAXS) and the helical pitch of the collagen molecule by x-ray diffraction (XRD). Raman scattering gives information on the conformation of the collagen molecule. Finally, x-ray fluorescence (XRF) enables the detection of calcium and other elements potentially present in mineral.


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SAXS/XRD characterization of mineralized collagen Mineralized collagen in the form of bone has been investigated by X-ray scattering methods

since mid of the last century25-28. X-ray scattering generally provides information on the arrangement of atoms in crystalline phases, molecules and supramolecular units at the length scale of 0.1 to about 100 nm. X-ray diffraction (XRD) applied to mineralized collagen mainly provides information on the crystalline HA particles, such as the lattice spacing therein and preferred orientations of the crystallographic axes. Two-dimensional detectors are used to record X-ray scattering patterns, as shown for non-mineralized collagen in Figure 3b and for mineralized collagen in Figure 3c. The center of Figure 3b shows a series of Bragg peaks at small angles indicating the staggering of collagen molecules, while the center of Figure 3c shows an anisotropic signal originating from the crystalline HA particles. In mineralized collagen the large differences in electron density between mineral and organic phases lead to a much stronger signal from the mineral particles and therefore the signal from the collagen is most often not detected. The crystallographic orientation of HA crystals in bone can been measured by means of neutron and X-ray diffraction29. By means of X-ray pole figure analysis it has been found that the c-axis of HA generally orients parallel to the longitudinal axis of bone (bone axis) and that a significant amount of c-axis is oriented in other directions19, 30. Such interpretations highly depend on the X-ray beam size and the exposed bone volume (beam size times sample thickness), since the scattering signal always is an averaged information from this volume. As a consequence, nanofocus beam stations are continuously developed at several synchrotron sources, including ESRF in Grenoble or PETRA III in Hamburg, where spot sizes can now reach to 100 nm range for SAXS. XRD has been used in the 1980s to study the size of HA crystals in calcified cartilage and cortical bone of rat and compared with that of synthetic poorly crystalline


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hydroxyapatite31. The mean length of mineral particles (L parameter) can be calculated by determining the full width at half maximum intensity of the (002) peak (inner ring in Figure 3c) by applying the Scherrer equation16. The location of the HA particles within the organic matrix, i.e. the distribution of the amount among the gap zones or between fibrils, is an ongoing topic of XRD investigations on bone and has been studied already some decades ago32. Small angle X-ray scattering (SAXS) has been used to characterize mineral particle thickness (T parameter) as well as the average orientation of the particles in a given bone volume (ρ parameter) since the early 1990s33-36. A recent review on imaging the nanostructure of bone and dentin through X-ray scattering provides more detailed information on the evaluation of these parameters16. The application of X-ray scattering measurements to mineralized tissues has been further developed by extending the measuring area by scanning the beam across the samples, i.e. by scanning SAXS15, 36-37. Modern instrumentation, enabling small beam sizes and short exposure times, has therefore made XRD as well as SAXS to imaging methods38. During the last decade SAXS has been more and more related to clinically relevant research questions39, such as the characterization of the bone nanostructure near implants40 or the influence of strontium on the bone nanostructure41. Several scanning SAXS studies have been performed on diseased bone, such as from patients exhibiting osteogenesis imperfecta42-44 or from bone healing models45-46. SAXS has recently been developed to be used in a tomographic fashion and provide threedimensional imaging47-49. These methods are computationally very challenging and are so far mostly in the proof-of-principle state. Earlier approaches to SAXS tomography are less demanding in computational time but require specimens with isotropic or axial symmetric structure50-51 or serial sectioning of the specimen52. These developments exhibit an exciting perspective for the future.


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Raman spectroscopic characterization of collagen-based tissues Vibrational spectroscopy has been widely applied to study bone materials53-60. In this context

Infra-red and Raman spectroscopies were crucial in providing details of bone composition, mineral crystallinity and level of carbonation61, collagen structure and amount of covalent crosslinks. More recently, polarized Raman approach has been applied to simultaneously analyze structural and chemical information of collagen fibrils in human osteonal bone in a single experiment11, 62-64. In particular, the 3D arrangement of collagen fibrils in osteonal lamellae was assessed. By analyzing the anisotropic intensity of the amide I Raman band of collagen as a function of the orientation of the incident laser polarization, different parameters related to the orientation of the collagen fibrils and the degree of alignment of the fibrils can be derived12. Outcomes of this work have further contributed to study collagen within ancient parchment manuscripts and in particular helped to quantify collagen degradation (fibrous vs gelatin)65. Furthermore, in vivo Raman spectroscopy was instrumental in assessing various mineral phases involved in early stages of bone formation66-67. Chemical composition and fibrillar organization are the major determinants of osteonal bone mechanics; however, there are few methodologies commonly applied to investigate bone on the micro scale, which are not able to concurrently describe both factors. In order to further explore the intrinsic properties of bone material across multiple length scales, it is necessary to apply experimental techniques that are not only able to provide information on several levels of the hierarchical structure, but can also monitor the in situ real-time responses of the structures to the application of external stimuli. In this experimental context, simultaneous microfocus X-ray and Raman scattering permit the acquisition of structural and chemical information across multiple


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length scales spanning from the molecular up to the macroscopic level24, 68. These techniques, accompanied with in situ mechanical testers, deliver highly complementary information regarding the compositional and structural hierarchy allowing next generation of in situ tissue characterization of bone samples.

4.

A dedicated beamline for multi-scale characterization: µSpot at BESSY II The microfocus beamline at BESSY II (µSpot) Germany has an accessible energy range 1.9–

30 keV, provided by a fixed exit double crystal monochromator with two sets of crystals (Si 111 and Si 311) and a MoBC multilayer. The energy resolution can be varied between DE/E = 10-4 (Si 311) and DE/E = 10-2 (MoBC multilayer)69. For scanning microbeam SAXS and XRD, both a small beam at the sample position and a low divergence are required at a reasonable flux. Three setups are available for scanning microbeam at the microfocus beamline at BESSY II, the simultaneous SAXS, XRD, the simultaneous SAXS, XRD, X-ray Fluorescence (XRF) and the simultaneous SAXS, Raman spectroscopy. The simultaneous SAXS/XRD experimental setup is based on a beam-defining pinhole, a guard pinhole, a sample stage and a two-dimensional position-sensitive CCD-detector MarMosaic 225 (Mar USA Evanston, USA). The sample stage consists of a xyz translation stage and a θ-2θ goniometer. There is an additional small xyz scanning stage with linear encoders (resolution 100 nm) on top of the goniometer for microbeam scanning. Furthermore, there is a small manually changeable goniometer used as sample holder to tilt the sample. The whole goniometer, as well as the CCD-detector, can be moved independently along the beam direction, and the CCD-detector can also be moved vertically to maximize the accessible q range. For simultaneous SAXS/XRD/XRF measurements, there is an additional fluorescence detector


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ASAS-SDD (KETEK Germany) with 100 mm2 sensitive area and about 167.4 eV energy resolution. The fluorescence detector is fixed on the 2θ goniometer arm. For simultaneous SAXS Raman measurements the customized Video Fiber Optical Probe ((v)FOP) plate is also mounted on the 2θ goniometer arm. To adjust the alignment of the Raman FOP with the X-ray beam there are additional three independent motors in x-, y-, z-direction.

The setup allows excitation and collection of the Raman spectra through a probe, which is connected with 20m long optical fiber to a Renishaw spectrometer placed outside the hutch (Fig. 2).

Figure 2: Schematic representation of the integration of the Raman probe head (FOP) into the Xrays beam path.

The remote probe can be inserted into the beam line and has its focus of the 45 deg collimation mirror with a 2 mm hole coaxially aligned with the passing synchrotron radiation and this allows simultaneous collection of Raman and X-ray data. The optical fiber-coupling of the remote probe


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to the laser and spectrometer allows the use of the laser beam with around 25-30 microns for the micro-focus and 100 microns for experiments with lower resolution. The customized (v)FOP achieves a spot size of ca. 26µm for 532nm (200mW frequency doubling Nd:YAG Laser) and 30µm for 785nm (100mW point focus diode laser) at a long working distance of 50mm. The laser power on the sample can be regulated by a set of motorized ND filters (Neutral Density Filter) for laser power attenuation between 100% and 0.00005. The stigmatic single-pass spectrometer with high throughput is equipped with an automatic Rayleigh Edge-filter exchange unit allowing spectral record from 150 cm-1 (10% cut-off) and with a motorized grating system for high spectral resolution: 1800 1/mm optimized for 532nm excitation wavelength and a 1200 1/mm grating for the 785nm. The CCD array detector (576 x 384 Pixel) operates at -70°C cooled down by a peltier-element. System operation and spectral analysis were performed using the software package Renishaw WiRE version 3.4.

5.

Materials and Results The turkey leg tendons (tibialis cranialis) originate from turkeys sacrificed after 20-25

weeks and were sectioned with a cryomicrotome into slices with a thickness of about 170 µm. After cutting sections from the area where mineralized and non-mineralized tendon are connected, the slices were directly placed on a specimen slide and freeze dried for about 12 hours. The slices were mounted onto the sample holder at the µSpot beamline (Figure 4a). For the presented measurements, we used the MoBC multilayer monochromator, 30 micron beam size and an energy of 15 keV. First, a transmission scan in region of interest (ROI) was performed using a photodiode. This supplies absorption information of the specimen, and is used for further evaluation on background correction. In the next step, a SAXS pattern and Raman


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spectrum were recorded simultaneously for each scan point in a chosen ROI, the Raman spectrum was saved as a separate file. For simultaneous SAXS/XRD measurements, the MarMosaic CCD-detector was moved as closer as possible to sample and recorded SAXS/XRD pattern simultaneously.

The SAXS/XRD data analysis performed using software DPDAK (directly programmable data analysis kit70). This software is based on a plug-in structure and allows individual extension in accordance with the requirements of the user. It can be used for online data reduction and analysis during the experiment in order to interactively optimize experimental design and also for offline data analysis. These data reduction processes involve instrument geometry calibration, correction of raw data, one- or two-dimensional integration, as well as peak fitting and further analysis tools, including the extraction of certain parameters.


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Figure 3: Simultaneous SAXS/XRD and Raman scattering experiment on the mineralized turkey leg tendon. (a) Schematic drawings show the integration of the Raman FOB of two laser wavelengths (785nm and 532nm) into the X-Ray diffraction facility at µSpot beamline (BESSY II). The arrangement of the instruments allows SAXS, XRD and Raman measurements being performed on the same sample spot and under same temperature and humidity conditions. (b), (c) and (d) show a representative scattering patterns of SAXS on collagen, XRD on mineralized tendon and Raman spectra of both materials, respectively.

The study on the mechanical response of mineralized collagen from turkey leg tendon (TLT) to changes of relative humidity is reported elsewhere10. Briefly, results indicate that water plays a fundamental role in stabilizing the structure of the collagen molecule and its removal results in conformational changes of the collagen molecule, which is effectively shortened, producing large tensile stresses. The force generation mechanism is independent from the presence of the mineral and the force generation found is already significant at relatively low osmotic pressure changes which might occur even inside a fully hydrated environment. The contraction of collagen puts mineral particles under compression leading to strains in the percent range, which implies localized compressive loads in mineral of up to 800 MPa. Whereas the loads on mineral can be monitored through XRD the conformational changes of collagen are evident in protein related Raman peaks71. Figure 4 shows the results of simultaneous SAXS/XRD and Raman measurements on a partly mineralized turkey leg tendon (Figure 4a). Figure 4b displays the integral of the v1 PO43- Raman peak (920cm-1 – 990cm-1) indicating the distribution of phosphate vibrational units in the analyzed sample.

Figure 4c shows the mineral particle thickness (T

parameter) as evaluated from the SAXS signal and Figure 4d the mean lengths of the mineral


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crystallites (L parameter). Both parameters determined from X-ray scattering show a rather homogenous distribution within the mineralized region (T varies between 2.2 and 2.6 nm while L varies between 20 and 25 nm), but show an abrupt edge at the interface towards the nonmineralized area. Both parameters are within the typical range for mineralized collagen72.

a

Optical

Raman

b

Phosphate

Mineralized part

5mm c

X-Ray scattering

T parameter

d

X-Ray scattering

L parameter

Figure 4: Simultaneous and co-localized X-rays and Raman scattering data obtained by scanning an interface between mineralized and non-mineralized turkey leg tendon cross-section. (a) light microscopy image of the sample, showing the non-mineralized part (left) and the mineralized part (right), (b) integral of the v1 PO43- Raman peak showing the presence of carbonated hydroxyapatite, (c) X-rays scattering results on the same area like indicated by the marked ROI in (a) showing the thickness (c) and the length (d) of the apatite crystals. The step size in (b), (c) and (d) is 200µm.


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6.

Conclusion Correlative imaging is a promising tool to elucidate structure-function and even structure-

disease relationships. In-situ simultaneous collection of Raman and X-ray signals provides timeand space-resolved multi-scale information. Especially if external stimuli, such as changes in humidity, temperature or mechanical stress, are applied to the specimen, it is essential that different imaging modalities are acquired simultaneously since the modification of the specimen does allow ex-situ combination of these techniques. Complex sample environments needed to control the external stimuli are imposing constraints on the beamline design and also light and xray beams need to be aligned for the measurement. While the operation of this technology has been demonstrated here for the imaging of mineralized TLT, it may be applied for a wide variety of anisotropic complex materials.

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Multiscale Analysis of Mineralized Collagen Combining X-ray

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