Mg Biodegradation Mechanism Deduced from the Local Surface Environment under Simulated Physiological Conditions.

Although certified magnesium-based implants are launched some years ago, the not well-defined Mg degradation mechanism under physiological conditions makes it difficult to standardize its use as a degradable biomaterial for a wide range of implant applications. Among other variables influencing the Mg degradation mechanism, monitoring the pH in the corrosive solution and, especially, at the corroding interface is important due to its direct relation with the formation and stability of the degradation products layer. The interface pH (pH at the Mg/solution interface) developed on Mg-2Ag and E11 alloys are studied in situ during immersion under dynamic conditions (1.5 mL min-1 ) in HBSS with and without the physiological amount of Ca2+ cations (2.5 × 10-3 m). The results show that the precipitation/dissolution of amorphous phosphate-containing phases, that can be associated with apatitic calcium-phosphates Ca10-x (PO4 )6-x (HPO4 or CO3 )x (OH or ½ CO3 )2-x with 0 ≤ x ≤ 2 (Ap-CaP), promoted in the presence of Ca2+ generates an effective local pH buffering system at the surface. Thus, high alkalinization is prevented, and the interface pH is stabilized in the range of 7.6 to 8.5.

Influence of the amount of intermetallics on the degradation of Mg-Nd alloys under physiological conditions.

The influence of amount of intermetallics on the degradation of as-extruded Mg-Nd alloys with different contents of Nd was investigated via immersion testing in DMEM+10% FBS under cell culture conditions and subsequent microstructural characterizations. It is found that the presence of intermetallic particles Mg41Nd5 affects the corrosion of Mg-Nd alloys in two conflicting ways. One is their negative role that their existence enhances the micro-galvanic corrosion. Another is their positive role. Their existence favours the formation of a continuous and compact corrosion layer. At the early stage of immersion, their negative role predominated. The degradation rate of Mg-Nd alloys monotonously increases with increasing the amount of intermetallics. Mg-5Nd alloy with maximum amount of intermetallics suffered from the most severe corrosion. With the immersion proceeding (≥7 days), then the positive role of these intermetallic particles Mg41Nd5 could not be neglected. Owing to the interaction between their positive and negative roles, at the later stage of immersion the corrosion rate of Mg-Nd alloys first increases with increasing the content of Nd, then reaches to the maximum at 2 wt. % Nd. With a further increase of Nd content, a decrease in corrosion rate occurs. The main corrosion products on the surfaces of Mg-Nd alloys include carbonates, calcium-phosphate, neodymium oxide and/or neodymium hydroxide. They are amorphous at the early stage of immersion. With the immersion proceeding, they are transformed to crystalline. The existence of undegradable Mg41Nd5 particles in the corrosion layer can enhance the crystallization of such amorphous corrosion products.

In Vivo Simulation of Magnesium Degradability Using a New Fluid Dynamic Bench Testing Approach.

The degradation rate of magnesium (Mg) alloys is a key parameter to develop Mg-based biomaterials and ensure in vivo-mechanical stability as well as to minimize hydrogen gas production, which otherwise can lead to adverse effects in clinical applications. However, in vitro and in vivo results of the same material often differ largely. In the present study, a dynamic test bench with several single bioreactor cells was constructed to measure the volume of hydrogen gas which evolves during magnesium degradation to indicate the degradation rate in vivo. Degradation medium comparable with human blood plasma was used to simulate body fluids. The media was pumped through the different bioreactor cells under a constant flow rate and 37 °C to simulate physiological conditions. A total of three different Mg groups were successively tested: Mg WE43, and two different WE43 plasma electrolytically oxidized (PEO) variants. The results were compared with other methods to detect magnesium degradation (pH, potentiodynamic polarization (PDP), cytocompatibility, SEM (scanning electron microscopy)). The non-ceramized specimens showed the highest degradation rates and vast standard deviations. In contrast, the two PEO samples demonstrated reduced degradation rates with diminished standard deviation. The pH values showed above-average constant levels between 7.4-7.7, likely due to the constant exchange of the fluids. SEM revealed severe cracks on the surface of WE43 after degradation, whereas the ceramized surfaces showed significantly decreased signs of corrosion. PDP results confirmed the improved corrosion resistance of both PEO samples. While WE43 showed slight toxicity in vitro, satisfactory cytocompatibility was achieved for the PEO test samples. In summary, the dynamic test bench constructed in this study enables reliable and simple measurement of Mg degradation to simulate the in vivo environment. Furthermore, PEO treatment of magnesium is a promising method to adjust magnesium degradation.

In vitro evaluation of the ZX11 magnesium alloy as potential bone plate: Degradability and mechanical integrity.

Considering the excellent biocompatibility of magnesium (Mg) alloys and their better mechanical properties compared to polymer materials, a wrought MgZnCa alloy with low contents of Zn (0.7 wt%) and Ca (0.6 wt%) (ZX11) was developed by twin roll casting (TRC) technology as potential biodegradable bone plates. The degradability and cell response of the ZX11 alloy were evaluated in vitro, as well as the mechanical integrity according to tensile tests after immersion. The results revealed a slightly higher degradation rate for the rolled ZX11, in comparison to that of the annealed one. It was mainly caused by the deformation twins and residual strain stored in the rolled alloy, which also seemed to promote localized degradation, thereby leading to a relatively fast deterioration in mechanical properties, especially the fracture strain/elongation. In contrast, after the annealing treatment, the alloy showed relatively lower strength, yet a lower degradation rate and quite stable elongation during the initial weeks of immersion were observed. More importantly, the ZX11 alloy, regardless of the annealing treatment, showed good in vitro cytocomopatibility regarding human primary osteoblasts. The assessment indicates the rolled alloy as a good choice for implantation sites where relatively high mechanical strength is needed during the early implantation, while the annealed alloy is a potential candidate for the sites which demand stable mechanical integrity during service. STATEMENT OF SIGNIFICANCE: The development of magnesium alloys as bone implants demands low degradation rate to gain not only a slow hydrogen evolution, but also a stable mechanical integrity during service. The present study develops a micro-alloyed MgZnCa alloy via twin roll casting (TRC) technology. It exhibited limited cytotoxicity, fairly low degradation rate and comparable strength to the reported Mg-1Zn-5Ca alloy which has been used as bone screws in clinical trials, indicating the great potential application as biodegradable bone implants. Furthermore, it showed good mechanical integrity during immersion to support the defect healing. Our results can aid other researchers to evaluate the mechanical integrity of biodegradable materials and to pay more attention to the effect of degradation behaviour on mechanical integrity of materials.

Proteome analysis of human mesenchymal stem cells undergoing chondrogenesis when exposed to the products of various magnesium-based materials degradation.

Treatment of physeal fractures (15%-30% of all paediatric fractures) remains a challenge as in approximately 10% of the cases, significant growth disturbance may occur. Bioresorbable Magnesium-based implants represent a strategy to minimize damage (i.e., load support until bone healing without second surgery). Nevertheless, the absence of harmful effects of magnesium-implants and their degradation products on the growth plate should be confirmed. Here, the proteome of human mesenchymal stem cells undergoing chondrogenesis was evaluated when exposed to the products of various Magnesium-based materials degradation. The results of this study indicate that the materials induced regulation of proteins associated with cell chondrogenesis and cartilage formation, which should be beneficial for cartilage regeneration.

Different effects of single protein vs. protein mixtures on magnesium degradation under cell culture conditions.

Bovine serum albumin (BSA) or fetal bovine serum (FBS), as the protein component, is usually added into solution to study the influence of proteins on Mg degradation. However, the specific character of proteins used and the interaction between organic molecules in FBS do not draw enough attention. This study investigated the influence of BSA, fibrinogen (Fib) and FBS on Mg degradation in Hanks' balanced salt solution without (HBSS) or with calcium (HBSSCa) and Dulbecco's modified eagle medium Glutamax-I (DMEM). The results reveal that the effect of BSA, Fib and FBS on the degradation rate of Mg is time- and media-dependent, as a result of the overlap of protein adsorption, binding/chelating to ions and interaction between organic molecules. The binding/chelating of proteins and/or the possible effect of proteins on the kinetics of products formation lead to the formation of different degradation precipitates on Mg surface in HBSS. The interaction between proteins and Ca2+/PO43- accelerates the formation of Ca-P salts in HBSSCa and DMEM, thereby impeding the degradation of Mg. Moreover, the interplay between organic molecules and the specific character of proteins are highlighted by the cooperative (in media + FBS) or competitive (in DMEM + BSA + Fib) effect of proteins in the presence of more kinds of proteins and the different effect of BSA and Fib on the degradation of Mg. Therefore, the addition of proteins to testing medium is necessary for in vitro tests and DMEM + 10% FBS is recommended as the in vitro testing medium to present an in vivo-like degradation for Mg. STATEMENT OF SIGNIFICANCE: The present study emphasizes the difference between proteins, and the difference between single protein and protein mixture in view of the effect on Mg degradation. The results highlight the importance of the interaction between proteins in media, which can increase or decrease the degradation of Mg compared to the single protein. It can aid other researchers to understand the effect of proteins on Mg degradation and to pay more attention to the interaction of organic molecules on Mg degradation when more kinds of organic molecules are used in medium, especially for FBS. The submitted work could be of significant importance to other researchers working in the related fields, thus appealing to the readers of Acta Biomaterialia.

Improved In Vitro Test Procedure for Full Assessment of the Cytocompatibility of Degradable Magnesium Based on ISO 10993-5/-12.

Magnesium (Mg)-based biomaterials are promising candidates for bone and tissue regeneration. Alloying and surface modifications provide effective strategies for optimizing and tailoring their degradation kinetics. Nevertheless, biocompatibility analyses of Mg-based materials are challenging due to its special degradation mechanism with continuous hydrogen release. In this context, the hydrogen release and the related (micro-) milieu conditions pretend to strictly follow in vitro standards based on ISO 10993-5/-12. Thus, special adaptions for the testing of Mg materials are necessary, which have been described in a previous study from our group. Based on these adaptions, further developments of a test procedure allowing rapid and effective in vitro cytocompatibility analyses of Mg-based materials based on ISO 10993-5/-12 are necessary. The following study introduces a new two-step test scheme for rapid and effective testing of Mg. Specimens with different surface characteristics were produced by means of plasma electrolytic oxidation (PEO) using silicate-based and phosphate-based electrolytes. The test samples were evaluated for corrosion behavior, cytocompatibility and their mechanical and osteogenic properties. Thereby, two PEO ceramics could be identified for further in vivo evaluations.

Adsorption of Proteins on Degradable Magnesium-Which Factors are Relevant?

Although the adsorption of proteins on the Mg surface was ascribed to be the main reason for the effect of proteins on magnesium (Mg) degradation, few studies about the adsorption of proteins on the Mg surface were performed due to the labile circumstances during immersion. In the present study, the adsorption of bovine serum albumin (BSA) and fibrinogen (Fib) on the Mg surface during and after immersion was extensively investigated in different media for the first time. The results revealed that BSA and Fib showed a similar adsorption trend on the Mg surface during and after immersion, and they adsorbed more on the Mg surface in Hank's balanced salt solution (HBSS) than in Dulbecco's modified Eagle medium Glutamax-I (DMEM). The possible influence factors for protein adsorption, such as pH, surface roughness, and wettability, were considered to elucidate different adsorption in HBSS and DMEM. It was found that the participation of Ca2+ in the formation of degradation products largely affected the degradation rate of Mg, changed surface roughness, compactness, and surface charge during immersion, which largely suppressed the adsorption of proteins on the Mg surface.

The effect of osteoblasts on the surface oxidation processes of biodegradable Mg and Mg-Ag alloys studied by synchrotron IR microspectroscopy.

High-resolution analytical methods, including synchrotron infrared microspectroscopy combined with wavelength-dispersive X-ray emission spectroscopy were applied to study the structure and chemical composition of the oxidized layer of pure and Ag-alloyed Mg exposed to cell culture media without and with osteoblasts. Comparative analysis has been done on pure Mg immersed in two different cell culture media: Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), whereas Mg-xAg binary alloys (x = 2, 4, 6, 8 wt%) were studied after immersion in DMEM. It is shown that the physicochemical formation of degradation products as well as the activity of the biological component is influenced by the addition of silver. It could be demonstrated that the presence of Ag in the Mg alloy enhances the chemical reaction between Mg and C to form amorphous and/or crystalline MgCO3 on account of CaCO3. As a consequence, the further available P and Ca react easily to form Mg-poor amorphous calcium phosphate phases. Osteoblasts actively adjusted these phases towards hydroxyapatite-like phases.

Magnesium degradation under physiological conditions - Best practice.

This review focusses on the application of physiological conditions for the mechanistic understanding of magnesium degradation. Despite the undisputed relevance of simplified laboratory setups for alloy screening purposes, realistic and predictive in vitro setups are needed. Due to the complexity of these systems, the review gives an overview about technical measures, defines some caveats and can be used as a guideline for the establishment of harmonized laboratory approaches.

Influence of the Microstructure and Silver Content on Degradation, Cytocompatibility, and Antibacterial Properties of Magnesium-Silver Alloys In Vitro.

Implantation is a frequent procedure in orthopedic surgery, particularly in the aging population. However, it possesses the risk of infection and biofilm formation at the surgical site. This can cause unnecessary suffering to patients and burden on the healthcare system. Pure Mg, as a promising metal for biodegradable orthopedic implants, exhibits some antibacterial effects due to the alkaline pH produced during degradation. However, this antibacterial effect may not be sufficient in a dynamic environment, for example, the human body. The aim of this study was to increase the antibacterial properties under harsh and dynamic conditions by alloying silver metal with pure Mg as much as possible. Meanwhile, the Mg-Ag alloys should not show obvious cytotoxicity to human primary osteoblasts. Therefore, we studied the influence of the microstructure and the silver content on the degradation behavior, cytocompatibility, and antibacterial properties of Mg-Ag alloys in vitro. The results indicated that a higher silver content can increase the degradation rate of Mg-Ag alloys. However, the degradation rate could be reduced by eliminating the precipitates in the Mg-Ag alloys via T4 treatment. By controlling the microstructure and increasing the silver content, Mg-Ag alloys obtained good antibacterial properties in harsh and dynamic conditions but had almost equivalent cytocompatibility to human primary osteoblasts as pure Mg.

A study of degradation resistance and cytocompatibility of super-hydrophobic coating on magnesium.

Calcium stearate based super-hydrophobic coating was deposited on plasma electrolytic oxidation (PEO) pre-treated magnesium substrate. The pre-treated magnesium and super-hydrophobic coating covered sample were characterized by scanning electron microscopy, X-ray diffraction and electrochemical corrosion measurements. The cytocompatibility and degradation resistance of magnesium, pre-treated magnesium and super-hydrophobic coating were analysed in terms of cell adhesion and osteoblast differentiation. The results indicate that the calcium stearate top coating shows super-hydrophobicity and that the surface is composed of micro/nanostructure. The super-hydrophobic coating covered sample shows higher barrier properties compared with the PEO pre-treated magnesium and bare magnesium. Human osteoblast proliferation, but not differentiation is enhanced by the PEO coating. Contrary, the super-hydrophobic coating reduces proliferation, but enhances differentiation of osteoblast, observable by the formation of hydroxyapatite. The combination of corrosion protection and cell reaction indicates that this system could be interesting for biomedical applications.

Influence of Dy in solid solution on the degradation behavior of binary Mg-Dy alloys in cell culture medium.

Rare earth element Dy is one of the promising alloying elements for magnesium alloy as biodegradable implants. To understand the effect of Dy in solid solution on the degradation of Mg-Dy alloys in simulated physiological conditions, the present work studied the microstructure and degradation behavior of Mg-Dy alloys in cell culture medium. It is found the corrosion resistance enhances with the increase of Dy content in solid solution in Mg. This can be attributed to the formation of a relatively more corrosion resistant Dy-enriched film which decreases the anodic dissolution of Mg.

Behavior of bone cells in contact with magnesium implant material.

Magnesium-based implants exhibit several advantages, such as biodegradability and possible osteoinductive properties. Whether the degradation may induce cell type-specific changes in metabolism still remains unclear. To examine the osteoinductivity mechanisms, the reaction of bone-derived cells (MG63, U2OS, SaoS2, and primary human osteoblasts (OB)) to magnesium (Mg) was determined. Mg-based extracts were used to mimic more realistic Mg degradation conditions. Moreover, the influence of cells having direct contact with the degrading Mg metal was investigated. In exposure to extracts and in direct contact, the cells decreased pH and osmolality due to metabolic activity. Proliferating cells showed no significant reaction to extracts, whereas differentiating cells were negatively influenced. In contrast to extract exposure, where cell size increased, in direct contact to magnesium, cell size was stable or even decreased. The amount of focal adhesions decreased over time on all materials. Genes involved in bone formation were significantly upregulated, especially for primary human osteoblasts. Some osteoinductive indicators were observed for OB: (i) an increased cell count after extract addition indicated a higher proliferation potential; (ii) increased cell sizes after extract supplementation in combination with augmented adhesion behavior of these cells suggest an early switch to differentiation; and (iii) bone-inducing gene expression patterns were determined for all analyzed conditions. The results from the cell lines were inhomogeneous and showed no specific stimulus of Mg. The comparison of the different cell types showed that primary cells of the investigated tissue should be used as an in vitro model if Mg is analyzed. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 165-179, 2017.

Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation.

Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10wt% of gadolinium (Mg-10Gd) and Mg with 2wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants.

The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells.

Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys) is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells) are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity.

Intramedullary Mg2Ag nails augment callus formation during fracture healing in mice.

UNLABELLED: Intramedullary stabilization is frequently used to treat long bone fractures. Implants usually remain unless complications arise. Since implant removal can become technically very challenging with the potential to cause further tissue damage, biodegradable materials are emerging as alternative options. Magnesium (Mg)-based biodegradable implants have a controllable degradation rate and good tissue compatibility, which makes them attractive for musculoskeletal research. Here we report for the first time the implantation of intramedullary nails made of an Mg alloy containing 2% silver (Mg2Ag) into intact and fractured femora of mice. Prior in vitro analyses revealed an inhibitory effect of Mg2Ag degradation products on osteoclast differentiation and function with no impair of osteoblast function. In vivo, Mg2Ag implants degraded under non-fracture and fracture conditions within 210days and 133days, respectively. During fracture repair, osteoblast function and subsequent bone formation were enhanced, while osteoclast activity and bone resorption were decreased, leading to an augmented callus formation. We observed a widening of the femoral shaft under steady state and regenerating conditions, which was at least in part due to an uncoupled bone remodeling. However, Mg2Ag implants did not cause any systemic adverse effects. These data suggest that Mg2Ag implants might be promising for intramedullary fixation of long bone fractures, a novel concept that has to be further investigated in future studies. STATEMENT OF SIGNIFICANCE: Biodegradable implants are promising alternatives to standard steel or titanium implants to avoid implant removal after fracture healing. We therefore developed an intramedullary nail using a novel biodegradable magnesium-silver-alloy (Mg2Ag) and investigated the in vitro and in vivo effects of the implants on bone remodeling under steady state and fracture healing conditions in mice. Our results demonstrate that intramedullary Mg2Ag nails degrade in vivo over time without causing adverse effects. Importantly, radiographs, µCT and bone histomorphometry revealed a significant increase in callus size due to an augmented bone formation rate and a reduced bone resorption in fractures supported by Mg2Ag nails, thereby improving bone healing. Thus, intramedullary Mg2Ag nails are promising biomaterials for fracture healing to circumvent implant removal.

Degradation testing of Mg alloys in Dulbecco's modified eagle medium: Influence of medium sterilization.

This work studies the in vitro degradation of Mg alloys for bioabsorbable implant applications under near physiological conditions. For this purpose, the degradation behaviour of Mg alloys in Dulbecco's modified eagle medium (DMEM) which is a commonly used cell culture medium is analysed. Unfortunately, DMEM can be contaminated by microorganisms, acidifying the medium and accelerating the Mg degradation process by dissolution of protective degradation layers, such as (Mgx,Cay)(PO4)z. In this paper the influence of sterilization by applying UV-C radiation and antibiotics (penicillin/streptomycin) is analysed with two implant material candidates: Mg-Gd and Mg-Ag alloys; and pure magnesium as well as Mg-4Y-3RE as a reference.

Magnesium degradation influenced by buffering salts in concentrations typical of in vitro and in vivo models.

Magnesium and its alloys have considerable potential for orthopedic applications. During the degradation process the interface between material and tissue is continuously changing. Moreover, too fast or uncontrolled degradation is detrimental for the outcome in vivo. Therefore in vitro setups utilizing physiological conditions are promising for the material/degradation analysis prior to animal experiments. The aim of this study is to elucidate the influence of inorganic salts contributing to the blood buffering capacity on degradation. Extruded pure magnesium samples were immersed under cell culture conditions for 3 and 10 days. Hank's balanced salt solution without calcium and magnesium (HBSS) plus 10% of fetal bovine serum (FBS) was used as the basic immersion medium. Additionally, different inorganic salts were added with respect to concentration in Dulbecco's modified Eagle's medium (DMEM, in vitro model) and human plasma (in vivo model) to form 12 different immersion media. Influences on the surrounding environment were observed by measuring pH and osmolality. The degradation interface was analyzed by electron-induced X-ray emission (EIXE) spectroscopy, including chemical-element mappings and electron microprobe analysis, as well as Fourier transform infrared reflection micro-spectroscopy (FTIR).