rus
Issue #4/2023
P. E. Timchenko, E. V. Timchenko, D. A. Dolgushkin, O. O. Frolov, A. N. Nikolaenko, L. T. Volova, A. Yu. Ionov
Features of the Spectral Surface Estimation of Titanium Implants for Animals
Features of the Spectral Surface Estimation of Titanium Implants for Animals
DOI: 10.22184/1993-7296.FRos.2023.17.4.326.336
The paper presents the study results relating to the material state of the implants made of titanium alloy and coated with chitosan. The implants have been studied before and after preclinical use in animals. A feature of this research method is the use of Raman scattering spectroscopy with a high sensitivity in the region of 400–1 800 cm−1. Confirmation of the implant surface study results was obtained using the scanning electron microscopy. The details of spectral changes are taken as an indirect estimate of the complete biodegradation of the implant coating after one month.
The paper presents the study results relating to the material state of the implants made of titanium alloy and coated with chitosan. The implants have been studied before and after preclinical use in animals. A feature of this research method is the use of Raman scattering spectroscopy with a high sensitivity in the region of 400–1 800 cm−1. Confirmation of the implant surface study results was obtained using the scanning electron microscopy. The details of spectral changes are taken as an indirect estimate of the complete biodegradation of the implant coating after one month.
Теги: chitosan gaussian function deconvolution implant lda analysis lda-анализ raman scattering spectroscopy spectrum statistical analysis деконволюция функции гаусса имплант спектр спектроскопия рамановская статистический анализ хитозан
Features of the Spectral Surface Estimation of Titanium Implants for Animals
P. E. Timchenko1, E. V. Timchenko1, D. A. Dolgushkin2, O. O. Frolov1, A. N. Nikolaenko2, L. T. Volova2, A. Yu. Ionov1
Korolev Samara National Research University, Samara, Russia
Samara State Medical University, Institute of Experimental Medicine and Biotechnology, Samara, Russia
The paper presents the study results relating to the material state of the implants made of titanium alloy and coated with chitosan. The implants have been studied before and after preclinical use in animals. A feature of this research method is the use of Raman scattering spectroscopy with a high sensitivity in the region of 400–1 800 cm−1. Confirmation of the implant surface study results was obtained using the scanning electron microscopy. The details of spectral changes are taken as an indirect estimate of the complete biodegradation of the implant coating after one month.
Keywords: Raman scattering spectroscopy, Gaussian function deconvolution, spectrum, statistical analysis, chitosan, implant, LDA analysis
Received on: 27.01.2023
Accepted on: 27.02.2023
Introduction
The optical research methods are widely used in the field of biomedical problems [1, 2]. Among optical methods, it is possible to place emphasis on the IR spectroscopy, as well as the Raman scattering spectroscopy that is a non-invasive and operational analytical method [3]. IR spectroscopy [4] is a well-established method aimed at transmission with the absorption estimation that is not applicable for opaque biological objects.
Another promising analytical method the surface elemental composition is scanning electron microscopy. This method allows to analyze the surface and elemental composition of the sample [5].
The Raman scattering spectroscopy in combination with the mathematical methods of analysis and scanning electron microscopy can be used in the production technology of combined products for tissue engineering, transplantology, and reconstructive surgery.
In some cases, to achieve a clinical result, it is necessary either to limit the implant material contact with the microorganism medium, or, on the contrary, to apply a bioactive coating. Such an implant coating can create favorable conditions for the post-implantation period, stimulate the regenerative processes, and prevent the development of inflammatory complications.
An urgent issue is to perform the follow-up study of the chitosan-based implant coatings, since it has biological activity, as well as the ability to activate the regenerative processes in tissues [6–13]. The study process is enhanced by the combination of Raman scattering spectroscopy and scanning electron microscopy methods [14–16].
Therefore, the aim of this research was to develop an indirect evaluation of the complete coating biodegradation of titanium implants based on the surface spectral study results in the animal experiments.
Material and methods
The preclinical experimental studies on animals were performed at the biotechnological center “BioTech” of the Federal State Budgetary Educational Institution of Higher Education “Samara State Medical University of the Ministry of Healthcare of Russia”. 20 mature laboratory Wistar rats were implanted with the chitosan-coated VT6 titanium samples in both shoulder blades. The terms for animal withdrawal from the experiment were considered 1 month and 1 week after the surgery. Accordingly, the first surgery intervention on one shoulder blade of the animal was performed 1 month prior to slaughter, and the second surgery on the other shoulder blade was performed 1 week prior to slaughter. The VT6 titanium-based samples were made using the selective laser sintering technology (Pavlov First Saint-Petersburg State Medical University). The chitosan-contained coating was applied to the cylindrical bars to place them in the region of the rat scapular spine. Previously, a through hole was made in the bone using a cylindrical dental bur. The bar shape and dimensions made it possible to rigidly and immovably fix it in the formed bone window. Chitosan has antiseptic properties and is able to participate in the regenerative processes in the peri-implant region. Moreover, the coating can serve as a temporary separating film for a deeper implant coating the effect of which is required in the peri-implant region during the late time period. The chitosan-contained coating was applied to the samples at the Chair of Polymer and Crystal Physics of the Lomonosov Moscow State University.
Pressurized coating of the cylindrical bars made of VT6 titanium alloy was carried out at the room temperature with the constant stirring for 2 days. Prior to commencement of the preclinical studies, the samples were subjected to the staged sterilization (Fig. 1).
Prior to the experimental study, the sample surface was assessed using a JED‑2300 focused-beam scanning microscope (TokyoBoeki, Japan) with the AnalysisStation 3.63.01 software. Deposition was performed using an EMITECHK450X device. We also studied the sample surface before implantation using the Raman scattering spectroscopy.
During an experimental study, the cylindrical bars made of VT6 titanium alloy coated with chitosan were implanted in the rat scapular spine while forming a hole in it with a bur for compact implant placement in the bone tissue (Fig. 2). The wounds were sutured in layers. The animals were subject to the follow-up examination with withdrawal from the experiment after 1 week and 1 month.
The samples obtained were re-examined after appropriate processing using the Raman scattering spectroscopy. The method was implemented with a stand. It included a semiconductor laser (LML‑785.0RB‑04), an optical Raman scattering module (PBL 785), a spectrograph (Sharmrock SR‑303i) with an integrated digital camera (ANDOR DV‑420A-OE) cooled down to –60 °C, and a computer [17].
The spectrograph provided a wavelength resolution of 0.15 nm at a low intrinsic noise level. The laser radiation power of 200 mW within the exposure times used (30 sec) did not lead to any changes in the samples causing changes in the spectrum.
The Raman spectra were registered using an optical probe that was placed above the object at a distance of 7 mm [18]. In this paper, the Raman spectra were analyzed in the range of 400–1 800 cm−1, where the main lines of various organic compounds were observed. To exclude the autofluorescence contribution in the Raman spectrum, we subtracted the fluorescent component of the polynomial approximation with additional filtering of random noise effects. The Raman spectra were processed and analyzed using the WolframMathematica 12.2 software package.
During the mathematical measurement result processing using the Raman scattering spectroscopy, the Gaussian function deconvolution method was used, combined with the logit regression and statistical analysis method (LDA).
Results and discussion
When visually inspecting the cylindrical bars made of VT6 titanium alloy with a chitosan-based coating, it could be noted that their bases in the saw-cut section were uneven and had a non-uniform relief. The sample was covered with a whitish film that was sometimes peeled off (Fig. 3). When examining the samples at magnifications of 1 000–2 000, it was found that the coating was represented by two different types of areas (Fig. 4).
A large sample area (at least 3 / 4) was occupied by the coating represented by the whitish granular crystals of various sizes and shapes. They were tightly adhered to each other and to the sample surface. These areas had an extremely uneven relief (Fig. 5a).
Another type of coating areas was smoother, covered with a transparent film, under which the granular whitish crystals were also visualized (Fig. 5b).
It should be noted that we previously performed the scanning electron microscopy of control VT6 titanium samples prior to coating application. Neither films nor whitish crystals were found on the surface of control samples. Availability of such inhomogeneous areas on the chitosan-coated samples may be due to the powder deposition technological features.
It was worth noting that the coating areas, similar to the film, were often peeled off. Fig. 6 shows such a case, with the whitish crystals remaining fixed to both the film and the sample underneath.
The Raman scattering spectroscopy of the samples was performed over time, namely prior to implantation, as well as 1 week and 1 month after the animal withdrawal from the experiment. Figure 7 shows the normalized Raman spectra of the samples under study. It can be seen that the chitosan-coated VT6 titanium samples prior to implantation have the significant spectral lines ~1 006 cm−1 (Aromatic ring breathing of phenylalanine νs (C–C)), ~1 260−1 (Amide III – Due to C-N stretching and N-H bending), ~1 414 cm−1 (CH deformation), ~1 558 cm−1 (Amide II Parallel / Antiparallel β-sheet structure), and ~1 748 cm−1 (ν(C=O ester group), phospholipids (Lipid assignment)).
However, after implantation and subsequent sample extraction, when examining their surface after 1 week and 1 month, the spectral changes in the line intensities occur in the entire spectral range under study.
As can be seen from this figure, the line intensity at 1 414 cm−1 is decreased, with the reduced intensity of the lines at 1 260 cm−1 that may indicate the chitosan biodegradation as early as 1 week after implantation.
To increase the information value of the obtained Raman spectra, a nonlinear regression analysis of the spectra was performed consisted in their decomposition into the spectral lines. The full spectrum region 380–1 780 cm−1 was divided for analysis into 4 spectral contours: 1 – 380 – 508 (adjR2 = 0.9999), 2 – 508 – 1136 (adjR2 = 0.9978), 3 – 1 136 – 1 491 (adjR2 = 0.9998), 4 – 1 491 – 1 781 cm‑1 (adjR2 = 0.9998).
The results obtained after a linear discriminant spectrum analysis, consisting in their decomposition into the spectral lines, were used for further analysis and visualization of the results by the classification method using the logistic regression in reduced and LDA modes (Fig. 8a, b).
The resulting discriminatory model makes it possible to classify the measured objects with an accuracy of 87.0%. To assess the biodegradation degree of the titanium implant coating, a complex criterion can be used based on the relative intensity of 6 lines: 458 cm−1, 497 cm−1, 648 cm−1, 728 cm−1, 1 006 cm−1, 1 414 cm−1.
The statistical analysis helped to identify the Raman spectra lines that determined the main difference between the studied groups. It was found that the most significant differences between the groups before and 1 month after implantation occurred on the lines 1 414 cm−1 (CH deformation) and 1 006 cm−1 (Aromatic ring breathing of phenylalanine νs (C–C)).
Conclusion
The following research results were obtained. During the study of cylindrical bars made of VT6 titanium alloy using the scanning electron microscopy, it was found that their surface had two types of heterogeneous coating areas. At least 3 / 4 of the surface area was occupied by the regions represented by the whitish granular crystals of various sizes and shapes, tightly linked to each other and the sample surface. The other type of coating regions was smoother and covered with a transparent film, under which the granular whitish crystals were also visualized. The film often peeled off from the implant. We related the available heterogeneous coating regions with the technological features of chitosan application to the implants.
Using the Raman scattering spectroscopy, it was found that before implantation the studied chitosan-coated samples had the significant spectral lines: ~1 006 cm−1 (Aromatic ring breathing of phenylalanine νs (C–C)), ~1 260 cm−1 (Amide III – Due to C-N stretching and N-H bending), ~1 414 cm−1 (CH deformation), ~1 558 cm−1 (Amide II Parallel / Antiparallel β-sheet structure), and ~1 748 cm−1 (ν(C=O ester group), phospholipids (Lipid assignment)).
The spectral changes in the surfaces of the studied samples after their implantation into the rate scapula were found. It was shown that intensity of the lines ~1 260 cm−1 (Amide III – Due to C-N stretching and N-H bending), ~1 414 cm−1 (CH deformation) was decreased significantly already 1 week after implantation that indicated the beginning of early chitosan biodegradation and, accordingly, the expected manifestation of its biological effects.
87.0% of cross-validated grouped observations were classified correctly. The Wilks’ lambda was used to check the significance that was 0.268 for all two discriminant functions (Chi-square = 23.017, statistical significance = 0.000). To assess the coating biodegradation degree of titanium implants, we used a complex criterion based on the relative intensity of 6 lines: 458 cm−1, 497 cm−1, 648 cm−1, 728 cm−1, 1 006 cm−1, 1 414 cm−1.
P. E. Timchenko1, E. V. Timchenko1, D. A. Dolgushkin2, O. O. Frolov1, A. N. Nikolaenko2, L. T. Volova2, A. Yu. Ionov1
Korolev Samara National Research University, Samara, Russia
Samara State Medical University, Institute of Experimental Medicine and Biotechnology, Samara, Russia
The paper presents the study results relating to the material state of the implants made of titanium alloy and coated with chitosan. The implants have been studied before and after preclinical use in animals. A feature of this research method is the use of Raman scattering spectroscopy with a high sensitivity in the region of 400–1 800 cm−1. Confirmation of the implant surface study results was obtained using the scanning electron microscopy. The details of spectral changes are taken as an indirect estimate of the complete biodegradation of the implant coating after one month.
Keywords: Raman scattering spectroscopy, Gaussian function deconvolution, spectrum, statistical analysis, chitosan, implant, LDA analysis
Received on: 27.01.2023
Accepted on: 27.02.2023
Introduction
The optical research methods are widely used in the field of biomedical problems [1, 2]. Among optical methods, it is possible to place emphasis on the IR spectroscopy, as well as the Raman scattering spectroscopy that is a non-invasive and operational analytical method [3]. IR spectroscopy [4] is a well-established method aimed at transmission with the absorption estimation that is not applicable for opaque biological objects.
Another promising analytical method the surface elemental composition is scanning electron microscopy. This method allows to analyze the surface and elemental composition of the sample [5].
The Raman scattering spectroscopy in combination with the mathematical methods of analysis and scanning electron microscopy can be used in the production technology of combined products for tissue engineering, transplantology, and reconstructive surgery.
In some cases, to achieve a clinical result, it is necessary either to limit the implant material contact with the microorganism medium, or, on the contrary, to apply a bioactive coating. Such an implant coating can create favorable conditions for the post-implantation period, stimulate the regenerative processes, and prevent the development of inflammatory complications.
An urgent issue is to perform the follow-up study of the chitosan-based implant coatings, since it has biological activity, as well as the ability to activate the regenerative processes in tissues [6–13]. The study process is enhanced by the combination of Raman scattering spectroscopy and scanning electron microscopy methods [14–16].
Therefore, the aim of this research was to develop an indirect evaluation of the complete coating biodegradation of titanium implants based on the surface spectral study results in the animal experiments.
Material and methods
The preclinical experimental studies on animals were performed at the biotechnological center “BioTech” of the Federal State Budgetary Educational Institution of Higher Education “Samara State Medical University of the Ministry of Healthcare of Russia”. 20 mature laboratory Wistar rats were implanted with the chitosan-coated VT6 titanium samples in both shoulder blades. The terms for animal withdrawal from the experiment were considered 1 month and 1 week after the surgery. Accordingly, the first surgery intervention on one shoulder blade of the animal was performed 1 month prior to slaughter, and the second surgery on the other shoulder blade was performed 1 week prior to slaughter. The VT6 titanium-based samples were made using the selective laser sintering technology (Pavlov First Saint-Petersburg State Medical University). The chitosan-contained coating was applied to the cylindrical bars to place them in the region of the rat scapular spine. Previously, a through hole was made in the bone using a cylindrical dental bur. The bar shape and dimensions made it possible to rigidly and immovably fix it in the formed bone window. Chitosan has antiseptic properties and is able to participate in the regenerative processes in the peri-implant region. Moreover, the coating can serve as a temporary separating film for a deeper implant coating the effect of which is required in the peri-implant region during the late time period. The chitosan-contained coating was applied to the samples at the Chair of Polymer and Crystal Physics of the Lomonosov Moscow State University.
Pressurized coating of the cylindrical bars made of VT6 titanium alloy was carried out at the room temperature with the constant stirring for 2 days. Prior to commencement of the preclinical studies, the samples were subjected to the staged sterilization (Fig. 1).
Prior to the experimental study, the sample surface was assessed using a JED‑2300 focused-beam scanning microscope (TokyoBoeki, Japan) with the AnalysisStation 3.63.01 software. Deposition was performed using an EMITECHK450X device. We also studied the sample surface before implantation using the Raman scattering spectroscopy.
During an experimental study, the cylindrical bars made of VT6 titanium alloy coated with chitosan were implanted in the rat scapular spine while forming a hole in it with a bur for compact implant placement in the bone tissue (Fig. 2). The wounds were sutured in layers. The animals were subject to the follow-up examination with withdrawal from the experiment after 1 week and 1 month.
The samples obtained were re-examined after appropriate processing using the Raman scattering spectroscopy. The method was implemented with a stand. It included a semiconductor laser (LML‑785.0RB‑04), an optical Raman scattering module (PBL 785), a spectrograph (Sharmrock SR‑303i) with an integrated digital camera (ANDOR DV‑420A-OE) cooled down to –60 °C, and a computer [17].
The spectrograph provided a wavelength resolution of 0.15 nm at a low intrinsic noise level. The laser radiation power of 200 mW within the exposure times used (30 sec) did not lead to any changes in the samples causing changes in the spectrum.
The Raman spectra were registered using an optical probe that was placed above the object at a distance of 7 mm [18]. In this paper, the Raman spectra were analyzed in the range of 400–1 800 cm−1, where the main lines of various organic compounds were observed. To exclude the autofluorescence contribution in the Raman spectrum, we subtracted the fluorescent component of the polynomial approximation with additional filtering of random noise effects. The Raman spectra were processed and analyzed using the WolframMathematica 12.2 software package.
During the mathematical measurement result processing using the Raman scattering spectroscopy, the Gaussian function deconvolution method was used, combined with the logit regression and statistical analysis method (LDA).
Results and discussion
When visually inspecting the cylindrical bars made of VT6 titanium alloy with a chitosan-based coating, it could be noted that their bases in the saw-cut section were uneven and had a non-uniform relief. The sample was covered with a whitish film that was sometimes peeled off (Fig. 3). When examining the samples at magnifications of 1 000–2 000, it was found that the coating was represented by two different types of areas (Fig. 4).
A large sample area (at least 3 / 4) was occupied by the coating represented by the whitish granular crystals of various sizes and shapes. They were tightly adhered to each other and to the sample surface. These areas had an extremely uneven relief (Fig. 5a).
Another type of coating areas was smoother, covered with a transparent film, under which the granular whitish crystals were also visualized (Fig. 5b).
It should be noted that we previously performed the scanning electron microscopy of control VT6 titanium samples prior to coating application. Neither films nor whitish crystals were found on the surface of control samples. Availability of such inhomogeneous areas on the chitosan-coated samples may be due to the powder deposition technological features.
It was worth noting that the coating areas, similar to the film, were often peeled off. Fig. 6 shows such a case, with the whitish crystals remaining fixed to both the film and the sample underneath.
The Raman scattering spectroscopy of the samples was performed over time, namely prior to implantation, as well as 1 week and 1 month after the animal withdrawal from the experiment. Figure 7 shows the normalized Raman spectra of the samples under study. It can be seen that the chitosan-coated VT6 titanium samples prior to implantation have the significant spectral lines ~1 006 cm−1 (Aromatic ring breathing of phenylalanine νs (C–C)), ~1 260−1 (Amide III – Due to C-N stretching and N-H bending), ~1 414 cm−1 (CH deformation), ~1 558 cm−1 (Amide II Parallel / Antiparallel β-sheet structure), and ~1 748 cm−1 (ν(C=O ester group), phospholipids (Lipid assignment)).
However, after implantation and subsequent sample extraction, when examining their surface after 1 week and 1 month, the spectral changes in the line intensities occur in the entire spectral range under study.
As can be seen from this figure, the line intensity at 1 414 cm−1 is decreased, with the reduced intensity of the lines at 1 260 cm−1 that may indicate the chitosan biodegradation as early as 1 week after implantation.
To increase the information value of the obtained Raman spectra, a nonlinear regression analysis of the spectra was performed consisted in their decomposition into the spectral lines. The full spectrum region 380–1 780 cm−1 was divided for analysis into 4 spectral contours: 1 – 380 – 508 (adjR2 = 0.9999), 2 – 508 – 1136 (adjR2 = 0.9978), 3 – 1 136 – 1 491 (adjR2 = 0.9998), 4 – 1 491 – 1 781 cm‑1 (adjR2 = 0.9998).
The results obtained after a linear discriminant spectrum analysis, consisting in their decomposition into the spectral lines, were used for further analysis and visualization of the results by the classification method using the logistic regression in reduced and LDA modes (Fig. 8a, b).
The resulting discriminatory model makes it possible to classify the measured objects with an accuracy of 87.0%. To assess the biodegradation degree of the titanium implant coating, a complex criterion can be used based on the relative intensity of 6 lines: 458 cm−1, 497 cm−1, 648 cm−1, 728 cm−1, 1 006 cm−1, 1 414 cm−1.
The statistical analysis helped to identify the Raman spectra lines that determined the main difference between the studied groups. It was found that the most significant differences between the groups before and 1 month after implantation occurred on the lines 1 414 cm−1 (CH deformation) and 1 006 cm−1 (Aromatic ring breathing of phenylalanine νs (C–C)).
Conclusion
The following research results were obtained. During the study of cylindrical bars made of VT6 titanium alloy using the scanning electron microscopy, it was found that their surface had two types of heterogeneous coating areas. At least 3 / 4 of the surface area was occupied by the regions represented by the whitish granular crystals of various sizes and shapes, tightly linked to each other and the sample surface. The other type of coating regions was smoother and covered with a transparent film, under which the granular whitish crystals were also visualized. The film often peeled off from the implant. We related the available heterogeneous coating regions with the technological features of chitosan application to the implants.
Using the Raman scattering spectroscopy, it was found that before implantation the studied chitosan-coated samples had the significant spectral lines: ~1 006 cm−1 (Aromatic ring breathing of phenylalanine νs (C–C)), ~1 260 cm−1 (Amide III – Due to C-N stretching and N-H bending), ~1 414 cm−1 (CH deformation), ~1 558 cm−1 (Amide II Parallel / Antiparallel β-sheet structure), and ~1 748 cm−1 (ν(C=O ester group), phospholipids (Lipid assignment)).
The spectral changes in the surfaces of the studied samples after their implantation into the rate scapula were found. It was shown that intensity of the lines ~1 260 cm−1 (Amide III – Due to C-N stretching and N-H bending), ~1 414 cm−1 (CH deformation) was decreased significantly already 1 week after implantation that indicated the beginning of early chitosan biodegradation and, accordingly, the expected manifestation of its biological effects.
87.0% of cross-validated grouped observations were classified correctly. The Wilks’ lambda was used to check the significance that was 0.268 for all two discriminant functions (Chi-square = 23.017, statistical significance = 0.000). To assess the coating biodegradation degree of titanium implants, we used a complex criterion based on the relative intensity of 6 lines: 458 cm−1, 497 cm−1, 648 cm−1, 728 cm−1, 1 006 cm−1, 1 414 cm−1.
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