Isaac Afara | University of Eastern Finland (original) (raw)
Papers by Isaac Afara
Molecules
The aim of the study was to optimize preprocessing of sparse infrared spectral data. The sparse d... more The aim of the study was to optimize preprocessing of sparse infrared spectral data. The sparse data were obtained by reducing broadband Fourier transform infrared attenuated total reflectance spectra of bovine and human cartilage, as well as of simulated spectral data, comprising several thousand spectral variables into datasets comprising only seven spectral variables. Different preprocessing approaches were compared, including simple baseline correction and normalization procedures, and model-based preprocessing, such as multiplicative signal correction (MSC). The optimal preprocessing was selected based on the quality of classification models established by partial least squares discriminant analysis for discriminating healthy and damaged cartilage samples. The best results for the sparse data were obtained by preprocessing using a baseline offset correction at 1800 cm−1, followed by peak normalization at 850 cm−1 and preprocessing by MSC.
Biomechanical indentation testing of equine articular cartilage. Testing protocol consisted of si... more Biomechanical indentation testing of equine articular cartilage. Testing protocol consisted of single 7.5% strain step.
Near infrared spectroscopic (NIRS) measurements of equine articular cartilage. NIRS measurements ... more Near infrared spectroscopic (NIRS) measurements of equine articular cartilage. NIRS measurements are paired with reference parameters calculated from biomechanical and histological measurements. These reference parameters reflect the biomechanical response, chemical composition and internal structure of the articular cartilage samples.<br><b>Change history: </b>Following publication, it was discovered that a number of samples in nirs_and_references.mat were mislabelled. This has been corrected in version 2.
Indendation testing of articular cartilage consisting of three cumulative 7.5% strain steps and f... more Indendation testing of articular cartilage consisting of three cumulative 7.5% strain steps and four cycles of dynamic sinusoidal loading (at 2% amplitude using frequencies of 0.1, 0.25, 0.625, 0.833, 1.0, and 2.0 Hz).
Dataset contains histological reference measurements of equine articular cartilage, measured with... more Dataset contains histological reference measurements of equine articular cartilage, measured with fourier-transform infrared microspectroscopy.
Biophotonics Congress: Biomedical Optics Congress 2018 (Microscopy/Translational/Brain/OTS)
We present preliminary findings on multivariate modeling between cartilage biomechanical properti... more We present preliminary findings on multivariate modeling between cartilage biomechanical properties and spectroscopic measurements, performed in arthroscopic surgery and laboratory. Also, generic multivariate regression techniques were compared.
Biophotonics Congress: Optics in the Life Sciences Congress 2019 (BODA,BRAIN,NTM,OMA,OMP)
We introduce classification models based on partial least squares discriminant-analysis (PLS-DA) ... more We introduce classification models based on partial least squares discriminant-analysis (PLS-DA) for estimating cartilage integrity (assessed by OARSI grade) based on mid-infrared spectra of cartilage matrix. The best model achieved accuracy of 84%.
Biophotonics Congress: Optics in the Life Sciences Congress 2019 (BODA,BRAIN,NTM,OMA,OMP)
In this study, we demonstrate the potential of mid-infrared (MIR) and near infrared (NIR) spectro... more In this study, we demonstrate the potential of mid-infrared (MIR) and near infrared (NIR) spectroscopies to reveal and differentiate between superficial changes in articular cartilage (AC) after mechanical or enzymatic degradation.
Biophotonics Congress: Biomedical Optics Congress 2018 (Microscopy/Translational/Brain/OTS)
Near-infrared spectroscopy is a promising tool for estimating the composition and biomechanical p... more Near-infrared spectroscopy is a promising tool for estimating the composition and biomechanical properties of meniscus. Here, we developed a predictive model was created for mapping meniscus biomechanical properties based on near-infrared spectroscopy.
World Academy of Science, Engineering and Technology, International Journal of Biotechnology and Bioengineering, 2017
This paper reviews the current status of the application of optical non-destructive methods, part... more This paper reviews the current status of the application of optical non-destructive methods, particularly infrared (IR) and near infrared (NIR), in the evaluation of the physiological integrity of articular cartilage. It is concluded that a significant amount of work is still required in order to achieve specificity and clinical applicability of these methods in the assessment and treatment of dysfunctional articular joints.
IEEE Journal of Biomedical and Health Informatics, 2021
Traditional sleep staging with non-overlapping 30-second epochs overlooks multiple sleep-wake tra... more Traditional sleep staging with non-overlapping 30-second epochs overlooks multiple sleep-wake transitions. We aimed to overcome this by analyzing the sleep architecture in more detail with deep learning methods and hypothesized that the traditional sleep staging underestimates the sleep fragmentation of obstructive sleep apnea (OSA) patients. To test this hypothesis, we applied deep learning-based sleep staging to identify sleep stages with the traditional approach and by using overlapping 30-second epochs with 15-, 5-, 1-, or 0.5-second epoch-to-epoch duration. A dataset of 446 patients referred for polysomnography due to OSA suspicion was used to assess differences in the sleep architecture between OSA severity groups. The amount of wakefulness increased while REM and N3 decreased in severe OSA with shorter epoch-to-epoch duration. In other OSA severity groups, the amount of wake and N1 decreased while N3 increased. With the traditional 30-second epoch-to-epoch duration, only smal...
Articular cartilage is a connective tissue that enables smooth movements between bones in articul... more Articular cartilage is a connective tissue that enables smooth movements between bones in articulating joints. Cartilage consists of extracellular matrix (ECM) and chondrocytes – the cells responsible for synthesis of the ECM. The ECM consists of type II collagen, proteoglycans, water, and some other minor components. Cartilage is prone to degenerative joint conditions, such as osteoarthritis, due to its weak repair capacity resulting from a lack of vascular, neural, and lymphatic networks. Osteoarthritis causes erosion of the cartilage matrix and therefore inhibits its function, resulting in joint pain, loss of mobility, and significant global socioeconomic burden. Currently, surgical treatment of cartilage pathologies is carried out during arthroscopy with variable outcomes. This variability occurs due to the subjective nature of arthroscopy, which relies on manual palpation and visual evaluation of the tissue surface. Diffuse optical spectroscopy in the near-infrared spectral reg...
CARTILAGE
Objective Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining signifi... more Objective Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by ( a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and ( b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state. Design The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identifie...
The scourge of malaria infection continues to strike hardest against pregnant women and children ... more The scourge of malaria infection continues to strike hardest against pregnant women and children in Africa and South East Asia. For global elimination, testing methods that are ultrasensitive to low-level ring-staged parasitemia are urgently required. In this study, we used a novel approach for diagnosis of malaria infection by combining both electronic ultraviolet-visible (UV/vis) spectroscopy and near infrared (NIR) spectroscopy to detect and quantify low-level (1-0.000001%) ring-staged malaria-infected whole blood under physiological conditions uisng Multiclass classification using logistic regression, which showed that the best results were achieved using the extended wavelength range, providing an accuracy of 100% for most parasitemia classes. Likewise, partial least-squares regression (PLS-R) analysis showed a higher quantification sensitivity (R2 = 0.898) for the extended spectral region compared to UV/vis and NIR (R2 = 0.806 and 0.556, respectively). For quantifying different-stage blood parasites, the extended wavelength range was able to detect and quantify all thePlasmodium falciparum accurately compared to testing each spectral component separately. These results demonstrate the potential of a combined UV/vis-NIR spectroscopy to accurately diagnose malaria-infected patients without the need for elaborate sample preparation associated with the existing mid-IR approaches.
Biomedical Optics Express
Optical properties of biological tissues in the NIR spectral range have demonstrated significant ... more Optical properties of biological tissues in the NIR spectral range have demonstrated significant potential for in vivo diagnostic applications and are critical parameters for modelling light interaction in biological tissues. This study aims to investigate the optical properties of articular cartilage as a function of tissue depth and integrity. The results suggest consistent wavelength-dependent variation in optical properties between cartilage depth-wise zones, as well as between healthy and degenerated tissue. Also, statistically significant differences (p<0.05) in both optical properties were observed between the different cartilage depth-wise zones and as a result of tissue degeneration. When taken into account, the outcome of this study could enable accurate modelling of light interaction in cartilage matrix and could provide useful diagnostic information on cartilage integrity.
Acta Biomaterialia
The lateral resolution of infrared spectroscopy has been inadequate for accurate biochemical char... more The lateral resolution of infrared spectroscopy has been inadequate for accurate biochemical characterization of the cell microenvironment, a region regulating biochemical and biomechanical signals to cells. In this study, we demonstrate the capacity of a high-resolution Fourier transform infrared microspectroscopy (HR-FTIR-MS) to characterize the collagen content of this region. Specifically, we focus on the collagen content in the cartilage cell (chondrocyte) microenvironment of healthy and osteoarthritic (OA) cartilage. Human tibial cartilage samples (N = 28) were harvested from 7 cadaveric donors and graded for OA severity (healthy, early OA, advanced OA). HR-FTIR-MS was used to analyze the collagen content of the chondrocyte microenvironment of five distinct zones across the tissue depth. HR-FTIR-MS successfully showed collagen content distribution across chondrocytes and their environment. In zones 2 and 3 (10 - 50% of the tissue thickness), we observed that collagen content was smaller (P < 0.05) in early OA compared to the healthy tissue in the vicinity of cells (pericellular region). The collagen content loss was extended to the extracellular matrix in advanced OA tissue. No significant differences in the collagen content of the chondrocyte microenvironment were observed between the groups in the most superficial (0-10%) and deep zones (50-100%). HR-FTIR-MS revealed collagen loss in the early OA cartilage pericellular region before detectable changes in the extracellular matrix in advanced OA. HR-FTIR-MS-based compositional assessment enables a better understanding of OA-related changes in tissues. This technique can be used to identify new disease mechanisms enabling better intervention strategies. STATEMENT OF SIGNIFICANCE: : Osteoarthritis (OA) is the most common degenerative joint disease causing pain and disability. While significant progress has been made in OA research, OA pathogenesis is still poorly understood and current OA treatments are mainly palliative. This study demonstrates that high-resolution FTIR microspectroscopy (HR-FTIR-MS) can characterize OA-induced compositional changes in the cell microenvironment (pericellular matrix) during the early disease stages before tissue changes in the extracellular matrix become apparent. This technique may further enable the identification of new OA mechanisms and improve our current understanding of OA pathogenesis, thus, enabling the development of better treatment methods.
Nature Protocols
Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and l... more Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and label-free assessment of biological materials. Compared to mid-infrared spectroscopy, NIR spectroscopy excels in penetration depth, allowing intact biological tissue assessment, albeit at the cost of reduced molecular specificity. Furthermore, it is relatively safe compared to Raman spectroscopy, with no risk of laser-induced photothermal damage. A typical NIR spectroscopy workflow for biological tissue characterization involves sample preparation, spectral acquisition, pre-processing and analysis. The resulting spectrum embeds intrinsic information on the tissue’s biomolecular, structural and functional properties. Here we demonstrate the analytical power of NIR spectroscopy for exploratory and diagnostic applications by providing instructions for acquiring NIR spectra, maps and images in biological tissues. By adapting and extending this protocol from the demonstrated application in connective tissues to other biological tissues, we expect that a typical NIR spectroscopic study can be performed by a non-specialist user to characterize biological tissues in basic research or clinical settings. We also describe how to use this protocol for exploratory study on connective tissues, including differentiating among ligament types, non-destructively monitoring changes in matrix formation during engineered cartilage development, mapping articular cartilage proteoglycan content across bovine patella and spectral imaging across the depth-wise zones of articular cartilage and subchondral bone. Depending on acquisition mode and experiment objectives, a typical exploratory study can be completed within 6 h, including sample preparation and data analysis. This protocol describes how to perform near-infrared spectroscopy and imaging of connective tissues. Detailed guidelines are provided for sample preparation, spectral acquisition and data pre-processing and analysis, with example applications.
Journal of Orthopaedic Research
This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and t... more This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and their effect on joint homeostasis and function of the equine carpus. In nine adult Shetland ponies, the cartilage in the radiocarpal and middle carpal joint of one front limb was grooved (blunt or sharp randomized). The ponies were subjected to an 8-week exercise protocol and euthanized at 39 weeks. Structural and compositional alterations in joint tissues were evaluated in vivo using serial radiographs, synovial biopsies, and synovial fluid samples. Joint function was monitored by quantitative gait analysis. Macroscopic, microscopic, and biomechanical evaluation of the cartilage and assessment of subchondral bone parameters were performed ex vivo. Grooved cartilage showed higher OARSI microscopy scores than the contralateral sham-operated controls (p < 0.0001). Blunt-grooved cartilage scored higher than sharp-grooved cartilage (p = 0.007) and fixed charge density around these grooves was lower (p = 0.006). Equilibrium and instantaneous moduli trended lower in grooved cartilage than their controls (significant for radiocarpal joints). Changes in other tissues included a threefold to sevenfold change in interleukin-6 expression in synovium from grooved joints at week 23 (p = 0.042) and an increased CPII/C2C ratio in synovial fluid extracted from blunt-grooved joints at week 35 (p = 0.010). Gait analysis outcome revealed mild, gradually increasing lameness. In conclusion, blunt and, to a lesser extent, sharp grooves in combination with a period of moderate exercise, lead to mild degeneration in equine carpal cartilage over a 9-month period, but the effect on overall joint health remains limited.
Molecules
The aim of the study was to optimize preprocessing of sparse infrared spectral data. The sparse d... more The aim of the study was to optimize preprocessing of sparse infrared spectral data. The sparse data were obtained by reducing broadband Fourier transform infrared attenuated total reflectance spectra of bovine and human cartilage, as well as of simulated spectral data, comprising several thousand spectral variables into datasets comprising only seven spectral variables. Different preprocessing approaches were compared, including simple baseline correction and normalization procedures, and model-based preprocessing, such as multiplicative signal correction (MSC). The optimal preprocessing was selected based on the quality of classification models established by partial least squares discriminant analysis for discriminating healthy and damaged cartilage samples. The best results for the sparse data were obtained by preprocessing using a baseline offset correction at 1800 cm−1, followed by peak normalization at 850 cm−1 and preprocessing by MSC.
Biomechanical indentation testing of equine articular cartilage. Testing protocol consisted of si... more Biomechanical indentation testing of equine articular cartilage. Testing protocol consisted of single 7.5% strain step.
Near infrared spectroscopic (NIRS) measurements of equine articular cartilage. NIRS measurements ... more Near infrared spectroscopic (NIRS) measurements of equine articular cartilage. NIRS measurements are paired with reference parameters calculated from biomechanical and histological measurements. These reference parameters reflect the biomechanical response, chemical composition and internal structure of the articular cartilage samples.<br><b>Change history: </b>Following publication, it was discovered that a number of samples in nirs_and_references.mat were mislabelled. This has been corrected in version 2.
Indendation testing of articular cartilage consisting of three cumulative 7.5% strain steps and f... more Indendation testing of articular cartilage consisting of three cumulative 7.5% strain steps and four cycles of dynamic sinusoidal loading (at 2% amplitude using frequencies of 0.1, 0.25, 0.625, 0.833, 1.0, and 2.0 Hz).
Dataset contains histological reference measurements of equine articular cartilage, measured with... more Dataset contains histological reference measurements of equine articular cartilage, measured with fourier-transform infrared microspectroscopy.
Biophotonics Congress: Biomedical Optics Congress 2018 (Microscopy/Translational/Brain/OTS)
We present preliminary findings on multivariate modeling between cartilage biomechanical properti... more We present preliminary findings on multivariate modeling between cartilage biomechanical properties and spectroscopic measurements, performed in arthroscopic surgery and laboratory. Also, generic multivariate regression techniques were compared.
Biophotonics Congress: Optics in the Life Sciences Congress 2019 (BODA,BRAIN,NTM,OMA,OMP)
We introduce classification models based on partial least squares discriminant-analysis (PLS-DA) ... more We introduce classification models based on partial least squares discriminant-analysis (PLS-DA) for estimating cartilage integrity (assessed by OARSI grade) based on mid-infrared spectra of cartilage matrix. The best model achieved accuracy of 84%.
Biophotonics Congress: Optics in the Life Sciences Congress 2019 (BODA,BRAIN,NTM,OMA,OMP)
In this study, we demonstrate the potential of mid-infrared (MIR) and near infrared (NIR) spectro... more In this study, we demonstrate the potential of mid-infrared (MIR) and near infrared (NIR) spectroscopies to reveal and differentiate between superficial changes in articular cartilage (AC) after mechanical or enzymatic degradation.
Biophotonics Congress: Biomedical Optics Congress 2018 (Microscopy/Translational/Brain/OTS)
Near-infrared spectroscopy is a promising tool for estimating the composition and biomechanical p... more Near-infrared spectroscopy is a promising tool for estimating the composition and biomechanical properties of meniscus. Here, we developed a predictive model was created for mapping meniscus biomechanical properties based on near-infrared spectroscopy.
World Academy of Science, Engineering and Technology, International Journal of Biotechnology and Bioengineering, 2017
This paper reviews the current status of the application of optical non-destructive methods, part... more This paper reviews the current status of the application of optical non-destructive methods, particularly infrared (IR) and near infrared (NIR), in the evaluation of the physiological integrity of articular cartilage. It is concluded that a significant amount of work is still required in order to achieve specificity and clinical applicability of these methods in the assessment and treatment of dysfunctional articular joints.
IEEE Journal of Biomedical and Health Informatics, 2021
Traditional sleep staging with non-overlapping 30-second epochs overlooks multiple sleep-wake tra... more Traditional sleep staging with non-overlapping 30-second epochs overlooks multiple sleep-wake transitions. We aimed to overcome this by analyzing the sleep architecture in more detail with deep learning methods and hypothesized that the traditional sleep staging underestimates the sleep fragmentation of obstructive sleep apnea (OSA) patients. To test this hypothesis, we applied deep learning-based sleep staging to identify sleep stages with the traditional approach and by using overlapping 30-second epochs with 15-, 5-, 1-, or 0.5-second epoch-to-epoch duration. A dataset of 446 patients referred for polysomnography due to OSA suspicion was used to assess differences in the sleep architecture between OSA severity groups. The amount of wakefulness increased while REM and N3 decreased in severe OSA with shorter epoch-to-epoch duration. In other OSA severity groups, the amount of wake and N1 decreased while N3 increased. With the traditional 30-second epoch-to-epoch duration, only smal...
Articular cartilage is a connective tissue that enables smooth movements between bones in articul... more Articular cartilage is a connective tissue that enables smooth movements between bones in articulating joints. Cartilage consists of extracellular matrix (ECM) and chondrocytes – the cells responsible for synthesis of the ECM. The ECM consists of type II collagen, proteoglycans, water, and some other minor components. Cartilage is prone to degenerative joint conditions, such as osteoarthritis, due to its weak repair capacity resulting from a lack of vascular, neural, and lymphatic networks. Osteoarthritis causes erosion of the cartilage matrix and therefore inhibits its function, resulting in joint pain, loss of mobility, and significant global socioeconomic burden. Currently, surgical treatment of cartilage pathologies is carried out during arthroscopy with variable outcomes. This variability occurs due to the subjective nature of arthroscopy, which relies on manual palpation and visual evaluation of the tissue surface. Diffuse optical spectroscopy in the near-infrared spectral reg...
CARTILAGE
Objective Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining signifi... more Objective Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by ( a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and ( b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state. Design The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identifie...
The scourge of malaria infection continues to strike hardest against pregnant women and children ... more The scourge of malaria infection continues to strike hardest against pregnant women and children in Africa and South East Asia. For global elimination, testing methods that are ultrasensitive to low-level ring-staged parasitemia are urgently required. In this study, we used a novel approach for diagnosis of malaria infection by combining both electronic ultraviolet-visible (UV/vis) spectroscopy and near infrared (NIR) spectroscopy to detect and quantify low-level (1-0.000001%) ring-staged malaria-infected whole blood under physiological conditions uisng Multiclass classification using logistic regression, which showed that the best results were achieved using the extended wavelength range, providing an accuracy of 100% for most parasitemia classes. Likewise, partial least-squares regression (PLS-R) analysis showed a higher quantification sensitivity (R2 = 0.898) for the extended spectral region compared to UV/vis and NIR (R2 = 0.806 and 0.556, respectively). For quantifying different-stage blood parasites, the extended wavelength range was able to detect and quantify all thePlasmodium falciparum accurately compared to testing each spectral component separately. These results demonstrate the potential of a combined UV/vis-NIR spectroscopy to accurately diagnose malaria-infected patients without the need for elaborate sample preparation associated with the existing mid-IR approaches.
Biomedical Optics Express
Optical properties of biological tissues in the NIR spectral range have demonstrated significant ... more Optical properties of biological tissues in the NIR spectral range have demonstrated significant potential for in vivo diagnostic applications and are critical parameters for modelling light interaction in biological tissues. This study aims to investigate the optical properties of articular cartilage as a function of tissue depth and integrity. The results suggest consistent wavelength-dependent variation in optical properties between cartilage depth-wise zones, as well as between healthy and degenerated tissue. Also, statistically significant differences (p<0.05) in both optical properties were observed between the different cartilage depth-wise zones and as a result of tissue degeneration. When taken into account, the outcome of this study could enable accurate modelling of light interaction in cartilage matrix and could provide useful diagnostic information on cartilage integrity.
Acta Biomaterialia
The lateral resolution of infrared spectroscopy has been inadequate for accurate biochemical char... more The lateral resolution of infrared spectroscopy has been inadequate for accurate biochemical characterization of the cell microenvironment, a region regulating biochemical and biomechanical signals to cells. In this study, we demonstrate the capacity of a high-resolution Fourier transform infrared microspectroscopy (HR-FTIR-MS) to characterize the collagen content of this region. Specifically, we focus on the collagen content in the cartilage cell (chondrocyte) microenvironment of healthy and osteoarthritic (OA) cartilage. Human tibial cartilage samples (N = 28) were harvested from 7 cadaveric donors and graded for OA severity (healthy, early OA, advanced OA). HR-FTIR-MS was used to analyze the collagen content of the chondrocyte microenvironment of five distinct zones across the tissue depth. HR-FTIR-MS successfully showed collagen content distribution across chondrocytes and their environment. In zones 2 and 3 (10 - 50% of the tissue thickness), we observed that collagen content was smaller (P < 0.05) in early OA compared to the healthy tissue in the vicinity of cells (pericellular region). The collagen content loss was extended to the extracellular matrix in advanced OA tissue. No significant differences in the collagen content of the chondrocyte microenvironment were observed between the groups in the most superficial (0-10%) and deep zones (50-100%). HR-FTIR-MS revealed collagen loss in the early OA cartilage pericellular region before detectable changes in the extracellular matrix in advanced OA. HR-FTIR-MS-based compositional assessment enables a better understanding of OA-related changes in tissues. This technique can be used to identify new disease mechanisms enabling better intervention strategies. STATEMENT OF SIGNIFICANCE: : Osteoarthritis (OA) is the most common degenerative joint disease causing pain and disability. While significant progress has been made in OA research, OA pathogenesis is still poorly understood and current OA treatments are mainly palliative. This study demonstrates that high-resolution FTIR microspectroscopy (HR-FTIR-MS) can characterize OA-induced compositional changes in the cell microenvironment (pericellular matrix) during the early disease stages before tissue changes in the extracellular matrix become apparent. This technique may further enable the identification of new OA mechanisms and improve our current understanding of OA pathogenesis, thus, enabling the development of better treatment methods.
Nature Protocols
Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and l... more Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and label-free assessment of biological materials. Compared to mid-infrared spectroscopy, NIR spectroscopy excels in penetration depth, allowing intact biological tissue assessment, albeit at the cost of reduced molecular specificity. Furthermore, it is relatively safe compared to Raman spectroscopy, with no risk of laser-induced photothermal damage. A typical NIR spectroscopy workflow for biological tissue characterization involves sample preparation, spectral acquisition, pre-processing and analysis. The resulting spectrum embeds intrinsic information on the tissue’s biomolecular, structural and functional properties. Here we demonstrate the analytical power of NIR spectroscopy for exploratory and diagnostic applications by providing instructions for acquiring NIR spectra, maps and images in biological tissues. By adapting and extending this protocol from the demonstrated application in connective tissues to other biological tissues, we expect that a typical NIR spectroscopic study can be performed by a non-specialist user to characterize biological tissues in basic research or clinical settings. We also describe how to use this protocol for exploratory study on connective tissues, including differentiating among ligament types, non-destructively monitoring changes in matrix formation during engineered cartilage development, mapping articular cartilage proteoglycan content across bovine patella and spectral imaging across the depth-wise zones of articular cartilage and subchondral bone. Depending on acquisition mode and experiment objectives, a typical exploratory study can be completed within 6 h, including sample preparation and data analysis. This protocol describes how to perform near-infrared spectroscopy and imaging of connective tissues. Detailed guidelines are provided for sample preparation, spectral acquisition and data pre-processing and analysis, with example applications.
Journal of Orthopaedic Research
This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and t... more This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and their effect on joint homeostasis and function of the equine carpus. In nine adult Shetland ponies, the cartilage in the radiocarpal and middle carpal joint of one front limb was grooved (blunt or sharp randomized). The ponies were subjected to an 8-week exercise protocol and euthanized at 39 weeks. Structural and compositional alterations in joint tissues were evaluated in vivo using serial radiographs, synovial biopsies, and synovial fluid samples. Joint function was monitored by quantitative gait analysis. Macroscopic, microscopic, and biomechanical evaluation of the cartilage and assessment of subchondral bone parameters were performed ex vivo. Grooved cartilage showed higher OARSI microscopy scores than the contralateral sham-operated controls (p < 0.0001). Blunt-grooved cartilage scored higher than sharp-grooved cartilage (p = 0.007) and fixed charge density around these grooves was lower (p = 0.006). Equilibrium and instantaneous moduli trended lower in grooved cartilage than their controls (significant for radiocarpal joints). Changes in other tissues included a threefold to sevenfold change in interleukin-6 expression in synovium from grooved joints at week 23 (p = 0.042) and an increased CPII/C2C ratio in synovial fluid extracted from blunt-grooved joints at week 35 (p = 0.010). Gait analysis outcome revealed mild, gradually increasing lameness. In conclusion, blunt and, to a lesser extent, sharp grooves in combination with a period of moderate exercise, lead to mild degeneration in equine carpal cartilage over a 9-month period, but the effect on overall joint health remains limited.