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FTIR Attenuated Total Reflection (ATR) spectroscopy as a tool to investigate extracellular matrix
Abstracts / Bone 44 (2009) S253–S338
Background: The aim of this study was to develop a clinically relevant surgically created defect model in ulna's pigeon and to characterize the healing process at different time points using histological methods. Methods: Seventeen 1-year-old pigeons were used. Under general anaesthesia, the surface of ulna and radius were exposed by a ventral surgical approach. A segmental fracture was created with a bone saw, and a 5–8 mm segment of mid-ulna diaphysis was removed. To follow the healing process, animals were humanely euthanized at 1, 2, 4 and 6 weeks and the defects and surrounding tissues prepared for histological and histomorphometrical analyses. Results: The healing process could be detected in the histological investigations and showed comparable scoring parameters in all animals (Fig. 1). At 2 and 4 weeks, it was observed appositional bone formation on the defect walls that was supplemented by appositional and membranous bone formation between and within particles, particularly those nearest the defect walls. At 6 weeks, a remodeling process was detectable with a decrease in cartilage and an increase in mineralized tissue. Conclusion: Using this model, the basic cellular events of fracture healing can be investigated and the effects of local systemic factors and conditions assessed.
S313
P159 FTIR Attenuated Total Reflection (ATR) spectroscopy as a tool to investigate extracellular matrix N. Hassler⁎, R. Thaler, F. Varga, K. Klaushofer, E. Paschalis Ludwig Boltzmann Institute of Osteology, Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Med. Dept. Hanusch Hospital, Vienna, Austria FTIR attenuated total reflection (ATR) spectroscopy is increasingly used for investigations of processes at or near a surface. Very sensitive techniques are needed, particularly if layers of extracellular matrices are studied with respect to the relative amount of secondary structural elements of proteins present in the matrix. A main part of extracellular matrix produced by osteoblasts consists of collagen, and its maturation, i.e. the cross-linking, influences bone development and strength, as well as mineralization commencement [1]. In this work, we present the principal features and advantages of the ATR technique compared to transmission spectroscopy, such as: (i) Since ATR spectroscopy is a surface sensitive technique and cells secrete the matrix to the surface where they attach, the matrix instead of the cells is predominantly detected by the IR beam. (ii) Weak signals of the matrix are enhanced by applying the multiple reflections technique. In a first step we measured FTIR ATR spectra of dried extracellular matrix produced in culture dishes by the osteoblast-like cell line MC3T3-E1 to compare them with spectra of matrix deposited by cells on the measuring element. The single beam sample reference (SBSR) method [2] was used to eliminate water vapour interference and baseline drifts. Preliminary results showed that the spectral quality and obtained information is similar to that achieved by the more often employed transmission spectroscopy. As a result, the use of a flow-through chamber with independently accessible reference and sample compartments allows the real-time evaluation of the kinetics of matrix deposition in aqueous environment. Furthermore, it enables the study of orientational effects in the matrix because measurements are performed with polarized IR light. Work was supported by FWF project P20646-B11. [1] M. Yamauchi, Collagen: The major matrix molecule in mineralized tissues, in Calcium and Phosphorus in Health and Disease, J.J.B. Anderson, S.C. Garner (eds.), CRC Press: New York, 1996, 127–141. [2] U. P. Fringeli, In situ infrared attenuated total reflection membrane spectroscopy, in Internal Reflection Spectroscopy, Theory and Applications, F. M. Mirabella (Ed.), Marcel Dekker: New York, 1992, 255–324. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.585
Fig. 1. Histological overview of bone formation in surgically created defects: A, 1 week: B, 2 weeks; C, 4 weeks; D, 6 weeks.
Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.584
P160 Calcium-sensing receptor-dependent and independent mechanisms are involved in the increased osteoblast replication and survival by strontium ranelate O. Fromiguéa,*, E. Haÿa, A. Barbaraa, C. Petrelb, E. Traiffortb, M. Ruatb, P. Mariea a Laboratory of Osteoblast Biology and Pathology, INSERM U606, University Paris 7, Paris cedex 10 b Cellular and Molecular Neurobiology Laboratory, UPR 9040 CNRS, IFR 2118, Gif-sur-Yvette, France Age-related osteopenia is characterized by a negative balance between bone resorption and formation. The anti-osteoporotic treatment strontium ranelate reduces bone resorption and promotes bone formation. Here, we investigated the implication of the calcium sensing receptor (CaSR) in the response to strontium ranelate using
Background: The aim of this study was to develop a clinically relevant surgically created defect model in ulna's pigeon and to characterize the healing process at different time points using histological methods. Methods: Seventeen 1-year-old pigeons were used. Under general anaesthesia, the surface of ulna and radius were exposed by a ventral surgical approach. A segmental fracture was created with a bone saw, and a 5–8 mm segment of mid-ulna diaphysis was removed. To follow the healing process, animals were humanely euthanized at 1, 2, 4 and 6 weeks and the defects and surrounding tissues prepared for histological and histomorphometrical analyses. Results: The healing process could be detected in the histological investigations and showed comparable scoring parameters in all animals (Fig. 1). At 2 and 4 weeks, it was observed appositional bone formation on the defect walls that was supplemented by appositional and membranous bone formation between and within particles, particularly those nearest the defect walls. At 6 weeks, a remodeling process was detectable with a decrease in cartilage and an increase in mineralized tissue. Conclusion: Using this model, the basic cellular events of fracture healing can be investigated and the effects of local systemic factors and conditions assessed.
S313
P159 FTIR Attenuated Total Reflection (ATR) spectroscopy as a tool to investigate extracellular matrix N. Hassler⁎, R. Thaler, F. Varga, K. Klaushofer, E. Paschalis Ludwig Boltzmann Institute of Osteology, Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Med. Dept. Hanusch Hospital, Vienna, Austria FTIR attenuated total reflection (ATR) spectroscopy is increasingly used for investigations of processes at or near a surface. Very sensitive techniques are needed, particularly if layers of extracellular matrices are studied with respect to the relative amount of secondary structural elements of proteins present in the matrix. A main part of extracellular matrix produced by osteoblasts consists of collagen, and its maturation, i.e. the cross-linking, influences bone development and strength, as well as mineralization commencement [1]. In this work, we present the principal features and advantages of the ATR technique compared to transmission spectroscopy, such as: (i) Since ATR spectroscopy is a surface sensitive technique and cells secrete the matrix to the surface where they attach, the matrix instead of the cells is predominantly detected by the IR beam. (ii) Weak signals of the matrix are enhanced by applying the multiple reflections technique. In a first step we measured FTIR ATR spectra of dried extracellular matrix produced in culture dishes by the osteoblast-like cell line MC3T3-E1 to compare them with spectra of matrix deposited by cells on the measuring element. The single beam sample reference (SBSR) method [2] was used to eliminate water vapour interference and baseline drifts. Preliminary results showed that the spectral quality and obtained information is similar to that achieved by the more often employed transmission spectroscopy. As a result, the use of a flow-through chamber with independently accessible reference and sample compartments allows the real-time evaluation of the kinetics of matrix deposition in aqueous environment. Furthermore, it enables the study of orientational effects in the matrix because measurements are performed with polarized IR light. Work was supported by FWF project P20646-B11. [1] M. Yamauchi, Collagen: The major matrix molecule in mineralized tissues, in Calcium and Phosphorus in Health and Disease, J.J.B. Anderson, S.C. Garner (eds.), CRC Press: New York, 1996, 127–141. [2] U. P. Fringeli, In situ infrared attenuated total reflection membrane spectroscopy, in Internal Reflection Spectroscopy, Theory and Applications, F. M. Mirabella (Ed.), Marcel Dekker: New York, 1992, 255–324. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.585
Fig. 1. Histological overview of bone formation in surgically created defects: A, 1 week: B, 2 weeks; C, 4 weeks; D, 6 weeks.
Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.584
P160 Calcium-sensing receptor-dependent and independent mechanisms are involved in the increased osteoblast replication and survival by strontium ranelate O. Fromiguéa,*, E. Haÿa, A. Barbaraa, C. Petrelb, E. Traiffortb, M. Ruatb, P. Mariea a Laboratory of Osteoblast Biology and Pathology, INSERM U606, University Paris 7, Paris cedex 10 b Cellular and Molecular Neurobiology Laboratory, UPR 9040 CNRS, IFR 2118, Gif-sur-Yvette, France Age-related osteopenia is characterized by a negative balance between bone resorption and formation. The anti-osteoporotic treatment strontium ranelate reduces bone resorption and promotes bone formation. Here, we investigated the implication of the calcium sensing receptor (CaSR) in the response to strontium ranelate using