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The in vitro formation of a calcified extracellular matrix on biomaterials by rat bone marrow cells
Abstracts from Imaging of Cartilage and Bone Meeting,
99
June 1991
The in vitm formation of a calcified extracellular matrix on hiomaterials by rat bone marrow cells. J.D. de Bruijn, C.P.A.T. Klein, K. de Groot, J.E. Davies* and C.A. van Blitterswijk Biomaterials Research Group, Lab Otobiol. & Biocomp., Bld 25, University Hospital Leiden, Rijnsburger 10, 2333 AA Leiden, The Netherlands *Centre for Biomaterials, Faculty of Dentistry, University of Toronto, Canada Since the development of an in vitro bone forming system by Maniatopoulos (1988), interface reactions with biomaterials can be studied without the complexities of the in vivo environment. With this system the early bone-bonding phenomena were studied on three types of plasma sprayed hydroxyapatite (HA). They were distinguished in amorphous, amorphouscrystalline, and crystalline HA by X-ray diffraction. After irradiation, primary rat bone marrow cells were cultured on the materials. The culture medium consisted of a-MEM supplemented with 15% FBS, antibiotics, and freshly added lo-* M dexamethasone, 10 mM B-glycerophosphate, and 50 @ml ascorbic acid. At 18 days of culture the cells were processed for scanning and transmission electron microscopy. The deposition of a globular matrix was observed on the materials in which collagen fibres were integrated. At the interface between crystalline HA and the calcified extracellular matrix an electron-dense layer was seen, as known from in vivo studies. This electrondense layer was also present on amorphous and amorphouscrystalline HA, but regularly also an amorphous zone was seen interposed between this layer and the HA. This zone was devoid of collagen and was composed of both inorganic and organic material. From these results it is suggested that, depending on their crystallinity, calcium phosphates will display different interfacial reactions with bone tissue.
In-situ hybridisation of cytoplasmic mRNA in tissue sections of cartilage and bone. A.J. Freemont Department of Pathological Sciences, University of Manchester, Manchester Ml3 9PT In-situ hybridisation is a technique that can be used to detect specific molecules of messenger RNA (mRNA) within the cytoplasm of cells. mRNA consists of a chain of bases. For every base there is a complimentary base to which it will bind. A string of such complimentary bases in the correct sequence will bind strongly to the corresponding part of mRNA molecules. If some of the bases on the string do not match those in the mRNA molecule the overall binding will be weaker. In-situ hybridisation exploits this property of RNA; that is, that a probe, consisting of complimentary bases, will bind to a section of an mRNA molecule, and the more closely the sequence of bases in the probe matches that of the n-RNA being studied the stronger the bond between the probe and the mRNA. The hybridisation conditions can be varied to ensure that only probes that very closely match sequences of cytoplasmic mRNA will remain bound in the tissue. Probes can be synthesised by organisms (usually bacteria) genetically engineered to contain a segment of DNA that codes for the probe, or they may be synthesised by machines. Probes synthesised by organisms tend to be much longer than those synthesised by machine, the former being typically 1,000 bases
(lkb) long, whereas the latter are typically between 20 and 30 bases long. Whatever the length or nature of the probe, the principle underlying in-situ hybridisation is that tissue sectioning exposes cytoplasmic mRNA within tissue cells making the mRNA available for binding with probes applied to the surface of the section. Just as in immunohistochemistry, the probe can be “tagged” with a label that can be visualised, thus disclosing the position within the tissue of the mRNA to which the probe has bound. In-situ hybridisation can identify the cells that contain specific mRNA, but this is not necessarily evidence of synthesis of the final gene product. In-situ hybridisation should, ideally, complement, or be complemented by, immunohistochemical studies of the final gene product. In-situ hybridisation is dependent upon the preservation of mRNA within the tissue. RNA is stable, being susceptible only to specific degradative enzymes. Following cell death, RNases are released within the cell, which disassemble the mRNA molecules within l-2 hours, rendering them undetectable. It is, therefore, essential to neutralise RNases as soon as possible after tissue is removed from the body; this is simply done by fixation. If quantitation of mRNA is to be attempted, fixation must occur as quickly as possible so that mRNA loss is kept to a minimum. Finally, comparative quantitation of mRNA is a sensitive marker of lethal and sublethal cell damage. In the Osteoarticular Pathology laboratories of Manchester University we have started to use in-situ hybridisation to examine the pathology of cartilage and bone and some examples of this will be presented.
Localiition of IL-6 and osteopontin mRNA by in situ hybridisation in bone sections. A.J. Littlewood, K.H. Merry, R.A. Dodds and M. Gowen Bath Institute for Rheumatic Diseases, Trim Bridge, Bath BAI IHD and University of Bath In situ hybridisation with 3sS-labelled riboprobes was used to locate specific mRNA species in bone sections representing various stages of the remodelling cycle. Sections were cut from osteoarthritic osteophytic bone invaded by granulation tissue. This tissue was rich in osteoclasts, giant cells, and associated mononuclear cells, as well as cells at various stages of the osteoblast lineage. We were able to demonstrate specific hybridisation of IL-6 and osteopontin antisense mRNA. Decalcification of sections followed by proteinase K treatment ensured specific binding with only background traces in sections containing sense probes. The sections retained good cell morphology throughout the study. IL-6 mRNA was present in all cells, but particularly high expression was located in osteoblasts and osteoclasts. The osteopontin antisense probe bound to osteoclasts, giant cells, and selective mononuclear cells, although it did not react with osteoblasts in this tissue. This method has the potential to provide valuable information about cells and their function within the remodelling cycle. The unexpected expression of both a cytokine and a bone matrix protein by osteoclasts raises interesting questions about the functions of this cell in bone remodelling.
X-ray diffraction studies of collagen in cartilage. D.W.L. Hukins Department of Medical Biophysics, University of Manchester, Stopford Building, Manchester Ml3 9PT
99
June 1991
The in vitm formation of a calcified extracellular matrix on hiomaterials by rat bone marrow cells. J.D. de Bruijn, C.P.A.T. Klein, K. de Groot, J.E. Davies* and C.A. van Blitterswijk Biomaterials Research Group, Lab Otobiol. & Biocomp., Bld 25, University Hospital Leiden, Rijnsburger 10, 2333 AA Leiden, The Netherlands *Centre for Biomaterials, Faculty of Dentistry, University of Toronto, Canada Since the development of an in vitro bone forming system by Maniatopoulos (1988), interface reactions with biomaterials can be studied without the complexities of the in vivo environment. With this system the early bone-bonding phenomena were studied on three types of plasma sprayed hydroxyapatite (HA). They were distinguished in amorphous, amorphouscrystalline, and crystalline HA by X-ray diffraction. After irradiation, primary rat bone marrow cells were cultured on the materials. The culture medium consisted of a-MEM supplemented with 15% FBS, antibiotics, and freshly added lo-* M dexamethasone, 10 mM B-glycerophosphate, and 50 @ml ascorbic acid. At 18 days of culture the cells were processed for scanning and transmission electron microscopy. The deposition of a globular matrix was observed on the materials in which collagen fibres were integrated. At the interface between crystalline HA and the calcified extracellular matrix an electron-dense layer was seen, as known from in vivo studies. This electrondense layer was also present on amorphous and amorphouscrystalline HA, but regularly also an amorphous zone was seen interposed between this layer and the HA. This zone was devoid of collagen and was composed of both inorganic and organic material. From these results it is suggested that, depending on their crystallinity, calcium phosphates will display different interfacial reactions with bone tissue.
In-situ hybridisation of cytoplasmic mRNA in tissue sections of cartilage and bone. A.J. Freemont Department of Pathological Sciences, University of Manchester, Manchester Ml3 9PT In-situ hybridisation is a technique that can be used to detect specific molecules of messenger RNA (mRNA) within the cytoplasm of cells. mRNA consists of a chain of bases. For every base there is a complimentary base to which it will bind. A string of such complimentary bases in the correct sequence will bind strongly to the corresponding part of mRNA molecules. If some of the bases on the string do not match those in the mRNA molecule the overall binding will be weaker. In-situ hybridisation exploits this property of RNA; that is, that a probe, consisting of complimentary bases, will bind to a section of an mRNA molecule, and the more closely the sequence of bases in the probe matches that of the n-RNA being studied the stronger the bond between the probe and the mRNA. The hybridisation conditions can be varied to ensure that only probes that very closely match sequences of cytoplasmic mRNA will remain bound in the tissue. Probes can be synthesised by organisms (usually bacteria) genetically engineered to contain a segment of DNA that codes for the probe, or they may be synthesised by machines. Probes synthesised by organisms tend to be much longer than those synthesised by machine, the former being typically 1,000 bases
(lkb) long, whereas the latter are typically between 20 and 30 bases long. Whatever the length or nature of the probe, the principle underlying in-situ hybridisation is that tissue sectioning exposes cytoplasmic mRNA within tissue cells making the mRNA available for binding with probes applied to the surface of the section. Just as in immunohistochemistry, the probe can be “tagged” with a label that can be visualised, thus disclosing the position within the tissue of the mRNA to which the probe has bound. In-situ hybridisation can identify the cells that contain specific mRNA, but this is not necessarily evidence of synthesis of the final gene product. In-situ hybridisation should, ideally, complement, or be complemented by, immunohistochemical studies of the final gene product. In-situ hybridisation is dependent upon the preservation of mRNA within the tissue. RNA is stable, being susceptible only to specific degradative enzymes. Following cell death, RNases are released within the cell, which disassemble the mRNA molecules within l-2 hours, rendering them undetectable. It is, therefore, essential to neutralise RNases as soon as possible after tissue is removed from the body; this is simply done by fixation. If quantitation of mRNA is to be attempted, fixation must occur as quickly as possible so that mRNA loss is kept to a minimum. Finally, comparative quantitation of mRNA is a sensitive marker of lethal and sublethal cell damage. In the Osteoarticular Pathology laboratories of Manchester University we have started to use in-situ hybridisation to examine the pathology of cartilage and bone and some examples of this will be presented.
Localiition of IL-6 and osteopontin mRNA by in situ hybridisation in bone sections. A.J. Littlewood, K.H. Merry, R.A. Dodds and M. Gowen Bath Institute for Rheumatic Diseases, Trim Bridge, Bath BAI IHD and University of Bath In situ hybridisation with 3sS-labelled riboprobes was used to locate specific mRNA species in bone sections representing various stages of the remodelling cycle. Sections were cut from osteoarthritic osteophytic bone invaded by granulation tissue. This tissue was rich in osteoclasts, giant cells, and associated mononuclear cells, as well as cells at various stages of the osteoblast lineage. We were able to demonstrate specific hybridisation of IL-6 and osteopontin antisense mRNA. Decalcification of sections followed by proteinase K treatment ensured specific binding with only background traces in sections containing sense probes. The sections retained good cell morphology throughout the study. IL-6 mRNA was present in all cells, but particularly high expression was located in osteoblasts and osteoclasts. The osteopontin antisense probe bound to osteoclasts, giant cells, and selective mononuclear cells, although it did not react with osteoblasts in this tissue. This method has the potential to provide valuable information about cells and their function within the remodelling cycle. The unexpected expression of both a cytokine and a bone matrix protein by osteoclasts raises interesting questions about the functions of this cell in bone remodelling.
X-ray diffraction studies of collagen in cartilage. D.W.L. Hukins Department of Medical Biophysics, University of Manchester, Stopford Building, Manchester Ml3 9PT