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Application of a high-resolution electron microscopy technique to characterisation of a bone-implant interface
91
Abstracts from Imaging of Cartilage and Bone Meeting, June 1991
Gardner, D.L.; O’Connor, P.; Oaks. K. J. Anat. 132~267-282:
1981.
Middleton. J.F.S.; Hunt, S.; Oates. K. Cell Tissue Res. 29vW-475: 1988. Morgan, A.J. Scanning Electron Microscopy. SEM Inc.; 197% 635-648.
aggregates in articular cartilage and their association with cartilage damage.
Crystal
A. Hayes, S.E. Clift,* I.G. Turner, P.A. Dieppet and B. Harris School of Materials Science, *School of Mechanical Engineering, University of Bath, Bath BA2 7AY fDept. of Rheumatology, Royal Infirmary, Bristol The distribution, morphology, and internal organisation of large crystal aggregates within articular cartilage have important consequences for its structural integrity. However, before a quantitative assessment of the stress-concentrating effects of crystal deposits can be performed, it is necessary to have available data on their size, shape, and distribution. Such observations are reported here. Specimens of articular cartilage were taken from human post-mortem knee joints. Small samples taken from crystal-rich areas were freeze-fractured and freeze-dried prior to examination in a JEOL JSM 35-C scanning electron microscope. Energy dispersive analysis and X-ray diffraction were used to analyse the composition and structure of the crystals. Calcium pyrophosphate crystals were identified in both dense and loosely packed arrangements. Densely packed aggregates, up to 700 pm in diameter, were observed in isolation deep within the cartilage layer, but not associated with any overt damage. It thus appears that such aggregates may not induce mechanical damage of the articular cartilage, as previously expected. However, smaller aggregates, having a circular crosssection of average diameter 50-100 pm, were often observed in clusters separated by a layer of cartilage at least 100 pm thick, and were always associated with a fibrillated articular surface. Less regularly shaped, densely packed aggregates were also observed following the line of cracks emanating from such a fibrillated surface, whereas more regularly shaped aggregates occurred at the base of such cracks. In contrast, loosely packed aggregates comprising mineral interspersed with organic material were also observed in close association with surface fibrillation. These aggregates were generally found to be less than 50 pm in diameter. These results provide important evidence concerning the location and distribution of the crystal aggregates within the cartilage layer and their association with overt cartilage damage. Such data can now be used in conjunction with numerical stress analysis techniques, such as finite element analysis, to clarify the hypothesis that the onset of degeneration of articular cartilage is mediated in part by mechanical processes.
lage collagen, type IX, has revealed the multilayered nature of capsules of the basket-like structures that enclose and include the chondrocytes from rat chondrosarcoma tissue. These units are equivalent to similar structures, “chondrons,” which can be isolated as intact entities from adult cartilage. In sections of mature rat cartilage, type IX collagen is localised in corresponding pericellular regions. Although in young rat cartilage type IX appears, by conventional immunofluorescence microscopy, to be evenly distributed throughout the matrix, the removal of fluorescent flare and the use of sequential optical sectioning through the specimen by confocal microscopy shows that much of this fluorescence is again localised preferentially around the chondro-cytes, which are much more numerous in the young than in the mature cartilage. We have also used these methods to demonstrate that cathepsin B is produced by rat chondrosarcoma chondrocytes which are not dedifferentiated and that two patterns of lysosoma1 localisation occur, punctate and network forms. Further studies into cartilage structure and degradation are in progress in which the diverse capabilities of scanning confocal microscopy are proving to be of great use.
Surface analysis of femoral hip prostheses. I.G. Turner School of Materials Science, University of Bath, Bath BA2 7AY Loosening of prosthetic components remains a long-term problem associated with total joint replacement. It is recognised that problems can arise between the metal stem and the surrounding tissues. An understanding of the surface properties of such devices is therefore important. Prosthetic femoral stems are commercially available with either a highly polished or a satin finished surface. Production of a satin finish involves blasting the metal stem with one of a number of possible types of abrasive particle. Recently concern has been expressed that some of the blasting medium may be retained on the surface of prosthetic stems produced in this way. In this study, seven new hip prostheses were obtained from different manufacturers. These comprised two stainless steel, two cobalt chrome, and three titanium femoral components. Transverse sections were taken from each stem and prepared for examination in the electron microscope. The chemical compositions of the central area of these sections and the surface of each stem were analysed and compared using energy dispersive analysis (EDA). The results showed that particles containing aluminium were found on the surface of three stems; particles containing silicon were found on two of the stems and evidence of particles containing aluminium and silicon was found on two of the stems. These findings indicate that some of the particle blasting medium is being retained on the surface of the prosthetic stems. The clinical consequences of this may need to be considered.
The application of scanning confocal microscopy in cartilage research. S.F. Wotton, R.E. Jeacocke, R.A. Maciewicz, R.J. Wardale and V.C. Duance Muscle and Collagen Research Group, Department of Veterinary Medicine, Langford, Bristol BSI 8 7DY Laser scanning confocal microscopy achieves improved resolution over conventional light microscopy and provides an optical sectioning facility which is especially useful for immunofluorescence localisation studies. Immunofluorescence labelling with antibodies to the carti-
Application of a high-resolution electron microscopy technique to characterisation of a bone-implant interface. Z.B. Luklinska and W. Bonfield Department of Materials, Queen Mary and Westfield College, University of London High-resolution electron microscopy technique has been well established in material science for resolving lattice planes and studying defects or other imperfections on an ultrastructure
98
Abstracts from Imaging of Cartilage and Bone Meeting,
scale. The technique is also able to characterise the continuity of the interface between the two dissimilar materials and hence predict their potential long-term stability as judged by the fine lattice plane details. The importance of the ultrastructure at the interface between a foreign body and its adjacent tissue has become increasingly recognised as a rate controlling factor of a new tissue growth in the area of biomaterials (Tracy & Doremus 1988; Lausmaa et al. 1988; Bjursten et al. in press). The paper evaluates the technique in application to implantbone interface and reports on the results from a bone-analogue composite based on high density polyethylene (PE), reinforced with 40% by volume of synthetic hydroxyapatite ceramic (HA) after 1, 3, and 6 months of implantation periods into condyles of mature rabbits. The removed implants surrounded by tissue were first fixed, dehydrated, and embedded before final preparation for initial scanning electron microscope examination, followed by investigation of the thin sections of a high-resolution elecron microscope. Fully undecalcified thin sections were examined. Some of them were stained using uranyl acetate and osmium tetroxide. Selected area diffraction studies were also carried out in the transmission mode. There are strict requirements for the specimens as well as for the instruments when high-resolution imaging is performed in order to secure the necessary stability of a specimen, its electron beam transparency, and imaging contrast in an electron microscope operating at high-resolution level. The paper demonstrates the potential ability of the technique in resolving 0.817 nm lattice fringes of (100) plane across the interface and by doing so helps in understanding crystallographic and morphological processes taking place at the interface between the implanted material and the new growth tissue on an ultrastructure scale. References Thomsen,P.
Personal communication. Lausmaa, Y.; Ratner, B.D., ed. Surface characterization sterdam: Elsevier; 1988. Tracy, B.M.; Doremus, R.H. J.
of biomaterials.
Glass-ionomer cement implantibone interface and rat tissue culture models. P.V. Hatton, I.M. Brook and L.M. Jonck* University of Shefield, UK *Centre for Bone Biology, University of Pretoria,
Am-
in primate
South Affica
The ultrastructure of the undisturbed bone/Glass-ionomer cement (GICo, V-OS Ionos Medizinische Producte GmBH & Co., KG Germany) interface is reported in a primate baboon in vivo culture model and in an in vitro rat calvarial model. TEM-linked X-ray microanalysis (energy dispersive) EDAX was used to aid characterisation of the bone/implant interface. For in vivo culture set GIC and autogenous cancellous bone were placed into Plexiglas diffusion chambers and implanted for 42 days into prepared holes of the femora of baboons. The in vitro model consisted of 14-day primary bone organ cultures derived from neonate rat calvaria set GIC being placed directly upon the calvarial bone or between the bone and periosteum. Specimens were prepared using a modified cryofixation and freeze-substitution procedure prior to embedding in resin. Active tissue proliferation and differentiation occurred in both types of culture. Osteoblast-like cells colonised and elaborated a collagen-containing extracellular matrix directly onto the surface of the GIC.
June 1991
EDAX confirmed the glass and matrix components of the GIC and should prove a useful tool to study the movement of ions and stability of the bone-implant bond. This study was facilitated by support from the Royal Society, England.
Better growth and differentiation of isolated rat osteoblasts cultured upon Bioglass compared with several other glass substrates. C.C. Groot, W.C.A. Vrouwenvelder* and K. de Groat* Laborator? for Cell Biology and Histology *Department of Biomaterials, Biomaterials Rijnsbergerweg 10, bld 55, 2333 AA Leiden,
Research Group, The Netherlands
Bioglass has been described in the literature as one of the possible candidates for bone replacement. It has unique properties responsible for the excellent bonding to bone when implanted in vivo. However, most of the knowledge concerning Bioglass is derived from in vivo experiments, and a lot of questions of a cell biological nature remain to be resolved. We investigated the growth and differentiation of isolated osteoblasts in relation to different substrates, hoping that our findings would shed some light upon the properties of Bioglass. We isolated osteoblasts from 19-day-old fetal calvaria by sequential collagenase digestion according to a standard procedure. 50 p,l of the obtained cell suspension were applied upon homogeneous Bioglass slides (code 4585: 45% SiO,, 24.5% Na,O, 24.5% CaO, 6% P,O,,) (Hench 1971), 1, 8 mm diameter, or slides of nonreactive glasses (NRG) (soda/silica/lime glass, quartz glass, coverslips) at a density of 5 X IO5 cells/ml for morphological studies (i.e., scanning electron microscopy, immunocytochemistry for collagen I and II, osteocalcin, and a specific osteoblast-associated antigen (OAA)). For the determination of biochemical parameters (DNA content, alkaline phosphatase activity) the cells were seeded at a density of 1 X lo6 cells/ml. After 2 h 800 ~1 of culture medium were added (aMEM + 5% FCS). The cells were kept in a humified incubator (95% H,O in air, 5% CO,) for up to 12 days. The medium was changed every 72 h in the first six days, then every 48 h. The osteoblasts cultured upon Bioglass generally showed better differentiation characteristics when compared with the NRG cultures. Upon Bioglass the cells showed more dorsal ruffles and a more “stand-off” morphology, which resulted in a denser cell layer. The alkaline phosphatase activity and the immunocytochemical markers (osteocalcin, collagen I, and OAA) were more evenly distributed over the culture, whereas in the NRG cultures these markers only could be detected in clusters of cells, probably because a favourable microenvironment was created inside the clusters. The growth rate, expressed as the amount of DNA per culture, was also better for the Bioglass cultures. From our observations we concluded that isolated rat osteoblasts proliferate and differentiate better when cultured upon Bioglass compared with nonreactive glasses. The development of a thin layer of calcium phosphate, which has the characteristics of hydroxyapatite, upon the surface of the Bioglass slides might be one of the causes of these differences.
Reference Hench. L.L. J. Biom.
Mat.
Res. Symp.
2:117-141;
1971.
Abstracts from Imaging of Cartilage and Bone Meeting, June 1991
Gardner, D.L.; O’Connor, P.; Oaks. K. J. Anat. 132~267-282:
1981.
Middleton. J.F.S.; Hunt, S.; Oates. K. Cell Tissue Res. 29vW-475: 1988. Morgan, A.J. Scanning Electron Microscopy. SEM Inc.; 197% 635-648.
aggregates in articular cartilage and their association with cartilage damage.
Crystal
A. Hayes, S.E. Clift,* I.G. Turner, P.A. Dieppet and B. Harris School of Materials Science, *School of Mechanical Engineering, University of Bath, Bath BA2 7AY fDept. of Rheumatology, Royal Infirmary, Bristol The distribution, morphology, and internal organisation of large crystal aggregates within articular cartilage have important consequences for its structural integrity. However, before a quantitative assessment of the stress-concentrating effects of crystal deposits can be performed, it is necessary to have available data on their size, shape, and distribution. Such observations are reported here. Specimens of articular cartilage were taken from human post-mortem knee joints. Small samples taken from crystal-rich areas were freeze-fractured and freeze-dried prior to examination in a JEOL JSM 35-C scanning electron microscope. Energy dispersive analysis and X-ray diffraction were used to analyse the composition and structure of the crystals. Calcium pyrophosphate crystals were identified in both dense and loosely packed arrangements. Densely packed aggregates, up to 700 pm in diameter, were observed in isolation deep within the cartilage layer, but not associated with any overt damage. It thus appears that such aggregates may not induce mechanical damage of the articular cartilage, as previously expected. However, smaller aggregates, having a circular crosssection of average diameter 50-100 pm, were often observed in clusters separated by a layer of cartilage at least 100 pm thick, and were always associated with a fibrillated articular surface. Less regularly shaped, densely packed aggregates were also observed following the line of cracks emanating from such a fibrillated surface, whereas more regularly shaped aggregates occurred at the base of such cracks. In contrast, loosely packed aggregates comprising mineral interspersed with organic material were also observed in close association with surface fibrillation. These aggregates were generally found to be less than 50 pm in diameter. These results provide important evidence concerning the location and distribution of the crystal aggregates within the cartilage layer and their association with overt cartilage damage. Such data can now be used in conjunction with numerical stress analysis techniques, such as finite element analysis, to clarify the hypothesis that the onset of degeneration of articular cartilage is mediated in part by mechanical processes.
lage collagen, type IX, has revealed the multilayered nature of capsules of the basket-like structures that enclose and include the chondrocytes from rat chondrosarcoma tissue. These units are equivalent to similar structures, “chondrons,” which can be isolated as intact entities from adult cartilage. In sections of mature rat cartilage, type IX collagen is localised in corresponding pericellular regions. Although in young rat cartilage type IX appears, by conventional immunofluorescence microscopy, to be evenly distributed throughout the matrix, the removal of fluorescent flare and the use of sequential optical sectioning through the specimen by confocal microscopy shows that much of this fluorescence is again localised preferentially around the chondro-cytes, which are much more numerous in the young than in the mature cartilage. We have also used these methods to demonstrate that cathepsin B is produced by rat chondrosarcoma chondrocytes which are not dedifferentiated and that two patterns of lysosoma1 localisation occur, punctate and network forms. Further studies into cartilage structure and degradation are in progress in which the diverse capabilities of scanning confocal microscopy are proving to be of great use.
Surface analysis of femoral hip prostheses. I.G. Turner School of Materials Science, University of Bath, Bath BA2 7AY Loosening of prosthetic components remains a long-term problem associated with total joint replacement. It is recognised that problems can arise between the metal stem and the surrounding tissues. An understanding of the surface properties of such devices is therefore important. Prosthetic femoral stems are commercially available with either a highly polished or a satin finished surface. Production of a satin finish involves blasting the metal stem with one of a number of possible types of abrasive particle. Recently concern has been expressed that some of the blasting medium may be retained on the surface of prosthetic stems produced in this way. In this study, seven new hip prostheses were obtained from different manufacturers. These comprised two stainless steel, two cobalt chrome, and three titanium femoral components. Transverse sections were taken from each stem and prepared for examination in the electron microscope. The chemical compositions of the central area of these sections and the surface of each stem were analysed and compared using energy dispersive analysis (EDA). The results showed that particles containing aluminium were found on the surface of three stems; particles containing silicon were found on two of the stems and evidence of particles containing aluminium and silicon was found on two of the stems. These findings indicate that some of the particle blasting medium is being retained on the surface of the prosthetic stems. The clinical consequences of this may need to be considered.
The application of scanning confocal microscopy in cartilage research. S.F. Wotton, R.E. Jeacocke, R.A. Maciewicz, R.J. Wardale and V.C. Duance Muscle and Collagen Research Group, Department of Veterinary Medicine, Langford, Bristol BSI 8 7DY Laser scanning confocal microscopy achieves improved resolution over conventional light microscopy and provides an optical sectioning facility which is especially useful for immunofluorescence localisation studies. Immunofluorescence labelling with antibodies to the carti-
Application of a high-resolution electron microscopy technique to characterisation of a bone-implant interface. Z.B. Luklinska and W. Bonfield Department of Materials, Queen Mary and Westfield College, University of London High-resolution electron microscopy technique has been well established in material science for resolving lattice planes and studying defects or other imperfections on an ultrastructure
98
Abstracts from Imaging of Cartilage and Bone Meeting,
scale. The technique is also able to characterise the continuity of the interface between the two dissimilar materials and hence predict their potential long-term stability as judged by the fine lattice plane details. The importance of the ultrastructure at the interface between a foreign body and its adjacent tissue has become increasingly recognised as a rate controlling factor of a new tissue growth in the area of biomaterials (Tracy & Doremus 1988; Lausmaa et al. 1988; Bjursten et al. in press). The paper evaluates the technique in application to implantbone interface and reports on the results from a bone-analogue composite based on high density polyethylene (PE), reinforced with 40% by volume of synthetic hydroxyapatite ceramic (HA) after 1, 3, and 6 months of implantation periods into condyles of mature rabbits. The removed implants surrounded by tissue were first fixed, dehydrated, and embedded before final preparation for initial scanning electron microscope examination, followed by investigation of the thin sections of a high-resolution elecron microscope. Fully undecalcified thin sections were examined. Some of them were stained using uranyl acetate and osmium tetroxide. Selected area diffraction studies were also carried out in the transmission mode. There are strict requirements for the specimens as well as for the instruments when high-resolution imaging is performed in order to secure the necessary stability of a specimen, its electron beam transparency, and imaging contrast in an electron microscope operating at high-resolution level. The paper demonstrates the potential ability of the technique in resolving 0.817 nm lattice fringes of (100) plane across the interface and by doing so helps in understanding crystallographic and morphological processes taking place at the interface between the implanted material and the new growth tissue on an ultrastructure scale. References Thomsen,P.
Personal communication. Lausmaa, Y.; Ratner, B.D., ed. Surface characterization sterdam: Elsevier; 1988. Tracy, B.M.; Doremus, R.H. J.
of biomaterials.
Glass-ionomer cement implantibone interface and rat tissue culture models. P.V. Hatton, I.M. Brook and L.M. Jonck* University of Shefield, UK *Centre for Bone Biology, University of Pretoria,
Am-
in primate
South Affica
The ultrastructure of the undisturbed bone/Glass-ionomer cement (GICo, V-OS Ionos Medizinische Producte GmBH & Co., KG Germany) interface is reported in a primate baboon in vivo culture model and in an in vitro rat calvarial model. TEM-linked X-ray microanalysis (energy dispersive) EDAX was used to aid characterisation of the bone/implant interface. For in vivo culture set GIC and autogenous cancellous bone were placed into Plexiglas diffusion chambers and implanted for 42 days into prepared holes of the femora of baboons. The in vitro model consisted of 14-day primary bone organ cultures derived from neonate rat calvaria set GIC being placed directly upon the calvarial bone or between the bone and periosteum. Specimens were prepared using a modified cryofixation and freeze-substitution procedure prior to embedding in resin. Active tissue proliferation and differentiation occurred in both types of culture. Osteoblast-like cells colonised and elaborated a collagen-containing extracellular matrix directly onto the surface of the GIC.
June 1991
EDAX confirmed the glass and matrix components of the GIC and should prove a useful tool to study the movement of ions and stability of the bone-implant bond. This study was facilitated by support from the Royal Society, England.
Better growth and differentiation of isolated rat osteoblasts cultured upon Bioglass compared with several other glass substrates. C.C. Groot, W.C.A. Vrouwenvelder* and K. de Groat* Laborator? for Cell Biology and Histology *Department of Biomaterials, Biomaterials Rijnsbergerweg 10, bld 55, 2333 AA Leiden,
Research Group, The Netherlands
Bioglass has been described in the literature as one of the possible candidates for bone replacement. It has unique properties responsible for the excellent bonding to bone when implanted in vivo. However, most of the knowledge concerning Bioglass is derived from in vivo experiments, and a lot of questions of a cell biological nature remain to be resolved. We investigated the growth and differentiation of isolated osteoblasts in relation to different substrates, hoping that our findings would shed some light upon the properties of Bioglass. We isolated osteoblasts from 19-day-old fetal calvaria by sequential collagenase digestion according to a standard procedure. 50 p,l of the obtained cell suspension were applied upon homogeneous Bioglass slides (code 4585: 45% SiO,, 24.5% Na,O, 24.5% CaO, 6% P,O,,) (Hench 1971), 1, 8 mm diameter, or slides of nonreactive glasses (NRG) (soda/silica/lime glass, quartz glass, coverslips) at a density of 5 X IO5 cells/ml for morphological studies (i.e., scanning electron microscopy, immunocytochemistry for collagen I and II, osteocalcin, and a specific osteoblast-associated antigen (OAA)). For the determination of biochemical parameters (DNA content, alkaline phosphatase activity) the cells were seeded at a density of 1 X lo6 cells/ml. After 2 h 800 ~1 of culture medium were added (aMEM + 5% FCS). The cells were kept in a humified incubator (95% H,O in air, 5% CO,) for up to 12 days. The medium was changed every 72 h in the first six days, then every 48 h. The osteoblasts cultured upon Bioglass generally showed better differentiation characteristics when compared with the NRG cultures. Upon Bioglass the cells showed more dorsal ruffles and a more “stand-off” morphology, which resulted in a denser cell layer. The alkaline phosphatase activity and the immunocytochemical markers (osteocalcin, collagen I, and OAA) were more evenly distributed over the culture, whereas in the NRG cultures these markers only could be detected in clusters of cells, probably because a favourable microenvironment was created inside the clusters. The growth rate, expressed as the amount of DNA per culture, was also better for the Bioglass cultures. From our observations we concluded that isolated rat osteoblasts proliferate and differentiate better when cultured upon Bioglass compared with nonreactive glasses. The development of a thin layer of calcium phosphate, which has the characteristics of hydroxyapatite, upon the surface of the Bioglass slides might be one of the causes of these differences.
Reference Hench. L.L. J. Biom.
Mat.
Res. Symp.
2:117-141;
1971.