1,721,212 research outputs found
Combining confocal and BSE SEM imaging for bone block surfaces
No full textThe present report presents a method for the correlation of qualitative and quantitative BSE SEM imaging with confocal scanning light microscopy (CSLM) imaging modes applied to bone samples embedded in PMMA. The SEM has a proper digital scan generator: we leave the BSE image unchanged, and match the CSLM image to it, because the CSLM scan mechanism is not digital, though the signal is digitised. Our overlapping program uses a linear transformation matrix which projects one system to the other, calculated by finding three corresponding points in BSE and CSLM pictures. BSE images are empty where cells and osteoid are present. Fluorescence mode CSLM fills in these gaps. The combination images enhance our understanding of what is going on - and re-establish the need for good cellular preservation
Alkaline phosphatase cytochemistry in confocal scanning light microscopy for imaging the bone marrow stroma.
No full textThis paper reports on the use of alkaline phosphatase cytochemistry and combined conventional and confocal reflection and fluorescence scanning light microscopic modes in the study of human marrow stroma. It was found that the end product of the enzyme reaction using Napthol AS phosphate as substrate and Fast Blue BB as coupler reflected the 633 nm (red) light from a Helium-Neon laser. Serial optical sections suitable for 3-D reconstruction and selectively depicting the marrow reticulum cells could be obtained from thick glycol methacrylate sections reacted for Alkaline phosphatase. Furthermore, the yellow background of uncoupled diazonium salt over cytochemically unreactive structures in the same specimens and fields was used for imaging haemopoietic cell mass by operating the microscope at 488 nm (argon ion laser, blue-green). These methods may offer advantages in the investigation of the bone marrow stroma and its interplay with haemopoiesis and osteogenesis in normal and disease conditions
Conventional and confocal epi-reflection and fluorescence microscopy of the rat kidney in vivo
No full textTo visualize superficial and accessible renal tubule cells functioning in situ and to relate what we can 'see' to what we know of their function from more invasive in vivo or less direct in vitro studies means applying and adapting recent advances in epifluorescence and confocal microscopy to improve image resolution and to combine this with the use of fluorescent labels to monitor the handling of specific molecules by the proximal and distal renal tubule cells in vivo. Doing this in living tissue is novel, especially in the kidney. Application of confocal microscopy to the imaging of living tissue, as opposed to isolated cells, has not been widely reported. The kidney surface has been imaged before using the confocal microscope and in preliminary studies we have extended this by using a different confocal system with and without fluorescence. While the studies published up to now have been morphological, comparing standard renal (structural) histology of surface glomeruli and renal tubules with the corresponding in vivo confocal images, more dynamic, real-time studies have been limited. Individual red blood cells can be seen flowing around the peritubule capillary network and nucleated white blood cells can also be distinguished. Tubule cells, endothelial cells, the proximal tubule cell brush border and cell mitochondria can be visualized. Filtration and secretion can be observed, and the early and late parts of the proximal tubule distinguished, and the distal tubule recognized. Localization of fluorescently labeled insulin to the luminal brush border and progressive uptake of label and distribution within proximal tubule cells toward the basolateral (blood side) membrane can be demonstrated. The possibility of monitoring hemodynamic changes and tracking the filtration, uptake, secretion and absorption of fluorescently tagged molecules, as well as intracellular fluorescence, e.g. calcium or pH is an exciting prospect and is ripe for detailed exploration
3D microscopy of bone and bone cells: new views of bone wounds by looking directly at the implant interface
No full textISBN 978-80-210-7159-9Large numbers of studies of implants in bone have been made to assess bone bonding to a variety of materials, but especially titanium and in the context of dental implants, and nearly always employing sections of the implant to bone interface. The present report focuses on novel approaches in which we either separate the boundary and look straight at it, as against sectioning it, or look through the surrounding tissue straight at the implant with 3D light microscopy: in both cases we see a much greater proportion of the interface. Cylindrical or screw Ti implants, 3.2 mm diameter, were placed through the proximal medial tibial plateau in three month old rabbits, and retrieved at intervals from 7 to 365 days (1). Bones were fixed in glutaraldehyde, embedded in PMMA and implants sectioned longitudinally. Tissues near implants were studied using reflected light microscopy, confocal LM and, after carbon coating, compositional contrast BSE SEM and quantitative BSE (qBSE). Suitable blocks were prepared to permit study of the implant surface through the surrounding PMMA-embedded bone or marrow environment using direct view 3D LM methods and confocal LM. We also studied Ti framed glass window implants used for intra-vital microscopy retrieved at up to two years, prepared by PMMA embedding or maceration for SEM (2). Later, hemisectioned implants were removed from the PMMA and the half beds recoated for BSE SEM. PMMA facing more difficult-to-remove implants was sectioned again to produce quarter beds, imaged using BSE SEM of uncoated samples at 50Pa chamber pressure before and after staining with ammonium tri-iodide for cells and soft tissues. Plasma ashing was used to remove PMMA, cells and matrix from implant beds to visualise the nearest mineralised tissue surface with 3D BSE SEM. Conversely, sequential HCl and NaOCl etching was used to remove all bone components and produce a non-bone space cast for study of osteocytes and their canalicular processes. The relatively smooth finish of Ti implants mostly permitted separation of PMMA with all included tissue elements, the resin exactly replicating original machining marks. Direct viewing of the bed showed the exact extent of true bone contact, varying from very small areas with reticular, immature, highly cellular woven bone in early healing to large tracts of mature bone at 6 and 12 months - with the greatest coverage on smooth cylinder forms. Extensive bone-free patches bordered by scalloped osteoclastic resorption lacunae outlines indicated recent removal: similarly sized bone-covered patches had a lower mineralisation density, indicating recent re-formation, i.e., remodelling-turnover. Mature bone contained oriented osteocyte lacunae with canaliculi near the implant demonstrating growth from the implant. Tri-iodide staining additionally disclosed both osteoid and cells contacting the implant, with large numbers of multinucleated osteoclasts where bone was missing. Some regions showed direct contact of adipocytes and small blood vessels. Highly mineralised acellular material was often found in contact with the implant. Whereas some of this might be categorised as cement line matrix, we believe that we demonstrate the occurrence of an undescribed repair phase involving a type of dystrophic calcification akin to the cleft or gap sealing and healing seen elsewhere in bone and cartilage (3, 4). References 1. Boyde A, Wolfe LA, Bone Engineering, (Davies JE ed.), em squared Inc, Toronto, 321-331, 2000. 2. Boyde A, et al. Scanning 17, 72–85, 1995. 3. Boyde A, J Anat., 203,173-189, 2003. 4. Boyde A et al., J Anat., 225, 436-446, 2014
Novel mechanism identified in early implant healing in rabbit bone by direct viewing of the interface.
No full textTitanium implants in rabbit tibias were assessed by removing the metal and looking straight at the tissue interface boundary. Cylindrical or screw Ti implants, 3.2 mm diameter, placed in proximal tibias in 3 month old rabbits were retrieved at intervals from 1 week to 1 year [1]. Bones were fixed in glutaraldehyde, embedded in PMMA and implants sectioned longitudinally for reflected LM, confocal LM and compositional contrast BSE SEM. Hemisectioned implants were removed from the PMMA and the half beds recoated for BSE SEM. Other cases were sectioned to produce quarter beds and imaged using BSE SEM of uncoated samples at 50Pa chamber pressure before and after staining with ammonium tri-iodide to visualise cells and non-mineralised matrix components. In addition, plasma ashing was used to remove PMMA, cells and matrix from implant beds to investigate the nearest mineralised tissue surface with 3D BSE SEM. Conversely, HCl and NaOCl etching was used to remove all bone components and produce a non-bone space cast for study of osteocytes and their canalicular processes. The relatively smooth finish of the unetched, as-machined Ti implants mostly permitted separation of PMMA with all included tissue elements, the resin exactly replicating original machining marks. Direct viewing of the bed showed the exact extent of true bone contact, varying from very small areas with reticular, immature, highly cellular woven bone in early healing to large tracts of mature bone at 6 and 12 months - with the greatest areal coverage on smooth cylinder forms. Extensive bone-free patches bordered by scalloped osteoclastic resorption lacuna outlines indicated recent removal: similarly sized bone-covered patches had a lower mineralisation density, indicating recent re-formation, i.e., remodelling-turnover. Mature bone contained oriented osteocyte lacunae with canaliculi near the implant demonstrating growth from the implant. Tri-iodide staining additionally disclosed both osteoid and cells contacting the implant, with large numbers of multinucleated osteoclasts where bone was missing. Some regions showed direct contact of adipocytes and small blood vessels. Extensive patches of highly mineralised acellular material were often found in contact with the implant metal in earliest healing stages. Some of this might be categorised as cement line matrix. However, we consider that we demonstrate the occurrence of an undescribed repair phase involving a type of dystrophic calcification resembling the high density mineral infill crack closure mechanism seen in articular calcified cartilage and bone [2] and the formation of high density mineralised protrusions from the tidemark into hyaline articular cartilage [3]. References 1. Boyde A, Wolfe LA, in Bone Engineering, (Davies JE ed.), em squared Inc, Toronto, 321-331, 2000. 2. Boyde A, J Anat., 203,173-189, 2003. 3. Boyde A et al., J Anat., 225, 436-446, 2014
Marrow stromal (Western-Bainton) cells: identification, morphometry, confocal imaging and changes in disease.
No full textOsteoconduction in large macroporous hydroxyapatite ceramic implants: evidence for a complementary integration and disintegration mechanism
No full text‘The MSK Grand National: What we have learnt about the musculoskeletal system from studying racehorses’.
No full textBone Research Society Annual Meeting Liverpool 13-15 April 2023 Programme Page 6 [Joint meeting with 50th ECTS Congress] www.boneresearchsociety.org Invited Lecture IL1 Friday 14th April 2023 ‘The MSK Grand National: What we have learnt about the musculoskeletal system from studying racehorses’. Alan Boyde, London U.K. I reviewed studies I have conducted over many years and jointly with many colleagues concerning calcified tissues in horses from: Scanning electron microscopy of primary membrane bone. Boyde A, Hobdell MH. Z Zellforsch Mikrosk Anat. 1969;99(1):98-108. doi: 10.1007/BF00338800. Through: Coronal cementogenesis in the horse. Jones SJ, Boyde A. Archs Oral Biol. 1974 Aug;19(8):605-14. doi: 10.1016/0003-9969(74)90128-9. To: The Bone Cartilage Interface and Osteoarthritis. Boyde A. Calcif Tissue Int. 2021 Sep;109(3):303-328. doi: 10.1007/s00223-021-00866-9. The PowerPoint slides are condensed into a PDF available on the QMRO site which I have also made available in the format of a 2024 and 2025 Calendar
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