3D microscopy of bone and bone cells: new views of bone wounds by looking directly at implant to tissue interfaces.
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Introduction Large numbers of studies of implants in bone have been made mainly from the viewpoint of assessing 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. We describe a different tack in which we separate the boundary and look straight at it, as against sectioning it, thus seeing a much greater proportion of it. We treat the implant as a probe into the bone wound environment. Materials and Methods 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). Later, hemisectioned implants were removed from the PMMA and the half beds recoated for BSE SEM. PMMA facing more intransigent 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. 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. 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, 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. 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). Results and Discussion 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.
AuthorsBOYDE, A; Wolfe, LA; Matrix Biology Ireland 2014
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