Evidence that Osteocytes in Autogenous Bone Fragments can Repair Disrupted Canalicular Networks and Connect with Osteocytes in de novo Formed Bone on the Fragment Surface
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Autogenous bone fragments generated during surgery (e.g. implant site preparation) accelerate bone formation by the release of a large variety of growth factors from the extracellular matrix and the cells contained within. Osteocytes, whether viable or apoptotic, within such fragments are able to recruit osteoclasts to a site of bone remodelling. Here, using correlative scanning electron microscopy, we provide compelling evidence that at one week healing in the Sprague Dawley rat tibia, following surgery (and/or the placement of a bone-anchored implant), autogenous bone fragments support bone formation on their surface. Furthermore, osteocytes within the autogenous fragments are frequently able to repair the disrupted canalicular networks and appear to connect with osteocytes (or osteoblastic-osteocytes) in the de novo formed bone on the surface of the fragment.
KeywordsAutogenous bone Early implant healing Osteocyte Scanning electron microscopy
Autogenous bone, still considered the gold standard for most bone graft applications, osseointegrates with the surrounding bone and undergoes vascularisation , and is believed to provide optimal osteoconductive, osteoinductive, and osteogenic properties . A number of growth factors affecting bone formation and resorption may be released from the extracellular matrix and cells (e.g. osteoblasts) in autogenous bone grafts including transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) , vascular endothelial growth factor (VEGF), receptor activator of nuclear factor kappa-β ligand (RANKL), and osteoprotegerin (OPG) . Osteoclasts on the surface of autogenous fresh rib grafts are observed in close contact with the cytoplasmic processes of apparently viable osteocytes within the graft . Viable osteocytes have also been reported within a microvascular fibula flap used in combination with an additively manufactured osteosynthesis plate after 33 months in the human mandible . Both viable and apoptotic osteocytes can recruit osteoclasts to sites of bone remodelling . In the context of peri-implant healing, it is claimed that autogenous bone fragments accelerate bone formation and offer the possibility of earlier implant loading ; however, bone fragments generated during implant site preparation are believed to be devoid of osteocytes [9, 10]. While it may be true that the destructive nature of the drilling process could render osteocytes non-viable, we report compelling evidence that such an assumption may not hold true in all cases. Here, we demonstrate that osteocytes in autogenous bone fragments can potentially restore disrupted canalicular networks and connect with osteocytes in the bone formed on the surface of such fragments.
Materials and Methods
Bone-implant blocks embedded in LR White resin (London Resin Company, UK) were obtained from in vivo animal experiments conducted previously in our group. All specimens represented an early healing stage, i.e. 6–7 days, following the placement of commercially pure (Grade 4) titanium (cp-Ti) implants with and without different commonly applied surface modifications in Sprague Dawley rat tibia. Resin embedded bone-implant blocks were polished using 400–4000 grit SiC paper and examined in a Quanta 200 environmental SEM (FEI Company, The Netherlands) operated in the backscattered electron (BSE) mode at 20 kV accelerating voltage, 0.5 Torr water vapour pressure. Elemental analysis was performed using energy dispersive X-ray spectroscopy (INCA EDX system, Oxford Instruments GmbH, Wiesbaden, Germany) performed at 15–20 kV accelerating voltage, 10 mm working distance, 40 µm aperture size, and 0–10 keV spectral energy range. Elemental maps for the Kα X-ray emission lines for calcium (~3.691 keV), phosphorus (~2.013 keV), and carbon (~0.277 keV) are shown. The osteocyte lacuno-canalicular network was exposed by resin cast etching for direct visualisation . Briefly, the polished resin embedded bone-implant blocks were sequentially immersed in 9% ortho-phosphoric acid and 5% sodium hypochlorite. After overnight drying, the samples were Au sputter-coated (10 nm), and examined in an Ultra 55 FEG SEM (Leo Electron Microscopy Ltd, UK) operated in the secondary electron (SE) mode at 5 kV accelerating voltage and 5 mm working distance.
Discussion and Conclusions
Implant surfaces considered to exhibit a higher osteogenic potential display a higher degree of bone-implant contact or direct bone apposition, often referred to as contact osteogenesis. As a consequence of such an osteogenic potential, precursor cells, i.e. mesenchymal stem cells (MSCs) are recruited to an implant (or a surgical defect) site, resulting in high amounts of rapidly formed woven bone. This early-formed tissue, however, lacks a well-ordered structure . Gradually, this tissue is removed by osteoclastic activity and is replaced by organised, lamellar bone—a process that progresses at a considerably slower pace in comparison to the formation of woven bone.
Particulate, decellularised bone has been used as an effective scaffold for bone repair . Small bone fragments in healing bone sites, generated during surgery, lend themselves to a unique scenario where they serve as osteoinductive surfaces and provide attachment sites for bone forming cells, i.e. osteoblasts. It is assumed that canalicular networks associated with the osteocytes nearest to the fragment surface are disrupted and/or destroyed during the surgical procedure. Incorporation of autologous bone grafts occurs through a process termed creeping substitution (a slow, near-complete resorption of the graft with simultaneous deposition of new, viable bone) . Contrary to the fate of cortical autografts, where mature osteocytes degenerate in the early stages following transplantation , when autogenous bone fragments are generated in the defect during the drilling process without exposure to an ex vivo environment, the osteocytes possibly remain viable and functional.
Here we demonstrate, using correlative scanning electron microscopy (SEM) techniques, that (i) autogenous bone fragments contribute towards osteogenesis within healing surgical defects, e.g. in the vicinity of bone-anchored implants, and (ii) osteocytes within autogenous bone fragments are frequently observed to restore a close physical proximity with osteocytes (osteoblastic-osteocytes) in new bone formed on the surface of these fragments, through interconnecting canaliculi that contain cytoplasmic extensions of osteocytes. However, it is not known whether the restored interconnectivity between osteocyte canaliculi in old and new bone plays any role in transmitting biochemical signals or transporting biomolecules involved in osteocyte function. Although certain implant surfaces are believed to exhibit an enhanced osteogenic potential and stronger mechanical interlocking with the surrounding bone tissue [19, 20], it is assumed that the presence of autogenous bone fragments within a healing surgical defect, and the prevalence thereof, is a function of the surgical technique. Therefore, the physico-chemical properties of the implant surface have no direct bearing on the osteopromotive potential of such bone fragments. Nevertheless, bone drilling may induce osteocyte death, particularly as a function of time , and temperature .
Further experiments are required to ascertain the viability and the eventual fate of the osteocytes in autogenous bone fragments. Such information may be of benefit in optimising surgical and drilling techniques in order to minimise their destructive effects on the implantation site. The presence of bone fragments at experimental implantation sites, particularly within implant threads, and their osteogenic potential have wide-ranging implications on peri-implant healing. It may be appreciated that, at least during early healing, such autogenous bone fragments (and the associated de novo formed bone) contribute to the overall amount of mineralised tissue found within the healing defect in addition to bone apposition directly on the implant surface.
This study was supported by the Swedish Research Council (Grant K2015-52X-09495-28-4), the BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Region Västra Götaland, the ALF/LUA Research Grant “Optimization of osseointegration for treatment of transfemoral amputees” (ALFGBG-448851), the IngaBritt and Arne Lundberg Foundation, the Dr. Felix Neubergh Foundation, Promobilia, the Hjalmar Svensson Foundation, the Wilhelm and Martina Lundgren Vetenskapsfond, and the Materials Science Area of Advance at Chalmers and the Department of Biomaterials, University of Gothenburg. The authors wish to thank Prof. Peter Thomsen for many helpful discussions and Dr. Sarunas Petronis for kindly providing the specimens used in this work.
Compliance with Ethical Standards
Conflict of interest
Furqan A. Shah and Anders Palmquist declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Human and Animal Rights and Informed Consent
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.
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