Histochemistry and Cell Biology

, Volume 149, Issue 4, pp 433–447 | Cite as

Histochemical examination on the peri-implant bone with early occlusal loading after the immediate placement into extraction sockets

  • Yoshiki Ikeda
  • Tomoka Hasegawa
  • Tomomaya Yamamoto
  • Paulo Henrique Luiz de Freitas
  • Kimimitsu Oda
  • Akiko Yamauchi
  • Atsuro Yokoyama
Original Paper


Early and immediate loading of dental implants has become a routine procedure in dental practices throughout the world, but the histological feature of peri-implant bone has not been fully understood. Therefore, we aimed to elucidate the histological response of peri-implant bone bearing the early occlusal loading using rat models. Four-week-old male Wistar rats were subjected to extraction of their maxillary left first molars and had titanium implants inserted immediately into the post-extraction sockets. In experimental groups at 1 week after placement, implants were loaded for 1 or 2 weeks by adding adhesive resin on the top of the screws. In control groups, no adhesive resin was added to the implants. After 1 or 2 weeks with loading, rats were fixed with an aldehyde solution for histochemical assessment. Newly-formed bone adhered broadly to the implant surface in both the control and experimental groups. The experimental group loaded for 2 weeks showed thicker trabeculae between the implant threads compared to those in the control group. Osteopontin- and osteocalcin-positive cement lines, which are histological hallmarks of bone remodeling, were narrow and smooth in the experimental groups, while featuring a complex meshwork with thick scalloped lines in the control groups. The index of sclerostin-positive osteocytes located close to implants loaded for 2 weeks was significantly lower than in controls, suggesting that osteoblast activity was preserved. Summarizing, our experimental model suggested that early implant loading increases trabecular thickness in the peri-implant bone tissue in a process that involves the regulation of bone remodeling.


Dental implant Early occlusal loading Bone Osseointegration 



The authors thank Dr. N. Amizuka and H. Hongo for their invaluable suggestions for histochemical analyses.


This study was partially supported by Grants (23659893) from Japanese Society for the Promotion of Science (Yokoyama A).

Compliance with ethical standards

Conflict of interest

No conflict of interest is declared.


  1. Albrektsson T, Branemark PI, Hansson HA, Lindström J (1981) Osseointegrated titanium implants. Requirements for ensuring a long-lasting direct bone-to-implant anchorage in man. Acta Orthop Scand 52:155–170CrossRefPubMedGoogle Scholar
  2. Amizuka N, Takahashi N, Udagawa N, Suda T, Ozawa H (1997) An ultrastructural study of cell–cell contact between mouse spleen cells and calvaria-derived osteoblastic cells in a co-culture system for osteoclast formation. Acta Histochem Cytochem 30:351–362CrossRefGoogle Scholar
  3. Amizuka N, Kwan MY, Goltzman D, Ozawa H, White JH (1999) Vitamin D3 differentially regulates parathyroid hormone/parathyroid hormone-related peptide receptor expression in bone and cartilage. J Clin Invest 103(3):373–381CrossRefPubMedPubMedCentralGoogle Scholar
  4. Amizuka N, Shimomura J, Li M, Seki Y, Oda K, Henderson JE, Mizuno A, Ozawa H, Maeda T (2003) Defective bone remodelling in osteoprotegerin-deficient mice. J Electron Microsc (Tokyo) 52(6):503–513CrossRefGoogle Scholar
  5. Amizuka N, Li M, Hara K, Kobayashi M, de Freitas PH, Ubaidus S, Oda K, Akiyama Y (2009) Warfarin administration disrupts the assembly of mineralized nodules in the osteoid. J Electron Microsc (Tokyo) 58(2):55–65. CrossRefGoogle Scholar
  6. Amizuka N, Hongo H, Sasaki M, Hasegawa T, Suzuki R, Tabata C, Ubaidus S, Masuki H, Guo Y, de Freitas PH, Oda K, Li M (2012) The distribution of osteocytic lacunar-canalicular system, and immunolocalization of FGF23 and sclerostin in osteocytes. J Oral Biosci 54(1):37–42. CrossRefGoogle Scholar
  7. Barone A, Covani U, Cornelini R, Gherlone E (2003) Radiographic bone density around immediately loaded oral implants. Clin Oral Implants Res 14(5):610–615CrossRefPubMedGoogle Scholar
  8. Blanco J, Mareque S, Liñares A, Pérez J, Muñoz F, Ramos I (2013) Impact of immediate loading on early bone healing at two-piece implants placed in fresh extraction sockets: an experimental study in the beagle dog. J Clin Periodontol 40(4):421–429. CrossRefPubMedGoogle Scholar
  9. Cameron HU, Pilliar RM, MacNab I (1973) The effect of movement on the bonding of porous metal to bone. J Biomed Mater Res 7:301–311. CrossRefPubMedGoogle Scholar
  10. Dai J, Cheng N, Miron RJ, Shi B, Cheng X, Zhang Y (2014) Effect of decreased implant healing time on bone (re)modeling adjacent to plateaued implants under functional loading in a dog model. Clin Oral Investig 18(1):77–86. CrossRefPubMedGoogle Scholar
  11. De Smet E, Jaecques SV, Wevers M, Jansen JA, Jacobs R, Sloten JV, Naert IE (2006) Effect of controlled early implant loading on bone healing and bone mass in guinea pigs, as assessed by micro-CT and histology. Eur J Oral Sci 114(3):232–242. CrossRefPubMedGoogle Scholar
  12. de Freitas PH, Hasegawa T, Takeda S, Sasaki M, Tabata C, Oda K, Li M, Saito H, Amizuka N (2011) Eldecalcitol, a second-generation vitamin D analog, drives bone minimodeling and reduces osteoclastic number in trabecular bone of ovariectomized rats. Bone 49(3):335–342. CrossRefPubMedGoogle Scholar
  13. Degidi M, Piattelli A, Gehrke P, Felice P, Carinci F (2006) Five-year outcome of 111 immediate nonfunctional single restorations. J Oral Implantol 32(6):277–285. CrossRefPubMedGoogle Scholar
  14. Eriksson RA (1984) Heat-induced bone tissue injury. An in vivo investigation of heat tolerance of bone tissue and temperature rise in the drilling of cortical bone (Thesis). Sweden: University of Goteborg, pp 1–112Google Scholar
  15. Eriksson RA, Albrektsson T (1984) The effect of heat on bone regeneration. J Oral Maxillofac Surg 42(11):705–711CrossRefPubMedGoogle Scholar
  16. Esaki D, Matsushita Y, Ayukawa Y, Sakai N, Sawae Y, Koyano K (2012) Relationship between magnitude of immediate loading and peri-implant osteogenesis in dogs. Clin Oral Implants Res 23(11):1290–1296. CrossRefPubMedGoogle Scholar
  17. Frost HM (1994) Wolff’s Law and bone’s structural adaptations to mechanical usage: an overview for clinicians. Angle Orthod 64(3):175–188.<0175:WLABSA>2.0.CO;2Google Scholar
  18. Fujii N, Kusakari H, Maeda T (1998) A histological study on tissue responses to titanium implantation in rat maxilla: the process of epithelial regeneration and bone reaction. J Periodontol 69(4):485–495. CrossRefPubMedGoogle Scholar
  19. Futami T, Fujii N, Ohnishi H, Taguchi N, Kusakari H, Ohshima H, Maeda T (2000) Tissue response to titanium implants in the rat maxilla: ultrastructural and histochemical observations of the bone-titanium interface. J Periodontol 71(2):287–298. CrossRefPubMedGoogle Scholar
  20. Guo Y, Li M, Zhusheng L, Yamada T, Sasaki M, Hasegawa T, Hongo H, Tabata C, Suzuki R, Oda K, Yamamoto T, Kawanami M, Amizuka N (2012) Immunolocalization of sclerostin synthesized by osteocytes in relation to bone remodeling in the interradicular septa of ovariectomized rats. J Electron Microsc (Tokyo) 61(5):309–320. CrossRefGoogle Scholar
  21. Haga M, Fujii N, Nozawa-Inoue K, Nomura S, Oda K, Uoshima K, Maeda T (2009) Detailed process of bone remodeling after achievement of osseointegration in a rat implantation model. Anat Rec (Hoboken) 292(1):38–47. CrossRefGoogle Scholar
  22. Haga M, Nozawa-Inoue K, Li M, Oda K, Yoshie S, Amizuka N, Maeda T (2011) A morphological analysis on the osteocytic lacunar canalicular system in bone surrounding dental implants. Anat Rec (Hoboken) 294(6):1074–1082CrossRefGoogle Scholar
  23. Hasan I, Rahimi A, Keilig L, Brinkmann KT, Bourauel C (2012) Computational simulation of internal bone remodelling around dental implants: a sensitivity analysis. Comput Methods Biomech Biomed Eng 15(8):807–814. CrossRefGoogle Scholar
  24. Hasegawa T, Amizuka N, Yamada T, Liu Z, Miyamoto Y, Yamamoto T, Sasaki M, Hongo H, Suzuki R, de Freitas PH, Yamamoto T, Oda K, Li M (2013) Sclerostin is differently immunolocalized in metaphyseal trabecules and cortical bones of mouse tibiae. Biomed Res 34(3):153–159CrossRefPubMedGoogle Scholar
  25. Hasegawa T, Endo T, Tsuchiya E, Kudo A, Shen Z, Moritani Y, Abe M, Yamamoto T, Hongo H, Tsuboi K, Yoshida T, Nagai T, Khadiza N, Yokoyama A, de Freitas PH, Li M, Amizuka N (2017) Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes. J Oral Biosci 59(1):55–62. CrossRefGoogle Scholar
  26. Hongo H, Hasegawa T, Sasaki M, Suzuki R, Yamada T, Shimoji S, Yamamoto T, Amizuka N (2012) Bone-orchestrating cells, osteocytes. Hokkaido J Dent Sci 32:93–103Google Scholar
  27. Imai Y, Yokoyama A, Yamamoto S, Obata T, Iizuka T, Kohgo T, Shindoh M (2006) Peri-implant tissue after osseointegration in diabetes in rat maxilla. J Oral Biosci 48(1):54–61CrossRefGoogle Scholar
  28. Kameo Y, Adachi T (2014) Interstitial fluid flow in canaliculi as a mechanical stimulus for cancellous bone remodeling: in silico validation. Biomech Model Mechanobiol 13(4):851–860. CrossRefPubMedGoogle Scholar
  29. Kameo Y, Adachi T, Hojo M (2011) Effects of loading frequency on the functional adaptation of trabeculae predicted by bone remodeling simulation. J Mech Behav Biomed Mater 4(6):900–908. CrossRefPubMedGoogle Scholar
  30. Leucht P, Kim JB, Wazen R, Currey JA, Nanci A, Brunski JB, Helms JA (2007) Effect of mechanical stimuli on skeletal regeneration around implants. Bone 40(4):919–930. CrossRefPubMedGoogle Scholar
  31. Matsunaga S, Shirakura Y, Ohashi T, Nakahara K, Tamatsu Y, Takano N, Ide Y (2010) Biomechanical role of peri-implant cancellous bone architecture. Int J Prosthodont 23(4):333–338PubMedGoogle Scholar
  32. McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A (1993) Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HSglycoprotein) proteins in rat bone. J Bone Miner Res 8(4):485–496. CrossRefPubMedGoogle Scholar
  33. McKenzie JA, Silva MJ (2011) Comparing histological, vascular and molecular responses associated with woven and lamellar bone formation induced by mechanical loading in the rat ulna. Bone 48(2):250–258. CrossRefPubMedGoogle Scholar
  34. Oda K, Amaya Y, Fukushi-Irie M, Kinameri Y, Ohsuye K, Kubota I, Fujimura S, Kobayashi J (1999) A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissuenonspecific alkaline phosphatase. J Biochem 126(4):694–699CrossRefPubMedGoogle Scholar
  35. Ohashi T, Matsunaga S, Nakahara K, Abe S, Ide Y, Tamatsu Y, Takano N (2010) Biomechanical role of peri-implant trabecular structures during vertical loading. Clin Oral Investig 14(5):507–513. CrossRefPubMedGoogle Scholar
  36. Ozawa H (1985) Ultrastructural concepts on biological calcification; focused on matrix vesicles. J Oral Biosci 27:751–774Google Scholar
  37. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR (1987) Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2(6):595–610. CrossRefPubMedGoogle Scholar
  38. Poole KE, van Bezooijen RL, Loveridge N, Hamersma H, Papapoulos SE, Löwik CW, Reeve J (2005) Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J 19(13):1842–1844. CrossRefPubMedGoogle Scholar
  39. Romanos GE, Nentwig GH (2006) Immediate versus delayed functional loading of implants in the posterior mandible: a 2-year prospective clinical study of 12 consecutive cases. Int J Periodontics Restorative Dent 26(5):459–469PubMedGoogle Scholar
  40. Romanos GE, Toh CG, Siar CH, Wicht H, Yacoob H, Nentwig GH (2003) Bone-implant interface around titanium implants under different loading conditions: a histomorphometrical analysis in the Macaca fascicularis monkey. J Periodontol 74(10):1483–1490. CrossRefPubMedGoogle Scholar
  41. Ruff C, Holt B, Trinkaus E (2006) Who’s afraid of the big bad Wolff?: “Wolff’s law” and bone functional adaptation. Am J Phys Anthropol 129(4):484–498. CrossRefPubMedGoogle Scholar
  42. Sasaki M, Hongo H, Hasegawa H, Suzuki R, Zhusheng L, de Freitas PH, Yamada T, Oda K, Yamamoto T, Li M, Totsuka Y, Amizuka N (2012) Morphological aspects of the biological function of the osteocytic lacunar canalicular system and of osteocyte-derived factors. Oral Sci Int 9:1–8CrossRefGoogle Scholar
  43. Sasaki M, Hasegawa T, Yamada T, Hongo H, de Freitas PH, Suzuki R, Yamamoto T, Tabata C, Toyosawa S, Yamamoto T, Oda K, Li M, Inoue N, Amizuka N (2013) Altered distribution of bone matrix proteins and defective bone mineralization in klotho-deficient mice. Bone 57(1):206–219. CrossRefPubMedGoogle Scholar
  44. Scherft JP (1972) The lamina limitans of the organic matrix of calcified cartilage and bone. J Ultrastruct Res 38(3):318–331. Google Scholar
  45. Scherft JP (1978) The lamina limitans of the organic bone matrix: formation in vitro. J Ultrastruct Res 64(2):173–181. CrossRefGoogle Scholar
  46. Shibata Y, Tanimoto Y (2015) A review of improved fixation methods for dental implants. Part I: surface optimization for rapid osseointegration. J Prosthodont Res 59(1):20–33. CrossRefPubMedGoogle Scholar
  47. Shirakura M, Fujii N, Ohnishi H, Taguchi Y, Ohshima H, Nomura S, Maeda T (2003) Tissue response to titanium implantation in the rat maxilla, with special reference to the effects of surface conditions on bone formation. Clin Oral Implants Res 14(6):687–696CrossRefPubMedGoogle Scholar
  48. Slaets E, Naert I, Carmeliet G, Duyck J (2009) Early cortical bone healing around loaded titanium implants: a histological study in the rabbit. Clin Oral Implants Res 20(2):126–134. CrossRefPubMedGoogle Scholar
  49. Stokholm R, Isidor F, Nyengaard JR (2014) Histologic and histomorphometric evaluation of peri-implant bone of immediate or delayed occlusal-loaded non-splinted implants in the posterior mandible—an experimental study in monkeys. Clin Oral Implants Res 25(11):1311–1318. CrossRefPubMedGoogle Scholar
  50. Traini T, Neugebauer J, Thams U, Zöller JE, Caputi S, Piattelli A (2009) Peri-implant bone organization under immediate loading conditions: collagen fiber orientation and mineral density analyses in the minipig model. Clin Implant Dent Relat Res 11(1):41–51. CrossRefPubMedGoogle Scholar
  51. van Bezooijen RL, Roelen BA, Visser A, van der Wee-Pals L, de Wilt E, Karperien M, Hamersma H, Papapoulos SE, ten Dijke P, Löwik CW (2004) Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J Exp Med 199(6):805–814. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Vandamme K, Naert I, Geris L, Vander Sloten J, Puers R, Duyck J (2007) The effect of micro-motion on the tissue response around immediately loaded roughened titanium implants in the rabbit. Eur J Oral Sci 115(1):21–29. CrossRefPubMedGoogle Scholar
  53. Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, Shpektor D, Jonas M, Kovacevich BR, Staehling-Hampton K, Appleby M, Brunkow ME, Latham JA (2003) Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 22(23):6267–6276. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Wirth AJ, Müller R, van Lenthe GH (2012) The discrete nature of trabecular bone microarchitecture affects implant stability. J Biomech 45(6):1060–1067. CrossRefPubMedGoogle Scholar
  55. Wolff J (1986) The law of bone remodeling. Springer, Berlin Heidelberg New York (translation of the German 1892 edition)CrossRefGoogle Scholar
  56. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95(7):3597–3602CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zarb GA, Albrektsson T (1985) Nature of implant attachments. In: Branemark PI, Zarb C, Albrektsson T (eds) Tissue-integrated prostheses osseointegration in clinical dentistry. Quintessence Publishing Co., Chicago, pp 89–98Google Scholar
  58. Zarb GA, Albrektsson T (1991) Osseointegration: a requiem for periodontal ligament? Int J Periodontal Restor Dent 11(2):88–91Google Scholar
  59. Zhang X, Torcasio A, Vandamme K, Ogawa T, van Lenthe GH, Naert I, Duyck J (2012) Enhancement of implant osseointegration by high-frequency low-magnitude loading. PLoS One 7(7):e40488. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yoshiki Ikeda
    • 1
    • 2
  • Tomoka Hasegawa
    • 2
  • Tomomaya Yamamoto
    • 3
  • Paulo Henrique Luiz de Freitas
    • 4
  • Kimimitsu Oda
    • 5
  • Akiko Yamauchi
    • 1
  • Atsuro Yokoyama
    • 1
  1. 1.Oral Functional Prosthodontics, Department of Oral Functional Science, Graduate School of Dental Medicine and Faculty of Dental MedicineHokkaido UniversitySapporoJapan
  2. 2.Developmental Biology of Hard Tissue, Graduate School of Dental Medicine and Faculty of Dental MedicineHokkaido UniversitySapporoJapan
  3. 3.Department of Dentistry, Japan Self-Defense ForcesHanshin HospitalKawanishiJapan
  4. 4.Department of DentistryFederal University of SergipeLagartoBrazil
  5. 5.Showa HospitalFukuokaJapan

Personalised recommendations