Academic presentation on osseointegration of dental implants. A brief outline on surface modification, alveolar bone biology and phases of osseointegration

1. OSSEOINTEGRATION
RAKESH CHANDRAN

2. ‘a direct structural and functional connection between ordered, living bone and the
surface of a load-bearing implant’
Osseointegration definition
Bosshardt et al 2017. Osseointegration of titanium, titanium alloy and zirconia dental implants. Perio 2000

3. IMPLANT
Macroscopic structure
Microscopic structure
BONE
Cellular level
osteoprogenitor cells
osteoblasts and lining cells
osteocytes
osteoclasts
Molecular level
minerals
hormones
growth factors
cytokines
extracellular matrix proteins

4. IMPLANT DESIGN AND SURFACE CHARACTERISTICS

5. Different types of implant designs and surface characteristics
Barfeie et al 2015. Implant surface characteristics and their effect on osseointegration. BDJ

6. Surface chemistry (inorganic or organic)
Surface energy/charge and wettability
Surface topography (micro and nano-topography)
Implant surface modification
Feller L et al 2015. Cellular responses evoked by different surface characteristics. Biomed Res Int

7. • exposure of the implant surface to an electrolytic environment and to ionic
exchange can lead to adsorption and conformation of proteins which play a part
in platelet adhesion
• electrochemical deposition, ion implantation, anodisation, plasma based fluorine
ion application are examples of chemical methods of surface modification
• anodisation leads to a substantial thickness of the titanium oxide layer which may
increase osseointegration
Surface chemistry
Feller L et al 2015. Cellular responses evoked by different surface characteristics. Biomed Res Int

8. • defined as the excess energy at the surface of a material compared to the bulk
• quantifies the disruption of intermolecular bonds that occur when a surface is
created
• this extra energy provides the driving force for the adhesion to the surrounding
tissues.
• so an active implant surface provides the required conditions for starting the
desired interaction with the cellular environment.
• a negatively charged surface promotes better osseointegration
• surface energy of an implant can be modified by oxidisation, chemical and
topographical modification and by plasma treatment.
Surface energy/charge
Feller L et al 2015. Cellular responses evoked by different surface characteristics. Biomed Res Int

9. • largely dependent on surface energy
• describes the ability of a liquid to maintain contact with a solid surface
• determined by a balance between adhesive and cohesive forces
• adhesive forces between a liquid and a solid cause a liquid drop to spread across
the surface
• cohesive forces within the liquid cause the drop to ball up
• calculated from the contact angle that is formed between the drop and the
surface.
Surface wettability
Feller L et al 2015. Cellular responses evoked by different surface characteristics. Biomed Res Int

10. Examples of additive processes
Hydroxyapatite (HA) and other calcium phosphate coatings.
Titanium plasma-sprayed (TPS) surfaces.
Ion deposition.
Examples of subtractive processes
Electropolishing
Mechanical polishing
Blasting
Etching
Oxidation
Techniques to alter surface topography
Wennerberg and Albrektsson 2009. Effects of titanium surface topography on bone integration. Clin Oral Impl Res

11. Classification of surface roughness of implant surfaces
Albrektsson and Wennerberg 2004. Oral implant surfaces. Int J Prosthodont

12. Different types of implant designs and surface characteristics
Barfeie et al 2015. Implant surface characteristics and their effect on osseointegration. BDJ
Bosshardt et al 2017. Osseointegration of titanium, titanium alloy and zirconia dental implants. Perio 2000
• moderately rough implant surfaces are believed to deliver better osseointegration
compared with smooth surfaces however, results from different studies vary.
• currently, pure titanium is the ideal material for implants.
• ceramics have the potential to become the next generation of dental implants but
currently there is not sufficient scientific evidence for routine use of ceramic
implants.
• more standardised high quality prospective studies are required

13. Implant surface characteristics influence cellular responses such as cell adhesion,
proliferation, differentiation and migration, thus affecting osseointegration
Feller L et al 2015. Cellular responses evoked by different surface characteristics. Biomed Res Int

14. BONE

15. IMPLANT
Macroscopic structure
Microscopic structure
BONE
Cellular level
osteoprogenitor cells
osteoblasts and lining cells
osteocytes
osteoclasts
Molecular level
minerals
hormones
growth factors
cytokines
extracellular matrix proteins

16. Nanci A 2013. Ten Cate’s Oral Histology. 8th Ed. Mosby
Origin of bone cells- osteoblasts

17. MAJOR SIGNALING PATHWAYS
• Wnt pathway (been shown to control the differentiation of both osteoblasts and
osteoclasts)
• notch signaling (cell fate division and homeostatic maintenance, the notch
pathway is believed to be important in osteogenesis because of notch1–BMP-2
interactions that promote osteogenic differentiation)
• hedgehog proteins (plays a role in many embryonic processes and also is involved
with the maintenance of stem cells in adults)
• TGF-β/BMP superfamily
• fibroblast growth factors
TRANSCRIPTION PATHWAYS
• Runt-related transcription factor 2/core-binding factor alpha 1 (Runx2/cbfα-1)
• osterix
Mesenchymal stem cells differentiate into mature osteoblasts
Seitz et al 2013. Repair and Grafting of Bone. Plastic surgery

18. Receptor signaling typically leads to the formation or modification of biochemical
intermediates and/ or activation of enzymes, and ultimately to the generation of active
transcription factors that enter the nucleus and alter gene expression.
Gene expression is the process in which information from a gene is used by the cell to
produce a functional product, typically a protein.

19. DNA replication and RNA transcription and translation. Khan academy. www.khanacademy.org. Assessed 25 Feb 2017

20. Response to a signal. Khan academy. www.khanacademy.org. Assessed 25 Feb 2017
The mRNA leaves the
nucleus and enters the
cytosol. There, it directs
synthesis of a protein,
indicating which amino
acids should be added to
the chain. This step is
called translation

21. • Type 1 collagen
• Cell adhesion proteins (osteopontin, fibronectin, thrombospondin)
• Calcium-binding proteins (osteonectin, bone sialoprotein)
• Proteins involved in mineralization (osteocalcin)
• Enzymes (collagenase, alkaline phosphatase)
• Growth factors (IGF-1, TGF-β, PDGF)
• Cytokines (IL-1, IL-6, RANKL)
Proteins concentrated from serum
• β2-microglobulin
• Albumin
Proteins of the bone matrix
Seitz et al 2013. Repair and Grafting of Bone. Plastic surgery

22. Nanci A 2013. Ten Cate’s Oral Histology. 8th Ed. Mosby
Osteoblasts, lining cells, osteocytes and osteoclasts

23. TRAP, tartrate-resistant acid phosphatase
Nanci A 2013. Ten Cate’s Oral Histology. 8th Ed. Mosby
Origin of bone cells- osteoclasts

24. Risteli et al 2012 Bone and mineral metabolism
RANKL stimulates osteoclast activation by inducing secretion of protons and lytic
enzymes into a sealed resorption vacuole formed between the basal surface of the
osteoclast and the bone surface. Acidification of the vacuole leads to activation of
tartrate- resistant acid phosphatase (TRACP) and cathepsin K (Cat K)

25. Nanci A 2013. Ten Cate’s Oral Histology. 8th Ed. Mosby
Osteoblasts, lining cells, osteocytes and osteoclasts

26. OSSEOINTEGRATION

27. Phase 1: hemostasis
Phase 2: inflammatory phase
Phase 3: proliferative
Phase 4: remodelling
Series of events leading to osseointegration
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

28. Phase 1: hemostasis (minutes to hours)
Phase 2: inflammatory phase (after 10 minutes to a few days)
Phase 3: proliferative (few days to a few weeks)
Phase 4: remodelling (few days to a few years)
Series of events leading to osseointegration- duration
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

29. Phase 1: hemostasis (from trauma to the degranulation of platelets)
Phase 2: inflammatory phase (from degranulation of platelets to attachment of
fibroblasts)
Phase 3: proliferative (from attachment of fibroblasts to formation of woven bone)
Phase 4: remodelling (removal of woven bone and formation of lamellar bone)
Series of events leading to osseointegration- initiating and concluding events
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

30. • trauma to bone
• growth and differentiation factors are unmasked and liberated from their heparin
binding domains by heparin hydrolases from blood platelets
• platelets release
• platelet derived growth factor (PDGF)
• transforming growth factor (TGF) β1 and β2
• insulin like growth factor (IGF)
• vascular endothelial growth factor (VEGF)
• basic fibroblast growth factor (bFGF)
• coagulative and vasoactive agents
• platelets initiate polymerization of fibrinogen to form an extracellular matrix by
thrombin (extrinsic system) and the intrinsic clotting cascade (Hageman Factor)
• platelets aggregate and form a white thrombus closing the vascular leak.
Phase 1- hemostasis
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

31. • implant surface interacts with water molecules and ions- this can change the
charge pattern of the surface
• this is followed by plasma proteins present at high concentrations like albumin,
globulins or fibrin
• these will slowly be replaced by proteins with a lower concentration, but a higher
affinity for the surface such as vitronectin or fibronectin
Phase 1- hemostasis
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

32. • platelets bind to vitronectin. This activates platelets, converting the major platelet
integrin αVβ₃ from a resting state to an active conformation.
• platelets also bind to collagen with glycoprotein receptors
• surface bound fibrin on the metal surface of the implant can bind platelets over
the glycoprotein receptor to the titanium implant surface.
• this binding results in activation and degranulation of the platelets
• the release of cytokines from degranulating platelets is the beginning of the
inflammatory phase.
Phase 1- hemostasis
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

33. Cell to cell communication- Osseointegration. Quintessence Publishing

34. • degranulated platelets release
• TGF-β
• PDGF
• bFGF
• bradykinin (increases the vascular permeability)
• histamine (increases blood flow, decreases the blood stream velocity and
induces hyperaemia).
• innate host defence systems are activated
• molecular (e.g. complement system)
• cellular elements (PML/ PMN and macrophages)
Phase 2: Inflammatory phase
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

35. • diapedesis is initiated by loose adhesion of lectins in the inner lining of blood
vessels.
• mediated by adhesion of L-selectin on the leucocyte with E-selectin on the
endothelial side
• later stronger adhesion molecules such as intercellular adhesion molecule-1
(ICAM-1), ICAM-2 and the vascular cell adhesion molecule-1 (VCAM-1) catch the
leukocyte out of the blood stream, binding to integrins on the leucocyte
• PMN produce elastase and collagenase which helps them in digesting the basal
lamina of the blood vessel and pass beyond the basal lamina.
• endothelial cells open a small gap, and the leukocyte migrates in amoeboid
fashion through the gap.
• PMLs kill bacteria through reactive radicals (oxygen species and hydroxygroups,
chlorine radicals and hypochlorites) which are also toxic for the host cells and the
healthy tissue surrounding the wound.
Phase 2: Inflammatory phase- PML
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

36. • first published by a German pathologist Karl Donath in 1992
• although neutrophils are recruited on the basis of a pure wound healing
phenomena, macrophages are only recruited if a biomaterial is present
• macrophages are found frequently on the surface of titanium oral implants and
justify the concept of osseointegration being a foreign body reaction, because oral
implants are in themselves foreign bodies.
Concept of foreign body equilibrium
Trindade et al 2015. Current Concepts for the Biological Basis of Dental Implants. Oral Maxillofac Surg Clin North Am

37. a) osseointegration is the result of a foreign body reaction that, with the right
intensity in the inflammatory response, will balance itself out and allow for bone
to grow on the implant surface
b) similar to soft tissue implants, which end up encapsulated in poorly enervated
and vascularized fibrous tissue, dental implants also become surrounded by
condensed bone that is very poor in vascularization and enervation, the typical
result of a foreign body reaction that has reached equilibrium
c) the ongrown bone may be seen as a manner of shielding off the foreign entity
from the tissues, that is, as a protective mechanism.
Concept of foreign body equilibrium
Albrektsson et al 2014. Is marginal bone loss around oral implants the result of a provoked foreign body reaction?

38. • PML are relatively short-lived and are replaced by lymphocytes and macrophages.
• macrophages remove tissue debris
• macrophages also secrete TIMPs (tissue inhibitors of metalloproteinases) which
antagonize the digesting enzymes of PMN and therefore protect the extracellular
matrix proteins like proteoglycans. These in turn can protect growth factors which
are stored in the extracellular matrix.
• macrophages secrete angiogenic and fibrogenic growth factors.
• high concentration of fibronectin allows attachment of fibroblasts
• cells can hereupon crawl into the wound
• this is the beginning of the proliferative phase
Phase 2: Inflammatory phase- macrophages
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

39. Cell to cell communication- Osseointegration. Quintessence Publishing

40. FORMATION OF NEW ECM
• this newly formed tissue is called granulation tissue.
• macrophages release FGF which leads to migration of fibroblasts from the
surrounding healthy tissue into the blood clot.
• metalloproteinases degrade the blood derived fibrin in the clot und uncover
integrin binding sites in the fragments.
• to replace the degraded provisional clot matrix, they produce insoluble cellular
fibronectin and other insoluble proteins of the extracellular matrix like collagens,
vitronectin, decorin and other proteoglycans.
Phase 3: Proliferative phase- the formation of new ECM, angiogenesis and woven bone
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

41. Feller et al 2015. Cellular Responses Evoked by Different Surface Characteristics of Intraosseous Titanium Implants.
Phase 3- focal adhesion

42. ANGIOGENESIS
• in parallel, angiogenesis is stimulated by hypoxia.
• hypoxia attracts macrophages, which are able to survive under hypoxic conditions
by adjusting their metabolism to an oxygen independent generation of ATP. In
macrophages, vascular endothelial growth factor (VEGF) expression is stimulated
by an intracellular transcription factor called hypoxia inducible factor (HIF-1).
• In response to VEGF, pericytes detach from the outer walls of the vessel. These
cells use matrix metalloproteinases to digest the basal lamina around the vessels.
• The pericytes give rise to the new endothelial progenitor cells, which migrate to
the place of low oxygen tension where they are chemotactically attracted by the
chemokine stromal cell derived factor (SDF-1) which is produced by cells in the
wound. This process is called homing of endothelial cells.
• The cells proliferate to form condensed groups and they arrange themselves to
form tubes and these tubes are connected to an existing blood vessel.
• A new vascular loop is created and blood can flow through.
Phase 3: Proliferative phase- the formation of new ECM, angiogenesis
Terheyden et al 2012. Osseointegration – communication of cells. Clin Oral Impl Res.

43. FORMATION OF WOVEN BONE
• new bone formation begins with the secretion of a collagen matrix by osteoblasts
• depending on the process of ossification (endochondral or intramembraneous),
this can be collagen type II or type III, which is ultimately replaced by collagen
type I.
• bone formation within the alveolar process is a process of intramembranous
ossification, starting by the secretion of collagen type III.
• this matrix is subsequently mineralized by hydroxyapatite.
• then nanometer-sized, uniaxially oriented hydroxyapatite crystal plates are
formed within the collagen fibres.
• removal of the woven bone by osteoclasts is the beginning of remodelling and
thus the fourth and last phase.
Phase 3: Proliferative phase
Bosshardt et al 2017. Osseointegration of titanium, titanium alloy and zirconia dental implants. Perio 2000

44. Cell to cell communication- Osseointegration. Quintessence Publishing

45. • osteoclasts appear in the wound after a few days.
• primary bone implant contacts are removed.
• lamellar bone formed after remodelling
• in contrast to woven bone which is oriented parallel to the titanium surface in the
grooves of the threads, lamellar bone attaches rather to the tips of the
macrothreads.
Phase 4: remodelling phase
Bosshardt et al 2017. Osseointegration of titanium, titanium alloy and zirconia dental implants. Perio 2000

46. Cell to cell communication- Osseointegration. Quintessence Publishing

47. Thank you for your attention