Bone basics for dentists

BONE

RAKESH CHANDRAN

Bone is a complex organ composed of multiple specialized tissues (osseous, periosteum/ endosteum, and bone marrow) that act synergistically and serve multiple functions.

Definition

Lang and Lindhe 2015. Clinical Periodontology and Implant Dentistry, Sixth Edition.

• Development of bone• Microstructure of bone• Composition of bone• Formation of osteoblasts• Mineralisation of bone• Formation of osteoclasts• Resorption of bone• Macrostructure of bone• Volume changes in bone• Bone healing

Presentation outline

Derivatives of the branchial arch system

Ten Cate 2013. Oral Histology 8th Edition

Intramembranous - clavicle, mandible, maxilla and skullEndochondral- mandibular condyle, long bones and vertebrae

Ossification

Endochondral ossification

http://teambone.com/education-basic/basic-biology-of-bone/

The Growth Plate

The growth plate is divided into multiple zones. These zones include the reserve zone, proliferative zone, and the hypertrophic zone. The reserve zone is the area closest to the secondary center of ossification. It has epiphyseal vessels the pass through this area but do not provide it with oxygen and keeps the oxygen tension low in this area. It has no known function with respect to longitudinal growth.

Endochondral ossification


Development of the mandible


Spread of mandibular ossification away from Meckel’s cartilage at the lingula

Development of the mandible


Development of the maxilla


Development of the maxilla


• Primary palate from the frontonasal and medial nasal processes• Secondary palate from three outgrowths; the nasal septum that grows downward

from the frontonasal process along the midline, and two palatine shelves or processes

• Commences between 7 and 8 weeks of gestation and completes around the 3rd month.

Mature or adult bones, whether compact or trabecular, are histologically identical in that they consist of microscopic layers or lamellae. Three distinct types of layering are recognized: circumferential, concentric, and interstitial

Microstructure of bone

Periosteum

Endosteum

The fibrous, non‐mineralized lining of the medullary cavity is the endosteum. Osteoblasts form in the endosteum and begin the formation of endosteal bone.

Hierarchical structure of bone

Launey et al 2010. On the mechanistic origin of bone.

A actinomycetemcomitans size: ~1 μm

~50mm

~100 μm

~50 μm

Hierarchical structure of bone

Launey et al 2010. On the mechanistic origin of bone.

~10 μm

~1 μm

~300nm

~1nm

Hydroxyapatite: ~ 25 nm in length, ~ 15 nm in width,~ 2–5 nm in thickness.

Endotoxin size: ~2.5nm

Nanci A 2013. Ten Cate’s Oral Histology. 8th Ed. Mosby

Bone cells

Osteoblasts and osteocytes

Lindhe 2015. Clinical Periodontology. 6th Ed

Cell communication in bone


• Local strain gradients provide the information that guides osteoclasts and osteoblasts during bone remodeling.

• This information is sensed by the osteocyte network, as reduced or increased canalicular fluid flow.

• The osteocyte network then signals to the osteoclasts and osteoblasts on the bone surface, thereby recruiting them to resorb or produce bone matrix.

New cell communication system Involved in the coupling of bone formation and bone resorption. • expression of ephrin B2 and its receptor ephrin B4

(EphB4) in osteoclasts and osteoblasts, respectively, and have revealed that ephrinB2-EphB4 bidirectional signalling links the suppression of osteoclast differentiation to the stimulation of bone formation.

• Early studies noted that direct, noxious mechanical stimulation of the periosteum produced painful percepts in human subjects (Inman and Saunders, 1944), and indeed some more recent literature highlights the prevailing opinion that pain from bone is generally not perceived unless the periosteum is involved.

• However, injection of irritants into the medullary cavity is also very painful, as is needle aspiration of bone marrow, and this pain is distinct from that associated with disruption of the periosteum. Thus, it appears that both the periosteum and the marrow cavity of bones must be innervated by primary afferent neurons capable of transducing and transmitting nociceptive information.

• There are many studies that have reported the existence of primary afferent neurons that innervate bone, and it has become clear that most of these sensory neurons have a morphology and molecular phenotype consistent with a role in nociception.

• Some of these nerve fibres were in close apposition with blood vessels

Physiology of bone pain.

Nencini and Ivanusic 2016

Composition of bone


Skeleton (storage), gut (uptake), kidney (secretion).

Parathyroid hormone (PTH): Action = Ca increase in circulation• stimulates bone resorption (osteoclasts)• stimulates renal reabsorption of Ca• stimulates synthesis of active vitamin D

Calcitonin (CT): Action = Ca decrease in circulation• counteracts all PTH actions

1,25-dihydroxyvitamin D: Action = Ca (& Pi) increase in circulation• stimulates Ca and Pi uptake from food (through gut)

Fibroblast Growth Factor, FGF23: Action = Pi decrease in circulation• reduces renal reabsorption of Pi, inhibits synthesis active 1,25 vitamin D

Bone - calcium and phosphate metabolism

1. Collagen Type 1• major organic bone component• basic structural unit is a triple helix

molecule.• crosslinks between the chains and

between the molecules give collagen its strength.

2. Non collagenous bone proteins

PROTEINS OF BONE MATRIX

Non collagenous bone proteins: bone formation role/function

Protein Bone formation function

Bone specific alkaline phosphatase (BAP)

Osteoblastic enzyme involved in bone formation (probably plays a role in bone matrix maturation)

Osteonectin Binds calcium and is involved in regulation of mineralization

Osteocalcin (OC) or Bone gla protein

Binds calcium and attracts osteoclasts. Major structural protein of the bone matrix.

Bone Sialoprotein(BSP)

Its role has been associated with mineral crystal formation.Major structural protein of the bone matrix.

Matrix gla protein(MGP)

Binds calcium. In bone, its production is increased by vitamin D. Found in in bone, heart, kidney and lung.

Proteoglycans Role is unclear.

Non collagenous bone proteins: bone resorption role/function

Protein Bone resorption function

Tartrate resistant isoenzymeacid phosphatase (TRAP5b)

Synthesized by active osteoclasts. TRAP5b catalyzes the formation of reactive oxygen species (ROS), that degrade bone matrix products in resorbing osteoclasts.

Cathepsin K Main osteoclastic protease, responsible for bulk degradation of Type I Collagen.

Matrix Metalloproteinases(MMPs)

MMP-2, -9, -13, -14 participate in the degradation of the collagenous bone matrix, resulting in epitope ICTP (carboxyterminal telopeptide of type I collagen).

Lysozymal enzymes Enzymes secreted by the osteoclastsResponsible for mineral dissolution in an acid environment

Calcitonin receptor Osteoclastic receptor for calcitoninInhibitor of osteoclast activity.

Protein Bone resorption function

RANK (NF-κB ) Osteoclastic receptor for sRANKL

sRANKL Soluble Receptor Activator of Nuclear Factor (NF)-κB Ligand. Binds to RANK and is the main stimulatory factor for the formation of mature osteoclasts.

OPG (Osteoprotegerin)OCIF (Osteoclast Inhibiting Factor)OBF (Osteoclast Binding Factor)

Key factor in inhibition of osteoclast differentiation and activity.Binds and acts as decoy receptor for sRANKL.

Osteopontin Cell-binding protein, synthesized by osteoblasts, that anchors osteoclasts to mineralized matrix.

Sclerostin Synthesized by osteocytes, inhibits bone formation by regulating osteoblast function and promoting osteoblast apoptosis.

Non collagenous bone proteins: bone resorption role/ function

Source of pluripotent mesenchymal stem cells• bone lining cells• inner cellular layer of the periosteum• pericytes in the walls of smallest blood vessels (Corselli et al. 2010).

Osteoblast differentiation



Osteoblast differentiation

MAJOR SIGNALING PATHWAYS• Wnt pathway (been shown to control the differentiation of both osteoblasts and

osteoclasts)• TGF-β/BMP superfamily• 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)

• 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

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 (functional messenger RNA (mRNA)).

Signal transduction. Wikipedia. https://en.wikipedia.org/wiki/Signal_transduction. Assessed 25 Feb 2017

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

Three distinct periods of osteoblast phenotype development 1. proliferation2. maturation and extra-cellular matrix synthesis3. matrix mineralization.

Mineralisation

Proliferation• express genes that support proliferation and several genes encoding for

extracellular matrix proteins, such as type I collagen and fibronectin. • BMP-2 and BMP-5 play a significant role in increasing alkaline phosphatase

activity, osteocalcin synthesis and parathyroid hormone (PTH) responsiveness.Maturation and extra-cellular matrix synthesis

• The active bone-matrix-secreting osteoblasts are cuboidal cells, with a large Golgi apparatus and an abundant rough endoplasmic reticulum, and are provided with regions of plasma membrane specialized in the trafficking and secretion of vesicles that facilitate the deposition of bone matrix

• high synthesis of alkaline phosphatase, • osteoblasts synthesize several proteins that are associated with the mineralized

matrix in vivo , including sialoprotein, osteopontin and osteocalcinMineralisation

• cellular levels of alkaline phosphatase mRNA decline• 50%–70% of mature osteoblasts undergo apoptosis• the remainder can differentiate into lining cells or osteocytes or transdifferentiate

into cells that deposit chondroid bone

Bone formation stimulators & inhibitors

Protein Stimulation Inhibition

Systemic PTHPTHrPSex-steroids (Estrogen, Testosterone)

Growth hormoneIGF-1Thyroid hormoneLeptin

Glucocorticoidsβ-2 adrenergicLeptinSerotonin

Local Transforming growth factor-ßBMP-2Platelet-derived growth factorsEndothelial growth factor (EGF)Vascular EGFIGF-1Fibroblast growth factors (FGF-2)ProstaglandinsWnt-LRP5/6

SclerostinNogginInterleukin-1ß,7Interferon- ƳTumor necrosis factorsDickkopfs (DKK-1)

Osteoclast differentiation


TRAP, tartrate-resistant acid phosphatase

Osteoclast differentiation

1. Attachment of osteoclasts to the mineralized surface of bone via integrins2. Creation of a sealed acidic microenvironment (resorption pit) through action of the

proton pump, which demineralizes bone and exposes the organic matrix 3. Degradation of the exposed matrix by the action of released enzymes, such as acid

phosphatase and cathepsin B4. Endocytosis at the ruffled border of organic degradation products5. Translocation of degradation products in transport vesicles and extracellular release

along the membrane opposite the ruffled border (transcytosis)

Osteoclast Cell• Responsible for mineral dissolution (by acid environment and lysozymal enzyme

action below ruffled border).• Responsible for collagen break down (Cathepsin K and MMP pathways).

Osteoclasts- sequence of resorptive events

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)

Bone resorption

Bone resorption stimulators & inhibitors

Protein Stimulation Inhibition

Systemic PTHPTHrP1,25 (OH2)D3Thyroid hormoneβ-2 adrenergic

CalcitoninSex steroids (Estrogen, Testosterone)

Local RANKLMacrophage-CSFGranulocyte macrophage-CSFInterleukin-1,6,7,11,15,17Interferon-ƳTumor necrosis factor-α (TNF-α)Fibroblast growth factorsProstaglandins

Osteoprotegerin (OPG)Transforming growth factor-ß (TGF-β)Interferon-ƳInterleukin-4,10, 13,18Interleukin-1 receptor antagonist

Bone remodelling timeline

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• (A and B). Remodeling of bone in a multicellular bone unit starts with osteoblastic activation of osteoclast differentiation, fusion, and activation. When resorption lacunae are formed, the osteoclasts leave the area and mononucleated cells of uncertain origin appear and “clean up” the organic matrix remnants left by the osteoclast, also possibly forming the cement line (dotted line) at the bottom of the lacunae

• (C). During the resorption process, coupling factors, including insulin-like growth factor–I and transforming growth factor–β, are released from the bone-extracellular matrix, and these growth factors contribute to the recruitment of osteoblasts to the resorption lacunae and their activation.

• (D). The osteoblasts will then fill the lacunae with new bone; when the same amount of bone is formed as is being resorbed, the remodeling process is finished, and the mineralized extracellular matrix will be covered by osteoid and a single-cell layer of osteoblasts

The bone- remodelling cycle

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The alveolar process forms with the eruption of teeth and growth of the jaws After tooth extraction, the alveolar process gradually resorbs

The alveolar process

Beagle 2013. Surgical Essentials of Immediate Implant Dentistry. John Wiley & Sons, Inc

Alveolar process

Antonio Nanci. Ten Cate's Oral Histology, 8th Edition. Elsevier Canada

Cortical plate (buccal and lingual)

Alveolar bone (bone lining the alveolus)

Spongiosa (trabecular bone)

Bundle bone


Haversian system of trabecular bone

Bundle bone

Sharpeysfibres

PDL Cementum

• The aspect of bone which lines the tooth socket is also referred to as the bundle bone.

• The extrinsic collagen fiber bundles of the periodontal ligament (PDL) embed into this area of bone. The bundle bone usually exhibits a course-fibered texture and contains fewer intrinsic collagen fibrils than that in lamellar bone (Avery, 1994).

Facial bone (buccal plate) thickness in the maxillary anterior region

Facial bone(bundle bone and cortical plate)


CBCT of the maxillary lateral incisor

Bundle bone

Width of bundle bone varies between 0.1 mm and 0.4 mm (Schroeder 1986).

When the buccal bone plate is ≤ 0.5mm it may be solely comprised of bundle bone.

Since bundle bone is a tooth dependent structure, the loss of teeth will result in the loss of bundle bone

Chen & Darby 2016. The relationship between facial bone wall defects and dimensional alterations of the ridge following flapless tooth extraction in the anterior maxilla. Clin Oral Implants Res.

Horizontal or buccolingual ridge reduction• approximately 5 to 7 mm (almost 50% of the initial ridge width)• reduction occurs over a period of 6 to 12 months• most of the changes occur during the first 2 months (Chen and Darby 2016)

(short window to place implants).

Vertical or apicocoronal height reduction• 2.0 to 4.5 mm

Dimensional changes of the extraction socket

Al-Sabbagh & Kutkut 2015. Immediate Implant Placement. Dent Clin N AmChen & Darby 2016. The relationship between facial bone wall defects and dimensional alterations of the ridge

following flapless tooth extraction in the anterior maxilla. Clin Oral Implants Res.

Based on the volume of remaining mineralized bone, the edentulous sites may, according to Lekholm and Zarb (1985), be classified into five different groups. In groups A and B, substantial amounts of the ridge still remain, whereas in groups C, D, and E, only minute amounts of hard tissue remain.

Evaluation of bone volume

Lekholm and Zarb 1985

Qualitative evaluation of bone quality

Lekholm and Zarb 1985

• First 24 hours are characterized by the formation of a blood clot in the socket• 2–3 days the blood clot is gradually replaced with granulation tissue• 4–5 days, the epithelium from the margins of the soft tissue starts to proliferate

to cover the granulation tissue in the socket. • 1 week after extraction, the socket contains granulation tissue and young

connective tissue, and osteoid formation is ongoing in the apical portion of the socket.

• After 3 weeks, the socket contains connective tissue and there are signs of mineralization of the osteoid. The epithelium covers the wound.

• After 6 weeks of healing, bone formation in the socket is pronounced and trabeculae of newly formed bone can be seen.

• the process by which woven bone was replaced by lamellar bone and marrow, that is remodeling, was slow and exhibited great individual variation and may take years to be completed.

Wound healing following tooth extraction

Trombelli et al 2008. Modeling and remodeling of human extraction sockets.

• The mechanisms of healing and incorporation of autogenous bone grafts are universal, irrespective of the donor site.

• Autogenous bone grafts are comprised of cortical or cancellous bone (or both)• The factors affecting the healing of bone are the

• amount of cellular marrow transplanted with the bone graft, • the vascularity of the tissue bed and • the attainment of graft stability

Autogenous bone grafts and their healing

Marx, RE 2007. Bone and bone graft healing. Oral Maxillofacial Surg Clin N Am

• During the 1st week, platelets are responsible for regulating bone regeneration. The platelets secrete several growth factors, namely –PDGF; TGFβ-1 and EGF.

• Most osteocytes within the autogenous bone grafts die off, as they are encased in a mineral matrix and the harvesting of the graft disrupts their delicate canalicular blood supply . However, those osteocytes (in their lacunae) situated within 0.3 mm of a perfusion surface appear to survive.

• New bone formation occurs by the process of osteogenesis, which is induced by the surviving osteoblasts and marrow stem cells within the autogenous bone. These cells are open to the local environment and survive by oxygen and nutritional diffusion (plasmatic circulation) until the graft becomes revascularizedby capillary ingrowth.

• By day 3, capillaries are seen to penetrate into the graft and osteocompetent cells undergo proliferation.


Healing of cancellous bone grafts

• By day 7, the platelets are exhausted and contribute little to further healing. Macrophages are attracted to the site by the initial hypoxic state in the area, as well as by chemotaxis by the platelets. The macrophages also secrete growth factors until the graft is fully vascularized between day 14 and 21.

• The vascularization of the graft provides oxygen and nutrients to the osteocompetent cells, which then synthesize and secrete osteoid.

• During the process of revascularization, osteoclasts also arrive from the circulation. The osteoclasts resorb the mineral matrix (this includes mineral matrix surrounding the osteocytes) and allow the release of growth factors (BMP and IGF-1 and 2), which induce osteoinduction and allows maturation of the graft.

• Cancellous bone grafts are initially weaker due to their spongy, trabecular architecture, however these grafts continue to gain strength.

Healing of cancellous bone grafts


• Large cortical block bone grafts with their compact architecture, form new bone mainly through the processes of osteoinduction and osteoconduction from the adjacent bone margins.

• Cortical grafts require significant resorption by osteoclasts, before osteoblasts commence new bone formation – this process is referred to as “creeping substitution”.

• Cortical bone grafts are strong initially, but weaken overtime – before regaining strength. There is also a significant loss in dimension as a result of the resorption process that takes place.

• Cortical grafts require proper fixation and have a high risk of sequestration in contaminated areas.

Healing of cortical block bone grafts

Buser, D 2009. 20 Years of Guided Bone Regeneration in Implant Dentistry

• Bone is a complex organ composed of multiple specialized tissues serve multiple functions.

• Except for the condylar and coronoid processes, the rest of the maxillofacial structure undergoes intramembranous ossification.

• Bone is composed mainly of inorganic hydroxyapatite and it plays a major role in calcium homeostasis.

• The balance of serum ionized calcium blood concentration results from a complex interaction between parathyroid hormone (PTH), vitamin D, and calcitonin.

• The bone remodeling cycle involves a complex series of sequential steps that involves osteoclasts in the “resorption” phase followed by a “reversal” phase and finally a “formation” phase that involves osteoblasts, which lay down matrix.

• Remodeling of bone is slow and exhibited great individual variation and may take years to be completed.

• Sound knowledge of bone anatomy, physiology and wound healing are essential for surgeons to perform successful periodontal and implant surgical procedures.

Conclusion

Thank you for your attention

Bone basics for dentists

Healthcare

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