OSTEOCYTES: CLINICAL RELEVANCE

Jenneke Klein-Nulend

Vraag 1.

Wat zijn osteocyten, en wat is hun functie?

Bone structure is mechanically meaningful

Disuse leads to bone loss

MICROGRAVITY

Environment of unloading leads to bone loss

Severe spinal osteoporosis

Man, 42 yr Man, 90 yr Mosekilde, 1990

Age-related bone loss

Julius Wolff (1892)

Mechanical adaptation bones adapt their mass and structure to the loading conditions to optimize their load bearing capacity

Use it or lose it

OSTEOCYTES, MECHANOTRANSDUCTION, AND BONE REMODELING

Mechanical adaptation

bone mass and architecture

local mechanical

signal

effector cells

external load

stimulus

osteocytes

Antwoord op Vraag 1. Wat zijn osteocyten, en wat

is hun functie?

Vraag 2.

Hoeveel % van alle botcellen is osteocyt?

Waar liggen osteocyten in bot, en

waarom is hun lokatie in bot belangrijk?

Antwoord op vraag 2.

95% van alle botcellen is osteocyt

De lokatie van osteocyten in bot is van belang om hun mechanosensorische

functie uit te oefenen.

Vraag 3.

Welke signaalfactoren worden door osteocyten uitgescheiden?

Antwoord op vraag 3.

Osteocyten scheiden o.a. RANK-L af.

Vraag 4.

Wat is de relatie tussen botadaptatie en botremodellering

in volwassen bot?

Poliomyelitis and bone remodeling

Haversian and Volkmann channels

loaded unloaded

•  Bone MASS how much/how little bone

•  Bone ALIGNMENT orientation along principal loading

directions

MECHANICAL ADAPTATION relates to:

IN ADULT HUMAN BONE ADAPTATION OCCURS DURING REMODELING

Antwoord op vraag 4.

In volwassen humaan bot treedt botadaptatie op tijdens het proces van

botremodellering.

OSTEOCYTES, MECHANOTRANSDUCTION,

AND BONE REMODELING

Hypothesis

REMODELING IS GUIDED BY (DAILY) LOADING

Finite element model

Smit et al. J Bone Min Res 17, 2000

Equivalent strain distribution

Loaded remodeling osteon

Loading of remodeling bone leads to opposite strain fields in the wall of the cutting cone and the closing cone.

Decreased strain occurs in front of the cutting cone, where osteoclasts are activated.

Elevated strain occurs around the closing cone, where osteoblasts are activated.

marrow

reversal zone

closing cone

osteoclasts in cutting cone

bone forming osteoblasts

old bone

new bone

Ruimerman et al. J Biomech 38, 2005

Computer simulation of bone remodeling

10 MPa 1 Hz

2x2 mm2 OCY density 1600 mm-2

3 osteoclasts

Computer simulation model

Mechanical signal

Ruimerman et al. J Biomech 38, 2005

Vraag 5.

Wat gebeurt er met de richting van de “cutting cone” als de

richting van de belasting verandert?

Rotated load 30°

Loading direction

Ruimerman et al. J Biomech 38, 2005

Antwoord op vraag 5.

De richting van de “cutting cone” verandert mee met de belastingsrichting.

20% Reduced load 20% Increased load Loading magnitude

No load

Ruimerman et al. J Biomech 38, 2005

Conclusion

DAILY LOADING EXPLAINS BONE TUNNELING

● loading direction: orientation of the tunnel ● loading magnitude: amount of refilling

LOADING Deformation

Flow of canalicular fluid around the osteocytes

Mechanosensing by the osteocytes

Production of soluble factors

Bone remodeling by the osteoblasts/osteoclasts

OPTIMAL BONE ARCHITECTURE AND DENSITY

THE BONE MECHANOSENSORY SYSTEM

lc ocy ocy

load

flow

CANALICULAR FLUID FLOW

ocy: osteocyte lc: lining cell

OSTEOCYTES OSTEOBLASTS PERIOSTEAL FIBROBLASTS

APPLICATION OF

FLUID FLOW

OB PF

time (min) time (min)

1500 1500

1000 1000

500 500

0 0 0 0 0 15 15 30 30 45 45 60 60

PFF Con

PFF Con

time (min)

Con

OCY

1500

1000

500

0 15 30 45 60

PFF

PGE 2

, pg/

µg

DN

A Fluid flow stimulates PGE2 release

Osteocytes (OCY) release more PGE2 than osteoblasts (OB) and fibroblasts (PF)

Ajubi et al. BBRC 225, 1996

Fluid flow stimulates NO release by osteocytes

Klein-Nulend et al. BBRC 217, 1995

osteocytes

NO

2 nM

/103

cel

ls

minutes

con

PFF

240

120

0 15 30 45

fibroblasts

con

PFF

minutes

NO

2 nM

/103

cel

ls

240

120

0 15 30 45

Fluid flow-stimulated osteocytes:

� inhibit osteoclastogenesis via the release of soluble factors, resulting in decreased bone resorption.

Intercellular communication

� produce soluble factors that modulate proliferation and differentiation of osteoblasts.

LOADING

LOADING

NO

LOADING

LOADING

TREDMILL? NO

LOADING

LOADING

NO

NO

LOADING

LOADING

PGE2: OSTEOBLAST RECRUITMENT?

OSTEO- BLASTS

LOADING

LOADING

ADAPTIVE BONE REMODELING

ADAPTIVE BONE REMODELING

Sclerostin and

Van Buchem Disease (VBD)

“Mineralized Tissues in Oral and Craniofacial Science: Biological Principles and Clinical Correlates”

•  Genetic background: SOST gene

•  Its product sclerostin

•  The clinical features caused by SOST mutations - Van Buchem Disease

•  Therapeutic possibilities

Van Buchem Disease •  VBD first described in 1955 and originally named hyperostosis

corticalis generalisata

•  Extremely rare autosomal recessive sclerosing bone dysplasia (Vanhoenacker et al., 2000)

•  Increase in cortical bone thickness and density affecting the skull, mandible, and long bones

•  Classified as craniotubular hyperostosis (Beighton et al., 2007)

Van Hul et al., 1998

Prevalence •  Prevalence VBD is very low:

in the 90’s < 30 patients, predominantly in the Dutch population (reported by Van Buchem)

•  13 VBD patients in a highly inbred Dutch family with a common ancestor and living in a small ethnic isolated village (Van Hul et al., 1998)

Pedigree of the 13 VBD patients

Characteristic Features

Protruding chin High forehead

Thickened naseal area Facial nerve paralysis

Van Hul et al., 1998

Genetic background •  Mutations SOST gene chromosome 17q12-21 two similar diseases

(A) SCLEROSTEOSIS (much more severe) mutations in SOST coding region (B) VAN BUCHEM DISEASE 52-kb deletion ~35 kb downstream of the SOST gene

Chronological portraits of a patient with sclerosteosis from the age of 3 years onward.

She was born with syndactyly at both hands and developed facial palsy, deafness, facial distortion, and maxillary overgrowth during childhood.

By the age of 30, she had developed proptosis and elevated intracranial pressure due to overgrowth of the calvaria. Craniectomy was performed, but she died nevertheless because of elevated intracranial pressure at the age of 54 years Moester et al., 2010

Sclerostin, characteristics and expression

•  The SOST gene : 2 exons encoding 213-amino acid secreted sclerostin glycoprotein

•  Cystein-knot motif involved in dimerization and receptor binding and signaling peptide for secretion

•  SOST mRNA during embryogenesis expressed in many tissues

•  Sclerostin belongs to the evolutionary-conserved DAN (differential screening-selected gene aberrative in neuroblastoma) family of glycoproteins

•  Ability to affect the activity of several growth factors, including bone morphogenetic protein (BMP) and Wnts

Sclerostin in adult tissue

Postnatally in osteocytes, mineralized hypertrophic chondrocytes and cementocytes

Van Bezooijen et al., 2009

Mineralized hypertrophic chondrocytes Osteocytes

Cementocytes

• Osteocyte-derived secreted protein, • High sclerostin levels in lacunar-canalicular network

Winkler et al., 2003

Sclerostin in adult tissue

Expression in Van Buchem Disease

In VBD patients none of these cell types express sclerostin Winkler et al., 2003 Van Bezooijen et al., 2009

Van Bezooijen et al., 2009

Increased osteoid surface and lamellar bone

Active osteoblasts

Sclerostin as bone inhibitor:LRP/Wnt

Sclerostin binds to Wnt co-receptors LRP5 and LRP6, thereby antagonizing Wnt/β-catenin signaling by inhibiting β-catenin nuclear translocation and transcription of Wnt target genes

Nusse, 2005; Semënov et al., 2005

Sclerostin as bone inhibitor:BMP-7

Inhibition of BMP/Smad signaling by blocking intracellular BMP7 secretion in osteocytes

Krause et al., 2010

Mechanisms of action By keeping both Wnt/-

catenin and BMP7/Smad in check,

sclerostin plays an important role in maintaining bone homeostasis (A)

Without sclerostin, the negative feedback on osteoblast activity is absent, like in VBD,

which results in excessive bone

formation (B)

Van Buchem Disease - Clinical features - Thickened skull

-  Thickened mandible,

elongation and deformity

- Diaphyseal cortex of long bones à

narrowed medullary cavities

- Spine

- Pelvic bone

•  Disrupted bone contours due to subperiosteal osteophytes (exostoses), resulting in a rough surface

•  Hyperostosis of the skull leads to narrowing of the foramina, causing entrapment of –  7th cranial nerve, leading to facial palsy –  8th cranial nerve leading to deafness,

neurological pain, visual problems, and in some cases even blindness

•  Annual assessment from infancy is recommended for disturbed hearing, evidence of increased intracranial pressure, and nerve entrapment

Clinical features-general

Hyperostosis skull: nerve entrapment

•  Fractures and haematological changes are not found in VBD

•  Laboratory values are normal, except for several biochemical indices of bone turnover, such as elevated serum ALP levels

•  Serum procollagen 1 peptide, OC, and urinary type I collagen cross-linked N-telopeptide are increased (in several but not all cases)

Clinical features Van Buchem Disease - general

Orofacial bone and dental aspects

•  No evidence for direct effects on tooth development due to loss of function of SOST

•  Hyperostosis and hypercementosis could result in narrowing of the periodontal space or even ankylosis - a bone-like tissue connecting root dentin and alveolar bone

•  Tooth extraction may be difficult and management by an orthodontic or craniofacial team is recommended (Beighton et al., 2007)

Orofacial bone and dental aspects

However X-ray images from VBD patients do not show clear signs of ankylosis, although the identification of periodontal gaps is not always possible owing to the very dense radiopacity of the overlying bone

Van Bezooijen et al., 2009

Orofacial bone vs. tubular bone •  Prominent skull and mandibular bone growth in

osteosclerotic and VBD patients might be related by potential differences in “bone cells” at different skeletal sites?

•  Osteoclasts and osteocytes from craniofacial bones differ from osteoclasts and osteocytes in the long bones regarding the expression of molecules and sensitivity for loading (Zenger et al., 2010; Vatsa et al., 2008)

•  Calvarial bone and long bone also differ in composition, suggesting heterogeneity between osteoblasts from both skeletal sites

Orofacial bone vs. tubular bone

•  Osteoblasts of craniofacial bone (intramembranous bone of different embryological origin) more sensitive to loss of sclerostin?

•  Osteocytes from calvarial or jaw bone produce more sclerostin than osteoblasts in long bones?

•  Differences in the magnitude of mechanical loading on long bone versus craniofacial bone may also play a role

Therapeutic possibilities •  Surgical removal of excess bone

-technically difficult, sometimes dangerous (Marmary et al., 1989; Du Plessis, 1993)

•  Procedure might include:

–  Surgical decompression of entrapped cranial nerves –  Craniectomy for increased intracranial pressure –  Middle ear surgery for conductive hearing loss –  Reduction of mandibular overgrowth

•  Testing of relatives at risk is recommended: clinical appraisal, lateral skull radiography, and targeted mutation analysis for the deletion

•  These treatments aim to relief the symptoms, with no systemic approach to counteract the underlying hyperostosis

Surgical removal

Schendel, 1988

BEFORE

AFTER

Glucocorticoids •  Glucocorticoids attractive alternative to high risk

surgical procedures (Van Lierop et al., 2010)

•  Glucocorticoids inhibit osteoblast proliferation and differentiation and increase apoptosis (Weinstein et al., 1998)

•  Van Lierop et al. (2010) suggest that sclerostin is not only involved in bone formation, but also in bone resorption (exact mechanism yet to be explored)

•  Glucocorticoids could serve as an additional, systemic therapy in patients with increased risk of neurological complications due to bone overgrowth like Van Buchem Disease

Glucocorticoid inhibit bone formation by stimulating sclerostin

VBD

•  Preventing activation of bone lining cells •  Inactivation of active osteoblasts

Sclerostin antibody as a bone forming agent

•  Pharmacologic inhibition of sclerostin promising anabolic therapy for low bone mass-related disorders like osteoporosis

•  Inhibition of sclerostin by injection of antibodies has already been shown to increase bone formation, bone mass, and bone strength in animal models, including primates (Li et al., 2010; Ominsky et al., 2010)

•  A first phase I clinical study demonstrated that a single injection of a mAb against sclerostin increases bone formation markers and bone density, decreases bone resorption, and is well tolerated (Padhi et al., 2010)

Summary •  Sclerostin expressed in mineralizing cells

•  By keeping both Wnt/-catenin and BMP7/Smad in check, sclerostin plays an important role in maintaining bone homeostasis

•  Van Buchem Disease: Loss of SOST/sclerostin à abnormal bone formation skull, mandible and long bones

•  Intervention in sclerostin expression can stimulate or inhibit bone formation

Mechanical loading regulates sclerostin expression in osteocytes

•  Bone adapts mass and shape in response to mechanical loading or lack of loading.

•  Sclerostin is expressed in mechanosensitive osteocytes. Evidence for mechanoregulation of sclerostin expression was reported in mice and rats subjected to ulnar loading in vivo (Robling et al., 2008)

•  Modulation of sclerostin levels appears to be a finely tuned mechanism by which osteocytes coordinate regional and local osteogenesis in response to increased mechanical stimulation, perhaps via releasing the local inhibition of Wnt/Lrp5 signaling by sclerostin

•  Activation of the the Wnt/-catenin pathway in osteocytes occurs via a concerted mechanism.

•  Mechanical loading increases nitric oxide (NO) production as well as activates focal adhesion kinase (FAK) and the Akt signaling pathway, which results in β-catenin stabilization, followed by β-catenin translocation to the nucleus, and expression of β-catenin target genes such as CD44, connexin 43, cyclin Dd1, and c-fos (Santos et al., 2010).

Mechanical loading regulates sclerostin expression in osteocytes

•  Propagation of this signal occurs after induction of Wnt production by mechanical loading, which results in re-activation of the Wnt/-catenin signalling pathway (Santos et al., 2009)

•  Position of the osteocytes can affect production of sclerostin. Osteocytes close to the surface (probably more intense mechanical stimulation) mostly sclerostin-negative while osteocytes deeper in the tissue mostly sclerostin-positive

•  Osteocytes in the close proximity to an area of bone formation are also mostly sclerostin-negative (Poole et al., 2005), suggesting that not only new bone formation depends on sclerostin distribution, but also bone formation during remodeling might be dependent on the local position of sclerostin producing osteocytes

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Thanks to: Mel Bacabac Astrid Bakker Ton Bronckers Elisabeth Burger Steve Cowin Vanessa da Silva Jesus Delgado-Calle Vincent Everts Rik Huisvkes Richard Jaspers Petra Juffer Rishikesh Kulkarni Fred MacKintosh

Daisuke Mizuno Peter Nijweide Janice Overman Henk-Jan Prins Ronald Ruimerman Ana Santos Christoph Schmidt Cor Semeins Theo Smit Djien Tan Aviral Vatsa Marjoleine Willems