Over osteocyten, de klinische relevantie, Presentatie vanProf. dr. J. Klein Nulend op 18 november 2011 voor de Stichting IWO.

1. OSTEOCYTES:
CLINICAL RELEVANCE
Jenneke Klein-Nulend

2. Vraag 1.
Wat zijn osteocyten, en wat is
hun functie?

3. Bone structure
is mechanically
meaningful

4. Disuse leads to bone loss

5. MICROGRAVITY
Environment of
unloading leads
to bone loss

6. Severe spinal osteoporosis

7. Age-related bone loss
Man, 42 yr Man, 90 yr
Mosekilde, 1990

8. Mechanical adaptation
bones adapt their mass and
structure to the loading
conditions to optimize their
load bearing capacity
Julius Wolff
(1892) Use it or lose it

9. OSTEOCYTES, MECHANOTRANSDUCTION,
AND BONE REMODELING

10. Mechanical adaptation
external load
local bone mass and
mechanical architecture
signal
stimulus
osteocytes
effector
cells

11. Antwoord op Vraag 1.
Wat zijn osteocyten, en wat
is hun functie?

12. Vraag 2.
Hoeveel % van alle botcellen is
osteocyt?
Waar liggen osteocyten in bot, en
waarom is hun lokatie in bot
belangrijk?

13. 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.

14. Vraag 3.
Welke signaalfactoren worden
door osteocyten uitgescheiden?

15. Antwoord op vraag 3.
Osteocyten scheiden o.a. RANK-L af.

16. Vraag 4.
Wat is de relatie tussen
botadaptatie en botremodellering
in volwassen bot?

17. Poliomyelitis and bone
remodeling
loaded unloaded
Haversian and Volkmann channels

18. MECHANICAL ADAPTATION
relates to:
• Bone MASS
how much/how little bone
• Bone ALIGNMENT
orientation along principal loading
directions
IN ADULT HUMAN BONE ADAPTATION
OCCURS DURING REMODELING

19. Antwoord op vraag 4.
In volwassen humaan bot treedt
botadaptatie op tijdens het proces van
botremodellering.

20. OSTEOCYTES, MECHANOTRANSDUCTION,
AND BONE REMODELING

21. Hypothesis
REMODELING IS GUIDED
BY (DAILY) LOADING

22. Loaded remodeling osteon
Finite element model Equivalent strain
distribution
Smit et al. J Bone Min Res 17, 2000

23. 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.

24. Computer simulation of bone remodeling
osteoclasts in
cutting cone reversal
zone
bone forming
osteoblasts
closing
cone
marrow
new
bone old
bone
Ruimerman et al. J Biomech 38, 2005

25. Computer simulation model
Mechanical signal
10 MPa
1 Hz
2x2 mm2
OCY density 1600 mm-2 Ruimerman et al.
3 osteoclasts J Biomech 38, 2005

26. Vraag 5.
Wat gebeurt er met de richting
van de “cutting cone” als de
richting van de belasting
verandert?

27. Loading direction
30°
Rotated load
Ruimerman et al.
J Biomech 38, 2005

28. Antwoord op vraag 5.
De richting van de “cutting cone”
verandert mee met de belastingsrichting.

29. Loading magnitude 20% Reduced load 20% Increased load
No load
Ruimerman et al.
J Biomech 38, 2005

30. Conclusion
DAILY LOADING EXPLAINS
BONE TUNNELING
● loading direction: orientation of the tunnel
● loading magnitude: amount of refilling

31. THE BONE MECHANOSENSORY SYSTEM
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

32. CANALICULAR
load FLUID FLOW
ocy ocy lc
ocy: osteocyte
lc: lining cell
flow

33. OSTEOCYTES OSTEOBLASTS PERIOSTEAL
FIBROBLASTS

34. APPLICATION
OF
FLUID FLOW

35. Fluid flow stimulates PGE2 release
OCY OB PF
PFF PFF PFF
PGE2, pg/ µg DNA
Con 1500 Con
1500 Con 1500
1000 1000 1000
500 500 500
0 0 0
0 15 30 45 60 0 15 30 45 60 0 15 30 45 60
time (min) time (min) time (min)
Osteocytes (OCY) release more PGE2 than
osteoblasts (OB) and fibroblasts (PF)
Ajubi et al. BBRC 225, 1996

36. Fluid flow stimulates NO release
by osteocytes
osteocytes fibroblasts
PFF PFF
NO2 nM/103 cells
NO2 nM/103 cells
240 con 240 con
120 120
0 15 30 45 0 15 30 45
minutes minutes
Klein-Nulend et al. BBRC 217, 1995

37. Intercellular communication
Fluid flow-stimulated osteocytes:
Ÿ inhibit osteoclastogenesis via the
release of soluble factors, resulting
in decreased bone resorption.
Ÿ produce soluble factors that
modulate proliferation and
differentiation of osteoblasts.

38. LOADING
LOADING

39. LOADING
NO
LOADING

40. LOADING
NO
TREDMILL?
NO NO
LOADING

41. LOADING
PGE2:
OSTEOBLAST
RECRUITMENT?
LOADING

42. LOADING
OSTEO-
BLASTS
LOADING

43. ADAPTIVE BONE REMODELING

44. ADAPTIVE BONE REMODELING

45. Sclerostin
and
Van Buchem Disease (VBD)
“Mineralized Tissues in Oral and Craniofacial Science: Biological Principles and Clinical Correlates”

46. • Genetic background: SOST gene
• Its product sclerostin
• The clinical features caused by SOST
mutations - Van Buchem Disease
• Therapeutic possibilities

47. 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

48. 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)

49. Pedigree of the 13 VBD patients

50. Characteristic Features
Protruding chin
High forehead
Thickened naseal area
Facial nerve paralysis
Van Hul et al., 1998

51. 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

52. 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 theMoester et al., 2010
age of 54 years

53. 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

54. Sclerostin in adult tissue
Postnatally in osteocytes, mineralized hypertrophic
chondrocytes and cementocytes
Osteocytes Mineralized hypertrophic chondrocytes
Cementocytes
Van Bezooijen et al., 2009

55. Sclerostin in adult tissue
• Osteocyte-derived secreted protein,
• High sclerostin levels in lacunar-canalicular network
Winkler et al., 2003

56. Expression in Van Buchem Disease
In VBD patients none of
these cell types express
sclerostin
Winkler et al., 2003
Van Bezooijen et al., 2009
Increased osteoid
surface and lamellar
bone
Active osteoblasts
Van Bezooijen et al., 2009

57. 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

58. Sclerostin as bone inhibitor:BMP-7
Inhibition of BMP/Smad signaling by blocking
intracellular BMP7 secretion in osteocytes
Krause et al., 2010

59. 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)

60. Van Buchem Disease - Clinical features
- Thickened skull
- Thickened
mandible,
elongation and
deformity
- Diaphyseal cortex
of long bones à
narrowed
medullary cavities
- Spine
- Pelvic bone

61. Clinical features-general
• 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

62. Hyperostosis skull: nerve entrapment

63. Clinical features Van Buchem Disease -
general
• 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)

64. 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)

65. 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

66. 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

67. 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

68. 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

69. Surgical removal
BEFORE
AFTER
Schendel, 198

70. 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

71. Glucocorticoid inhibit bone formation
by stimulating sclerostin
VBD
• Preventing activation of bone lining cells
• Inactivation of active osteoblasts

72. 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)

73. 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

77. 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).

78. 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

79. .020 .00004 .0035
Relative gene expression 24
Control
23 Hip Frx
22 Hip OA
21
20
2 -1
2 -2
SOST FGF23 PHEX

80. Thanks to:
Mel Bacabac Daisuke Mizuno
Astrid Bakker Peter Nijweide
Ton Bronckers Janice Overman
Elisabeth Burger Henk-Jan Prins
Steve Cowin Ronald Ruimerman
Vanessa da Silva Ana Santos
Jesus Delgado-Calle Christoph Schmidt
Vincent Everts Cor Semeins
Rik Huisvkes Theo Smit
Richard Jaspers Djien Tan
Petra Juffer Aviral Vatsa
Rishikesh Kulkarni Marjoleine Willems
Fred MacKintosh