Osteocytes in Health and Disease

Nigel LoveridgeBone Research Group Cambridge

With the very grateful help of:Andy Pitsillides, Ken Poole, Brendon Noble,

Jonathan Reeve, Mitch Schaffler

OsteocytesOsteocytes

Entombed osteoblasts

Canalicular system acts as a syncitium with connections to the bone surface

Important as mechano-sensors

Detectors of matrix damage

Bone Remodelling CycleBone Remodelling Cycle

Osteocytes

Lynda Bonewald Sun Valley symposium 2005

OsteocytogenesisOsteocytogenesis

Diagrammatic representation of the possible stages on osteoblast differentiation1. Preosteoblast, 2. Preosteoblastic osteoblast, 3. Osteoblast, 4. Type 1 preosteocyte (osteoblastic osteocyte) 5. Type II preosteocyte (osteoid-osteocyte) 6. Type III preosteocyte, 7. Young osteocyte, 8. Old osteocyte.

Franz-Odenaal et al 2006

Possible Mechanisms for OsteocytogenesisPossible Mechanisms for Osteocytogenesis

A: Osteoblasts secrete matrix in all directions.

B: Each osteoblast is polarized in a different direction but stillsecretes matrix in one direction only.

C: One generation of osteoblasts buries the next generation.

D: The osteoblast to be embedded slows down its matrix production compared to neighbouring osteoblasts.

E: Matrix secretion does not embed cells.

Possible Mediators of OsteocytogenesisPossible Mediators of Osteocytogenesis

TGFβ and the Smad Pathway

Dental Matrix Protein I

Sclerostin

TGFβ and the Smad Pathway

Dental Matrix Protein I

Sclerostin

TGFβ and osteocytogenesisTGFβ and osteocytogenesis

Note the lack of staining in a mature osteocyte (arrow) and an osteoblast in the process of being embedded (arrowhead)

DMP-1DMP-1DMP-1 exclusively expressed in osteocytesSeems to be involved in maintenance of the lacuno-

canalicular system.Is reportedly responsive to loading (up-regulated)

SclerostinSclerostin

Sclerostin- secreted protein product of the SOST gene

Osteocyte specific and a powerful inhibitor of bone formation (BMP antagonist)

Demonstrated when it’s absence results in the high bone mass disorder sclerosteosis

Sclerostin & Bone FormationSclerostin & Bone Formation

Sclerostin/ Tol blue (G)

Control (H)

Alkaline phosphatase (I)

Double demeclocyline labels (J)

Distance from Closest Bone Surface

Sclerostin +ve

Sclerostin -ve

Wilcoxon p

Newly embedded osteocytes are sclerostin -ve

96.4 % of new osteocytes sclerostin negative within 16 days

Osteocytes and Bone HealthOsteocytes and Bone Health

Osteocytes required for bone health. In some cases osteocytes survive for many decades. In others osteocyte death is a cue for bone remodelling.

Osteocytes are considered to be the main mechano-sensors in bone.

Disruption of the canalicular system may result in cues to remodel damaged bone.

MicropetrosisMicropetrosis

Mineralised Osteocyte Lacunae

Mineralised Haversian

Canals

Courtesy Alan Boyde

Model : Rat Ulna Loading (Riggs/Lanyon Model)

ULNA olecranonprocessflexed carpus

radius humerus

ACTUATOR

Physiological Loading:• Short daily period of dynamic loading :

1200 cycles at 2 Hz; peak strain = 4000µε

For 10 days

Tension +

Compression -

Supra-Physiological Loading:• Short daily period of dynamic loading :

Physiological LoadingPhysiological Loading

A:- Loaded bone

B:- Contralateral control

Note the increased double calcein labelling seen in the loaded bone

Noble et al Am J Physiol 284:C934, 2003

NO and Bone TurnoverNO and Bone Turnover

NO inhibits bone resorption and stimulates bone formation

NO released in response to load

eNOS predominant isoform in normal bone, especially osteocytes

NO inhibits bone resorption and stimulates bone formation

NO released in response to load

eNOS predominant isoform in normal bone, especially osteocytes

Repeated episodes of bone loading in long-term culture:NO release increased during, but not after, loading

200

160

180

140

80

120

100

*

**

**

5 min post load

24 hrs post load

NO

Con

cent

ratio

n(%

con

trol)

0 (1st) 24 (2nd) 48 (3rd) 72 (4th)

Time (hrs) prior to load (no. of loading episodes)

Andy Pitsillides & colleagues

eNOS expression

mRNA

Protein

Andy Pitsillides & colleagues

Osteocytes release more NO than osteoblasts in response to mechanical strain in vitro7

6

5

4

3

2

1

0

Nitr

ite C

onc.

(µm

)

0

2

4

6

8

10

12 Strain-induced N

O release

(pm/ hr/ cell)

ControlStrain*

*

**

O’blastsOsteocytes O’cytesOsteoblasts

Relationship between loading-induced increases in G6PD activity and NO release

100

80

60

40

20

0

-205 15 25 35 45 55

R2 = 0.984P< 0.05

% in

crea

se in

NO

rele

ase

Egg typeWild typeMeat type

% increase in G6PD activity

Types of Cell DeathTypes of Cell Death

Necrosis: occurs in large areas of tissue and often provokes an inflammatory response. An example of this is avascular necrosis (osteonecrosis) where the blood supply fails.

Apoptosis: Is focal and does not provoke an inflammatory response. Can be either active through death receptors or passive through lack of cell survival agents.

Necrosis: occurs in large areas of tissue and often provokes an inflammatory response. An example of this is avascular necrosis (osteonecrosis) where the blood supply fails.

Apoptosis: Is focal and does not provoke an inflammatory response. Can be either active through death receptors or passive through lack of cell survival agents.

Physiological LoadingPhysiological LoadingNumber of apoptotic osteocytes with fragmented DNA

0

2.5

5

7.5

10

0 1000 2000 3000 4000 50000

1

2

3mean % apoptotic osteocytes

normal loaded

Peak strain magnitude

Noble et al Am J Physiol 284:C934, 2003

Supra-Physiological LoadingRat ulna: 10 days after fatigueRat ulna: 10 days after fatigue Rat ulna: ControlRat ulna: Control

Noble et al Am J Physiol 284:C934, 2003

Supra-physiologicalSupra-physiological

A:- Control bone

B:- Overloaded

C:- High power

A:- Control bone

B:- Overloaded

C:- High power

Noble et al Am J Physiol 284:C934, 2003

Supra-physiological loadingSupra-physiological loading

0

10

20

30

40

CONTROL LOADED

% osteocytes with fragmented DNA

0

10

20

30

40

CONTROL LOADED

% osteocytes with fragmented DNA

14 Days7 Days

Noble et al Am J Physiol 284:C934, 2003

Verborgt Verborgt et al, 2000et al, 2000

0

200

400

600

800

#/m

m2

(-)-M

dx

(+)-M

dx

E.la

c-M

dx

(-)-N

oMdx

(+)-N

oMdx

E.la

c-N

oMD

x

(-)-C

tl

(+)-C

tls

E.la

c-C

tl

Non-loaded control bone

Fatigued bone, away from Mdx

Fatigued bone, near Mdx

*

*

Apoptotic osteocytes associated with Apoptotic osteocytes associated with microcracks microcracks ((MdxMdx))

(-) = No TUNEL Staining(+) = TUNEL positive cellE.lac = Empty lacunae/

TUNEL positive debris

Apoptotic Apoptotic osteocytes osteocytes at at resorption resorption spacesspaces

RsSp

0

200

400

600

800

#/m

m2

(-) (+)

E.la

c

(-) (+) E

.lac

Nonloaded controlbone (no RsSp)

Fatigued bone,away from RsSp

Fatigued bone,near RsSp

*

*

(-) (+)

E.la

c

(-) = No TUNEL Staining(+) = TUNEL positive cellE.lac = Empty lacunae/

TUNEL positive debris

Osteocytes and Bone DiseaseOsteocytes and Bone DiseaseDunstan et al (1993) CTI 53:S113-S117

Post-menopausal Osteoporosis

Hip Fracture

Sclerosteosis

Osteoarthritis

Oestradiol changes during GnRH therapy

0

250

500

750

1000

1250

Oes

trad

iol l

evel

s (p

mol

/l)0 5 10 15 20 25

Time (Weeks)

11

10

9

8

7

6

5

4

3

2

1

Patient number

Tomkinson et al J Clin Endocrinol Metab 1997

Percentage of apoptotic osteocytes before and after treatment with GnRH analogue

p=0.008

0

5

10

15

20

%os

teoc

ytes

with

fr

agm

ente

d D

NA

Pre Post

Treatment

0

5

10

15

20

% o

steo

cyte

s w

ithfr

agm

ente

d D

NA

Pre Post

Treatment

Tomkinson et al J Clin Endocrinol Metab 1997

Ovariectomy in Rats

ShamOvxOvx + E2

2.5

5.0

7.5

10.0

12.5

Morphology Nick Translation

% Apoptosis

Tomkinson, et al J Bone Miner Res. 13:1243-50 (1998).

Hip Fracture Incidence Forecast in European Community

0100200300400500600700800900

1000

in th

ousa

nds

MenWomen

2000 2010 2020 2030 2040 2050

Density of eNOS+ve OsteocytesDensity of eNOS+ve Osteocytes

0

50

100

150

200 eNOS+ve osteocytes (no/mm2)

Inferior Superior

p=0.17

p=0.0004

p=0.0004

Control

Case

Location of eNOS+ve Osteocytes (Minimum)

Location of eNOS+ve Osteocytes (Minimum)

Inferior Region Superior Region

0

10

20

30

40

50 Distance from canal surface (µm)

0

10

20

30

40

50

60 Distance from canal surface (µm)

NOS -ve

NOS +ve

Case ControlControl Case

Superior aspect

Inferior aspectFormation of giant canals

Do not resorb

NONONO

NONO

eNOS positive

eNOS negative

CaseControl

SclerosteosisSclerosteosis

• Rare autosomal-recessive disease • Systemic skeletal syndrome bone mass• Markedly increased bone formation • Afrikaner population of South Africa • Clinically:

hyperostosis BMDnarrowing of skull syndactylycranial nerve compression tall stature nail dysplasia headachesfacial palsy strong teethhearing loss ICP

cortical thickness and cancellous bone volumebone formation rate

Sclerosteosis Sclerosteosis

• Stylomastoid foramen

Sclerosteosis Radiology: HandsSclerosteosis Radiology: Hands

SclerosteosisNormal Hand

Bone Histology in SclerosteosisBone Histology in Sclerosteosis

Hip OA and Femoral Neck Fracture

Femoral neck fracture is uncommon hip OA and it is has been suggested that hip OA offers protection in the form of increased cancellous bone strength.

In osteoporosis (OP) there is decreased bone mass whereas in hip OA bone mass is increased

In hip OA there is increased bone formation at more sites than in controls

Is this related to a decreased sclerostin expression

Comparison of Percentage Ct.Ar. and Cn.Ar.for all Biopsies

0

5

10

15

20

25

30

*%

Cortex Cancellous

* P < 0.01ControlOA Jordan et al ASBMR 1999

Sclerostin in active and inactive osteonsSclerostin in active and inactive osteons

Used adjacent ALP sections to mark forming (red) and quiescent (yellow) for analysis on the polarized light image.

For each subject, 10 forming and 10 quiescent osteons were measured. (where possible)

Bone surface undergoing active formationBone surface undergoing active formationNo difference in osteocyte density between OA and control groups.

In osteons undergoing bone formation density of sclerostin +ve osteocytes was higher:

OA: 229 mm2 ± 58 Control; 373 mm2 ± 56, p=0.02

No difference in density of sclerostin +ve osteocytes within quiescent osteons

% sclerostin +ve osteocytes within osteons undergoing bone formation higher in OA:

OA: 49.1% ± 7.0 Control: 74.2% ± 7.1 p= 0.01

Mean distance of sclerostin +ve osteocytes from the bone surface was higher in OA:OA: 64 µm ±5 Control: 48 µm ±5 p=0.024

Does the decreased sclerostin allow greater bone formation?

Does the decreased sclerostin allow greater bone formation?

Control osteon OA osteon

Sclerostin -ve Sclerostin +ve

SummarySummaryOsteocytes are embedded osteoblasts. Changing the rate of osteocytogenesis will have effects on bone formation

Osteocytes are important regulators of bone turnover especially in relation to mechanical loading & damage repair. It is possible that all bone turnover is regulated by the osteocyte network.

Failures in osteocyte activity may be responsible for some musculo-skeletal diseases

Osteocytes are embedded osteoblasts. Changing the rate of osteocytogenesis will have effects on bone formation

Osteocytes are important regulators of bone turnover especially in relation to mechanical loading & damage repair. It is possible that all bone turnover is regulated by the osteocyte network.

Failures in osteocyte activity may be responsible for some musculo-skeletal diseases

And now for a break to allow the brain to recover

And now for a break to allow the brain to recover

QuickTime™ and aYUV420 codec decompressor

are needed to see this picture.

Osteocytes in Health and DiseaseOsteocytesBone Remodelling CycleOsteocytogenesisPossible Mechanisms for OsteocytogenesisPossible Mediators of OsteocytogenesisTGF and osteocytogenesisDMP-1SclerostinSclerostin & Bone FormationOsteocytes and Bone HealthMicropetrosisPhysiological LoadingNO and Bone TurnoverTypes of Cell DeathPhysiological LoadingSupra-physiologicalSupra-physiological loadingOsteocytes and Bone DiseaseHip Fracture Incidence Forecast in European CommunityDensity of eNOS+ve OsteocytesLocation of eNOS+ve Osteocytes (Minimum)SclerosteosisSclerosteosisSclerosteosis Radiology: HandsBone Histology in SclerosteosisSclerostin in active and inactive osteonsBone surface undergoing active formationDoes the decreased sclerostin allow greater bone formation?SummaryAnd now for a break to allow the brain to recover