Introduction

CHAPTER 3
ANALYSIS OF BONE
REMODELING

Remember:
Remodeling occurs throughout
entire lifetime
Rate changes with age, location,
stress/strain

Most bone tissue in adults was

produced by remodeling

The A-R-F Sequence

The A-R-F Sequence

3 major stages of a BMU as it moves

through a cross-section
F
R
A

Activation - Resorption - Formation

formation

resorption

Activation

A-R-F +

Double BMU activation

The six phases of an osteon's lifetime

Originating
Haversian canal

Activation site

Quiescence

Formation

Activation
Resorption
Reversal

Mineralization

The A-R-F Sequence

The A-R-F Sequence

Six phases
1. activation: bone lining cells @ point of origin
retract from surface
attract osteoclasts
2. resorption: by osteoclasts (cutting cone)
advance ~ 40 m/day
~200 m
3. reversal: osteoblasts
recruited
~300 m
cement line formed

4. formation: osteoblasts form osteoid

Osteonal age

The A-R-F Sequence

shade = f(calcification)

6. quiescence: osteoblasts

rapidly at first, then gradually decreasing rate

average about 1-2 m/day

5. mineralization: hydroxyapatite

mineralization lag time ~ 10 days

primary mineralization: ~60% in few days
secondary mineralization: decreasing rate, ~6
months to complete
significance: new osteons have less mineral
than mature osteons - visible by back-scatter
SEM

bone lining cells

(BLC's)

resting
can be reactivated
not 1o cells used to repair fractures

Remodeling Cycle Duration

Histomorphometric Measurement
of Osteonal Remodeling

Times to complete
resorption: 3 weeks
reversal: 1-2 weeks
formation: 3 months

Technique
"fluorochrome label"
injected IV into living animal
binds to mineralizing bone
fluoresces in UV light

Frost called this cycle duration sigma ()

Significance: determines how fast bone
properties can be changed at a particular site

label administered twice

TL = time between labels, typically 10 days
cortical bone cross-sections ~ 100 m thick
"undemineralized" (so labels not lost)
assumes remodeling is in steady state

e.g, tetracycline (common antibiotic)

Measurement of Osteonal Remodeling

Measurement of Osteonal Remodeling

Measurements
1. Section area: results express per mm2 bone
2. Count resorption spaces (R's), NR, #/mm2
3. Perimeters of R's, mean = SR, mm
4. Count refilling BMUs (F's), NF
R
have osteoid seams
5. Perimeters of F's, mean = SF, m

Measurements
6. Distance between labels
doubly labeled BMUs
F,D
mean = DL, m
7. Mean wall thickness, tmw, m
completed osteons (no osteoid)
8. Osteoid seam thickness, tos, m

Never as clean in practice

Measurement of Osteonal Remodeling

F,S
F

F,D
C R
F
C

F
R
F,D

C F,D
C
F,D

F,S

Measurement of Osteonal Remodeling

Calculating BMU-Level results
mineral apposition rate: MF = DL / TL (m/day)
"mineral" because label lies at mineralization
front
in steady state = osteoid apposition rate
mineralization lag time: Tml = tos /MF (days)
time for osteoid to start mineralizing
BMU refilling time: TF = tmw / MF (days)
try to measure tmw in recently-formed osteons

Measurement of Osteonal Remodeling

Measurement of Osteonal Remodeling

Calculating BMU-Level results, cont'd.

BMU activation frequency
highly dependent on steady state condition
assume # BMUs activated/day = # starting
to refill
use a fundamental population relationship:

Calculating Tissue-Level results

Bone formation rate, VF
fractional amount of new bone made
per unit time

Population = Birthrate x Mean Lifetime

apply to refilling BMUs: NF = fa TF or

fa = NF / TF

BMUs/mm2/day

Measurement of Osteonal Remodeling

Example:

NF = 100/mm2
TL = 10 days
tos = 10 m

SF = 200 m
DL = 10 m
tmw = 100 m

appos. rate
MF = DL/TL = 10/10 = 1 m/day
mineral. lag time Tml = tos/MF = 10/1 = 10 days
refilling time
TF = tmw/MF = 100/1 = 100 days
activation freq. fa = NF/TF = 100/100 = 1 /mm2/day
bone form. rate VF = 0.365x10-3 MF SF fa
VF = 0.365x10-3 (1)(200)(1) = 0.073 mm2/mm2/yr

FYI: REMODELING MEASUREMENTS

ASSUMPTIONS

double fluorochrome labels, TL days apart

osteoid stained
A-R-F sequence occurs in parallel osteons
steady state!!!

MEASUREMENTS (start with section area)

1. # resorp. sp./area, NR
4. refill. perimeter, SF
2. resorp. perimeter, SR
5. inter-label dist., DL
3. # refill. BMUs/area, NF
6. wall thick., tmw
7. osteoid seam thickness, tos

VF = 0.365 x 10-3 MF SF NF

mm2/mm2/yr

mm3/mm3/yr

or
(volume fraction = area
fraction)
approx. same as bone turnover rate

Osteonal Remodeling Data

Table 3.1 Bone remodeling data for rib
Variable
Age, yrs
NF, #/mm2
NR, #/mm2
SF, m
MF, m/d
TF, d
fa, #/yr/mm2
tmw, m
VF, %/yr

Dogs

Rhesus
Monkeys
1.5-3.5 Adolescent
3.1+1.8
4.0+1.6
0.58+0.03 1.96+1.15
120+30
200+30
0.95+.26 1.3+.23
117+ 110 54+11
16+11
30+13
57+6
63+2
11.4+9.4 36+19

Humans
1-9
30-39
2.7+1.0 0.21+0.54
2.0+0.6 0.25+0.33
280+40 250+140
1.4+.46 1.1+.36
54+47 73+58
19+9
1.1+0.5
68+4
70+5
38+24 1.8+0.6

70-89
0.66+0.50
0.84+0.46
300+72
0.72+.20
109+55
2.2+1.1
66+7
4.4+1.5

FYI: REMODELING MEASUREMENTS

CALCULATIONS
mineral apposition rate
MF = DL/TL, [micrometers/day]
mineralization lag time
Tml = tos/MF, [days]
refilling period
TF = tmw/MF, [days]
BMU activation frequency or birth rate
NF = fa TF, fa = NF/TF, [BMU/mm2/day]

FYI: REMODELING MEASUREMENTS

TISSUE-LEVEL RESULTS

FYI: REMODELING MEASUREMENTS

LABEL ESCAPE ERROR

bone formation rate

VF = 0.365 x 10-3 MF SF NF,
[mm2/mm2/yr]
MF SF
If you know the rate of change of porosity
dp/dt = VR - VF
then you can calc. MR from
VR = 0.365 x 10-3 MR SR NR, [mm2/mm2/yr]

some BMUs only have one label because

they completed refilling before 2nd label was
given
they started refilling after the first label was
given
ratio of single to double labeled BMUs
N1L/N2L = 2 TL / (TF - TL)
true no. of refilling BMUs
NF = N1L + N2L

Bone Turnover

Remodeling Details

Remodeling turns over bone tissue

remove old bone, replace with new bone
osteonal Basic Multicellular Unit (BMU)

Activation Theories
Bone lining cells (Rodan)

formation

reversal

resorption

Activation
Activation Theories
Hormonal direct effect
increased bone turnover
parathyroid hormone
glucocorticoids

decreased bone turnover

vitamin D [1,25-(OH)2-vitamin D3]
calcitonin

bulk of evidence from cell culture expts.

eliminates structural signals

receptors -- PTH, Vit D3

change shape
expose bone
cAMP
release prostaglandins
cytologic loop

But: elegance is not evidence

Activation
Activation is Rate-Limiting Step
Better correlation between bone
turnover volume and activation
frequency than any other factor
Rate of remodeling cascade invariant

Resorption

Resorption

Osteoclasts -- mobile, multinucleated,

cousins of macrophages
attach
peripheral seal -- brush border
enzymatic dissolution of mineral and
collagen

Trabecular bone -- osteoclasts roam

over an area > contact area
Cortical bone -- tunnel straight ahead
in osteons
Reason / controlling factors unknown

Resorption

Resorption
Osteoclast lifespan difficult to
determine
evolving syncytium
nuclei enter and leave (or die)
nuclear lifespan (canine) ~11 days

Osteoclast speed
longitudinal -- 39 14 m/day
indirect, from labeling expts.
radial -- ~12 m/day
while opening canal

Reversal
Mononuclear phagocytes
?? old or recycled osteoclasts
related to circulating phagocytes
?? inflammation-remodeling link
?? RAP
may form cement line, prepare walls for
osteoid

Formation
also called Refilling

Osteoblasts -- mononuclear, immobile

marrow origin
not evolved from osteoclasts
attracted by disassembled collagen
dont survive well in bone marrow
transplants

Formation

Formation

Osteoblasts require mechanical loading

for complete differentiation
differentiation takes time -- ~2.5 days

Osteoblasts remain stationary as cutting

cone moves forward
begin cuboidal, covering 15 m2 of
bone surface
become shorter, flatter as refilling
progresses
bone lining cells cover 300 m2

2.5 days x 40 m/day = 100 m = approx.

length of reversal region

20X surface area only 5% survive

Formation

Mineralization

Single generation of osteoblasts

required to live for the entire
refilling period, ~3 months
cant explain lamellar layers
Multiple generations
? responsible for lamellar layers
consistent layer thickness and
different collagen orientation
unexplained

Follows deposition of unmineralized

matrix = osteoid
Mineralization time lag

Quiescence

Long-Term Remodeling Effects

Inactive period
osteoclasts disappear
osteoblasts become bone lining cells
Indeterminate duration
activation is a semi-random process
function of time, damage
accumulation, proximate injury,
metabolic activity

Skeletal Aging

~10 days

Primary mineralization
60% of final mineral amount
occurs within a few days

Secondary mineralization
occurs over 6 month period

Properties change as mineralization

Act Freq, No./mm2/yr

Activation frequency
varies with age (rib )
Childrens bone rapidly
turned over
softer strain
turnover

100

10

1
0

20

40

Age, yr

60

80

Long-Term Remodeling Effects

Long-Term Remodeling Effects

Osteonal Density

Osteonal Density

Limited by overlap -- new osteons

remove older ones
Activation location -- assumed random
Directional control -- principal stress
and/or material properties (easiest
tunnel)

Models built upon assumptions

Increased osteonal density with age
to a limited degree, f(osteonal size)

Age determinable by osteonal density

or percent of residual primary bone
good experimental correlation
archeological significance

(Kerley, 1965)