Properties

production

and

applications

of

camelid

single-domain

antibody

fragments

Camelids

produce

functional

antibodies

devoid

of

light

chains

of

which

the

single

N-terminal

domain

is

fully

capable

of

antigen

binding

These

single-domain

antibody

fragments

-LRB-

VHHs

or

Nanobodies

-RRB-

have

several

advantages

for

biotechnological

applications

They

are

well

expressed

in

microorganisms

and

have

a

high

stability

and

solubility

Furthermore

they

are

well

suited

for

construction

of

larger

molecules

and

selection

systems

such

as

phage

yeast

or

ribosome

display

This

minireview

offers

an

overview

of

-LRB-

-RRB-

their

properties

as

compared

to

conventional

antibodies

-LRB-

-RRB-

their

production

in

microorganisms

with

a

focus

on

yeasts

and

-LRB-

-RRB-

their

therapeutic

applications

Introduction

The

field

of

recombinant

antibody

technology

has

rapidly

progressed

during

the

last

two

decades

mainly

because

of

the

interest

in

their

human

therapeutic

use

The

ability

to

select

specific

human

antibodies

by

display

technologies

and

to

improve

their

affinity

stability

and

expression

level

by

molecular

evolution

has

further

boosted

the

field

Whole

antibodies

are

complex

molecules

that

consist

of

heavy

and

light

chains

-LRB-

Fig.

-RRB-

They

contain

an

N-linked

oligosaccharide

attached

to

the

second

heavy-chain

constant

domain

-LRB-

-RRB-

that

is

essential

for

antibody

effector

functions

such

as

antibody-dependent

cellular

cytotoxicity

-LRB-

ADCC

-RRB-

complement-dependent

cytolysis

-LRB-

CDC

-RRB-

and

for

retaining

a

long

serum

half-life

Fig.

diagram

of

conventional

-LRB-

a

-RRB-

and

heavy-chain

-LRB-

b

-RRB-

antibodies

and

fragments

thereof

Variable

domains

derived

from

the

antibody

heavy

-LRB-

VH

-RRB-

and

light

-LRB-

VL

-RRB-

chains

are

shaded

dark

gray

and

light

gray

respectively

whereas

constant

domains

-LRB-

CH

and

CL

-RRB-

are

not

shaded

Note

the

absence

of

the

light

chain

and

domain

in

heavy-chain

antibodies

Antibody

domains

that

pair

by

noncovalent

interactions

are

indicated

by

overlaying

them

The

B-subunits

of

naturally

pentamerizing

toxins

that

are

used

to

generate

pentabodies

are

indicated

as

hatched

spheres

Although

isolated

antibody

heavy

-LRB-

Utsumi

and

Karush

-RRB-

and

light

chains

-LRB-

Yoo

et

al.

-RRB-

can

retain

antigen-binding

specificity

their

affinity

and

solubility

is

often

reduced

-LRB-

Ward

et

al.

-RRB-

However

the

paired

N-terminal

variable

domains

of

heavy

-LRB-

VH

-RRB-

and

light

-LRB-

VL

-RRB-

chains

are

sufficient

for

antigen

binding

-LRB-

Sundberg

and

Mariuzza

-RRB-

Such

antibody

fragments

can

be

produced

as

monovalent

antibody

fragment

-LRB-

Fab

-RRB-

or

as

single-chain

Fv

-LRB-

scFv

-RRB-

where

the

VH

and

VL

domains

are

joined

by

a

polypeptide

linker

-LRB-

Fig.

-RRB-

Their

production

in

microbial

cells

is

often

cumbersome

especially

when

producing

multivalent

formats

because

of

the

requirement

for

domain

association

The

discovery

that

camelids

-LRB-

bactrian

camels

dromedaries

and

llamas

-RRB-

produce

functional

antibodies

devoid

of

light

chains

-LRB-

Hamers-Casterman

et

al.

-RRB-

formed

a

further

breakthrough

because

their

single

N-terminal

domain

-LRB-

VHH

also

referred

to

as

Nanobody

-RRB-

binds

antigen

without

requiring

domain

pairing

These

heavy-chain

antibodies

also

lack

the

domain

which

in

a

conventional

antibody

associates

with

the

light

chain

and

to

a

lesser

degree

interacts

with

the

VH

domain

-LRB-

Fig.

-RRB-

Although

single-domain

antibodies

later

were

also

identified

in

particular

cartilaginous

fish

-LRB-

Greenberg

et

al.

-RRB-

most

research

on

the

biotechnological

application

of

single-domain

antibodies

was

done

using

camelids

because

of

their

ease

of

handling

including

immunization

Methods

to

isolate

antigen-specific

VHHs

from

immune

-LRB-

Arbabi-Ghahroudi

et

al.

Van

der

Linden

et

al.

-RRB-

nonimmune

-LRB-

Tanha

et

al.

Yau

et

al.

Verheesen

et

al.

-RRB-

or

semisynthetic

-LRB-

Goldman

et

al.

-RRB-

libraries

using

phage

yeast

or

ribosome

display

are

now

well

established

For

further

reading

on

these

topics

we

refer

to

recently

published

reviews

-LRB-

Muyldermans

Dufner

et

al.

-RRB-

Properties

Sequence

analysis

-LRB-

Hamers-Casterman

et

al.

Muyldermans

et

al.

Vu

et

al.

Harmsen

et

al.

-RRB-

and

elucidation

of

the

crystal

structure

-LRB-

Desmyter

et

al.

Spinelli

et

al.

-RRB-

has

revealed

several

structural

features

of

VHH

domains

Similar

to

conventional

VH

domains

VHHs

contain

four

framework

regions

-LRB-

FRs

-RRB-

that

form

the

core

structure

of

the

immunoglobulin

domain

and

three

complementarity-determining

regions

-LRB-

CDRs

-RRB-

that

are

involved

in

antigen

binding

This

contrasts

with

shark

single-domain

antibodies

that

have

a

vestigial

that

does

not

contribute

to

antigen

binding

-LRB-

Streltsov

et

al.

-RRB-

As

compared

to

human

VH

domains

the

VHH

FRs

show

a

high

sequence

homology

of

more

than

and

their

structures

can

be

superimposed

-LRB-

Muyldermans

et

al.

Holliger

and

Hudson

-RRB-

The

most

characteristic

feature

of

VHHs

is

the

presence

of

amino

acid

substitutions

at

four

positions

-LRB-

positions

and

Kabat

numbering

-RRB-

that

are

conserved

in

conventional

VH

domains

and

that

are

involved

in

forming

the

hydrophobic

interface

with

VL

domains

Occasionally

antigen-binding

single-domain

antibody

fragments

that

lack

these

characteristic

substitutions

are

isolated

from

camelids

These

fall

into

two

groups

The

low-affinity

binders

isolated

from

a

nonimmune

library

originated

from

conventional

antibodies

presumably

because

of

the

polymerase

chain

reaction

crossover

cloning

artifact

as

they

were

linked

to

the

domain

-LRB-

Tanha

et

al.

-RRB-

We

refer

to

these

as

VHH-like

conventional

VHs

However

such

single-domain

antibody

fragments

with

conventional-like

sequences

that

bind

antigen

with

high

affinity

are

isolated

from

immune

libraries

with

the

high

efficiency

of

about

-LRB-

Conrath

et

al.

Saerens

et

al.

Harmsen

et

al.

-RRB-

which

equals

their

presence

in

unselected

libraries

-LRB-

Harmsen

et

al.

-RRB-

This

is

not

expected

when

such

clones

originate

from

a

cloning

artifact

Unlike

the

clones

isolated

by

Tanha

et

al.

-LRB-

-RRB-

these

clones

often

contain

a

hydrophilic

residue

-LRB-

mostly

arginine

-RRB-

at

position

This

substitution

is

probably

important

for

their

single-domain

nature

-LRB-

Desmyter

et

al.

-RRB-

because

conventional

antibodies

contain

a

highly

conserved

hydrophobic

residue

-LRB-

tryptophan

-RRB-

at

this

position

that

contacts

VL

This

suggests

that

these

represent

functional

VHH

domains

derived

from

recombination

of

conventional

VH

gene

segments

with

heavy-chain

constant

gene

segments

during

B

cell

maturation

This

was

confirmed

by

the

absence

of

the

domain

when

such

VHH

domains

were

reisolated

from

the

original

immune

repertoire

using

a

primer

-LRB-

De

Haard

unpublished

observation

-RRB-

Therefore

we

refer

to

these

as

conventional-like

VHH

domains

Although

the

increased

hydrophilicity

of

VHHs

predominantly

relies

on

the

aforementioned

changes

in

the

former

VL

interface

some

amino

acids

at

positions

that

form

a

slightly

hydrophobic

patch

on

conventional

VH

domains

that

contacts

-LRB-

Lesk

and

Chothia

-RRB-

are

also

changed

into

hydrophilic

residues

in

VHHs

-LRB-

Muyldermans

et

al.

Harmsen

et

al.

-RRB-

Furthermore

the

CDRs

of

VHHs

contain

some

characteristic

features

Firstly

the

N-terminal

part

of

is

more

variable

-LRB-

Vu

et

al.

Harmsen

et

al.

Nguyen

et

al.

-RRB-

Secondly

many

dromedary

VHHs

have

an

extended

that

is

often

stabilized

by

an

additional

disulfide

bond

with

a

cysteine

in

or

-LRB-

Muyldermans

et

al.

-RRB-

resulting

in

the

folding

of

the

loop

across

the

former

VL

interface

-LRB-

Desmyter

et

al.

-RRB-

A

particular

subfamily

of

llama

VHHs

-LRB-

-RRB-

also

contains

an

extended

that

is

stabilized

by

an

additional

disulfide

bond

with

a

cysteine

at

position

in

However

VHHs

of

this

subfamily

are

rarely

isolated

and

most

llama

VHHs

have

loops

similar

in

length

to

those

found

in

human

VHs

VHHs

have

many

advantages

for

biotechnological

applications

which

are

summarized

in

Table

An

important

advantage

is

their

high

microbial

production

level

-LRB-

see

next

section

-RRB-

Table

of

camelid

single-domain

antibody

fragments

as

compared

to

conventional

antibody

fragmentsAdvantageMolecular

basisFacile

genetic

manipulationSingle-domain

natureIncreased

functional

size

of

immune

librariesNo

decrease

in

library

size

because

of

reshuffling

of

VL

and

VH

domainsFacile

production

of

multivalent

formatsMore

flexible

linker

design

and

no

mispairing

of

VL

and

VH

domainsFacile

production

of

oligoclonal

preparations

from

single

cellsNo

mispairing

of

VL

and

VH

domainsHigh

physicochemical

stabilityEfficient

refolding

due

to

increased

hydrophilicity

and

single-domain

natureHigh

solubilityIncreased

hydrophilicityRecognition

of

hidden

antigenic

sitesSmall

size

and

extended

flexible

tissue

penetration

fast

clearanceSmall

sizeWell

expressedEfficient

folding

due

to

increased

hydrophilicity

and

single-domain

natureSee

text

for

references

Several

advantages

result

from

their

single

domain

nature

Thus

VHH

libraries

generated

from

immunized

camelids

retain

full

functional

diversity

This

contrasts

with

the

diminished

diversity

of

conventional

antibody

libraries

because

of

reshuffling

of

VL

and

VH

domains

during

library

construction

As

a

result

high-affinity

antigen-binding

VHHs

can

be

isolated

by

directly

screening

a

limited

number

of

clones

from

immune

libraries

without

prior

selection

using

display

technologies

-LRB-

Frenken

et

al.

Harmsen

et

al.

-RRB-

Furthermore

the

single-domain

nature

facilitates

subsequent

molecular

manipulation

For

example

for

many

applications

it

is

advantageous

to

engineer

monovalent

antibody

fragments

into

multivalent

formats

to

increase

functional

affinity

-LRB-

termed

avidity

-RRB-

or

to

produce

bispecific

antibody

fragments

that

can

simultaneously

bind

to

different

antigens

Such

molecules

-LRB-

diabodies

Fig.

-RRB-

can

be

produced

using

conventional

recombinant

antibodies

using

linkers

between

the

VH

and

VL

domains

of

a

specific

length

although

this

often

results

in

aggregation

and

reduced

affinity

because

of

mispairing

of

VH

and

VL

domains

-LRB-

Glockshuber

et

al.

Whitlow

et

al.

-RRB-

VHHs

are

more

suitable

for

production

of

such

formats

because

they

allow

more

flexible

linker

design

which

is

important

for

simultaneous

binding

of

multivalent

antigens

without

the

problems

posed

by

domain

mispairing

Thus

several

functional

trivalent-bispecific

VHHs

have

been

successfully

produced

-LRB-

Coppieters

et

al.

Roovers

et

al.

-RRB-

The

use

of

mixtures

of

a

limited

number

of

monoclonal

antibodies

-LRB-

oligoclonal

antibodies

-RRB-

is

advantageous

over

single

monoclonal

antibodies

for

particular

applications

such

as

toxin

neutralization

-LRB-

Nowakowski

et

al.

-RRB-

Because

of

regulatory

requirements

such

oligoclonals

are

preferentially

produced

from

single

cells

Again

VHHs

are

predicted

to

be

more

suitable

for

single-cell

production

of

oligoclonals

because

of

the

absence

of

domain

mispairing

although

this

is

yet

to

be

demonstrated

experimentally

Contrary

to

conventional

antibodies

VHHs

have

been

shown

to

remain

functional

at

°C

-LRB-

Van

der

Linden

et

al.

-RRB-

or

after

incubation

at

high

temperatures

-LRB-

Van

der

Linden

et

al.

Perez

et

al.

-RRB-

This

high

apparent

stability

is

mainly

attributed

to

their

efficient

refolding

after

chemical

or

thermal

denaturation

and

to

a

lesser

extent

because

of

an

increased

resistance

against

denaturation

-LRB-

Perez

et

al.

Dumoulin

et

al.

Ewert

et

al.

-RRB-

The

increased

apparent

stability

is

probably

due

to

an

increased

hydrophilicity

of

the

former

VL

interface

region

because

a

camelized

human

VH

fragment

that

contains

several

of

the

hallmark

hydrophilic

amino

acid

residues

of

VHHs

was

more

stable

than

the

original

VH

fragment

-LRB-

Davies

and

Riechmann

-RRB-

whereas

decamelization

of

a

VHH

to

mimic

a

VH

domain

reduces

its

thermodynamic

stability

-LRB-

Conrath

et

al.

-RRB-

In

addition

to

these

specific

mutations

the

packing

of

extended

loops

against

this

former

VL

interface

contributes

to

domain

stability

-LRB-

Bond

et

al.

-RRB-

Furthermore

refolding

of

VHHs

only

requires

domain

refolding

whereas

conventional

antibodies

also

require

association

of

VH

and

VL

domains

VHHs

can

also

recognize

antigenic

sites

that

are

normally

not

recognized

by

conventional

antibodies

such

as

enzyme

active

sites

-LRB-

Lauwereys

et

al.

De

Genst

et

al.

-RRB-

and

conserved

cryptic

epitopes

-LRB-

Stijlemans

et

al.

-RRB-

This

facilitates

their

use

as

enzyme

inhibitors

or

in

diagnosis

of

trypanosome

infections

The

ability

to

recognize

these

recessed

antigenic

sites

has

been

attributed

to

their

smaller

size

and

the

ability

of

the

extended

loop

to

penetrate

into

such

sites

-LRB-

Desmyter

et

al.

De

Genst

et

al.

-RRB-

It

is

interesting

to

note

that

this

structure

function

relation

is

also

observed

in

a

rare

example

of

a

broadly

reactive

human

mAb

that

recognizes

the

recessed

and

conserved

cavity

of

human

immunodeficiency

virus

type

by

virtue

of

an

extended

-LRB-

Zwick

et

al.

-RRB-

With

respect

to

antigen

binding

the

single-domain

nature

could

be

a

disadvantage

for

binding

to

small

antigens

such

as

haptens

and

peptides

because

these

normally

bind

in

a

groove

or

cavity

at

the

VH

VL

interface

-LRB-

Sundberg

and

Mariuzza

-RRB-

Indeed

llamas

immunized

with

clenbuterol

developed

conventional

but

not

heavy-chain

antibodies

against

this

hapten

-LRB-

Lange

et

al.

-RRB-

However

hapten

and

peptide-binding

VHHs

have

been

successfully

isolated

using

strong

selection

systems

-LRB-

Spinelli

et

al.

Yau

et

al.

Alvarez-Rueda

et

al.

Harmsen

et

al.

-RRB-

The

affinities

of

VHHs

are

generally

comparable

to

those

of

conventional

antibody

fragments

-LRB-

Muyldermans

et

al.

-RRB-

Occasionally

VHHs

with

affinity

constants

-LRB-

KD

-RRB-

as

low

as

pM

are

isolated

-LRB-

Saerens

et

al.

De

Genst

et

al.

Harmsen

et

al.

-RRB-

which

equals

the

affinity

ceiling

proposed

for

natural

antibodies

-LRB-

Sundberg

and

Mariuzza

-RRB-

Because

of

their

small

size

of

about

kDa

VHHs

rapidly

pass

the

renal

filter

which

has

a

cutoff

of

about

kDa

resulting

in

their

rapid

blood

clearance

In

addition

the

small

size

results

in

a

fast

tissue

penetration

This

is

advantageous

for

targeting

of

VHHs

coupled

to

toxic

substances

to

tumors

-LRB-

Cortez-Retamozo

et

al.

-RRB-

in

vivo

diagnosis

using

imaging

and

treatment

of

snake

bites

-LRB-

Harrison

et

al.

-RRB-

However

for

other

therapeutic

applications

such

as

treatment

of

infectious

or

inflammatory

diseases

the

short

serum

half-life

of

about

h

-LRB-

Cortez-Retamozo

et

al.

Harmsen

et

al.

-RRB-

is

a

disadvantage

Production

in

microorganisms

Although

a

fully

active

nonglycosylated

IgG

was

recently

produced

at

high

level

in

Escherichia

coli

most

functional

complete

antibodies

can

only

be

efficiently

produced

using

mammalian

cells

especially

when

their

appropriate

glycosylation

is

required

for

therapeutic

applications

However

antibody

fragments

that

lack

the

Fc

with

its

N-linked

oligosaccharide

are

preferably

produced

in

microbial

systems

-LRB-

Arbabi-Ghahroudi

et

al.

-RRB-

These

have

a

shorter

development

time

from

gene

to

product

and

require

simple

well-established

fermentation

conditions

that

can

be

performed

on

large-scale

resulting

in

costs

of

goods

that

can

be

as

low

as

per

gram

heterologous

protein

-LRB-

Estell

-RRB-

Most

large-scale

microbial

production

systems

are

based

on

E.

coli

yeasts

or

filamentous

fungi

Production

in

E.

coli

can

be

done

by

secretion

into

the

oxidizing

periplasmic

space

or

expression

in

the

reducing

cytosol

The

latter

requires

the

often

cumbersome

refolding

of

antibody

fragments

-LRB-

Arbabi-Ghahroudi

et

al.

-RRB-

Using

eukaryotic

microbial

hosts

antibody

fragments

are

generally

produced

by

targeting

to

the

secretory

pathway

This

enables

efficient

disulfide

bond

formation

addition

of

N-linked

oligosaccharide

and

secretion

of

soluble

correctly

folded

product

to

the

culture

medium

VHHs

have

often

been

produced

in

E.

coli

-LRB-

Arbabi-Ghahroudi

et

al.

Rahbarizadeh

et

al.

-RRB-

There

is

only

one

example

of

VHH

production

in

filamentous

fungi

which

resulted

in

limited

proteolytic

degradation

of

the

secreted

product

-LRB-

Joosten

et

al.

-RRB-

because

of

the

high

levels

of

proteases

secreted

by

filamentous

fungi

-LRB-

Gerngross

-RRB-

VHHs

have

also

often

been

produced

in

the

yeast

Saccharomyces

cerevisiae

-LRB-

Frenken

et

al.

Thomassen

et

al.

Van

der

Vaart

-RRB-

VHH

production

by

the

favored

yeast

expression

host

Pichia

pastoris

was

only

recently

described

-LRB-

Rahbarizadeh

et

al.

-RRB-

Occasionally

yeast-produced

VHHs

are

N-glycosylated

-LRB-

Frenken

et

al.

Harmsen

et

al.

-RRB-

This

can

affect

antigen

binding

-LRB-

Van

der

Vaart

et

al.

-RRB-

Furthermore

it

could

complicate

their

therapeutic

use

because

the

addition

of

yeast-specific

high-mannose

oligosaccharides

results

in

a

high

immunogenicity

and

decreased

serum

half-life

because

of

binding

to

specific

mannose

receptors

on

cells

of

the

reticulo-endothelial

system

-LRB-

Sethuraman

and

Stadheim

-RRB-

Although

VHHs

are

generally

well

produced

in

microorganisms

the

production

level

of

different

clones

can

vary

by

a

factor

of

-LRB-

Frenken

et

al.

Harmsen

et

al.

Van

de

Laar

et

al.

-RRB-

Several

VHH

sequence

patterns

can

be

associated

with

their

production

level

First

the

presence

of

a

potential

N-linked

glycosylation

site

often

increases

production

levels

in

yeast

-LRB-

Sagt

et

al.

-RRB-

Second

in

our

experience

-LRB-

Harmsen

unpublished

observations

-RRB-

conventional-like

VHHs

are

generally

produced

at

reduced

levels

in

yeast

This

contrasts

with

the

reported

efficient

production

in

E.

coli

of

VHH-like

VHs

-LRB-

Tanha

et

al.

-RRB-

but

is

consistent

with

the

increased

production

level

of

camelized

conventional

VH

domains

in

E.

coli

-LRB-

Davies

and

Riechmann

-RRB-

Third

the

presence

of

unpaired

C-terminal

cysteines

reduces

expression

levels

-LRB-

Simmons

et

al.

-RRB-

Fourth

replacement

of

hydrophobic

residues

of

conventional

VH

domains

normally

interacting

with

increased

scFv

production

in

E.

coli

-LRB-

Nieba

et

al.

-RRB-

suggesting

that

the

hydrophilic

mutations

that

naturally

occur

at

these

positions

in

VHHs

also

contributes

to

their

high

expression

level

However

there

are

many

examples

of

VHHs

that

differ

by

only

a

few

amino

acids

and

are

produced

at

highly

variable

levels

where

the

exact

amino

acid

change

responsible

for

the

difference

in

production

level

is

difficult

to

predict

-LRB-

Frenken

et

al.

Harmsen

et

al.

-RRB-

Furthermore

without

such

knowledge

VHH

production

can

be

improved

by

random

molecular

evolution

using

deoxyribonucleic

acid

shuffling

-LRB-

Van

der

Linden

et

al.

-RRB-

as

has

often

been

done

for

conventional

antibody

fragments

-LRB-

Dufner

et

al.

-RRB-

The

high

refolding

capability

of

VHHs

which

is

a

consequence

of

their

sequence

has

also

been

correlated

with

a

high

production

level

in

E.

coli

-LRB-

Jespers

et

al.

Olichon

et

al.

-RRB-

In

addition

to

the

nature

of

the

VHH

host

factors

affecting

VHH

production

have

been

identified

In

baker

's

yeast

the

specific

VHH

production

rate

is

correlated

with

growth

rate

-LRB-

Thomassen

et

al.

-RRB-

and

can

be

up

to

fivefold

increased

by

growing

on

ethanol

as

the

carbon

source

-LRB-

Van

de

Laar

et

al.

-RRB-

Supplementation

of

the

medium

with

sorbitol

casamino

acids

or

ethylenediamine

tetraacetic

acid

improves

VHH

production

by

P.

pastoris

-LRB-

Rahbarizadeh

et

al.

-RRB-

In

addition

to

monovalent

VHHs

several

expression

formats

for

the

production

of

VHH

multimers

have

been

described

-LRB-

Fig.

-RRB-

These

include

genetic

fusions

of

two

-LRB-

Conrath

et

al.

Harmsen

et

al.

-RRB-

or

three

VHHs

-LRB-

Coppieters

et

al.

Roovers

et

al.

-RRB-

that

either

recognize

different

antigens

or

the

same

repeating

antigen

to

increase

functional

affinity

Although

such

VHH

fusions

are

less

efficiently

produced

than

their

monovalent

versions

their

production

level

exceeds

that

of

their

conventional-antibody-based

fusion

counterparts

without

aggregation

or

low

solubility

However

antigen

binding

by

the

C-terminal

VHH

in

such

fusions

can

be

compromised

-LRB-

Conrath

et

al.

-RRB-

presumably

because

of

steric

hindrance

by

the

N-terminal

VHH

The

avidity

of

VHHs

has

also

been

strongly

increased

using

genetic

fusions

to

the

B-subunits

of

an

E.

coli

toxin

that

self-assembles

into

a

homopentamer

-LRB-

Zhang

et

al.

-RRB-

resulting

in

pentameric

recombinant

antibodies

-LRB-

pentabodies

Fig.

-RRB-

VHHs

on

their

own

can

not

recruit

effector

functions

such

as

ADCC

and

CDC

This

limits

their

therapeutic

application

Although

such

effector

functions

can

be

indirectly

recruited

using

bispecific

-LRB-

conventional

-RRB-

antibody

fragments

binding

to

host

immunoglobulin

-LRB-

Holliger

et

al.

-RRB-

it

may

be

more

efficient

to

recruit

these

functions

by

fusing

VHHs

to

host

Fc

domains

Production

of

such

functional

antibodies

requires

the

correct

glycosylation

of

the

domain

which

until

recently

could

only

be

accomplished

using

higher

eukaryotic

cells

-LRB-

Nguyen

et

al.

-RRB-

but

not

by

microbial

production

However

this

may

now

be

feasible

using

P.

pastoris

strains

with

an

engineered

glycosylation

machinery

that

are

able

to

produce

proteins

with

a

specific

human

glycoform

-LRB-

Hamilton

et

al.

-RRB-

Furthermore

transgenic

mice

containing

hybrid

llama/human

antibody

loci

that

contain

llama

V

regions

and

human

D

J

and

C

regions

have

recently

been

used

to

generate

human

heavy-chain

antibodies

in

mice

-LRB-

Janssens

et

al.

-RRB-

Therapeutic

applications

Although

VHHs

are

highly

suited

for

applications

that

require

a

high

stability

such

as

use

in

shampoo

for

the

prevention

of

dandruff

-LRB-

Dolk

et

al.

-RRB-

as

capturing

reagents

in

immunoaffinity

purification

-LRB-

Verheesen

et

al.

-RRB-

or

use

in

biosensors

-LRB-

Pleschberger

et

al.

-RRB-

we

would

like

to

focus

on

their

therapeutic

applications

which

are

more

challenging

Several

VHHs

are

now

being

studied

for

use

in

various

disease

areas

including

oncology

-LRB-

Revets

et

al.

-RRB-

and

in

infectious

inflammatory

and

neurodegenerative

diseases

-LRB-

Table

-RRB-

Table

of

therapeutic

applications

of

camelid

VHHsDiseasePathogenTarget

antigenVHH

valency

for

disease

targetAdditional

fusion

partnerReferenceSleeping

sicknessTrypanosomesVSG

oligomannoseMonovalentApolipoprotein

L-IBaral

et

al.

diarrheaRotavirusUnknownMonovalentNoneVan

der

Vaart

et

al.

diarrheaRotavirusUnknownMonovalentLactobacillus

cell-surface

anchorPant

et

al.

diarrheaE

fimbriaeMonovalentNoneHarmsen

et

al.

mutansI/II

adhesionMonovalentNoneKruger

et

al.

et

al.

meningitidisLPSMonovalentNoneEl

Khattabi

et

al.

CEAMonovalentβ-LactamaseCortez-Retamozo

et

al.

EGF

receptorBivalentAnti-albumin

VHHRoovers

et

al.

arthritis

TNFαBivalentAnti-albumin

VHHCoppieters

et

al.

disorders

α

-LRB-

-RRB-

SialoglycoproteinMonovalentNoneMuruganandam

et

al.

diseases

BaxMonovalentNoneGueorguieva

et

al.

VHHs

are

especially

suited

for

oral

immunotherapy

because

of

their

resistance

against

extremes

of

pH

and

the

capacity

to

bind

to

the

target

at

high

concentrations

of

chaotropic

agents

-LRB-

Dumoulin

et

al.

-RRB-

Administration

to

piglets

of

a

VHH

that

effectively

prevents

intestinal

attachment

of

E.

coli

bacteria

that

cause

diarrhea

resulted

in

poor

in

vivo

protection

-LRB-

Harmsen

et

al.

-RRB-

because

of

degradation

by

gastrointestinal

proteases

-LRB-

Harmsen

et

al.

-RRB-

However

by

selection

for

proteolytic

stability

a

VHH

could

be

isolated

from

the

original

library

that

was

not

degraded

in

vivo

-LRB-

Harmsen

et

al.

-RRB-

VHHs

that

successfully

prevented

diarrhea

caused

by

rotavirus

in

a

mouse

model

were

similarly

selected

for

resistance

against

the

acidic

environment

of

the

stomach

-LRB-

Van

der

Vaart

et

al.

-RRB-

Alternatively

VHH

proteolysis

can

be

prevented

by

local

VHH

production

using

natural

gut

commensal

bacteria

Thus

diarrhea

could

also

be

prevented

by

lactobacilli

that

produce

rotavirus-neutralizing

VHHs

fused

to

a

cell

surface

anchor

-LRB-

Pant

et

al.

-RRB-

Treatment

of

caries

caused

by

Streptococcus

mutans

with

VHHs

conferred

only

limited

protection

-LRB-

Kruger

et

al.

-RRB-

Because

these

VHHs

should

function

in

the

oral

cavity

the

low

level

of

protection

can

not

be

due

to

proteolytic

VHH

degradation

within

the

gastrointestinal

tract

The

short

serum

half-life

because

of

a

rapid

renal

clearance

limits

the

efficacy

of

VHHs

in

many

parenteral

applications

Therefore

VHHs

have

been

targeted

to

normally

long-lived

serum

proteins

such

as

albumin

-LRB-

Coppieters

et

al.

Roovers

et

al.

-RRB-

or

immunoglobulin

-LRB-

Harmsen

et

al.

-RRB-

using

bispecific

VHHs

recognizing

these

serum

proteins

in

addition

to

the

therapeutic

target

resulting

in

half-lives

that

equal

the

half-life

of

albumin

-LRB-

days

in

mice

-RRB-

and

immunoglobulin

-LRB-

days

-RRB-

An

alternative

well-known

approach

to

increase

serum

half-life

of

proteins

is

the

chemical

addition

of

polyethylene

glycol

-LRB-

PEG

-RRB-

Such

PEGylation

of

foot-and-mouth

disease

-LRB-

FMD

-RRB-

virus-neutralizing

VHHs

not

only

increased

serum

half-life

but

also

increased

in

vitro

neutralizing

potency

to

levels

above

that

of

the

hyperimmune

serum

-LRB-

Harmsen

et

al.

-RRB-

However

in

contrast

to

the

full

protection

afforded

by

the

hyperimmune

serum

these

VHHs

poorly

protected

guinea

pigs

from

FMD

viral

challenge

infection

suggesting

that

Fc-mediated

effector

functions

are

required

for

efficient

in

vivo

protection

-LRB-

Harmsen

et

al.

-RRB-

Nevertheless

many

diseases

were

successfully

treated

with

VHHs

in

the

absence

of

Fc-mediated

effector

functions

These

VHHs

either

are

used

as

targeting

devices

for

toxic

enzymes

or

block

a

specific

molecular

interaction

For

example

sleeping

sickness

was

successfully

treated

with

VHHs

that

bind

to

a

trypanosome

coat

protein

and

were

fused

to

the

apolipoprotein

enzyme

resulting

in

trypanosome

lysis

-LRB-

Baral

et

al.

-RRB-

In

oncology

a

VHH

directed

against

carcinoembryonic

antigen

was

used

for

targeting

the

genetically

fused

β-lactamase

to

tumor

cells

This

enzyme

then

converts

an

injected

nontoxic

prodrug

into

a

toxic

drug

in

the

vicinity

of

the

targeted

tumor

cells

leading

to

their

killing

-LRB-

Cortez-Retamozo

et

al.

-RRB-

Several

VHH

therapies

are

also

being

developed

for

treatment

of

oncology

or

inflammatory

diseases

based

on

blocking

molecular

interactions

VHHs

binding

to

epidermal

growth

factor

receptor

-LRB-

EGFR

-RRB-

can

block

epidermal

growth

factor

-LRB-

EGF

-RRB-

binding

to

its

receptor

which

can

be

used

to

treat

solid

tumors

-LRB-

Roovers

et

al.

-RRB-

Tenfold

more

potent

EGFR-binding

VHHs

could

be

obtained

by

construction

of

bivalent

formats

It

is

interesting

to

note

that

the

recently

approved

conventional

antibody

Panitumumab

directed

against

EGFR

also

blocks

EGF

binding

and

is

expected

to

give

poor

ADCC

and

CDC

-LRB-

Reichert

and

Valge-Archer

-RRB-

Furthermore

by

blocking

receptor

interaction

VHHs

binding

to

tumor

necrosis

factor-α

can

be

used

for

treatment

of

rheumatoid

arthritis

-LRB-

Coppieters

et

al.

-RRB-

The

potency

of

bivalent

formats

was

increased

as

compared

to

monovalent

VHHs

and

even

exceeded

the

potency

of

clinically

used

conventional

antibodies

both

in

vitro

and

in

a

murine

arthritis

model

Similarly

lipopolysaccharide

-LRB-

LPS

-RRB-

binding

VHHs

were

isolated

that

block

LPS

binding

and

signaling

to

host

cells

for

treatment

of

sepsis

-LRB-

El

Khattabi

et

al.

-RRB-

The

potential

immunogenicity

of

VHHs

could

compromise

their

parenteral

therapeutic

use

especially

in

treatments

that

require

repeated

injections

Until

now

multiple

injections

of

VHHs

have

not

shown

any

immunogenicity

in

mice

as

assessed

by

the

presence

of

specific

antibodies

T

cell

proliferation

or

cytokine

levels

-LRB-

Cortez-Retamozo

et

al.

Coppieters

et

al.

-RRB-

This

could

rely

on

their

high

sequence

homology

to

conventional

VH

domains

and

on

their

high

stability

because

aggregation

of

proteins

is

known

to

increase

immunogenicity

-LRB-

Hermeling

et

al.

-RRB-

If

necessary

technologies

developed

to

decrease

immunogenicity

of

mouse

monoclonal

antibodies

-LRB-

Presta

-RRB-

could

also

be

applied

to

VHHs

Alternatively

immunogenicity

could

be

reduced

by

the

use

of

conventional-like

VHHs

which

have

an

even

higher

structural

homology

to

conventional

VH

domains

For

their

use

in

targeting

drugs

across

the

blood

brain

barrier

-LRB-

BBB

-RRB-

into

the

brain

VHHs

were

selected

that

transmigrate

the

human

BBB

in

an

in

vitro

model

and

accumulate

in

the

brain

after

intravenous

injection

into

mice

-LRB-

Muruganandam

et

al.

-RRB-

These

could

be

used

for

treatment

of

neurological

disorders

Finally

Bax-specific

VHHs

have

been

expressed

in

the

cytoplasm

resulting

in

so-called

intrabodies

to

prevent

oxidative-stress-induced

apoptosis

that

is

implicated

in

several

neurodegenerative

diseases

-LRB-

Gueorguieva

et

al.

-RRB-

Because

of

their

stability

VHHs

are

especially

suited

for

intrabody

production

because

this

requires

expression

in

the

reducing

environment

of

the

cytoplasm

-LRB-

Gueorguieva

et

al.

Rothbauer

et

al.

-RRB-

Conclusions

Since

the

discovery

of

heavy-chain

antibodies

in

the

field

of

single-domain

antibody

fragments

has

been

rapidly

growing

VHHs

have

many

advantages

for

biotechnological

applications

They

can

be

economically

produced

in

microorganisms

and

have

a

high

stability

Furthermore

they

are

highly

suited

for

expression

as

multivalent

including

bispecific

formats

or

as

enzyme

fusions

This

permits

a

plug-and-play

approach

where

depending

on

the

target

biology

potency

can

be

increased

by

multivalent

constructs

or

bispecific

VHH

recognizing

two

different

targets

can

be

made

This

also

enables

the

tailor-made

design

of

serum

half-life

using

site-directed

PEGylation

or

by

targeting

to

long-lived

serum

proteins

using

bispecific

VHHs

Although

fusions

of

targeting

VHHs

to

Ig-binding

VHHs

or

Fc

can

be

used

to

recruit

effector

functions

most

current

research

on

VHHs

focuses

on

therapeutic

applications

where

such

effector

functions

are

not

required

Finally

conventional

whole

antibodies

occasionally

give

side

effects

because

of

their

bivalent

nature

which

can

result

in

target

cross-linking

or

the

presence

of

the

Fc

region

Evidently

such

side

effects

are

not

expected

to

occur

using

monovalent

VHHs

This

however

is

yet

to

be

confirmed

as

the

first

VHH

has

entered

phase

I

clinical

trials

in

-LRB-

-RRB-

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

microbial production
yeast
glycosylation


Biomechanical

Analysis

of

Reducing

Sacroiliac

Joint

Shear

Load

by

Optimization

of

Pelvic

Muscle

and

Ligament

Forces

Effective

stabilization

of

the

sacroiliac

joints

-LRB-

SIJ

-RRB-

is

essential

since

spinal

loading

is

transferred

via

the

SIJ

to

the

coxal

bones

and

further

to

the

legs

We

performed

a

biomechanical

analysis

of

SIJ

stability

in

terms

of

reduced

SIJ

shear

force

in

standing

posture

using

a

validated

static

simulation

model

This

model

contained

muscle

elements

ligaments

and

joints

in

trunk

pelvis

and

upper

legs

Initially

the

model

was

set

up

to

minimize

the

maximum

muscle

stress

In

this

situation

the

trunk

load

was

mainly

balanced

between

the

coxal

bones

by

vertical

SIJ

shear

force

An

imposed

reduction

of

the

vertical

SIJ

shear

by

resulted

in

increase

of

SIJ

compression

force

due

to

activation

of

hip

flexors

and

counteracting

hip

extensors

Another

reduction

of

the

vertical

SIJ

shear

force

resulted

in

further

increase

of

SIJ

compression

force

by

due

to

activation

of

the

transversely

oriented

M.

transversus

abdominis

and

pelvic

floor

muscles

The

M.

transversus

abdominis

crosses

the

SIJ

and

clamps

the

sacrum

between

the

coxal

bones

Moreover

the

pelvic

floor

muscles

oppose

lateral

movement

of

the

coxal

bones

which

stabilizes

the

position

of

the

sacrum

between

the

coxal

bones

-LRB-

the

pelvic

arc

-RRB-

Our

results

suggest

that

training

of

the

M.

transversus

abdominis

and

the

pelvic

floor

muscles

could

help

to

relieve

SI-joint

related

pelvic

pain

Introduction

The

human

body

uses

an

ingenious

framework

of

bones

joints

muscles

and

ligaments

for

posture

and

movement

In

upright

posture

the

trunk

load

passes

the

sacroiliac

joints

-LRB-

SIJ

-RRB-

The

orientation

of

the

SIJ

surfaces

however

is

more

or

less

in

line

with

the

direction

of

loading

which

induces

high

shear

forces

between

sacrum

and

coxal

bones

The

SIJ

have

a

strong

passive

viscoelastic

ligamentous

system

for

providing

stability

These

ligaments

are

vulnerable

for

creep

during

constant

trunk

load

and

need

to

be

protected

against

high

SIJ

shear

forces

From

a

biomechanical

point

of

view

an

active

muscle

corset

that

increases

the

compression

force

between

the

coxal

bones

and

the

sacrum

could

protect

the

ligamentous

system

and

support

the

transfer

of

trunk

load

to

the

legs

and

vice

versa

Interlocking

of

the

SIJ

may

be

promoted

by

transversely

oriented

muscles

e.g.

M.

transversus

abdominis

M.

piriformis

M.

gluteus

maximus

M.

obliquus

externus

abdominis

and

M.

obliquus

internus

abdominis

which

has

been

described

as

However

due

to

the

complex

lines

of

action

of

-LRB-

counteracting

-RRB-

muscles

and

ligaments

in

the

pelvic

region

it

is

difficult

to

demonstrate

the

contribution

of

transversely

oriented

muscles

to

SIJ

stability

in

vitro

as

well

as

in

vivo

In

the

past

a

number

of

biomechanical

models

of

the

lumbosacral

region

-LRB-

spine

and

pelvis

-RRB-

have

been

developed

to

study

the

aetiology

of

low

back

pain

-LRB-

LBP

-RRB-

in

relation

to

-LRB-

over

-RRB-

loading

of

the

lumbar

and

the

pelvis

Most

of

these

models

dealt

with

mechanical

stability

in

terms

of

and

compression

forces

between

-LRB-

lumbar

-RRB-

vertebrae

A

different

approach

is

to

relate

LBP

to

overloading

of

SIJ

and

nearby

ligaments

for

example

the

iliolumbar

ligaments

The

load

transfer

through

the

SIJ

was

studied

using

a

static

biomechanical

simulation

model

based

on

the

musculoskeletal

anatomy

of

the

trunk

pelvis

and

upper

legs

This

simulation

model

calculates

forces

in

muscles

ligaments

and

joints

that

are

needed

to

counterbalance

trunk

weight

and

other

external

forces

It

was

shown

that

this

simulation

model

underestimated

antagonistic

muscle

activity

but

a

good

agreement

was

found

for

agonist

muscle

activity

The

number

of

passive

structures

in

the

model

was

small

for

example

no

joint

capsules

were

incorporated

Therefore

the

model

was

only

valid

for

postures

in

which

of

the

joints

were

near

an

end

position

The

aim

of

the

present

study

was

to

determine

which

muscles

have

to

become

active

in

the

pelvic

simulation

model

when

there

is

an

imposed

reduction

of

the

vertical

SIJ

shear

force

Materials

and

Methods

The

Simulation

Model

The

present

study

was

performed

using

the

validated

simulation

model

as

described

by

Hoek

van

Dijke

et

al.

The

model

is

based

on

the

musculoskeletal

anatomy

of

the

trunk

pelvis

and

upper

legs

including

muscle

and

ligament

attachment

sites

cross-sectional

areas

of

muscles

and

the

direction

of

muscle

ligament

and

joint

reaction

forces

The

geometry

of

this

model

was

based

on

structures

extracted

from

MRI

slices

and

from

previously

published

data

on

lumbar

and

upper

geometry

Figure

illustrates

in

frontal

and

median

view

the

bones

on

which

muscle

and

ligament

forces

act

in

the

simulation

model

These

are

the

lowest

thoracic

vertebra

five

lumbar

vertebrae

the

sacrum

the

left

and

right

coxal

bones

and

the

left

and

right

femurs

The

vertebrae

are

treated

as

a

single

structure

The

arrangement

of

the

bones

depends

on

the

static

posture

for

which

muscle

forces

are

calculated

for

example

standing

with

or

without

trunk

flexion

A

description

of

the

model

equilibrium

its

optimization

scheme

and

validation

of

some

of

the

parameters

is

presented

in

the

Appendix

In

the

present

study

we

focus

on

the

compression

and

shear

forces

in

the

SIJ

These

forces

are

represented

as

perpendicular

vectors

The

normal

vector

of

the

SIJ

surface

has

an

oblique

direction

-LRB-

xyz

-RRB-

Compression

force

is

defined

along

this

normal

vector

and

can

only

vary

in

magnitude

One

of

the

two

components

of

the

SIJ

shear

force

was

defined

in

the

YZ-plane

-LRB-

xyz

-RRB-

This

force

is

denoted

as

the

vertical

SIJ

shear

force

Directions

of

the

SIJ

compression

force

and

the

SIJ

vertical

shear

force

in

the

YZ-plane

are

shown

in

Fig.

left

panel

Figure

shows

the

vectors

representing

the

most

important

muscle

and

ligament

forces

in

the

pelvic

region

superimposed

on

the

bones

Bone

shapes

are

for

illustration

purpose

only

they

are

not

part

of

the

simulation

model

In

total

the

model

contains

vectors

for

muscle

forces

vectors

for

ligament

forces

and

vectors

for

joint

forces

see

Table

for

a

list

of

all

the

structures

Figure

-LRB-

a

-RRB-

shows

the

bones

on

which

the

muscles

ligaments

and

joint

reaction

forces

act

in

the

frontal

plane

-LRB-

left

-RRB-

and

the

median

plane

-LRB-

right

-RRB-

the

lowest

thoracic

vertebra

five

lumbar

vertebrae

the

sacrum

the

left

and

right

coxal

bones

and

the

left

and

right

femurs

The

coordinate

system

is

defined

with

the

origin

halfway

between

the

rotation

centers

of

the

hip

joints

Axes

x

posterior

y

left

z

vertical

Panel

-LRB-

b

-RRB-

shows

superimposed

on

the

bones

the

vectors

representing

the

most

important

force

components

in

the

frontal

plane

-LRB-

left

-RRB-

and

the

median

plane

-LRB-

right

-RRB-

see

also

Table

The

labels

in

this

panel

refer

to

a

selection

of

the

muscle

structures

listed

in

Table

of

muscles

ligaments

and

joints

and

the

number

of

vectors

describing

the

forces

used

in

the

simulation

model

on

transferring

trunk

load

from

lumbar

spine

via

the

pelvis

to

the

upper

legs

-LRB-

unilateral

-RRB-

see

also

Fig.

of

structureNumber

of

adductor

and

lower

adductor

adductor

middle

and

lower

biceps

floor

floor

floor

gemellus

gemellus

gluteus

sacrum

and

ilium

gluteus

maximus

femur

and

trunk

gluteus

middle

and

lower

gluteus

middle

and

lower

obliquus

externus

and

dorsal

obliquus

internus

and

dorsal

obturatorius

obturatorius

and

lower

quadratus

quadratus

and

rectus

rectus

tensor

fasciae

transversus

planeBPosterior

sacroiliac

planeCSacrospinal

-LRB-

two

directions

-RRB-

and

compressionIISI

-LRB-

two

directions

-RRB-

and

compressionIIIHip

-LRB-

two

directions

-RRB-

and

compressionIVKnee

-LRB-

two

directions

-RRB-

and

compressionVPubic

Simulations

and

Data

Analyses

A

first

simulation

with

the

model

in

standing

posture

and

a

trunk

weight

of

N

showed

that

the

vertical

shear

SIJ

force

was

N

on

each

side

of

the

sacrum

To

find

the

muscles

that

promote

sacroiliac

joint

stability

the

maximum

value

for

the

vertical

SIJ

shear

force

was

decreased

in

steps

of

N

-LRB-

of

the

initial

vertical

SIJ

shear

force

-RRB-

Theoretically

lowering

of

the

imposed

vertical

SIJ

shear

force

to

N

could

induce

a

non-physiological

equilibrium

between

muscle

ligament

and

joint

forces

In

addition

when

the

model

was

set

in

flexion

the

force

in

the

iliolumbar

the

sacrotuberal

and

the

posterior

sacroiliac

ligaments

was

N

This

value

was

set

as

a

maximum

physiological

ligament

force

in

the

simulation

model

in

the

upright

position

to

prevent

overloading

of

the

pelvic

ligaments

that

were

implemented

in

the

model

The

following

criteria

were

defined

to

warrant

a

physiological

solution

for

muscle

and

ligament

forces.Muscle

tension

must

not

exceed

Lowering

of

the

maximum

vertical

SIJ

shear

force

must

result

in

reduction

of

the

total

SIJ

shear

force

-LRB-

combination

of

vertical

and

horizontal

shear

-RRB-

Ligament

force

must

not

exceed

N

A

muscle

was

included

for

further

analysis

when

it

produced

at

least

of

the

maximum

muscle

stress

during

the

simulation

For

all

muscles

the

maximum

muscle

stress

depended

on

the

calculated

minimum

muscle

stress

-LRB-

see

optimization

criterion

in

the

Appendix

-RRB-

Two

muscle

groups

were

analyzed

separately

-LRB-

-RRB-

the

muscles

that

increased

at

least

in

force

after

the

first

simulation

step

and

-LRB-

-RRB-

the

muscles

that

increased

at

least

times

in

force

after

completion

of

the

simulation

series

Results

Table

summarizes

the

muscle

-LRB-

de

-RRB-

activation

pattern

when

the

maximum

vertical

SIJ

shear

force

was

stepwise

decreased

Initially

vertical

SIJ

shear

force

was

N

-LRB-

on

each

side

of

the

sacrum

-RRB-

at

a

trunk

load

of

N

The

angle

between

the

normal

direction

of

the

SIJ

surface

and

the

direction

of

the

total

SIJ

reaction

force

was

indicating

that

mainly

vertical

shear

force

acted

through

the

SIJ

see

Fig.

Force

equilibrium

was

mainly

achieved

by

activation

of

M.

abdominal

oblique

-LRB-

internus

and

externus

-RRB-

M.

iliacus

M.

psoas

M.

rectus

abdominis

M.

rectus

femoris

M.

tensor

fasciae

latae

and

loading

of

the

sacrotuberous

ligament

Table

of

the

structures

that

stabilize

the

sacroiliac

joints

in

terms

of

lowered

shearReduction

of

sacroiliac

shearInitialPreset

value

-LRB-

N

-RRB-

SIJ

-LRB-

vertical

shear

-RRB-

-LRB-

N

-RRB-

M.

adductor

gluteus

medius

-LRB-

lower

-RRB-

gluteus

medius

-LRB-

middle

-RRB-

gluteus

medius

-LRB-

upper

-RRB-

gluteus

minimus

-LRB-

lower

-RRB-

gluteus

minimus

-LRB-

middle

-RRB-

gluteus

minimus

-LRB-

upper

-RRB-

obliquus

externus

obliquus

internus

obturatorius

psoas

-LRB-

lower

-RRB-

rectus

rectus

tensor

fasciae

transversus

sacroiliac

-LRB-

compression

-RRB-

-LRB-

horizontal

shear

-RRB-

SIJ

of

SIJ

reaction

force

-LRB-

-RRB-

muscle

tension

-LRB-

kPa

-RRB-

are

those

muscles

that

produced

at

least

of

the

maximum

muscle

stress

after

each

simulationThe

muscles

printed

italic

increased

at

least

in

force

after

the

first

simulation

step

The

muscles

printed

bold

increased

at

least

times

in

force

after

completion

of

the

simulation

seriesFigure

of

the

force

in

the

frontal

plane

exerted

by

the

right

ilium

through

the

SIJ

on

the

sacrum

as

a

reaction

to

trunk

load

Ftrunk

Panel

-LRB-

a

-RRB-

initial

loading

condition

without

limitation

of

the

vertical

shear

component

-LRB-

N

see

under

initial

in

Table

-RRB-

This

condition

led

to

loading

of

the

sacrotuberal

ligaments

Fsacrotuberal

lig

-LRB-

solid

thick

line

-RRB-

Panel

-LRB-

b

-RRB-

loading

condition

with

the

vertical

shear

component

preset

at

a

N

lower

level

than

the

initial

value

-LRB-

see

under

N

in

Table

-RRB-

This

condition

led

to

loading

of

the

sacrospinal

ligaments

Fsacrospinal

lig

-LRB-

solid

thick

line

-RRB-

Panel

-LRB-

c

-RRB-

loading

condition

with

the

vertical

shear

component

preset

at

a

N

lower

level

than

the

initial

value

-LRB-

see

under

N

in

Table

-RRB-

In

this

situation

SIJ

compression

force

increased

by

mainly

by

M.

transversus

abdominis

Ftransversus

abdominis

and

the

pelvic

floor

Fpelvic

floor

muscle

forces

The

location

of

these

muscles

is

schematically

drawn

by

the

thick

solid

lines

including

the

M.

pubococcygeus

the

M.

iliococcygeus

and

the

M.

coccygeus

-LRB-

as

drawn

from

the

mid

to

the

lateral

position

-RRB-

It

also

led

to

loading

of

the

iliolumbar

ligaments

to

the

maximum

allowed

force

Filiolumbar

lig

of

N

-LRB-

solid

thick

line

-RRB-

images

copyright

of

Primal

Pictures

Ltd.

When

the

maximum

vertical

SIJ

shear

force

was

decreased

from

to

N

in

steps

of

N

the

SIJ

compression

force

increased

by

about

Force

equilibrium

was

obtained

amongst

others

by

activation

of

some

of

the

muscles

with

a

hip

flexion

component

-LRB-

M.

adductor

longus

M.

iliacus

M.

pectineus

and

M.

sartorius

M.

rectus

femoris

-RRB-

and

some

of

the

counteracting

hip

extensors

-LRB-

MM

gluteus

medius

and

minimus

and

M.

piriformis

-RRB-

Most

of

these

muscles

became

-LRB-

more

-RRB-

active

after

we

lowered

the

maximum

vertical

SIJ

shear

force

by

N

This

led

to

unloading

of

the

sacrotuberous

ligaments

and

loading

of

the

sacrospinal

ligaments

The

angle

between

the

normal

direction

of

the

SIJ

surface

and

the

direction

of

the

total

SIJ

reaction

force

was

reduced

to

indicating

that

a

combination

of

reduced

vertical

SIJ

shear

and

increased

SIJ

compression

could

balance

the

trunk

load

on

the

sacrum

see

Fig.

Further

stepwise

reduction

of

the

vertical

SIJ

shear

force

resulted

in

a

sharp

rise

of

the

maximum

muscle

stress

The

simulation

series

ended

with

exceeding

the

maximum

physiological

muscle

stress

when

the

vertical

SIJ

shear

force

was

decreased

to

about

of

its

initial

value

Surprisingly

activation

of

some

of

the

hip

flexors

and

extensors

had

decreased

or

even

disappeared

This

was

not

the

case

for

the

MM

gluteus

medius

and

minimus

In

this

simulation

force

equilibrium

was

obtained

by

activation

of

the

transversely

oriented

M.

transversus

abdominis

-LRB-

ventral

to

the

SIJ

-RRB-

and

the

pelvic

floor

muscles

i.e.

the

M.

coccygeus

the

M.

iliococcygeus

and

the

M.

pubococcygeus

-LRB-

caudal

to

the

SIJ

-RRB-

This

resulted

in

further

reduction

of

the

angle

between

the

normal

direction

of

the

SIJ

surface

and

the

direction

of

the

total

SIJ

reaction

force

to

see

Fig.

This

indicates

that

the

SIJ

compression

force

which

increased

by

about

and

the

reduced

vertical

SIJ

shear

force

now

clamped

the

sacrum

between

the

coxal

bones

see

Fig.

The

MM

gluteus

medius

and

minimus

contributed

to

some

extent

to

this

increased

compression

due

to

a

distinct

force

component

in

the

transverse

direction

To

maintain

force

equilibrium

increased

SIJ

compression

led

to

loading

of

the

iliolumbar

and

posterior

sacroiliac

ligaments

to

the

preset

maximum

value

of

N.

Discussion

In

the

present

study

the

simulation

model

predicted

muscle

and

ligament

forces

in

the

pelvic

region

when

there

was

an

imposed

reduction

of

the

vertical

SIJ

shear

force

Initially

the

forces

acting

through

the

SIJ

were

mainly

vertical

shear

forces

see

Fig.

These

forces

were

not

only

caused

by

trunk

load

but

also

by

muscles

that

acted

in

the

longitudinal

direction

of

the

spine

for

example

the

M.

psoas

and

M.

rectus

abdominis

As

a

result

of

the

forward

bending

moment

the

sacrotuberous

ligament

was

loaded

This

large

ligament

protects

the

SIJ

against

excessive

flexion

of

the

sacrum

relative

to

the

coxal

bones

The

controlled

reduction

of

the

vertical

SIJ

shear

force

with

N

forced

some

muscles

that

act

as

hip

flexors

and

hip

extensors

to

become

active

Due

to

their

transverse

orientation

especially

the

MM

gluteus

medius

and

minimus

and

M.

piriformis

contributed

to

the

increased

compression

force

between

the

coxal

bones

and

the

sacrum

However

these

muscles

did

not

contribute

enough

to

self-bracing

of

the

SIJ

because

the

total

force

through

the

SIJ

still

mainly

acted

in

vertical

direction

When

the

vertical

SIJ

shear

was

further

reduced

to

about

of

its

initial

value

the

simulation

model

predicted

that

self-bracing

mainly

resulted

from

the

transverse

muscles

ventrally

-LRB-

M.

transversus

abdominis

-RRB-

and

caudally

-LRB-

pelvic

floor

-RRB-

to

the

SIJ

In

this

situation

some

of

the

hip

flexors

and

extensors

reduced

in

activity

for

example

the

M.

piriformis

Although

the

M.

piriformis

has

a

transverse

orientation

and

crosses

the

SIJ

its

contribution

was

minimized

by

the

simulation

program

because

this

muscle

also

induces

vertical

SIJ

shear

force

The

pelvic

floor

muscles

the

M.

coccygeus

and

M.

pubo

and

iliococcygeus

contribute

to

the

stabilization

with

respect

to

the

sacrum

It

has

been

suggested

that

this

stabilization

by

force

closure

has

an

analogy

with

a

classical

stone

arc

When

sideways

displacement

of

both

ends

of

the

arc

is

opposed

mechanical

equilibrium

of

the

stones

is

achieved

by

compression

forces

and

not

by

shear

forces

In

the

pelvis

the

pelvic

floor

muscles

may

help

the

coxal

bones

to

support

the

sacrum

by

compression

forces

while

shear

forces

between

sacrum

and

coxal

bones

are

minimized

see

Fig.

Note

that

the

SI

compression

force

is

defined

as

the

force

acting

perpendicular

to

the

SIJ

surface

Therefore

decreasing

or

increasing

this

force

will

not

alter

the

shear

forces

The

articular

surfaces

of

the

SIJ

are

irregular

which

results

in

bony

interdigitation

in

the

SI

joint

space

SIJ

shear

force

calculated

in

the

simulation

model

thus

reflects

the

combination

of

real

joint

friction

and

friction

due

to

this

intermingling

of

bones

The

real

joint

friction

forces

may

be

extremely

small

considering

the

extremely

low

coefficients

of

friction

between

the

articular

surfaces

The

majority

of

the

shear

force

is

effectuated

as

normal

contact

pressures

due

to

the

bony

interdigitation

in

the

SI

joint

space

It

was

not

possible

to

calculate

the

percentage

of

shear

in

terms

of

joint

friction

force

This

requires

a

more

detailed

description

of

the

SIJ

surfaces

Figure

of

pelvic

bones

supporting

the

trunk

with

a

classical

stone

arc

The

M.

transversus

abdominis

and

the

pelvic

floor

muscles

caudal

to

the

SIJ

mainly

oppose

lateral

movement

of

the

coxal

bones

Spinal

loading

is

transferred

mainly

by

compression

forces

through

the

SIJ

to

the

coxal

bones

and

further

down

to

the

legs

images

copyright

of

Primal

Pictures

Ltd.

The

simulation

model

predicts

that

simultaneous

contraction

of

the

M.

transversus

abdominis

and

pelvic

floor

muscles

i.e.

the

M.

coccygeus

the

M.

iliococcygeus

and

the

M.

pubococcygeus

contribute

to

lowering

of

the

vertical

SIJ

shear

forces

increasing

of

the

SIJ

compression

and

hence

increasing

of

the

SIJ

stability

We

emphasize

that

this

simulation

model

was

set

up

to

estimate

the

forces

acting

in

the

pelvic

region

under

static

conditions

and

that

the

outcome

of

the

simulations

must

be

interpreted

with

caution

Nevertheless

in

a

previous

study

co-contraction

was

shown

of

pelvic

muscles

and

M.

transversus

abdominis

This

result

and

the

prediction

of

our

simulation

model

suggest

that

a

protective

mechanism

against

high

SIJ

shear

forces

may

exist

in

humans

This

mechanism

has

been

investigated

in

vivo

and

in

vitro

The

contribution

of

the

M.

transversus

abdominis

to

SIJ

stability

was

shown

in

an

in

vivo

study

in

patients

with

LBP

An

in

vitro

study

in

embalmed

human

pelvises

showed

that

simulated

pelvic

floor

tension

increased

the

stiffness

of

the

pelvic

ring

in

female

pelvises

It

is

worthwhile

to

further

investigate

the

contribution

of

both

muscle

groups

simultaneously

not

only

during

stiffness

measurements

of

the

SIJ

but

also

during

lumbo-pelvic

stability

tests

based

on

increased

intra-abdominal

pressure

-LRB-

IAP

-RRB-

It

was

shown

that

the

pelvic

floor

muscles

in

combination

with

abdominal

muscles

and

the

diaphragm

may

control

and/or

sustain

IAP

to

increase

lumbar

spine

stability

as

well

In

the

present

study

the

ligament

forces

were

not

allowed

to

exceed

N

The

distribution

between

muscle

and

ligament

forces

depended

on

the

maximum

muscle

stress

as

formulated

in

the

first

optimization

scheme

as

presented

in

the

Appendix

Increasing

the

maximum

ligament

forces

might

result

in

a

lower

maximum

muscle

stress

which

could

lead

to

a

different

muscle

activation

pattern

to

stabilize

the

SIJ

A

small

sensitivity

test

however

showed

that

when

the

ligament

forces

were

allowed

to

exceed

the

N

up

to

N

and

in

a

next

step

up

to

N

the

model

calculated

a

similar

muscle

activation

pattern

The

outcome

of

the

present

study

also

depended

on

the

choice

of

optimization

criteria

and

the

magnitude

of

the

cross-sectional

areas

of

the

muscles

The

influence

of

different

criteria

was

previously

investigated

for

muscle

forces

in

the

leg

Indeed

various

choices

led

to

different

calculated

forces

but

the

obtained

solutions

were

qualitatively

similar

as

was

the

case

in

our

model

When

we

developed

the

model

other

optimization

criteria

were

also

tested

for

example

minimization

of

the

sum

of

muscle

forces

However

minimization

of

the

sum

of

squared

muscle

stresses

yielded

the

most

plausible

solutions

The

model

can

not

account

for

anatomical

variations

or

detailed

variation

in

muscle

attachment

sites

Obviously

direct

comparison

between

the

model

predictions

and

the

outcome

of

in

vivo

force

measurements

in

the

SIJ

are

not

available

so

there

is

no

data

to

confirm

the

outcome

of

the

present

study

Nevertheless

EMG

recordings

of

-LRB-

superficial

-RRB-

abdominal

and

back

muscles

in

various

postures

showed

higher

M.

abdominal

oblique

internus

activity

when

standing

upright

than

resting

on

one

leg

and

tilting

the

pelvic

backwards

This

muscle

is

considered

as

one

of

the

self-bracing

muscles

of

the

SIJ

It

was

hypothesized

that

when

standing

on

one

leg

the

shear

load

on

the

contralateral

SIJ

is

diminished

Posterior

tilt

of

the

pelvis

with

less

lumbar

lordosis

may

than

lead

to

less

M.

psoas

major

muscle

load

on

the

spine

meaning

less

shear

load

on

the

SIJ

These

findings

indirectly

support

our

findings

that

transversely

oriented

muscles

reduce

SIJ

shear

forces

We

emphasize

that

the

present

model

served

as

a

tool

to

investigate

the

general

relations

between

muscle

and

ligament

forces

in

the

pelvic

region

The

present

simulations

results

may

lead

to

the

development

of

a

new

SIJ

stabilizing

training-program

to

reduce

pain

induced

by

high

SIJ

shear

forces

The

effectiveness

of

such

a

program

however

can

only

be

tested

with

an

intervention

study

The

simulation

model

predicted

unloading

of

the

sacrotuberous

and

loading

of

the

iliolumbar

and

posterior

sacroiliac

ligaments

when

the

vertical

SIJ

shear

was

forced

to

reduce

This

loading

of

the

dorsal

ligaments

resulted

from

the

absence

of

transversely

oriented

muscles

at

the

dorsal

side

of

the

SIJ

to

counterbalance

activation

of

the

M.

transverse

abdominis

at

the

ventral

side

of

the

SIJ

Loading

of

the

iliolumbar

ligament

has

been

related

to

LBP

It

was

shown

that

in

sitting

position

the

stepwise

backward

movement

of

an

erect

trunk

-LRB-

from

upright

position

into

a

slouch

-RRB-

resulted

in

forward

flexion

of

the

spine

combined

with

backward

tilt

of

the

sacrum

relative

to

the

pelvis

It

was

shown

that

this

movement

into

a

sudden

or

sustained

slouch

might

cause

loading

of

the

well-innervated

iliolumbar

ligaments

near

failure

load

The

co-contraction

that

exists

between

the

deep

abdominal

M.

transversus

abdominis

and

the

deep

back

extensor

M.

multifidus

presumably

retains

lumbo-pelvic

stability

In

the

future

we

intend

to

extend

the

model

with

co-contraction

between

the

M.

transversus

abdominis

the

M.

multifidus

and

the

pelvic

floor

muscles

to

study

prevention

of

-LRB-

over

-RRB-

loading

of

pelvic

ligaments

at

different

static

postures

Conclusions

Effective

stabilization

of

the

SIJ

is

essential

in

transferring

spinal

load

via

the

SIJ

to

the

coxal

bones

and

the

legs

A

biomechanical

analysis

of

the

upright

standing

posture

showed

that

activation

of

transversely

oriented

abdominal

M.

transversus

abdominis

and

pelvic

floor

i.e.

M.

coccygeus

and

M.

pubo

and

iliococcygeus

muscles

would

be

an

effective

strategy

to

reduce

vertical

SIJ

shear

force

and

thus

to

increase

SIJ

stability

The

force

equilibrium

in

this

situation

induced

loading

of

the

iliolumbar

and

posterior

sacroiliac

ligaments

The

M.

transversus

abdominis

crosses

the

SIJ

and

clamps

the

sacrum

between

the

coxal

bones

Moreover

the

pelvic

floor

muscles

oppose

lateral

movement

of

the

coxal

bones

which

stabilizes

the

position

of

the

sacrum

-LRB-

the

pelvic

arc

-RRB-

O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O

sacroiliac joints
pelvis
pelvic floor muscles