articular cartilage Mesenchymal cell-based repair of ...

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Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage S Wakitani, T Goto, SJ Pineda, RG Young, JM Mansour, AI Caplan and VM Goldberg J. Bone Joint Surg. Am. 76:579-592, 1994.

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(pvrighi

Mesenchymal

I 994 by ihe Joiriia1

Cell-Based Defects

BY

SHI(IEYUKI

JOSEPh

WAKITANI. NI.

M.D.t.

MANSOUR.

PH.D.#.

Iii vestigation

at the

(111(1

of

Departitient

Osteochondral

ABSTRACT:

used

the

to repair

lar cartilage bits. Adherent

large,

full-thickness

GOTO.

M.D4.

I. CAPLAN.

I)epartnient

of Orthopaedics.

of Cleveland,

Mechanical

PH.D.’I.

and Aerospace

and

progenitor

cells

were

defects

of the

articu-

been liberated from connective tisdigestion, were grown in culture,

dispersed

collagen

transplanted

into

a large (three-by-six-millimeter), full-thickness millimeter) defect in the weight-bearing surface medial femoral condyle. The contralateral knee

and

(threeof the served

as a control: either the defect in that knee was or a cell-free collagen gel was implanted. The periosteal and the bone-marrow-derived showed

similar

patterns

cartilage

and

subchondral

tive

tissue

were

histological

of differentiation bone.

analyzed

grading

with

system

use and

Specimens

after

the

operation.

There

by mechanical

was

after transplantation, progenitor cells had

into

chondrocytes

throughout

repair cartilage was subsequently in a proximal-to-distal direction, weeks after transplantation, the

dral

was

bone *No

from

benefits

completely in any

form

repaired, have

been

without

received

or will

dif-

the autoluniformly the

loss

of

be received

Wepartment of Orthopaedic Surgery, National Defense Mcdical College. 3-2 Namiki. Tokorozawa 354. Japan. §Department of Orthopaedics. Case Western Reserve University School of Medicine. University Hospitals of Cleveland, 2074 Ahington Road. Cleveland, Ohio 44106. 9(Skeletal Research Center. Department of Biology. Case Western Reserve University. 2080 Adelbert Road. Cleveland, Ohio 44106-7080. #Department of Mechanical and Aerospace Engineering. Case Western Reserve University. Glennan Building. Room 615, Cleveland. Ohio 44106-7222.

NO.

4.

APRIL

Western

MDI.

Reserve

Research

Center,

Case

(Jniversitv

overlying

articular a useful

School

OHIO

Cleveland

cartilage.

of

D.V.Mj.

of Medicine.

University,

index

repair. Twenty-four reparative tissue

YOUNG.

CLEVELAND.

of Biology,

Reserve

data

G.

M.D.*.

Departtnent

Western

were

RANDELL

GOLDBERG.

The

of the

weeks both

mechanical

quality

testing

of the

long-term

after transplantation, the bone-marrow

periosteal cells was stiffer and less compliant tissue derived from the empty defects but less more compliant than normal cartilage. CLINICAL

The

RELEVANCE:

the repair of defects many disadvantages.

current

of the articular The transplantation

and

the the

than stiff

the and

modalities

for

cartilage have of progenitor

cells that will form cartilage and bone offers a possible alternative to these methods. As demonstrated in this report, autologous, bone-marrow-derived, osteochonprogenitor

cells

the

loss

or

can be isolated

of their

bone.

to

Sufficient

initiate

the

and the reformation tissues appear to

and

capacity

in vitro

grown

to differentiate

autologous repair

into

cells

of articular

can

be

cartilage

of subchondral bone. The repair undergo the same developmental

transitions

that

ular tissue of defects

in the embryo. of the articular

originally

repair

led

to the

formation

of artic-

This approach to the cartilage may have of large,

full-thickness

repair useful defects

de-

replaced until, at subchon-

a commercial

76-A.

Full-Thickness

applications in the of joint surfaces.

party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were grants-in-aid from the National Institutes of Health. tDepartment of Environmental Medicine, Osaka University Hospital Medical School. 2-2 Yamadaoke. Suita. Osaka 565. Japan.

VOl..

Case

M.

generated

with the cells the periosteum.

feds. This with bone twenty-four

PINEDA.

VICTOR

cartilage

testing

no apparent

As early as two weeks ogous osteochondral

J.

AND

without

to measure the until twenty-four

results obtained and those from

STEPHEN

dral

of repara-

ference between the from the bone marrow

differentiated

cells articular

of a semiquantitative

with employment of a porous indenter compliance of the tissue at intervals weeks

into

left empty

Incorporated

Cartilage*

Engineering,

periosteum that had sue by collagenase

gel,

Surgery.

of Large,

Skeletal

the

that had been created in the knees of rabcells from bone marrow, or cells from the

in a type-I

Joi,it

Repair

TATSUHIKO

Hospitals

atul

of Articular

ARNOLD

performed University

of Bats

It is well known that the capacity of articular cartilage for repair is limited. Injuries of the articular cartilage that do not penetrate the subchondral bone do not heal and usually progress to surface; a short-lived but it fails to provide

the degeneration tissue response sufficient cells

of the articular has been observed, and matrix to repair

even small defects’023235. Injuries that penetrate the subchondral bone undergo repair through the formation of tissues usually characterized as fibrous, fibrocartilaginous, or hyahine-hike cartilaginous, cies, the age of the animal, and the injury’4’6374’. However, these those

that

resemble

from normal biomechanically, and

extensive

tive tissues defects’3”.

hyahine and

hyaline

depending the location reparative

cartilage

on the speand size of tissues, even

histologically,

differ

cartilage both biochemically and by six months, fibrillation, fissuring,

degenerative

changes

of approximately half Similarly, the degenerated

occur

in the

repara-

of the full-thickness cartilage seen

in

579

1994


580

SHIGEYUKI

osteoarthrosis gressively attempts damaged totally ment

does deteriorates. to develop

usually There

undergo repair but have been a number

clinically

articular successful. is the

severe

not

useful

cartilage, Currently,

usual

clinical

degeneration

approach cartilage,

and long-term problems. including ing of the components. Biological resurfacing is an that could result in long-term tion.

Historically,

biological

joint, osteochondral, tion’52’45’. Experimental chondrocytes’3’427 ever, these methods

procedures

but these prosthetic

ofthe

for

the

but

it has short-term

alternative successful

of

loosen-

cartilage-shell studies have

the the

repair limited

transplantaused isolated

membrane43”. Howclinical application in

showing a living culture of the bone-marrow-derived the condylar defect (scale bar = fifty micrometers).

of defects availability

procedure. At our

laboratory.

osteochondral bone

marrow

defects bone

articular

marrow

cells

has

cently reported tor cells (which

been

from either proliferate

the

to form

bone

The

well

bone (grow)

for the useful

the

the

use

periosteum

for

the

presence

in periosteal

the isolation we referred

cells2235’2) these cells

investigated from method

cartilage.

progenitor

ability

have cells

as a biological

of the

chondral

we

progenitor

repair

lineage of joint

transitions tissue53.

cartilage

defects,

tologous

cells

a broad

they

may

also

range

we cells

of long grafts

chondrogenic the

embryonic

in the formation transplanted into

a practical

chondrogenic

source

potential.

gels have been used in the transplantation

cellular

of

recapitulate

provide

with

marrow

stem cells5”, as is that the stem

originally involved These cells, when

of auBecause

successfully of cells and

as delivery are of low

embedded autologous osteochondral from the periosteal membrane or bone bones

into

were

then

by six by three-millimeter), weight-bearing articular

a type-I

collagen

transplanted

into

full-thickness surface of the

gel. large

These (three

defects in the knee in rabbits.

I

was

cells,

released

by harvesting

The sequential repair studied by histological

tion

from

two

tion of the demonstrated

with

trypsin-EDTA

of these full-thickness and biomechanical

to twenty-four

weeks

planted

reformed by means basal cartilage.

cells

into

chondrocytes.

The

of endochondral

and

defects evalua-

after

osteochondral progenitor cells. the uniform differentiation

of

We

Materials

presumed

Sixty-eight

re-

progenistem

marrow or the periosteum; in culture without loss The

of

and

and

documented2’7557.

or cartilage.

capable

or of

of mesenchymal chondrocytes’2455’,

transplantaThis analysis of the im-

subchondra! replacement

plate of the

of osteo-

membrane

of osteochondral to as mesenchymal

are

mesenchymal

of the articular cartilage because of of donor material. Clearly. there

is a need to develop alternative technologies repair of cartilage in order to provide a clinically

of the use to articular

antigenicity59, progenitor

involved

FIG.

Phase micrograph then implanted into

vantage opposed

collagen vehicles

technology regenerahas

AL.

expression,

treatment and

ET

cells

to repair

infection

or periosteal have limited

proof

have not proved joint replace-

resurfacing

or

WAKITANI

of ad-

White

three

rabbits

Pennsylvania), were used

to

(100 THE

Research an The

injection milligrams JOURNAL


Methods

four-month-old

(Hazelton

weighing in this study.

by intramuscular hydrochloride

and

average rabbits of

OF

New Products,

Denver,

of 2.5 kilograms, were anesthetized

a mixture per milliliter BONE

Zealand

AND

of

JOINT

ketamine and 0.60 SURGERY

to

MESENCHYMAL

0.70

milliliter

(twenty

CELL-BASED

per

kilogram

milligrams

kilogram

of body

per

of body

REPAIR

milliliter

weight)

OF

weight) and

at the

and

0.30

time

ofthe

Derived

posed tracted,

of harvesting

of

time of transwere accom-

of heparin

rate

washed

was

(Sigma

twice

Chemical.

with

St. Louis,

per

milliliter).

New

York),

containing

balanced

Missouri)

and

calf serum antibiotics

selected

(JR Scientific, (penicillin G,

lots

Woodland, 100 units

solution centrifuged

of cells 10

was

to

each

determined

l0

nucleated

rabbit:

the

plastic serum)

with

Is-

of 10 per

cent

California) milliliter:

per

cells

were

acid

before

and

ampho-

the

a hemocytometer.

marrow

dish in complete at 37 degrees

phere first

acetic

cells

were

cultured

medium Celsius

of at ten

the

after

(10 per cent in a humidified

fetal calf atmos-

cells (Fig.

was com-

was

1). These

ficells

grew as a monolayer in nests that spread into neighboring nests. Approximately ten to fourteen days after seeding. the adherent cells were near confluence, at which time a five-minute millimolar eter before notation

the

EDTA, and incorporation and

troduction from which To vehicle,

cells exposure

handling

prepare the Pancogene

collagen, France)

obtained was

for one sterilized

were to

released 0.25 per

from cent

were

counted into the

in a hemocytomcollagen. Careful

were

of the cultured they had been

week on

dialyzed

required cells taken.

calf against

skin)

the

for the acid-soluble acid,

the

in-

rabbit

76-A,

NO.

4. APRIL

per

200

microliters concentracent type-I

delivery type-I pH

cartilage

defects

within

two

the

anteromedial

Periosteum-

per

Cells

was

of the

Celsius rupted through

cent

tibia

harvested

from

of each

collagenase

rabbit (CLS

and

incubated

2, 247

Biochemical, medium for three

with intermittent by vortexing, and a 100-micrometer

units

with

per

milli-

Freehold, New Jerhours at 37 degrees

agitation. The tissue was disthe liberated cells were passed Nitex filter (Tetko, Briar-

cliff Manor, New York), washed twice with Tyrode anced solution, and collected by centrifugation. The were then counted in a hemocytometer, seeded, in every

complete other

nearly

day.

medium. After

confluent,

The medium the adherent

they

were

trypsin-EDTA, lution in the mesenchymal

suspended in the same manner as the cells, and transplanted

Repair

Defects,

ofthe

A full-thickness millimeters wide,

and

and

Study

defect three

medial

femoral

condy!e,

ing three-millimeter In one knee, the

balcells and

was recells had

harvested

with

collagen-Ham F12 bone-marrow-derived autologously.

so-

Groups

(six millimeters long, three millimeters deep) was cre-

ated, through the articular cartilage chondral bone of the weight-bearing by the

and

drilling

into surface of two

the of

subthe

connect-

holes, each three millimeters deep. defect was filled with the collagen gel

containing the bone-marrow or the periosteum-derived mesenchymal cells. In the other knee, the defect either was filled with a collagen gel without cells or was left empty. Of the 136 knees in the study, thirty-one received

nine

served

empty,

as control

and

nineteen

cell-free All

3,

locomotion.

and the solution was light for one hour. To

Histological The

mesenchymal cells, mesenchymal knees

in which

served

collagen

rabbits

as control

gel was

were

operation and was observed

neutralize the hydrochloric acid, 100 microliters of twotimes concentrated Ham F12 medium was added to 100 microliters of collagen solution with gentle agitation at VOL.

ofthe

bone-marrow-derived received periosteum-derived

(Gatefoss#{233}, St. Priest,

hydrochloric

at 4 degrees Celsius, ice with ultraviolet

same

the

Mesenchymal

Periosteum

the

collagen solution S (0.3 per cent from

the dish by trypsin, one-

to ensure

into

Derived

become

from

in a 100-millimeter

seeding

at 1 x 10

F12 mixture (final per milliliter, 0.15 per

into

Preparation

Usually, obtained

bone-marrow-derived

days

suspended

transplanted

cultured placed

pletely replaced every other day thereafter. The change of medium at five days removed almost all of the cells that had not adhered to the culture dish. The typical broblastic

581

CARTILAGE

hours.

number

of 5 per cent carbon dioxide. The medium changed five days after seeding and was

appearance

were

gram) (Worthington sey) in Ham F12

resuspension Grand

microgram per milliliter), a small aliquot and the red blood cells were disrupted by cent

and

0.25

tericin B, 0.25 was removed,

4 per

dish

surface

100 micrograms

with

milliliter;

aspi-

streptomycin,

treatment

per

that 0.1

The

Tyrode

at 180 times gravity for five minutes. After in Ham F12 medium (Gibco Laboratories,

ARTICULAR

a temperature of 0 degrees Celsius. The cells were collected by centrifugation after removal from the culture

was

units

OF

of the collagen-Ham tions, 5 x 106 cells

Cells

(3000

DEFECTS

collagen). The mixture was allowed to gel in a carbondioxide incubator at 37 degrees Celsius for ten minutes. This gel that contained bone-marrow mesenchymal cells

with use of an 18-gauge needle a five-milliliter syringe containing

milliliter

fetal and

per

proximal-medial surface of each tibia was exthrough a small incision. The soft tissue was reand two milliliters of blood was aspirated from

the bone marrow was fastened to

land.

xylazine

Bone-Marrow-

Mesenchymal

The

FULL-THICKNESS

milliliter

the bone marrow or periosteum and at the plantation. The two operative procedures plished within a two-week period. Preparation

LARGE.

returned

were allowed to have an

the

thirty-seven cells, fortydefect

was

left

knees

in which

cages

after

implanted. to their

to move abnormal

the

freely. No animal gait or impaired

Evaluation rabbits

twenty-four weeks a carbon-dioxide

were

killed

after the chamber.

1994


at

two,

operation The entire

four,

twelve,

or

by placement knee was

in dis-

582

SHIGEYUKI

TABLE HIsT0L0;IcAL FOR

THE

I

GRADING DEFE-FS

OF

CARIII.A;E*

than Points

Cell morphology Hyaline cartilage

2 3

Mostly non-cartilage Non-cartilage only

Slightly

host

0

Surface

stain

(>3/4)

Moderate

Thickness

with

I

2

integrated

14

from smooth of the

lAverage

the

scale

described

thickness

et al..

cartilage

compared

of the

reparative

cartilage

macroscopically, of the femur was

cent

buffered

formalin

was

decalcified

with

for

defect.

The

0.5-molar

sections

then

Each

EDTA

with

that

specimen

containing

0.1-

were

stained

V. M. G.)

who

with

from

had

each animal by three of us

no knowledge

of

from which they had been obtained. was graded with use of a histologi-

scale,

which

is a modification

with

tissue that uninjured staining).

or the

toluidine

blue,

was normal, cartilage) to Surface

with

to

smooth).

The

0 (no

gap

cartilage) less than

cartilage). the

host

3

between

the

of

cartilage

was

and

host

donor lack

of the Repair

to 2 one-

Integration

adjacent

to 2 points (a complete refer to as dissociation).

the

of integration,

Tissue

of that

regularity,

degree was

described

compared 3 points or

from

by

0 (for

proportion

of

was

used.

and

served and

as the

An

The posicartilage and by a change

on a strip-recorder. a 1.5-millimeter

initial

uniform

the

preload

contact

cartilage.

softer

than

grams

porous

in-

grams

was

of two

between

The

normal was

normal,

and mate

cartilage.

then

deformation The

the thickness the compliance

of

the control

tip of the cartilage

inherent

compliance

THE

again the

K)URNAL

in-

came

test

load

to estibasis of

1/stiffness = k x indentation where k is an instrument confor all tests. give a relative measure of of the tissue, with emphasis of the single

tissue

digits)

stiffness),


which under

load

in an

=

resistance (usually (high

test

resulted

cartilage,

displacement

The compliance values mechanical properties numbers

additional

This

the cartilage were used of the cartilage on the

the equation: compliance depth/cartilage thickness, stant that was the same the

An

applied. of the

to an equilibrium.

Small the

were measured simultaneously. needle at the surface of the of calcified bone was identified

to ensure

on the

with the adjacent, (no metachromatic the

mesenchyunfilled deweeks after

slowly deformed under this preload and reached an equilibrium displacement after approximately one mmute. In some of the defects, more time was required to reach equilibrium displacement because the cartilage

of metachromatic graded

treated with periosteum-derived and five spontaneously healing, tissues were retrieved twenty-four

tip was

creased

scale is composed of five categories ranging from 0 to 14 points (Table

Matrix-staining.

surrounding

in the force335, as displayed In the indentation test,

of two

I). The cell morphology was graded from 0 (for tissue that was normal, compared with the adjacent, uninjured cartilage) to 4 points (when cartilaginous tissue was absent).

of the

displacement tion of the at the zone

indenter

from the center of sections were pre-

group sample

staining

smooth)

was

that of the surrounding average thickness was

Properties

applied

toluidine

study Each

Pineda et al.’. The and assigns a score

was

one-quarter

depth of the indentation were measured with use of procedures developed by Mow et al.3 and refined by Matsuura et al.3335 to estimate the compliance of the reparative cartilage. A needle-penetration technique was used to determine the thickness. The force on the needle and its

denter

acid and 0.005-molar sagittally, perpendicular

from each knee. The sections examined and scored independently

(S. W., S. J. P., and

cal

the

photographed. fixed with 10 per

week.

blue 0. These sections were obtained the defect, and as many as ten such

the

compared

and

one

molar epsilon-amino-n-caproic benzamidine and was sectioned

pared were

with

cartilage.

sected, examined The distal part

to the

by Pineda

area of the reparative cartilage defect.

surrounding

surface

than

the operation. The lateral, untreated condyles normal controls. The thickness of the cartilage

0

maximum

*Modjfied

the

from

had been mat cells, fects. The

host

edges integrated edge integrated edge

the

of less

smooth compared from 0 (when more

Microindentation was performed on the repair tissues from six defects that had been treated with bonemarrow-derived mesenchymal cells, five defects that

2

Neither

appears graded

cartilage

Mechanical

0 1

Both One

the

cartilage) which we

3

1/3-2/3 2/3

Integration adjacent

that donor

2

0 1

(>1/2-3/4)

Irregular (1/4-1/2) Severely irregular (21.7

>41.2

normal

cartilage

of the

lateral

too

results

value

could

not

be

determined

because

the

soft.

compared

with

those

at two

twenty-four weeks after inferior to the four-week were

in accord

with

weeks,

but

implantation, scores (Table

those

formed where the periosteum-derived cells had been p!aced in the defects sively more irregular compared with face in the bone-marrow-derived

at the II).

of macroscopic

and histological observations. The scores of the mesenchymal-cell groups were similar, except for for surface regularity. The surface of the cartilage

two that that

mesenchymal became progresthe cartilage surmesenchymal-cell

group. In the control

cell-free-collagen-gel

groups, inferior

the repair

cel!-implanted

Mechanical Compliance cartilage and in the groups marrow and

groups, Properties

compared

with

at each ofthe

the

scores that

interval Repair

empty-defect

indicated

mark-

in both

of the

of sampling. Tissue

values were determined for the norma! cartilage, from the same that had been treated with the the periosteum-derived ce!ls and

for

derived-cell

groups

compliant

and

histological

repair knees, bonein the

the

empty-defect ance values less

and

groups

5

8.8 4.0

condyle in the operatively treated knees and for the posterior and anterior portions of the repaired tissue of the same knees. Since all values for all specimens are provided, statistical analysis ofthe averages is not given.

edly

surface, and final stages of

In a!! groups, reformed.

bone-marrow

cells

defects

tThe

cartilage

staining

carti!age bone

surface

formed cells had

Grading

teum-derived

18.4

5.4

5

*Compliance

an appearof hya-

thinner than at twelve weeks staining was not observed

was very comparable

Histological

24.0

6.1

(control)

thinner

peared quite thin, metachromatic staining was absent in some regions, and the surface was more irregular than in the twelve-week specimens (Fig. 6, D). In the cellfree-collagen-gel and the empty-defect control groups, the tissue appeared

18.4

Average

after the Operation

mesenchymal

fects was slightly D). Metachromatic

of

and

Empty

was

Cartilage

2.7

Average

(Fig. 5, C). periosteum-

cartilage

surface

than the adjacent host all groups, subchondral

derived

Values*

5.1

cells

cartilage same thick-

appeared

articu!ar

thinner In

Twenty-four

a!so

metachromatic

brous tissue appeared but was patchy and

liance

Ant. Portion

that

staining

of the chondrocytes had with that of the chondrocytes

the

DEFECTS

Post. Portion

artic-

than

bone of the

group

line cartilage, the articular appeared to be more irregular. In the cell-free-collagen-ge! control

THE

OPERATION

Normal Cartilage

6

mesenchymal

3, C). The appearance with that of hyaline

host subchondral in the defects

most

OF THE

bone-marrow-

thinner

metachromatic

mesenchymal-cell that

TISSUE AFTER

No. of Specimens

Periosteum-derived mesenchymal

of

group cartilage (Fig. still comparab!e

as that of the The cartilage

than

WEEKS

Group

de

tissue

the Operation

in the

adjacent cartilage

derived

TWENTY-FOUR

III ThE

(Fig. 7, stain-

covering

abnormally faint or absent. The subchondral was completely replaced by new bone of the ness

FOR

Average

cartilage

cartilage,

VALUES

587

CARTILAGE

Comp

the

thick,

fibrous

of the host margin faint metachromatic

mesenchyma!-cell

of the of the

ARTICULAR

TABLE COMPLIANCE

ular surface.

derived

OF

Defect

extending to the B). We consistently

Twelve

DEFECTS

ad-

(Fig. 6, B). The appearance of comparable with that of hyathe surface was slightly irregu-

proximal

matrix

OF

control group (Table III). The complithe bone-marrow and the periosteum(more

control knees. These pair tissue was more

indicated stiff)

that than

that

the

repair

in the

values a!so indicated compliant (!ess stiff)

1994


tissue

was

empty-defect that than

the renormal

588

SHIGEYUKI

WAKITANI

FIG.

Schematic a collagen mensenchymal

drawings showing gel (upper-left panel) stem cell. TGF-

cartilage. Within repair cartilage

the sequence of events during the to the time of replacement of the 3 = transforming growth factor-beta.

a defect. was less

the posterior portion compliant than the

8

of the anterior

that were bone-articular in the

articular

cartilage

cartilage after

were

repaired

implantation

dral progenitor cells marrow or periosteal early

demonstrates weight-bearing

as

two

teochondral

of

weeks

progenitor

cells

had

was then replaced after transplantation,

ticular surrounding

host

first

report,

cells

differentiated in the

with

almost

these

but

united

with

vascularized the deeper

that

knowledge,

mesenchymal

underlying of the of the

autologous

full-thickness

reports

cells

and into

are

scarce,

we

have

by been of the the ar-

from

the

bone

was

This

differen-

chondrocytes

is

have

been

able

to grow

These

mesenchymal-stem-cell

Without cells, with

and

suitable

cartilage

experimental

this theory which the

cannot cartilage

in

rapidly

in defects We hypothesize

the

them

chickens, huhere, rabbits.

preparations

chondrocytes of the rabbit.

chondrocytes

noted

when tested in either in Although these progen-

from marrow harvested from rats, mice, dogs, goats, and, as described into condyle

of the

cells, which have both os-

culture mans, ferentiated femoral

in =

subchondral

laboratory

form is rep!aced by host-derived forming cells up to the bone-articular

progen-

of the

our

cells MSC

resurfacing

or periosteum-derived mesenchymal stem cells’,

donor

implantation

osteochondral defects,

cells

on

in the

bone. portion had

host.

from

stem cartilage.

bone up to the events resulted

cartilage

resulted

portion

replaced

by a gap

the

os-

into

deeper

completely

separated

cartilage, to our

cultured, into

of

been

remained

completely

of purified, tiation

had

cartilage

the

by twelve weeks, subchondral bone repaired without loss or alteration articular cartilage. In some specimens,

cartilage

always

repair

transplantation,

of the defects By four weeks

itor

of articular

by These

teogenic and chondral potential vivo or in vitro systems2223254’2. itor

hyaline-like osteochon-

after

the

defects

with

autologous

that had been isolated from bonetissue and grown in cell culture.

chondrocytes;

bone, and completely overlying

that large, region of

of mesenchymal by articular

replaced junction.

in effect,

Previous

The current investigation full-thickness defects of the

the

formation

that bone-marrow we have called

Discussion

of the

subsequently cartilage

bone, which, condyle.

condyle.

As

AL.

first four weeks, from the time of implantation resulting cartilage by donor host bone covered and BMPs = bone morphogenetic proteins.

portion. This was probably due to the kinematics of the rabbit knee and the different forces exerted on the postenor compared with the anterior aspect of the femora!

the

ET

of

tissue vascular cartilage

marking

dif-

the that

distal these

that

they

and bonejunction.

of these

donor

be verified; however, the speed is replaced by vascularized

bone is impressive and is comparable with of formation of endochondral bone, which scribed previously5. With this in mind, we

the process we have depropose the

following

which

a basis here

hypothetical for our

(Fig. 8). First, the

femoral

sequence

discussion creation

condyle

of the

establishes THE

of events,

of the

JOURNAL


observations

large an

OF

defect

osseous BONE

AND

forms

presented of the receptacle JOINT

distal into

SURGERY

which

MESENCHYMAL

CELL-BASED

host-bone-derived

bioactive

REPAIR

factors,

forming proteins,

growth factor-beta and are released. In addition,

post-injury lagenous

response serves to infuse delivery vehicle with bioactive

both These

bone we

LARGE.

such

FULL-THICKNESS

as trans-

morphogenetic suspect that

the

donor mesenchymal pathway, which

stem results

of embryonic-like cartilage Second, the continually ous receptacle causes the

the

Others

implanted colagents, provided

cytes are bioactive

change

rapidly

until

the

most

tissue become hypertrophic, its chondroclast-mediated tion.

The

the

invasion

resorption

macromolecules

stem cells

in these pathway places

in the cells into

highly

cartilage

sites

ofthe

host

that

of the

cartilage

contact

with

at the

articular

the synovial vial fluid. freely rapidly

the

enter

different neither

from the be resorbed

There

are

tracts

potent

which

are

proximal repair nor infiltrated important

bioactive

osteogenic which

to

rapthe

the subchondral

which This

has distal

been

tissue by the

and, thus, vasculature.

aspects

of our

support is already bone rapidly disburses factors,

transforming

of

the

most

often

can

hypoth-

tors

indeed, others49-’52

involves

either

the

in vivo

or the

in vitro

dylar defect, we would sorption substantia!ly

expect and to

VOL.

76-A,

NO.

4.

APRIL

a changing pattern in the proteoglycans, specificto comminuted

chondro-

through

this

lineage

to the

hypertrophic

state,

stem

ce!!s

rep!acement the replacement

will

enhance

and

contribute

of that cartilage by bone. events (endochondral

to

the

The sequence myelopoiesis)

of is

identical to that observed in the development of the embryonic joint, at the growth plate in growing animals, at sites of heterotopic implantation of deminera!ized bone at in vitro or in vivo sites bone-morphogenetic-protein

The

size

of

the

femoral

defects

condyle

that

in the

of either

native

were

created

current

study

in was

millimeters wide, six millimeters long, and meters deep. These defects, which composed

have

been

ously tion5,

or under conditions defects as large as

noted

to

undergo

or 71

repair

the

three

three milli40 to 50

either

condyle, studies cartilage spontane-

of tissue or cell augmentathose in our study have not

of or

jor

conversion It facchon-

to these articular con-

these cells to resist be unresponsive to

bioactive factors released from the osseous prediction is borne out by the observation ogous articular chondrocytes are retained

or chemical In particular,

family

cells into chondrocytes. target of these bioactive cells, not differentiated

drocytes; differentiated chondrocytes respond factors in a negative manner’538. Thus, when chondrocytes are transplanted into a full-thickness

identity

is not3.

studied

the bioassay developed by Urist for the purification of these fac-

of progenitor mesenchymal must be stressed that the tors is mesenchymal stem

the

to

been described previously, to our knowledge, as having undergone either spontaneous healing or substantial repair. The observed repair, although not perfect, is a ma-

the bone morphogenetic proteins4”’6. These factors function to convert osteochondral progenitor cells into chondrocytes; et al.7 and

but entities

exhibits synthesized

chondro-

culture or exposed of phenotype-specific

per cent of the weight-bearing surface of the are among the largest ever reported in repair in rabbits. Although small defects in articular

available and at-

growth-factor-beta

is affected,

articular

by

even in culture42. The in vivo hypertrophic cartilage is then the target for erosion and vascular invasion5. The presence of vascu!ature and new, host-derived mesen-

medial

milieu

the

and replacement slow process5559.

when

aggrecan6’#{176}’53. In contrast

progress

in

found in the synoallowed to move

that into cell quantity

589

CARTILAGE

cytes, when articu!ar cartilage chondrocytes are placed under identical in vitro circumstances, they continue to synthesize the aggrecan species that were previously synthesized in vivo. In contrast, mesenchymal stem cells that enter the chondrogenic lineage can rapidly

chips, or recombinant

cartilage

by the

ARTICULAR

shown

cartilage of the

chymal re-

the joint cartilage developed tissue, which is intrinsically

substantial Injured

the

defect,

is influenced

several

eses for which in the literature.

and

and the molecules the rabbits were

after the operation, into load-bearing

the tissue,

cartilage is not resorbed in a manner quite unlike

receptacle.

boundary

cavity Because

and mes-

and replacement into bone up

cartilage

in the

osseous

defect

or perivascularhost-derived cells

osseous

bone. The distal repair articular because it has differentiated

repair

host-derived

from the marrow this region. The

Third, the process of erosion changes the repair cartilage

natura!junction

of the

draw

vascularized

in the

OF

of these

developing composition

this tissue, in turn, e!icand vascular infiltra-

tissue

and rapidly fabricate the repair cartilage.

the chonfabrication

ally

cells

have introduced agents,

characteristics

milieu of the ossein the defect to

proximal

and erosion

by vascular

enchymal associated

cells into in the rapid

tissue. changing chondrocytes

DEFECTS

of full-thickness defects for months bone, when it occurs, is an extremely

from the site of the wound itself and systemically. agents initiate the immediate and rapid entrance

of the drogenic

idly

OF

rethe

defect. This that heterolin the depths

accomplishment. Two aspects

perimentation

of and

this

repair

require

improvement.

The

additional first

ex-

involves

the

progressive thinning of the repair tissue, which was discerned clearly by twenty-four weeks after transplantation. The mesenchymal-stem-cell preparation used for the periosteal-cell group showed some differences cornpared with the mesenchymal cells derived from the bone ness

marrow. These differences of the surface of the repair intervals. twenty-four

the smoothat the latest

respect to the weeks, although

periosteal-ce!! not all joints

sampling group

at

behaved paired

uniform!y, there surface to split and

bone-marrow-cell smooth, although area

for

improvement

With

involved cartilage

was a tendency fibri!late. With

group, the the cartilage involves

1994


for respect

repair cartilage became thin. the

integration

the reto the

remained The second of

the

590

SFIIGEYUKI

repair cartilage with substantial integration defects, pair

we and

the

observed host

cartilage occurred

of the host. Although around the edges of the

incomplete

integration

of the

at

locations.

Systematic

cartilage

experimentation the integration We realize

WAKITANI

some

is required to develop of these surfaces. that a detailed biochemical,

AL.

are due to the gait and motion which suggest that the posterior

re-

techniques

ET

for

to more

force

stressed

that

than the

the

anterior

rabbits

were

their cages immediately gradual weight-bearing,

immunohis-

of the cartilage cartilage.

It should

returned

after the coupled

be

activity

in

operation. In humans, with constant passive

motion47,

would

O’Driscoll tion may

et al.43” believed that constant passive mobe an important factor in the successful repair

typic character of the tissue that was formed and the sequence of events that occurred at the repair site. However, these studies will be conducted after we have optimized the repair of the defect by measurement of the

of full-thickness The procedures this report may

histological. here. mechanical

of the repair tissue. that the mechanical six

months

ences repair ment ent

and

treatment number

parame-

to evaluate

The indentation properties those

the qualities

data clearly of the repair of the

host

indicate tissue at

tissue.

two

locations.

These

mechanical

the

protocol

defects auto!ogous

grown

in vitro,

of the articular progenitor and

defects.

then

These

introduced cells,

differences

choice.

described method

cartilage. cells may

in of

A small be iso-

into

when

such defects, appear capable both articular cartilage and of inducing

Differ-

of

defects of the cartilage. to elicit repair that are lead to a clinically useful

of

full-thickness

the anterior and posterior aspects of the probably reflect the mechanical environrabbit knee, which is substantially differ-

in these

for

lated, testing

approached

between tissue of the

biomechanical

be

to full

tochemical, and molecular biological analysis, including in situ hybridization. is required to define the pheno-

macroscopic, ters reported We used

probably

rabbit joint, is subjected

massive

implanted

of differentiating the formation

in into of sub-

chondral bone. The procedures have considerable vance to the treatment of defects in the cartilage

releof

humans and provide the basis for repair technology that is capable areas of articular cartilage.

of a large

the development of regenerating

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of