Note: Descriptions are shown in the official language in which they were submitted.
''V~) 90~00~7 PCI~lJS90/01025
MATE~IALS ~ T~O~.S
~CKGRQ~ND OF T~E~Irn~ laE
1. Field of the I~ven~lon
This invention relate~ to a periodontal
ligament cell-attrastant ~actor useful for periodontal
and bone regeneration, The inv~ntion also relates to
regeneration methods, co~position3 and materials.
2. ~
~thods for bonding materials, such ac a
resin layer, to teeth and bone arQ described in Unlted
States Patents 4,382,792 granted May 10, 1983 and
4~600r383 granted July 15, 1986/ both issued to DoC~
S~ith et al.
A number o~ publications with a focus on
regeneration oP the periodontium have descri~ed
biologlcal factors and methods involved in the
formation of a n8w connectiv~ ~iS~ue a~achm~nts ~o
periodontally diseased or d~nuded root ~urfaces, such
as the publications di~cussed a~ follow~.
~'~olecular Factor~ Deter~ining Gingival
Tissué Interaction with Tooth Structure'~, Victor P.
Terranova et al., Journal of Periodontal Research
Yol., 17, 1982, pp. 530-533, de~cribes that the
attachment oP epithelial C811~ a~d ~ibroblast~ to
tooth sur~aces i~ stimulated byj respe~tively, th~
glycoprotsins laminin and ~ibronectin.
A review of the ~unction o~ polypeptid~
.
; ~ ` A `
-
--2--
growth factors and description of their potential role
in pQriodontal therapy to promote mi.gration of
periodontal ligament cells to dentin, as well as to
root surfaces preconditioned by treatment with
tetracycline HCl and/or citric acid, is given in:
"Extracellular Matrices amd Polypepkide
Growth Factors as Mediators o~ Functions of Cells of
the Periodonti~m", Victor P. Terranova et al~, J.
Periodontology, Vol. 58, No.6., ~une 1987, pp. 371~
380, the disclosure of which is incorporated herein by
reference.
United States Patent 4,702,734~ granted
October 27, 1987 to VD P~ Terrancva et al., the
disclo~ure o~ which is incorporated h~rein by refer-
ence, discloses a method ~or pro~oting periodontal
regen~ration, which includes exposing an area of tooth
sur~ace, preco~ditioning the area with a tetracycline
salt, applyin~ ~ibronectin (FN) and optionally
applying endothelial cell growth fac~or ~ECGF).
one ~ssential biological event in tissue
regeneration i5 specific cell directed movement
~chemotaxis). Chemotaxis is an essential fe ture o~
many biological processes in health and disease. A
specific example of chemotaxi~ is the move~ent of
endothellal ~ells in the process of n~ovascularization
as disclosed in:
. "~uman Endothelial Cells are Chemotactic to
Endothelial Cell Growth Factor and Heparin", V~ctor P.
Terranova et al., The Jou~nal o~ Cell Biology, Vol.
101, December 1985, pp. ~330-2334. Th~s publication,
the disclosur~ of which is incorporated herein by
reference, descr~es a chemotaxi~ assay.
Anot~er exampl~ is the description of
movement of tumor cells to form ~etastases in the
publication:
"~igration of Tumor Cell~ of Organ-derived
Chemoattractants~, ~rkki S. Hujanen et al, Cancer
PCT/US90/01025
Research, Vol. 45, Augu~t 1985, pp. 3517-3521.
The chemotactic propertle~ of laminin,
fibronectin and various polypeptides are described,
and laminin i~ shown to pro~ote chemotaxis and growth
of human gingival epithelial cells, in:
"Chemotaxis o~ Hu~an GingiYal Epithelial
Cells to Laminin", Vi~tor P. Terra~ova et al, J.
Periodontology, Vol. 57, No. 5, ~ay 1986, pp.311 317.
In the article:
~Biochemically Mediated Periodontal
Regeneration", Victor P~ Terranova et al., Journal of
Periodontal Research, Vol. 22, 1987, pp. ~48-251, the
chemotactic attraction of periodon~al ligament ~ells
to fibroneotin and endoth~lial c~ll gr~wth factor
bound to dentin i~ disclosed. Chemotactic prop~rties
of other protein and growth ~ac~ors such a~ pla~elet
deri~ed growth factor, are al30 discus~d.
The article:
"A 8iochemical Approach to Periodontal
Regeneration", Victor P. Terran~a et al., J.
Periodontolo~yt Yol. 58, ~oO 4, April 1987, pp. 247-
257, the disclosur~ o$ which i5 in~orpora~d herein by
reference, describes a new a~ay ~yGtem that test~ ~he
ability o~ a number of protein~ and polypeptide growth
factors applied on den~in to ~ti~ulat~ a chemotactic
and proli~erative response ~ro~ ~arious cell types.
The articl~: -
"~ e o~ a Recon~titut~d Ba~ement Membrane to
~easure Cell I~va ivenes~ and Select ~or ~ighly
Invasiv~ Tumor C~118~ Vi~tor P. ~erranoYa et al.,
Proc. ~atl. Acad. SCio ~SAg Vol. 83, January 1986, pp.
465 469, d~cribe~ the preparatio~ and use of
reconstituted ba~emQnt ~mbrane~ consi~ing of laminin
~nd type IV collagen recon~titutsd onto a d~k o~ type
I collagen, which ca~not be penetrated by fibroblasts
or epidermal cell~. '
~4--
SuMM~y-Q~ ~B~ v~TI~
The inv~ntion relate~ to a~ $solated
periodontal ligament cell-attractant: ~a~tor (~DL-CTX),
which ~ompris2s a protein obtainabl~ ~rom periodontal
ligament (PDL3 cells, Gaid factor having chemo-
attractant a~tivity to periodontal liga~ent cells.
~he inventiDn al80 r~lat~s ~o a composition
useful ~or periodontal regeneratlon which co~prises a
pharmaceutically acceptable amoun~ of said PDL-CTX
~ac~or and a pharmaceutically acc~ptable m~dium.
The in~ention further relate~ to a kit
useful for periodontal regeneration, which includles
said PDL-CTX ~actor.
Further, the invention relates to
improvem~nts in a method of periodontal regen¢ra~ion
or of bone re~eneration in which said PDL-CTX faator
i5 applied to the surface to be regen~rated.
In another embodiment, the i~vention relates
ZO to improvement~ in a method for inducing periodontal
cell ~igration on dentin, in which ~aid PD~-CTX factor
i~ applied to the dentin.
In a further embodim~nt the invention
relates to an improvement in a ~ethod ~or p~riodontal
regeneration which comprisss the step~ of:
(a) obtaining a specimen o~ a parent
popul~ion of periodontal ligament (PDL) ~ells from a
patient;
(b) growins the obtained PDL cells in a
tissue culture mediu~ to obtain a culture of the
patient~ PDL cells;
(c) selectin~ a sub-population oP PDL cells
of said culture which migrate throu~ a porous type I
collaqen barr~er by che~otaxi~ directed against said
PDL-CTX facto~.
Still ~urther, ~he invention relates to
improvements in a ~ethod oP periodontal regeneration
in which periodontal ligament cells are applied to the
- `'O~0/100l? PCT/US90/01025
-5-
root sur~ace oP a tooth, which comprises cov~r~ng ~he
tr~ated surface with an artif1ci~1 basement membrane
comprised of collagen.
The invention al~o xelates to a method of
periodontal regeneration, which comprise~:
(a) exposing a tooth ~urface to be treated:
(b~ applying to ~aid ~urfac:e a
pharmaceutically acceptable amcunt oi` sald PD~ CTX
factor in a pharmaceuti ally acceptable ~edium;
(c) applyiny a ~uspension o~ ~he patients
periodontal ligament cells to the ~urface; and
~d) covering the treated ~urface with an
arti~icial basement ~embrane compri~ing type I
collagen overlay~red with type IV collagen and
laminin.
~BI~F DESRIPTION O~_~EE_~A~I~GS
Figs. 1 to 5 are graphical repre~entations
of experi~ental data illustrating the che~otactic
attraction of PDL cells to ~h~ PD~-CTX factor.
Fig. 6 is a graphical representation of
experi~ental data illustrating the chemotactic
attraction o~ bone cell8 to the PDL-C~X factor.
Fig. 7 i~ a gxaphical represe~tation o~
experimental dat2 $11ustrating ~he chemotactio
attraction of parent and ~elect~d PD~ cells to the
PDL-CTX factorO
Fig. 8 i~ ~ graphical repre~ntation of
experi~sntal data illustrating 3H-thy~idine
in~orporatio~ into D~ in parent and~s~lected P~L
cells.
Fig. 9 i~ a chromatogram of ~ prot~in-
con~aining concentrate o~ PDL c~ll conditioned m~dia
precipitated with ammoniu~ ~ulfate.
. .
The repair of injury to the sum~ oP h~uman~
begins as soon a~ tissue damagQ ocaurc, an~ the
release of p~lypeptide grow~h faotors from i~jured
cells and inflammatory cells is a crit$cal part of
this process. Many o~ the pol~peptide growth factors
have been ~hown to be involved in tis ue repair. For
example, platelet derived growth ~actor (PDGF) and
transforming growth ~actor-B (TGF-B) are two
polypeptides that have been sh~wn ~o havQ an important
role. PDGF is initially rel~ased from the alpha-
granules of the platele~ and i~ a potent mitogen for
fibroblasts in the pre6~nce of either transforming
growth factor-~ (TGF-~) or epidermal growth factor
(EGF). Furthermore, PDGF stimulates tha production of
collagenase by fibrobla~s and thus contributes to the
re~odeling of matrix components, an es~en~ial feature
of tissus repair. TGF-B app~ars to have a particu-
larly important role in the repair process. This
peptide is ~ound in relatively high concentrations in
platelets, in activated T lymphocytes, as well as in
macrophages. TGF-B ~ti~ulates the formation o~
collagen in human or rodent fibroblast~ and when
injected subcutaneou~ly in newborn mice cause~ rapid
fibrotic and angiogenic responsa at thQ site of
injection. Ano~her recently di6covered source of TGF-
B is bone. TG~B ic pre~nt in bone in amount o~
almost 100 fold greater than ~ound in other ~oft
tissues. 1~ vitrb studies indicate that TGF-B can
control the ef~ects~of s~veral o~her polypept~de
growth ~actor including PDGF, TGF-~, EGF, AF~F
.~aci~ic ~ibr~b~a~t grow~ factor)~ BFGF (ba~ic
~ibroblast growth factor) and IL-2 (interleukin-2).
To fully understand the ~ec~anism of all o~ these
peptide growth ~actors it must be realized that they
act in sets or co~bination in which each peptide
modulate~ th~ e~fects o~ the next.
~lthough ~here ~ay be au~ocrine action of
these peptides in injured cells, it would appear that
---`VQ 90/10017 PCT/US90/01025
--7--
their paracrine action, driven by th~eir production and
release by various in~la~matory c~ll,s, accoun~s for
the key role of these pep~ides in th~e repair process.
However, autocrine function ~ay play an important role
as has been demonstrated in traumatized cultures of
artereal smooth ~usale c~115 which synthesi~ and
release peptides that resemble PDGF. The function of
many o~ these known peptide~ i~ r~lationship to c~lls
of the periodontium is presently under investigation.
In addition, other factors isolated from cem~tum have
the potential to be mitogenic for cells nf osseous
origin.
One ~f the least understood aspects o~
tissue repair is the bio~hemical factors that cuntrol
specific cell motilityO To expect a uniqus autocrine
factor that could he responsiblQ for initiation of
thi~ e~ent i~ not unreasonabl~. Recently~ an
"autocrine motility $actor" (A~F) has been id~nti~ied
~or melano~a cells. The factor was found to be
unique, bas~d on amino acid analy~ls and has a
molecular weight of 55~000O It~ action may be
associated with membrane changes in phospholipid
methylation. Si~i}ar membrane change~ have be~n
imp}icated in the motility o~ l~ukocykes. In other
systems marked inereas~s in p~ospholipid m~thylation
have been observed in hormonal e~fects upon
h~patocyte~ and bladd~r 1~pith~lial c~ o
. A nQwly de~Qlop~d a~say syste~ (d~scribed in
the above~mentioned publication ~A Bioche~ cal
Approa~h tn Periodontal Reyeneratlon~ 0~ VaP~
Terrano~a et al.) enables testing oP the potential
aativity of various biologi~al response modifiex~ on a
dentin substrate. Th~ as~ay ~ystem~ i~ di~ided into
two part~. ASSAY I allows the mea ure~ent o~ the
chemotactic activity of ~h~ te$t ~ub~tanc~ bound to
dentin. ~er~ the cell~ ~ust acti~ely move through a
filter (1~Nuclepore" [registered trademark, l00 ~m
thick, 8 ~ pore diameter) in respoll~e to b~ological
response ~od~fiers bound to dentin. In ASSAY II the
ability o~ the dentin-bound factor~ to stimulate
directed movement and proliferation of periodontal
tissue c~lls on dsntin ~ur~aces i~ D~easured. Using
~hese a~says ~t was demonstrated th2lt PDL cells
migrate to FN, ECGF and AFGF. In addition, FN, ECGF
and FGF induce a proli~erativ~ respon~e in PDL cells
}O grown on ~urface-deminQralized d~ntin. Gingival
epithelial c~lls were shown to miyrate to LM (la~inin)
and LM ~ragments. ~M wa~ also ~hown to sti~ulate
gingival epithelial cell proliferation on native
dentin surfaces. These ~n ~i~E~ findings sugge~t that
biological conditioning of the root surface (den~in)
may enhance me~nchymal cell attachm~nt and
proliferation. These events ~ubsequen~ly lead to an
improvsd h2aling a~ter periodon~al recon~tructlve
surgery.
TherQ is a need to develop more potent and
selecti~e che~oattractant factors ~or PD~ cells and
cells of osseou~ ori~in (bone cells), and ~or improved
~ethod~ for the treatment of root surfaces to
selectively enhance P~L cell repopulation of the
previously di~ea~ed root urface.
The PDL-CTX factor o~ the pre ent invention
is a potent new PDL cell and ~one cell attrac~ant
factor which i8 a protein isola~ed from, derived from
or obtainable from PDL cells. I~ may also be obtained
3Q by recombinant DNA me~hodolog~ or by peptide
~ynthesis, by technique~ ~nown ~or ~uch methodology or
synthesis.
It ha~ been found that the PDL-CTX factor of
th~ present invention is a pr~tein which is charac-
terized by periodontal ligament cell autocrine
motility and mitogenic activity. The pxotein factor
~ay be rec~vered fro~ a medi~n conditioned by PDL
c~lls (the medium obtaln~ a~ter being used in the
~ 90~00l? PCI/~1590/01025
tissue culture of the PDI, cells~. A concerltrate oi~
the protein may be r~covered a~ a precipitate by
treatment of said ~ediu~ with a ~alt 6uch as a
ammonium sulfate.
An e-~pecially use~ul PDL ~rx factor was
derived from periodontal ligament ce:Ll~ which were
sPlected from a plurality o~ periodontal ligament
cells ,, the selected periodontal ligament cells having
been sele~ted on the basiE/ of arl incrQased che~otactic
response to said ~ac:tor relative to the che~otactic
response of the other periodontal ligament cells of
said plurality o~ cell~.
The PDL-CTX f ac:tor has been ~ound ~o
comprise a protein having a molecular weight o~ 45,000
to about 55,000 daltons. Upon work-up ~urther the
protein was founà to have a ~olecular weight of about
50, 000 to about 53, 000 dalton~. E'urther, the protein
of the PDL-~rX factor is beli~ved to be~ a ltetramer.
Thus the u~eful factor ~ay c:ompris@ a monomer which
forms said tetra~er, sai~l ~ono~r having a Dlolec:ular
weight of about 12, 500 dalton~
The PDL-C:TX factor of the present invent:ion
has b~en characterized ~y an ~ nv~stigation the results
of which are summarized in ~:he drawing~ which ~re
explained ~urther in tlle ~ollowing discussion.
The graphical repr~;entation of data irl Fig.
1 shows the directed migration o~ PDI. t:sll~ to
conditioned media (509~) after inctabation at various
te311peratures, where migration i8 aseayed usir~g ASSAY I
and each assay point 3~epresents the mean +,/- s . d .
( standard d~viation) o~ tr~plica~ ;aDlpl s ;,
The data repr~serlted in Fig. ~ sh~w~
direc:tional migration o~ PDL cell~3 to 50% cond~tioned
media i~cubat~d w~th tryps~n for ~rarious times where
migration i~ assayed u~ing a5s~Y I and eac:h ~sE~ay
point represents the mean +~ ,d. o~ quadnaplicate
samples.
--10--
In FigNre 3, the graphlcal representation of
data shows direct~d migration o~ PD~ cell~, human
gingival ~pithelial cells and human gingival
fibroblasts to various concentrations (dilutlons) of
PD~ cell conditioned media wh~r~ ~igration is assayed
using ASS~Y I and each point repre~,~nts th~ ~ean +/-
s.d. of triplicate ~amples.
Figure 4 i6 a qraphical representation of
10 data whic~ show~ the effect of various concentrations
of TGF-~ and TGF-B on the che~otaxi~ cf PD~ cells
where migration is as~ayed u~ing ASSAY I, all factors
are allowed to bind.to TTC ~o~ditioned dentin (dentin
preconditioned in 100 mg/~l of tetraoycline ~ICl) or
15 30 minutes at 37-C in 100% humidity, PDL cell
conditio~ed ~edia is used ac a control and each assay
point represents the mean ~- s.d. of triplicate
samples.
Data repr~sented in Fig 5 show the effect of
20 various antibodies against extracellular matrix
component~ on migration of PDL cells to 50% PDL cell
cond~tioned ~edia where antibody dilutions are
incubated with the PDL cells abo~e the Nuclepore
filter, ~igration i~ assayed using ASSAY I and each
25 assay point is the mean ~/- ~.d. of triplicate
sa~ples.
The graphical representation of data in Fi~.
6 shows directed migration o~ human bone cells (ATCC
7009) to 50% PDL cell conditioned ~edia or TGF-~ wh~re
30 migration i~ aRsayed using A~SA~ I, both factors are
allowed to bind to pre~iously TTC conditioned dentin
for 30 minutes at 37C in 100% humidity and each assay
point represents the ~ean of duplicate samples not
dlffering by more than 10%.
In Fig. 7, the graphical representatio~ of
data shows directed migration of parent and ~elected
PDL cells to various dilution~ of condit~oned media
where election of PDL cell~ is accomplished by the
--`VO90/1001? PCT/US90/OtO25
methodology esta~lished in the above mentioned
publication "A Biochemical Approach to Periodvntal
Regeneration" of ~.P. Terranova et al.), migration is
assayed using ASSAY I and each assay~ point represents
the mean +/- s.d. of quadruplicate s~amples.
The data represented in Fi.g. 8 shows 3H
thymidine incorporation into DNA in parent and
selected PDL cells wherein the cell~ are cultivated
for 10 days and then labelled for 24 hours with 3H-TdR
50 ~Ci/ml in media containing ~N free serum, the cells
are removed with 0.1% ethylenediaminetetraacetic acid
~E~TA), 0.1% ~thylene glycol bi~(B-aminoethyl ether)-
N,N,N',N'-teteraacetic acid ~EGTA~ in a divalent
cation free PBS (phosphate-buffered saline), 3H-
radioactivity incorporated into the cell i~
quantitated using an LK~ scintillation counter and the
values represent the mean +/- 8.d. triplicate assays
with background incorporation subtracted.
~0 The chromatogra~ reproduced in ~ig. 9 i5 a
pro~ile resulting from PDL cell conditioned media
being subjected to tNH4)2S04 saltlng out procedure,
subjecting a concentrate containing the precipitated
protein to TSX55F (molecular sieve column) open colu~n
chromatography, and then ~ubjecting the eluted protein
having increased activity to rever~Q-phase high
performance liguid chromatography (HPLC)o
Our ~tudies indicate that there i~ a
æpecific *actor synthesized by PDL cells that is
ch~motactic for these cP.lls. When ~onditioned media
~rom cultures o~ hu~an P~ cell~ wa~ @xaminad for
che~oat~racta~t activity, we found a dos~ dep~ndent
xelationship for PDL cells. Other data suggested that
a specific polypeptid~ was r~sponsible for the
chemotactic acti~ity in the PDL ~11 conditioned
media~ Heating the media to 100 for 30 ~in dlecreased
in chemoattractant ac~ivl~y a~:did a 60 min incubation
with ~rypsin, indlcating a pol~peptide na~ure for the
-12-
responsible ~actor. Gingival ~ibrobla~ts and
~pithelial c~ did not re~pond to variou~
concentra~ions of the PDL cell conditioned media
indicating the response is ~ost l~klely not due to
other polypeptide growth factors acting on populations
of these cells that ~ay be present in the PDL
preparation~. Moreover, ~ntibodie~ against FN and LM
a~ well as EC~F, F~, or EGF did not inhibit ~he
chemotactic properties of the conditioned media for
both PDL and osseous cell~. TGF-~ enhancas the
activity of the PDL cell condltioned media but
antibodies directed ~gainst TGP-beta did not inhihit
the PDL ~actor-mediated chemotactic response in either
cell type. ~PLC Reverse Phase (C18 followed by C8)
chromatography has enabled us to isolate a 12,500
dalton peptide with high che~okactic and mi~ogenic
activity. Additional data ~rom open column
chromatography and HPLC support the peptide nature o~
this material. ~mino acid an~lysis and preliminary
NH2 t~rminal se~uence data indicate a uniqu~ peptide.
Information ~rom the NIH (U.S. National Institutes o~
Health) data banks indicate~ that ~herQ i5 no
ho~ologous ~eguencG to the peptide.
Ob~rvations indicate that PDL cell
recolonization o~ the root ~urface is necessary ~or
reyeneration, see Nyman, S. et. a~. (1982), J. ~lin
Periodontol 9:290, and Nyman, S., et. al. (1983)-
Textbook of Clinical Periodontolgy: Lindhe, J. (ed.),
pp. 410-432, Munks~arrd, Copenhagen. In view of this,
it is hypothe~ized that specific PDL cell ge~erated
factor~ play a si~nific~nt role in the migration and
proli~eration o~ a subpopulation of responsive PDL
cell~ and can contrlbute ~o ~he healing o~ this hard
soft tissu~ int~rface. These rasults should aid in
the development of therapies that could enhance the
biological procecses inYolved in the regeneration of
the periodontal liqament as well as the heallng at
, ~
--^ VO~O/lO~t7 ' ~; U Pcr/usso/0102s
-13-
other hard-soft tis ue interfac~.
Briefly~ our ~tudies have ~hown the
~ollowing:
(1) A chemotactic fac~or for E~D~ cells is
pre~ent in the conditioned media of cultures
of PDL cell~.
(2) This Eactor i~ heat labile: 100 ~or 30
minut~s.
~3~ This ~actor is tryp~in sen~itive.
(4) Gingival fibrobla~t~ are refractive to thi~
f~ctor.
(5) Gingival epithellal cells ar~ re~xactive to
this ~actor.
(6) Antibody against FN or L~ does not inhibit `:
th~ chemotactic propert~e~ of this :Eactor.
(7) Antibody against ~CGF, FGF or E~F does not
inhibit th~ chQmotacti~ prop~rties of this
factor.
~8) TGF ~ and TGF-~ do not sti~ulate chemotaxi~
o~ PDL cell~ to th~ ~ame degree a8 do~s the
factor.
(~) A ~ubpopulatiQn o~ PDL cells can be isolated
that are mor~ chemotac~ically responsive to
the factor.
Further data ~ndicate the ~ollowing-
(lA) When the 95% (NH4)2S04 precipitate i~
chro~atograph~d u~iny a mono P column with a
pH gxadient ~rom 9.S to 7.0 the material
elut~ at a p~ 9.3.
(2A) The active ~aterial ~r~ the FPLC ls
rechro~ato~raphed on a C8 r~verse phase
co~umn a ~ingl~ p~k o~ high biological
activity iæ observ~d with a xetention time
of 12 min~ .
(3A) 5DS Urea PA~E o~ the peak yields a sin~le
band ME=12,500~
(4A) Thi~ peptide has high biological activity
jJ ~
--14--
for PDL cells. Both chemotaxi~ and
proliferation are l:i~ulated.
(5A) Amino Acid analy6is using Pico Tag ~;ys~em is
given.
( 6A) Sequenca AnalyE~is for the f ~ rst e}ev0n NH2
terminal almino acid~ when checked at the NIH
data bank indieatQ~ a uniq;ue peptide.
The connectiY~ til5SllQ O~e the periodonitiu~
is a complex structur~ con~istlng of ihrGbla~ts
(gingival connective ti~ue fibroblasts and
periodontal ligament ~ibroblast~), gingival
epi thelium, vascul~r endothelial cell~3, ner-.re
proc:es~es, c:e~entu~n whicl~ omprised o~
cementoblasts with asso¢iated axtracellular 3~atrix,
alveolar bone and an ext~n~ive extri~cellular matrix
comprised o~ collageTl, glycoproteins and
proteoglycans. Ess~ntial biological processes
involved in periodontal regenerEItion include
chemotaxis, proli~era~ion and di~erentiation of cells
and structures such as blood vessels, a new connective
tissue attachment oP th tooth to th~ alveolar process
and the epithelial ~;eal to th~ ~oothO
PD$-CTX is believed to have an amino ac:id
sequ~nce fro~n th~ ~-ter~inal a~ ~hown in the ~ollowing
Table A whtch al80, indicate E~eq[u~nce dzta og known
polypeptide growth ~actor~ ~or comparison.
~ccordi2lgly,. th~ pr~ent inventiorl ~urther
relates to an i~olat~d p~riodol~tal ligament cell-
3 0 attracta~t protein obtainablQ ~ro~ huD~Ian periodontal
ligament cells, sald protein ~:omprising an initial
amino acid 6equerlce" fro~ th~ N-terminal, a~; follows:
Val Pro A~p Ser Ser Ala His Ly~ Ly~ Ala ..~ .
The present irlvention ~urther relates to an
isolat~d periodontal ligament c~ll attractant protein
ob~ainable from hu~an p~riodon~al ligament cells, ~aid
pro~ein comprising an a31ino a~::id s~quence, ending at
the C-terminal, as ~ollow~:
.
~ ................................................. .
.
- v~go/10017 PCT/US9~/01~25
-15-
... Pro Val Val Pro Ala Tyr ~la Pro Pro .
The present invention further relates to an isolated
periodontal ligament cell-attractant protein obtainable
~rom human periodontal ligament cells, said protein
having an amino acid sequence, from the N-terminal to
the C-terminal, substantially as follows:
V~ ~ ~ ~ ~ Ala ~5 ~ Ala ~ ~ Gln~ Y~ ~ Gly Gly ~ ~e
Gly ~ ~ Val ~ ~ ~ q~ Thr ~sn ~ Gly GlyGly ~ R~ Ala ~ h~
Gly ~ ~ ~5 q~ ~ ~ Gly Tyr ~ly V~ ~a ~ p Ile ~ Ala H~
Gly ~ 5er ~ Val Asp Yal Gly }~ A~ 1~ Val 'Val Ieiu ~ Ser l~p Val Asp
~la Gly ~ q~p Leu Ala ~is lys Ser Pr~ Ala Gly Val Ala Ala ~ys Ala Gly Gly
~eo Yal Val ~ ~la Iyr A~a E~ Pn~.
--1 6--
3 ~ &
3 ~ 3
3 ~ s ~ ~ ~ z
&
~1~ ~ 3
~1~ ~ fi
3 ~ ~ 3
..... ;. , ~: ;
.
.
' ~ ri
90/10017 PCT/US90/01025
-17-
The P~L-CTX factor o~ the invention is
isolated by ~eing separated Prom a natural ox
synthetic source, or concentrated or puri~ied. Thu~,
the PDL-CTX factor of th~ invention can be ln the ~on~
of a concentrate of ~aid ~actor, e.g. a concentrated
aqueous solution or di~persion o~ ~aild factor. The
PDL-C~X factor c~n suitably be obtained by tis~ue
culture of PDL cells in a cu~toma~y medium, following
which the medium contains th~ PDL-~TX ~actor. ~edia
containing the factor are referred to as PDL cell
conditioned media. Upon separat~on media may be
conc~ntrated, for example by hromatography,
precipitation (e~g. by means o~ salting out with a
salt such as ammonium sulfate), ce~trifugation,
electxophoresis, or co~binatio~s o~ the~e tech~iques.
A preferred procedure ~mploys a sequence of (l)
salting out with ammonium 6ulfate: t2) centri~ugatlon
and discarding th~ ~upernatant; repeatinq (l~ and (2);
(3) resuspending sediment; ~4) fractionating by
chromatography: (5) fxa~tionating th~ eluate
containing PDL-~TX by chromatography.
The separated or P~L- X factor or concen-
trate thereof i5 a compon~nt of ~h~ compo~ition of the
invention. A pharmaceutically zc~eptable amoun~ of
the PDL-CTX factor will vary dQp~nding upon th~
intend~d use, and d~pendi~g upun thQ activity of the
concenkrate. The acceptable amount al~o may ba in an
acceptable dosaye form for appli~a~io~ to te~th or to
bon~. The ~cceptable a~ount o~ the a~tor i~ combined
with a phar~aceutic~lly accep~able mediu~ to ~orm the
composition of the inv~ntion.
It ~ desirable to include in ths ~ompo-
~ition o~ th~ invention a pharmacologically a~ceptable
a~o~nt of a polypept~de growth ~actorO The gro~th
~actors ~ay be ~el~cted ~ro~ the one~ described where
they enhance reyeneration in co~bina~ion with ~h~ PDL-
CTX factorO The growth factor suitably ~ay ~o~sist of
-18-
transforming growth ~actor-~ (TGF-B~, platelet derived
growth facto~ (PDGF), and ~ixture~ thereo~.
Pre~erably the ~edium o~ the composition is
a~ueous. A ~uitable amount of the PDL-CTX ~actor is
an amoun~ of Prom about 10 picogramY~ to about 10
micro~ra~ per ~1 o~ th~ CO~pO5itiOIl.
The compos~tion ~ay ~urther compri~e TGF B
in an amount vf ~rom about ~0 pieog~.ams to about 10
microgram~ per ~1 of said composition. Al~ern~tively
the co~position ~ay comprise both TGF-B and PDGF, with
TGF B in an amount of ~rom about 10 picograms to about
10 microgra~3 p~r ml of sald compos~tion, and PDGF in
an amount o~ about 10 pico~rams to about 10 micrograms
per ml o~ said composition.
A kit in accordanc~ with the present inven-
tion comprises as individual component~:
(a) a pharmaceuticaly acceptable amount of
the PDL-CTX factor o~ the in~ntion in a pharmaceu-
tically acceptable medium; a~d
(b) a phar~acologically acceptable amount of
a polypeptid~ growth factor in a pharmacologically
acceptable medium.
The polypeptide growth ~actor o~ component (b) may be
selected ~o~m the group consisting of TGF~a, TGF-B,
PDGF, and mixtures ~h~reof~
The kit may Purther include a an i~dividual
co~pon~nt:
(c) a pharmacologically acceptabl2 amount of
a pol~peptide growth factor diff~rent from the
polypeptide growth factor o~ ~o~ponent ~b), in a
pharmacologically acceptable medium.
Component ~c) may be selected from the same growth
~actors a~ i5 component (b), but will dif~er ~rom the
particular growth factor wh~ch was selected from
component (b).
The media o~ co~ponents (a), (b~ and (c)
preferably is aqueous. The active factors of ~a), (b)
0g0/~00l7 PCT/USsO/01025
--19 -
and ~c~ may be present in the same a~ounts as speci-
fied above in relatio~ to the composition o~ the
present invention.
Further, the kit ~ay includ~ as~ agent which
demineralize~ enamel and~or d~ntin, to pre-condition
the tooth ~urface and to provide enhanced binding of
components ~a) ~o ~c), and impr~ed regeneration.
Pre~erably the further agent included is:
(d) an aqueou~ solution of tetracycline HCl,
preferably in a concentration of from 50 mg/ml to lOo
mg/ml; or
~ d-l) a~ agueou~ ~olution o~ citric acid.
Most preferably, $he furth~r agent i tetracycline
HCl.
The kit may also include, iP desir~d,
directions Por use, and appropriat~ materials for the
use, such as syringee and materials ~or sut.uring and
dress the treated area.
The kit of th~ invention containing compo-
nents ta)-(c) i~ also u~ul for bone regeneration.
Another embodi~ent of th~ pre~ent i~vention
reside~ in the u~e of an art~ficlal bas~ment mQmbrane
comprised of collagen to c~ver the tr~ated ~ur~ac~ oP
a tooth in a ~ethod of periodontal regeneratio~.
Follow~ng ~herapy which can include treatment o~ an
expo~ed, periodontally di~ea~ed root sur~ac~ with
che~otactic ~actor~, ~hQ treated ~ur~ace i~ ~overed
with the ~e~brane. Thi~ membrane act~ a5 a ~elective
barrier which inhibit~ cell~ which do no~ display a
~tron~ chQ~otactic respon~e to the gactor fro~
competing for adhesion a~d growth on th~ treated root
surfac~ w~th cells, such a~ ~he PDL cells, that ar2
~ective in the formation ~f a new ccnnectîve tissue
attachment to the treated root sur~ace.
~ preferred artific~al ba~ement ~e~brane
, i
comprises t~pe I collagen overlayer~d with type IV
collagen and la~inin. In th~ ~e~hod o~ using the
--20--
membrane, preferably the root surface i8 treated with
the PDL-CTX factor of the pre~ent invQntion, an~
dP.~irably ~aid root surfa~e i~ addil:ionally treated
with a polypeptide ~rowth factor ~elected from the
group conæisting of TGF-a, TGF B, PlX;F and mixtures
thereof O
The artificial b~sement me~brane also may be
us~d in the ~it of the present inve~ltion as a further
l o component .
The PDI.-CTX factcsr of the present invention
i~ ge2lerally useful in a method of per~ odontal or bone
regeneration. It i8 particularly useful for applying
to dentin in a ~ethod directed at induc~ g PDl~. c:ell
migration to the dentin. In ~uch methods, a par~icu-
larly preferred techa~ involves treatmQnt of the
diseased root with PDL cell~3 ~;eleoted ~rom the
patient ' 8 cells on the basis OI an increased response
to the PDL-CTX factor o~ the invention.
2 0 Thus ~ another embodimellt o~ the present
invention relates to a tec:hnique for selection of PDL
cells in connection with a method of periodontal
regeneration. In thi~ e~bodiment, the ~tep~ comprise:
(a) obtainirlg a . peci~nen of a parent
population of periodontal ligament (PDI.) c:ell~; from a
patient;
(b~ growir~g the obtained ~DI, cells in a
tissu~ cultur~ Dledium to obtain a culture oî the
patient ' ~ PDL cellæ; and
(c) selecting a ~ population of PDL cells
of said cultura wh~ch migrat~ through a porou~ type I
c:ollagen barrier by chemotaxis direc~ed against the
PD~-CTX fa::tor o~ the present invention.
In this techniqu~ of selecting PDL cells, it
is preferred that before step (c), an enriched
population of PDL cells s) the culture c)~ step (b) i~
obtained by incubating tha PDL cellE; of ~aid culture
in a ~ir;t cc)mpartment o~ a chamber having a ~econd
`~09V/10017 ~ J PCT/US90/01025
-21-
compartment containing a solution comprising the PDL
CTX factor, the ~ir~t and ~econd cha~bers being
separated by a se~iper~eable ~e~bran~, ~electing an
enriched sub-population of c~ wh~ch migra~e by
chemotaxis directed against ~aid PDL CTX factor, and
using said enriched ~ubpopulation ~o ~urkher select
therefxom cells wh~ch migrate through the collagen
barrier in step (~).
In a further embodiment of ~he present
invention, a prsferr d therapeutic procedure for
periodontal regen~ration comprise~:
~a) expo~ing a ~oo~h ~urface ~o be ~reated;
(b) applying to ~aid surface a
pharmaceutically acceptable amount of the PDL-CTX
factor of the present invention $n a pharmaceutically
acceptable medium;
(c) applying a ~uspen~lon o~ ~he patient's
periodontal liga~ent cell6 to the sur~ace: and
td) covering tha tr~ated ~urac~ with an
arti~icial basement ~embran~ co~prising type I
collagen overlayered with type IV collagen and
laminin.
It is ~urther pre~e~red in this therapeutic
procQdure ~hat before or ~imulkaneou~ly with ~tep (b~
there is additionally appli~d to said ~urface a
phar~aceutically acceptable amount of polypeptide
growth ~actor ~elect~d from the group consisting of
~GF-a, T~F-B, PDGF, and mixtures ~her~o~. Option~lly,
befoxe step (b) ther~ i~ applied to sald sur~ace an
aqueous solution o~ tetracyclinQ ~Cl or an aqu~ous,
saturated solution of citric acid, the application o~
te~racycline HCl being t~ preferred one.
The invention i~ further illustrated in the
~ollowing examples.
When conditioned ~edia fro~ culture~ o~
human PD~ cell~ are examined ~ox ch~moattract~nt
activi~y, it is ~ound that there i~ a dos~ dependent
relationship for both PDL cells and cell~ of osseous
origin as shown in the following Table I~
T~B~
% PDL
Conditioned
Media ~II.5~3a~
0 8 +/- 3 7 ~/- 2
1 16 +/~ 4 19 +/~ 3
22 ~/- 5 2~ 5
25 +/- 5 2~ 5
30 +/- 7 39 +/ 6
29 +/- 6 3~ 5
100 25 +/- 4 35 +/- 5
Assay conditions are 37CC, 100% humidity and a 6
hour in¢ubation using the mo~ d ~oyden Cha~ber Assay
System. ~he ~arrier in ~his as~ay is an 8 ~ pore
Nuclepore ~ er (polyvinyl parolodine free). For both
PDL cell~ and bone cells tha ~ilters are precoated with
gelatin at a concen~ration Or 5 ~g~filter. In addition,
when ASSAY I (assay for sp~cific cell migration) is
utiliz~d, conditioned media adsorbed to den~n produces
a dos~ dependent response or both PDL cells and bone
aell~, a~ shown in th~ ~ollowing Table II.
~a~
~ PDL
.ConditionQd . ~ ,
~ pDL Cells Bone Cells
o 3 ~- 3 1 +/-
1 10 ~- 2 15 +/- 3
20 ~/- 4 26 ~/ 4
34 +/- 5 3g ~- 9
"50 35 ~/- 6 39 ~J- 10
Assaying conditions were TTC conditioned dentin
(50 mg/block ~or 5 minutes at 37-C) w~th 5 ~1
,' ~
. '' .
PCT/US90/01025
-23-
application of conditioned ~edia for 30 minutes at 37~C
and 100~ humidlty. In addition, when the TTC condi-
tioned dentin blocks are further conclitioned by the
application of FN (50 ~ g/block) a small bu~ not
insignificant increase in PDL cell motility is observed.
~X~
~hen 50~ conditioned media (100% conditioned
media diluted 1 to 1 with ~EM ~ini~2l1 essential ~edia])
is incubat~d ~or 30 m~nutes at various temperatures,
loss of the factor's chemoattractant activity is noted
at hi~her te~peratures SFig. 1).
Since sensitivity to heat i8 an indicator of
protein charact~r, trypsin sensitivity of the concli-
tioned media is tested. Fifty percent conditionecl media
is incubated with purified trypsin 10 ~g/ml for various
time points. Incubation i~ stopped by the addition of
SBTI ~soybean tryp~in i~hibitor) at a lO ~old excess.
Che~oattracta~t activity i~ decrea~ed over a 60 minute
incubation with tryp~in (Fig. 2). SBTI by itsel~ had no
effect (either positive or n~ga~ive~ on PDL cell
migration.
The above data indic~te that ~he factor is of a
protQin (glycoprotein9 lipopro~ein) nature. ~i~ty
percent conditioned media i8 th~n dialyzed against MEM,
6 changes at 4-C. ~ol¢cular weigh~ cu~ of~ thQ dialysis
tubing is 6,000~ The activity is r~tained and i~
nondializabl~ abov~ ~r~ 6,000.
EXa~P~E ~
The ability.of other cell of ~he periodontium
to respond to 50~ condition2~ ~dia o~ PD~ ~ells is
~valuat~d. Both human gingival epithelial c~lls and
hu~an gingival ~ibrobla~t~ ~re te~ted ~or their ability
to respond to various dosage~ o~ conditioned ~edia (Fig.
3)O A~ po~itiv~ control~, LM i~ tested at 10 ~/block
for gingival ~pithelial cell~ ~nd FN is t~st~d at 100
g~block ~or gingival ~ibrobla~t~' Neither gingiYal
epithelial cells nor gln~ ibrobla~t6 re~pond to
-24~
cond~tioned media o~ PD~ call~.
In addition, it wa~ previouF,ly shown that
anti-ECGF inhibits ECGF indueed cheDIotaxi~ o~ PDL cellsO
As a further indication that P~L-CTX of the present
inv~ntion i8 a uniqu~ PDL cell ~ynthe~ized ~ac~or, the
ability of anti-~CGF, anti-FGF and alnt~-EGF to inhibit
50% conditioned media in a do e d~pe!ndent manner is
examined. No effect o~ ~ny antibodi~s on PD~ cell
movement i~ alter~d. Non-specif~c IgG (NSIgG) is also
evaluated, se~ the following Table III.
~LC
~u~r_p ~C~s~g~ ~ower Eield
Antibody
~ilY~iQn /U~ oÇE ~rtl~EQ~ ~nti-E~ g
0 38 +/~ 4 41 +/- 3 ~0 +/- 4 39 +/- 4
10_6 37 +/- 6 40 +~- 4 41 +/- 3 40 +/- 5
20 10-4 41 ~/- 5 33 ~/- 3 39 ~/- 5 41 ~
_2 43 +/_ 4 39 ~/- 2 40 ~/- 4 40 ~/- 5
_l 39 ~/_ ~ 39 ~/~ 4 41 +~- 3 38 +/- 4
In th~e assays the migratory respons~ o~ PDL
cell~ is ~Qasured in the presence o~ antibody dir~cted
again~t variou~ polypeptide ~itog~ to 50~ PDL cell
conditioned media . Anti~ody dilutions ( initial
conc:entration~ all ad~u~;ted to s~art with 100 mg protein
per ml media~ are incubated with the PDL cells above the
Nuclepore filter. Migration i~ aE;sayeci u~ing A5SAY I.
~ach assay point i~ the mean ~ 8,.tl. of triplica~e
pl~8 .
Sinc~ TGP-A i~ found in high lev~l in bone and
5~nCe both TGF~a and TGF-B are implicated in both
fibroblast proli~eration and che~otaxis, the abili~y o~
TGF~a and TGF-B and 50% conditioned media to generate a
chemotactic response in PDL cell~ i8 compared. A dose
dependent chemo1:actic response of PDL cells to 50% PDL
cell con~itioned media, T~;F~ and TGF-B is observed~
PD~ cell conditioned m~dia i~ significan~ly ~ore
PCT/US90/01025
-25-
effective as a chemoattractant (Fig. 4).
~E~.~
Previously it wa~ shown that a~finity purified
polyclonal monosp~ci~ic antibodies i~ibit ~peci~ic
factor induced chemotaxis. The ability of anti-LM and
anti-FN to inhib~t conditioned ~edia induced chemotaxis
of PDL cells is there~or examined. l'he additiQn oP
antibody (initial concentration oP lO0 ~g protein/ml~ in
a dose dep~ndent (logarithmic) fashion doe~ not i~hibit
50% conditioned media induced chemotaxi~ (Fig. 53.
5ince TGF-B is beliaved to be a "pa~regulin" the
ability of 1 and lO ng/block TGF/B ti augment 50% PDL
cell conditioned media (CM) is evaluated. In ~hi~
example, dentin blocks are conditioned wlth 50 mg/ml TTC
for 5 minutes at 37-C, rin~ed in cold PBS (6 x l minute
each rinse) then both TGF~ and 50% conditioned media
applied in 5 ~l aliguote~. For both 1 and lO ng/bloc~
o~ added TGF-~ an incr~as~ in PDh cell ~igration is
observed, a~ shown in th~ following Table IV.
~Y
50% CM l~L~E=~ lsl~L:~E~ +ln~ ~F--B ÇY~ E~
38 +/-4 23~/~ 3 25 ~/- 4 49 +/- 5 53 ~/- 6
This data ~upport~ ths observation ~hat TGF-B
acts in co~junction with other polypeptide gro~th
~actors. In addition, an~ibody dir~tQd agains~ TGF-B
inhibits the TGF-B induced ch~mo~ac~ic response but not
the conditioned media induc~d chemstacti~ re~ponse.
. . Si~ilar data with b0ne c~ ATCC ~merican T~p~
Culturs Collection) 700g and ~TCC 7051 ha~ been
obtained. It haR be~n found t~at:
(l) Bone cells r¢~pond to 50% PDL cell
condit1on~d media~ The response i~ dose d~pendent from
1% to 50%~ These a~says are perfor~d using both ASS~
I and using th~ Modified Boyden Cha~ber ~s ay~ The
chemotactlc r2 ponse generated by P~L cell conditioned
--26--
med~a on bone cells i8 so~ewhat higher than that
observed w~t~ PDL c~ (8~e above)O
(2) Antilbody ~tudieR indicated that the factor
in PDL rell conditiorled ~çldia i6l not ECGF, YGF, E:GF,
TGF-B, fragments of 1~ or E7~.
(3) A major dif~Eerence noted using ASS~Y I and
the Modif i ed Boyd~n Ch~mber Assay b~tween P~L cells and
bone cells is that bonQ cell~ re~pond more actively to
TGF-~ than do PDL c@ll~;.
When ATCC 7009 bone cells are ~valu~tad 1:o
respond to TGF-~ a dose dep~ndent rapid migration is
observed (Fig. 6).
~a~
An a~ ay sy~t63m that c:an be utilized to separate
subpopulation~ o~ cells based on their migratory
capacity (inva~i~e propen~ity) has been described (see
the above-mentioned article "U. e oi~ a Reconstituted
Basement NeD~ran~ to Mea~ure Cell ~nvasivenes~ and
Select ~or Highly Invasive q~or Cells'9 o~ V~ P.
Terranova et al). In thi~ a3~ay system a collag~en
barrier separate~; the upper and lower chaDlber~ of a
Modified Boyden Chamber. A collagen barrier is plac:ed
directly o~rer a . type ~V collagen coa~ad Nuclepore ~ilter
(1 llm pore diamet~r). C:ells that traverse the collagen
barrier attach to th~ coated filtex. P.fter various
time~3 oP incubat:Lon th2 f ilter~ are removed and the
attac::hed cells ~ubcultured. The~e observations have
b~en extended to generat~ populations o~ hu~asl PDL c~lls
3 0 which have the phenotypic characteristic of increased
che~notaxis toward PDL ~:ell c:onditioned ~nedia. When
these subpopulations are examined ~or their abil ity to
migrat~ and incorporate 3~ TdR into DNA, it is observed
that ~h~3y migrat~ more rapidly and have a high~r rate o~
3H-thymidine incorporation than do the parerlt population
( Fig . 7 and ~ ~ .
(NH4 ) 2S04 precipitation at 60% o~ the ~PDL cell
PCT~US~0/01025
-27-
conditioned media result~ in a lO0 fold enri~hment of
the Pactor.
~pplication o~ 5 ml o~ khe ~N~4)2S04 precipitate
to a mol~cular sieve column TSK55F equilibrated in Q.05
M Tris, 0.15 M ~aCl. pH 7.4, results in elution of a
pool o protein~olecular weight range 45,000 to 55,000)
with lOoO fold increased activity. Thi~ material is
applied to a reversad phase c-18 HPLC column and eluted
with a gradient o~ water/a~etonitrile (AN) starting with
lOo% water, 0% AN, reaching 0~ water, 100% ~N in a
retention time of 55 ~inute~). The active protein is
eluted in 24.77 minutes retention time, equivalent to a
~olecular weight of about 45,000 (see Fig. 9).
Also, when the material i~ applied to a
molecular sieve HPLC column (protein pack. 125), there
i~ obtained a band with a rQtention time of ll.56
minute~ for a protein having a molecular weight o~
53,000. This shows on SDS-PAGE (sodium dod~ayl sulfate-
poly~crylamide gel elec~rophoresl6) analysis with a Mr
of 51,000. Th~se analysis characterize the active PDL-
CTX materia}.
~AI~E~
Artificial basement ~embranes are prepared as
described in th~ above-~entioned article '~Use of a
Recon~tituted Ba~e~ent Me~brane to Measure cell
Invasiveness and Select for Highly Invasive Tu~or
Cells", Or V. P. Terranova ~t al. Typ~ I collagen is
cross-linked ~uch that on form~tion of a 13 ~m disc the
pore ~iz~ i~ no more than 5 ~m. ~his type I collagsn
matrix i~ th~n o~erlayered w~th ~00 ~g of type IV
collagen and 200 ~g o~ laminin d~persed in 0~05 ~ Trls,
0.15 M Na~l, p~ 7.4. After lyophilization, th~
~rtificial basem nt ~@mbrane~ ar~ sterilized (gas
stariliza~ion) and packaged in steril~ air ~igh~ plastic
wrap for ubseque~t u~e.
A su~p~nsion of PD~ cell~ in PBS is applied to
-28-
teeth using extract~d human teeth a~? a model. 5 x 106
selected PDL!eells in PBS are applied by use of a pipet
~n a drop-wise fashion. The cell$ are allowed to attach
to the root for 30 minute~. ~fter t:hi~ time perlod, the
teeth are tryp~inized and tha number of attached cell
~uan~itated by ~he uae of a cell pal~icles counter.
Routinely, 50~ aktachment of di~per~ed PDL cells to
tooth root structure i~generated (2.,5 x 106 cells per
tooth root)O
~E~
PERIO~ONT~ R~ENERATION PRO~$~U~
A. Obtaining and culturing patients' PDL oellsO
During the initial periodontal visit Wi~man-type
flap~ are raised in the gum~ of a patient and scrapings
~rom around the junation of the tooth and bone are
taken. The ~craping~ contain PDL call~. These
scrapings are cultured ~or growth o~ PDL cells as
follows.
A~ter washi~g in cold PBS, pH 7.4, the scrapings
are added to media containing collagenase-dispase at 100
mg/ml in 10 ml of an isotonic salt ~olution (~SS3
con~aining 100 mM NaCl, 60 mM mannitol, 25 mM Hepes, 10
mM ~aHC03, 6 m~ ~2HP04, 1~ CaC12, pH 7.4. Following
inc~bat$on ~or 90 ~inutes at 37-~, the contentæ are
vigoro~sly vortexed for two minute~. ~he medium is
removed and centri~uged at 500 x g at 4-C ~or 3 minutes .
The resulting cell pellet i~ resuspended and washed 3
times in DMEM Dulbecco ~odified minimal es~ential melin~
with 500 ~g~ml gsntamycin. The re~ulting cells ar then
added to type I collagen and FN Goated (300 ~g/dish
respectively) 35~ tissue culture dishes. The culture
medium consi~ts o~ Media NCTC 109 supplemented with 15%
fetal bovine ~erum (~BSi, 1% sodium pyruvate, 1% non-
e~sential amino acid and 25 ~gf ml gentamycin.
Unattached cells are re~ov~d after 24 hours of
incubation by decanting the medium and fresh medium is
added and changed daily. After con~luency is obtained
s -~l
- YO90/10017 PCT/US90/01025
-29-
(14 to 21 days), the cell~ are removed ~rom tissue
culture dishes by incubation with O.l~ EDTA and 0.01%
~rypsin in divalent cation-free PBS for 5 minutes. The
cells are then grown on the type I ~ollagen~FN coated
tissue culture dishes in the presenc~ of 1 ~g~ml anti LM
antibody~ This treatm~nt inhibit~ all epithelial cell
adherence. The ~edium i8 changed after 6 hours to NCTC
lO9 upplemented with 15~ FBS. Confluent cultures of
fibroblast-like cells ~PDL cells) are obtained in 14 to
2l days.
B. Selecting P~L cells having increas~d chemotactic
re~ponse to PDL-CTX ~actor:
A~ter obtaining culturQs of the patient's PD~
cells, the ~ost responsive cells are lsolated utilizing
khe following selection technique.
Twenty-~ive Modified Boyden Chambers are seeded
with 5 x 105 PDL cells in the upper chamber. After 6
hours of incubation with che~o~axis direc~ed against lO
9 M PDL-CTX, the cell that ~igrate all the way through
the Nuclepore ~ilter are collected and ~ub~cultured.
This is acco~plished as ~ollows.
The Nu~lepore filtQr~ are carefully removed and
placed top-si~e down on sterile glass ~lide~. The
under-sur~ace is then carePully scraped using a sterile
rubber policeman. Ten ~l o~ NCTC lO9 with 10% FBS is
next applied to the sur~ace and gently aspirated into a
pasteur pipette. Th~e lO Yl sample~ with re~oved PDL
ce~ls are transferred to a 3S mm tissue culture dish
which has pr~visusly been coated wi~h ~0 ~l of type I
collayen and lO ~g of FN. ~h~ ~dia volume is brought
up to 5 ml and the di~ im~ediately anchored at 37' C,
5% C2 for l20 minutes. After thi~ time, the
unattracted cell~ are re~oved by decanting the medium,
fresh media added; and the dish returned ko the
incubator. The culture dishe~ ax2 examined daily and
. ~
cell con~luency is expected a~ter l~ to 2~ days. ~hen
the ~ishe~ are confluent, the cells are removed using
f `~
-30-
0.1% ED~A, 0~01% ~ryp~in ~1:250) in divalent cation fxee
P~S, pH 7.4,'and transferr2d to T-75 tissu~ culture
flasks. In ordPr to generatQ an enric~ed population,
thi~ selection procedure $~ repeat~d ~our more times,
each time u ing ~rom the previou~ selection. Cells at
each selQct~on ar~ a~ ay~d ~or ~heir chemotactic
response (relatiYe to the parsnt population~ to PDL-CTX.
Additionally, porous type 1 collagen barriers
lo (loO ~ pore size) are ovQrlayed on l~elatin-coated ~ ~m
pore Nuclepore ~ilters and the election procedure
repeated. Here, the cells that ~igra~e through the
collagen barriers attach to the collagen-coated
Nuclepore ~ilters. The cells are collected and
subcultured.
C. PERIODONT~L THERAPY:
After the patientts ~elected PD~ cell population
i~ obtained, the patient i~ asked to return to the
o~fice for conv2ntional periodontal surgical therapy or
for any combination of the following procedures:
(1) Application o~ both TGF-B (in concentra~ion
range~ ~etween lO picograms per ml PBS and 10 micrograms
per ml P8S) ~ollowed by application o~ PDL-CTX in
concentrations betw~en lo pieograms and lo micrograms
per ~l ~S. Application i~ accomplished by dripping the
material onto the teeth by means o~ a pasteur pipette.
(2) The teeth ar~ previously treated with an
' ~qUeOUB solution o~ either satur~ted citria acid or
tetracycline HCl t50 ~g/~l to 100 mg/ml) ~or 5 minutes
aftar which they are rin~ed ~ time~ with PBS~
(3) Next the patient' B own highly re~ponsive PDL
cells are applied to the tooth surface~ by allowing a
Ruspen~ion of these cells (5 x lo6 p~r ~l PB~) to flow
onta the tooth ~tructure and remain undisturbed for a
period of ~0 minutes.
(4) The area treated as described abo~e is then
ov~rlayed ~ith an artiflcial basement ~embrane. This
ba~ement membrane is plac~d ~uch ~hat the type :~
'O90/10~17 PCT/US90/01025
-31-
collagen side is next to the tooth-bone ~urface while
the type IV collagen-laminin side is next to the soft
tissue. The membrane is placed suc~ that it extends
lO mm below the tooth-bone interface, lO mm to either
side ~right and left) of the area ~re!a~ed and lO mm
above the crest of the 60~t ti88U0 f~ap.
(5) Soft tissue flap~ are then su~ured 6uch that
the artiflcial ba~ement membrane i~ i~olded over the
crest of the ~of~ tis~ue flap and ecured to the soft
tissue with methylmethacrylate.
