CA2027583A1 - Periodontal and bone regeneration factor, materials and methods - Google Patents

Abstract

ABSTRACT

An isolated periodontal ligament cell-attractant factor (PDL-CTX), which comprises a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells.

Description

''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 `
-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 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 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 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.

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 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 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 ~

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 ... 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~.

3 ~ &

3 ~ 3 3 ~ s ~ ~ ~ z &
~1~ ~ 3 ~1~ ~ fi 3 ~ ~ 3 ..... ;. , ~: ;
.

.

' ~ ri 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 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 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 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 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 ,' ~
. '' .

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 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 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 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 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 (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 `~

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 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.

Claims (57)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An isolated periodontal ligament cell-attractant factor (PDL-CTX), which comprises a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells.

2. A factor according to claim 1, wherein said protein is characterized by periodontal ligament cell autocrine motility and mitogenic activity.

3. A factor according to claim 1, said protein having been derived from periodontal ligament cells.

4. A factor according to claim 3, wherein the protein was recovered from a medium conditioned by periodontal ligament cells.

5. A factor according to claim 4, wherein said protein was recovered as a precipitate by treatment of said medium with a salt.

6. A factor according to claim 3, wherein said factor was derived from periodontal ligament cells which were selected from a plurality of periodontal ligament cells, the selected periodontal ligament cells having been selected on the basis of an increased chemotactic response to said factor relative to the chemotactic response of the other periodontal ligament cells of said plurality of cells.

7. A factor according to claim 1, comprising a protein having a molecular weight of about 45,000 to about 55,000 daltons.

8. A factor according to claim 7, wherein said protein has a molecular weight of about 50,000 to about 53,000 daltons.

9. A factor according to claim 7, wherein said protein is a tetramer.

10. A factor according to claim 9, comprising a monomer which forms said tetramer, said monomer having a molecular weight of about 12,500 daltons.

11. A factor according to claim 1, wherein said factor was obtained by recombinant DNA methodology.

12. A factor according to claim 1, wherein said factor was obtained by peptide synthesis.

13. A composition useful for periodontal regeneration, which comprises:
(a) a pharmaceutically acceptable amount of an isolated periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells; and (b) a pharmaceutically acceptable medium.

14. A composition according to claim 13, further comprising a pharmacologically acceptable amount of a polypeptide growth factor.

15. A composition according to claim 14, wherein said polypeptide growth factor is selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

16. A composition according to claim 13, wherein said medium is aqueous.

17. A composition according to claim 16, wherein said PDL-CTX factor is in an amount of from about 10 picograms to about 10 micrograms per ml of said composition.

18. A composition according to claim 17, further comprising transforming growth factor-.beta. (TGF-.beta.) in an amount of from about 10 picograms to about 10 micrograms per ml of said composition.

19. A composition according to claim 17, further comprising platelet derived growth factor (PDGF) in an amount of from about 10 picograms to about 10 micrograms per ml of said composition.

20. A composition according to claim 17, further comprising transforming growth factor .beta. (TGF-.beta.) in an amount of from about 10 picograms to about 10 micrograms per ml of said composition, and platelet derived growth factor (PDGF) in an amount of about 10 picograms to about 10 micrograms per ml of said composition.

21. A kit useful for periodontal regeneration, comprising as individual components:
(a) a pharmaceutically acceptable amount of an isolated periodontal ligament cell attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells, in a pharmaceutically acceptable medium: and (b) a pharmacologically acceptable amount of a polypeptide growth factor in a pharmacologically acceptable medium.

22. A kit according to claim 21, wherein said polypeptide growth factor of component (b) is selected from the group consisting of transforming growth factor-(TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

23. A kit according to claim 22, further including as an individual component:
(c) a pharmacologically acceptable amount of a polypeptide growth factor different from the polypeptide growth factor of component (b), in a pharmacologically acceptable medium.

24. A kit according to claim 23, wherein said polypeptide growth factor of component (c) is selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

25. A kit according to claim 22, wherein the medium of component (a) and the medium of component (b) are aqueous.

26. A kit according to claim 25 wherein the PDL-CTX
factor of component (a) is in an amount of from about 10 picograms to about 10 microgram per ml of the medium.

27. A kit accordlng to claim 26, wherein component (b) comprises transforming growth factor .beta. according in an amount of from about 10 picograms to about 10 micrograms per ml of the medium.

28. A kit according to claim 26, wherein component (b) comprises platelet derived growth factor (PDGF) in an amount of from about 10 picograms to about 10 micrograms per ml of the medium.

29. A kit according to claim 26, wherein component (b) comprises transforming growth factor-.beta. (TGF-.beta.) in an amount of from about 10 picograms to about 10 micrograms per ml of the medium, and platelet derived growth factor (PDGF) in an amount of about 10 picrograms to about 10 micrograms per ml of the medium.

30. A kit according to claim 26, further including as an individual component:
(d) an aqueous solution of tetracycline HCL in a concentration of from 50 mg/ml to 100 mg/ml.

31. A kit according to claim 26, further including as an individual component:
(d-1) an aqueous, saturated solution of citric acid.

32. A kit according to claim 26, further including as an individual component:
(e) an artificial basement membrane comprised of collagen, for covering a treated portion of the surface of a tooth.

33. A kit according to claim 32, wherein said artificial basement membrane comprises type I collagen overlayered with type IV collagen and laminin.

34. A kit useful for a periodontal regeneration, comprising as individual components:
(1) a pharmaceutically acceptable amount of an isolated periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells, in a pharmaceutically acceptable aqueous medium;
(2) a pharmacologically acceptable amount of a polypeptide growth factor selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof, in a pharmacologically acceptable aqueous medium;

(3) an artificial basement membrane comprising type I collagen overlayered with type IV collagen and laminin/ for covering a treated portion of the surface of a tooth.

35. A kit according to claim 34, further including as an individual component:
(4) a pharmacologically acceptable amount of a polypeptide growth factor different from the polypeptide growth factor of component (2), said different polypeptide growth factor being selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

36. A kit according to claim 34, further including as an individual component:
an aqueous solution of tetracycline HCl in a concentration of from 50 mg/ml to 100 mg/ml.

37. A kit according to claim 35, further including as individual component:
(5) an aqueous solution of tetracycline HCl in a concentration of from 50 mg/ml to 100 mg/ml.

38. A kit according to claim 34, further including as an individual component:
an aqueous, saturated solution of citric acid.

39. In a method of periodontal regeneration, the improvement which comprises applying to the root surface of a tooth a pharmaceutically acceptable amount of an isolated periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells, in a pharmaceutically acceptable medium.

40. A method according to claim 39, which comprises additionally applying to said root surface a polypeptide growth factor selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

41. In a method of bone regeneration the improvement which comprises applying to the surface of bone a pharmaceutically acceptable amount of an isolated periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor also having chemoattractant activity to bone cells, in a pharmaceutically acceptable medium.

42. A method according to claim 41, which comprises additionally applying to said bone surface a polypeptide growth factor selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

43. In a method for inducing periodontal cell migration on dentin, the improvement which comprises applying to said dentin a pharmaceutically acceptable amount of an isolated periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells, in a pharmaceutically acceptable medium.

44. A method according to claim 43, which comprises additionally applying to said dentin a polypeptide growth factor selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta.
(TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

45. In a method for periodontal regeneration, the improvement which comprises the steps of:
(a) obtaining a specimen of a parent population of periodontal ligament (PDL) cells from a patient;
(b) growing the obtained PDL cells in a tissue culture medium to obtain a culture of the patient's PDL cells;
(c) selecting a sub-population of PDL cells of said culture which migrate through a porous type I collagen barrier by chemotaxis directed against a periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells.

46. A method according to claim 45, wherein before step (c), an enriched population of PDL cells of the culture of step (b) is obtained by incubating the PDL cells of said culture in a first compartment of a chamber having a second compartment containing a solution comprising a periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells, the first and second chambers being separated by a semipermeable membrane, selecting an enriched sub-population of cells which migrate by chemotaxis directed against said PDL-CTX factor, and using said enriched sub-population to further select therefrom cells which migrate through the collagen barrier in step (c).

47. In a method of periodontal regeneration in which periodontal ligament cells are applied to the root surface of a tooth, the improvement which comprises covering the treated surface with an artificial basement membrane comprised of collagen.

48. A method according to claim 47, wherein said artificial basement membrane comprises type I collagen overlayered with type IV collagen and laminin.

49. A method according to claim 48, wherein said root surface was treated with a periodontal ligament cell-attractant factor (PDL-CTX) which comprises a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells.

50. A method according to claim 49, wherein said root surface was additionally treated with a polypeptide growth factor selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof

51. A method of periodontal regeneration, which comprises:
(a) exposing a tooth surface to be treated;

(b) applying to said surface a pharmaceutically acceptable amount of an isolated periodontal ligament cell-attractant factor (PDL-CTX) comprising a protein obtainable from periodontal ligament cells, said factor having chemoattractant activity to periodontal ligament cells, in a pharmaceutically acceptable medium;
(c) applying a suspension of the patient's periodontal ligament cells to the surface; and (d) covering the treated surface with an artificial basement membrane comprising type I collagen overlayered with type IV collagen and laminin.

52. A method according to claim 51, wherein before or simultaneously with step (b) there is additionally applied to said surface a pharmaceutically acceptable amount of a polypeptide growth factor selected from the group consisting of transforming growth factor-.alpha. (TGF-.alpha.), transforming growth factor-.beta. (TGF-.beta.), platelet derived growth factor (PDGF), and mixtures thereof.

53. A method according to claim 52, wherein before step (b) there is applied to said surface an aqueous solution of tetracycline HCl.

54. A method according to claim 52, wherein before step (b) there is applied to said surface an aqueous, saturated solution of citric acid.

55. An isolated periodontal ligament cell-attractant protein obtainable from human periodontal ligament cells, said protein comprising an initial amino acid sequence, from the N-terminal, as follows:

.

56. An isolated periodontal ligament cell-attractant protein obtainable from human periodontal ligament cells, said protein comprising an amino acid sequence, ending at the C-terminal, as follows:

.

57. An isolated periodontal ligament cell-attractant protein obtainable from human periodontal ligament cells, said protein having an amino acid sequence, from the N-terminal to the C-terminal, substantially as follows:

.