Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BIOCOMPATIBLE HYDROXYAPATITE FORMULATIONS
AND USES ~l H ~:RFFOR
R~CKGROUND OF THF. Il~VF~TION
1. Field of the Invention
5This h~ iol1 generally relates to certain colllbi~alions of
ingly soluble calcium phos~h~lrs and applirqtion~ thereof. More
s~;r.r~lly, certain cqlrillm ph-)s~,h.~t.~ S may be colllbill~d with a liquid to form
a paste or slurry of hydroAya~alile, which has a variety of m.o lirql, dental and
other uses. Various binco,..~ 'il.le addiliv~;s may be incorporated into the paste
10 or slurry for various applications.
2. Desclil~tion of the Related Art
HyLoAyà~atite is a cqlri~lm l.hos~ mineral and is the plill~y
c~ 1 of hum~n bones and teeth. HydloAy~&lile is only one of a llulllbel
of such cqlrillm ph~l,h~p minerals, which have dirr. ling cqlrillm-to-phosph-qtr15 ratios, crystal structures, and physical cl~t~ ;~l;rs. Apatite is a general term
for a wide range of colll~ullds l~ ,sellted by the general formula
M2+lo(ZO43-)6Y-2, where M is a metal atom, particularly aLlcali or an qlkLqlinP
earth metal atom, and ZO4is an acid radical, where Z may be pho~horous,
arsenic, v~ -.., sulfur or silicon, or _ay be ~ulJ~Iilu~d in whole or part with
20 calllOl~t~ (CO32-). Y is an anion, usually halide, hydroxy or C&lbol~te.
A col,lbh~tion of ~ingly soluble cqlrillm ph-)sphqte salts,
especiqlly tetr~q.~-q-lr~ m phnsI)hqtr and a nother s~,&lingly soluble cq-lri~~mphosphqte salt, both in their solid state, in equilibriurn or quasi equilibrium in
an ~ eo~ or non-aqueous liquid phase such that both salts are in excess, can
25 ~ q-t~ hy~oA~àpalil~, i.e., Ca5ff'04)30H. If both cqlrinm phosphate s lts
are near equilibrium with the same s...ulated solution which additionally is
ted with respect to hy~oAyàp~ , then the co.,l~osilion will continue
to pl~;i~;~te hydloAya~àlil~. The ~l~;i~i~led Lydro~y~ali~ can be formed
either in vivo or ~ vivo (jn vitro) and may possess valyillg ~ ha,lir~l and
30 biological Ç~ s inrhlrlin~, ~,g" hald~ei,~, flexibility, ~,olo~ily, dissolution,
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biol~sol~lion, biodegradation, tissue adhesion, and replqr~m~nt by soft and hardtissues.
Hydlu~yapalile, as well as m~ifi~l forms thereof, is a naturally
OC~;Ullillg material in bone, teeth and some hl~ ~blate skeletons. Crystals of
5 hydro~y~alile are ennhedded in the lllallices of bone and tooth together with
cells and tissue matrix materials including fibrous ploteills such as cross-linked
collagen and mineral binding plol~hls such as certain gla proleills, dentin and
enamel. All ~elleblal~ and dentulous qnimqlc are capable of causing
mineralization of bone and tooth matrices through the pl~ci~i~lion of
10 hydro~ al~alil~ crystals under suitable physiological conditions of pH,
~lnpclalule and ionic conditions. The reslllting tissues are not highly cellularand display certain unique propcllies, such as .cignifirq-nt m~chqnirql slle
flexibility, physiological activity, and continual self-remodeling.
Recqll~e of its unique ~iopellies, hydro~yapalile is a highly
15 biocompàLible material. These unique pro~llies of hydlo~Lyapatil~ present in
bone have plolll~ted efforts to develop hll~Jl~ll~ made of hydro~yapatile,
c~,lalllicS and other similar hard calcium ~hosph~tP materials such as a- and ~-trirqlrillm phos~hat~,s. These hlll,l~t~ have been used to fill a wide range of
bony defects in oll]lo~lic and l~co~llu~;liv~ sulg~ies and in the allcholillg of20 tooth to bone (~, in periodontal, dental and ol~llo~na~ic applications). Manyphysical and ch~ v~t;A~;o..~ have been al~ d to create implants (i) with
ill~;l~d ,~rh,..,ir~l sLIe~lh in order to enable use of hydlo~y~d~ alone or
hydroxy~alile collll)osil~ implants in load bearing bone defect sites; (ii) withaltered ~lu~ily to allow better bone hl~ lUWlh such that the implant is ~rr~lively
25 hlcol~olaled in the newly formed bone tissue; and (iii) as granular forms to
allow parl~ing in surgical defect sites. In addition to these applications,
biological factors that are belic~d to cause the formation and growth of various
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cells implicated in bone formation have been used to produce hydroxyapatite
composite implants with "inductive" plopc,lies.
Hydrol~y~a~ile has other known applications, such as bone repair
and ~ incl~li7~tion of teeth. Such uses are shown in, for example, U.S. Patent
5 Nos. Re. 33,221 and Re. 33,161 to Brown et al. One specific example is the
repair of tooth lesions or cavities. When an h~ipielll lesion or cavity developson the surface of a tooth, the dentist traditionally fills the cavity that forms.
This procedure may pl~,nl the decay from spreading further, but does not
restore the tooth to its original state. A considerable amount of l.,sealch,
10 however, has been directed to the mineralization of hl~ip;ellt dental lesions. The
object of rell~ clillization is to deposit hydro~yapatile on the carie lesion such
that the dental enamel incorporates the l~-lro~ya~alile into its ~LIu~;lul~ at the
point of lesion. Thus, l~.,lAli7~~ion pl~ further tooth decay and reslul~s
the tooth. Generally, the ~upel~dlulated solutions or slurries used for
15 lel~clalization have been pl~p~d from a single form of calcium phosphate.
However, these solutions or slurries have been .~"c~ ctory for a variety of
reasons. For eYr '-, the amount of calril-m and phosphate ions available for
cn~l~lization in these su~l~lulat~d solutions is relatively low, Ihcl~by
requiring both an e~cessive volume of fluid and an excessive ,,u,~. of
20 applications.
The use of a single cqlrillm l)ho~phate, ~--Ca3(PO4)2, was
suggested for pulp capping in Driskell et al., rDevelopment of Ceramic and
Ceramic Composite Devices for Maxillofacial Application," J. Biomed. Mat.
Res. 6: 345-361 (1972) and the use of Ca4(PO4)20 was suggested by the
25 i~ tol~ in LADR Abstract. No. 120, J. Dent. Res. 54: 74 (1975) as a possiblepulp ca~p",g agent. These single cqlrillm phosphdle ce...~ , however, are
- inrapq~le of setting to a hard co~ y and suffer the same dlawbacks as
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single c-lri~m ph-sph~te lcll~i~ alizers. For example, they cannot IIIA;I~ a
relatively colls~ pH and do not have s~ffirie~t l~ ~lalization capacity.
EAp~.lic~lce with calcium-based implants for the replq-rf-m~nt of
skeletal tissue has also existed for many years. Most of these implants have
5 been in the form of prefabricated, sintered hydro~y~dlite in either granule orblock form. These plepalalions have several dlawl,acks, including a limited
ability to co,lro,lll to slff-hPtql defects, particularly in the case of blocks;inq.-lequAtP, structural integrity of granules (which do not bond together); and~iffirnlty in m~elin~ into an implant having the shape of ~I~;cs;l~g skeletal tissue.
Various forms of hydlu~y~?alile, mf-th~lc of their producti-)n,
coating of mPtqllir or other plo~ ir devices with hydro~y~?alilc,
cofo. ",~lqtir~n with other polymeric sl~b~ Pc, ~q,rl~l~;x;~g with biological tissue
uc~l c~ ellt~ such as collagen, co llbhldlioll with certain ph~-, ".Ar~l,l ;r Alagents of rliq~nostic or Illcld~culic uses, and mixing with biologically active
15 proteins or polypeptides have been desclil,ed in the scientific and patent
alulc. Since all such disclosures would be too llullll ~us to list, certain
examples relevant to the present invention are cited herein. U.S. Patent Nos.
4,795,467; 4,865,602; 4,992,226; 5,123,925; and 5,246,457 disclose mineral-
collagen llliAl~U~ p,~ p~alions, inr lutli~ mfth~lc of stcrilization, for bone repair
20 wL~lein the mineral COl~)Ol~cll can be sehPct~Pd from a group of tricalcium
pho~.h~tes or l~ ~dlJdlilcs with p" f~,~d particle size of bclwcell 100-2000
,um and the collagen is either mixed to provide a moldable formulation or
applied to coat the porous inlcl~lices within the ceramic particles. U.S. PatentNo. 5,204,382 and T..l. .,qlional Application WO 93/16657 disclose injectable
25 coll~iliol s cc...~ .g particulate ce.a,l~ic materials with sizes bcl~n 50-250
,um and collagen or another biocomrAtihle organic polymer. These
combinqtionc are intPn-lPd for repair and ~ t~ ;on of hard or soft tissues
through the dic~ ql;on of the biocc,...l,sl;l,le component leaving behind the
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ceramic particles. The patent liLla~le also describes admixil~ of biological
factors implicated in the regell~lalion of bone with hydro~y;.p~ s (including
coralline h~d,c,A~a~ali~ originally described by Holmes et al. in 1979 - Plasticand Reconstructive Surgery volume 63, page 626) or tricalcium phosphate
5 granules and the uses of such formulations in bone repair. All of these
disclosures relate to materials whc.em the hydro~y~a~ile is plefol.l,ed (as
octacalcium phosphate or ~-tricalcium ph-sphqtF salts) and is subs~ y
co,lll)osed of or mq-nl-fartllred from one essF ~liqlly homogeneous form of
c-q-lri~lm ph-)sphqtF salt with co.~ or i",l,u,ilies present due to or arising
10 from the raw material source or the mqnllfqr~lring method.
In Re. 33,221 and Re. 33,161, Brown and Chow have described
powders, pastes and slurries colll~ Sl~n ly soluble ca1rillm phosphate
salts, one of which is a,ll~drous tetr~r-q-lri~lm phospk~te, that are capable ofp~cipilaling hydro~y~ali~ under ~llbicill tell,~lalu,e conditions in a wide
15 range of liquid phases. In U.S. Patent Nos. 4,880,610; 5,047,031; 5,053,212;
5,129,905; and 5,178,845 and in T.~t~ ;onal Applications WO 92/02543, EP
0,347,028 A2, and EP 0,416,761 Al, COII.~IAIII~ et al. have disclosed certain insitu cqlrillm ph~sphqtP mineral mPth~c, inl;...-le ~Llules of cqlrillm and
ph~.~l h ~ , wL~I~ill the source of pl~ sl~h t~ is ~hr,~ph~,ic acid in a ~ lly
20 anh~ Jus form, co"l~os;lionc o~ d ~ ;~y, and their use in bone repair.
Co~.~l;..,l~ et al. also have described uses of such materials in coating of bone
,ros!l.rses. ~, U.S. Patent Nos. 5,164,187 and 5,188,670, and I,lt~lnational
Application EP 0,383,568 A2. U.S. Patent Nos. 5,034,059 and 5,231,169, and
Tn~ ;ol~l Application WO 93/12736 disclose C~ il~g and/or m~neralizing
25 collagen to produce physical ch~c~li~lics resembling bone. JP 1,111,762
icrlos~Ps a l",w~l or a powder ~lu,~ co,.l;~ tetracalcium phos~hate and
a kn~i~ solution to plCnluCe a lla,de~g composition which produces
o~al,a~i~ on contact with water. EP 436,499 provides a process for
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producing a c~lri1~m phosphate-type powder, as a highly active powder, as a
component in c~1ri11m phosphate-type hardening materials. These m~teri~1c may
be used to obtain a highly plastic, moldable material that haldtns under mild
conrlitionc. In U.S. Patent No. 5,068,122, Kokubo et al. have described a
5 process of fo~ g a bone-like bioactive film of hydro~y~alil~ on a desired
~ul~sll~lc surface without the use of heat L~ and through the pl.,cil,ilalion
of hydro~y~atilc from solutions co~ i~ a certain range of Ca2+ and HPO42-
ions. Liu et al. have desclibcd the use of tetr ~a1r~ n phosphate alone, or
together with a-t-ir~1ri~-m phosphate as a base, to produce c~1ri1-m phosphate
10 cP~ with relatively high surface pH that are considered beneficial for their
bioc~ ility in oll~opedic, dental and maxillofacial applir~tion~. See, U.S.
Patent No. 5,149,368. Unlike the plcfo~ cd hyd~v~dpalilcs, calcium
pho~l.h~l~ c~ col~ illg the various pl~;ulsol c~lci1-m pho~hale salts and
capable of l,l~i~itating hy~o~)alile provide the option of incoll,ol~lion with
15 bioactive s~b~ r~s such as pi~olcills to produce implants that induce active
.ge~ alion of tissues such as bone. Il~c~ lion with tissue matrix ploleills,
such as collagen, may result in ;",pl~"l~ that are desirable lllallices for growth
of tissues, inrlllllin~ bone and soft tissues. However, these lcr~l~,nces only
concern incoll,oldlion of bioactive or bioco.~.pal;1.1e pro~cills and related
20 ~.~b~l ~nrfs as a means to improve the ~o~llies of hy&~yal~alilc as an implant
material for tissue repair app1iration~.
Nulll~.rvus patents, patent applications and pllb1ir~tions describe
insl~ and their uses for and mPth~ of infl11~Pnrin~ tissue behavior,
including healing by the use of çlPct-ir-q1 and elecll~....a~n~otir fields. Invasive
25 implants that serve as electrodes as well as çxtPrnq11y placed electrodes have
been used to gen~la~c electrical or elecllo...-qgnPtir fields of direct or p111cating
nature in and around s~Pl~Pct~P(l tissue sites. The l~,l,ollcd results include
~llgll~f.~lsll;On or healing of bone rlaclules, rel-x~tion of muscle fibers,
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regel,elalion of soft tissues, and the like. One of the earliest related patents is
U.S. Patent No. 3,055,372. Several specific designs of devices with superior
utility in specific appli~- lion~ have since been descuil,ed inrlll(ling, for example,
devices used in the promotion of inglowlll of bones into pores of hydro~ya~
5 i"~lan~. ~, e.g., Shimim et al., Journal of Orthopedic Research vol. 6, pgs.
248-258. However, none of these ~. f.,lcl~ces discloses a formulation or device
designed to modulate and/or enh~l-re ~ rh~nir~l effects or the effects of
electrical stiml-l~tion in c~l-sin~ healing.
The available technologies and products, which utilize
10 Lydru~a~ali~ suffer from other co"~dl as well as placlical li.~ iol~. One
of the major conr~l li..,il;.~iol~ arises from the focus on using hy~oxya~alile
for tissue ,~,pl~rf.. -1 as described above. Typically, the implant is intPn-led to
either fill a tissue void and provide sllu~ support or to be incorporated into
newly formed tissue d~,eloped during the process of repair or regel~clalioll. As15 a con~eql~rnre~ little effort _as been devoted to lltili~in~ the bioco,.~ ihle
propellies of hydroxya~alile and developing formlll~tion~ whc.~y the
L~dlOx~a~àLi~ is ~~ol1Jcd or degraded in a mamler similar to certain synthetic
polymers and pl"~ci~ally rull~;lions as a bioc~....l.alihle matrix to localize
physiologically active materials at a ~l~del~ d site, or to achieve the
20 p~s~ l ;on of such materials in a physiological milieu in a controlled ma~l
resllltin~ in e ~h~nreci utility. The ~;,lactical limit~tions in pursuing the novel
fi~ ti-n~ of the present invention arise from several sig~;r~ technological
limit~tion~ .
First, current L~)x~a~ltil~ technology is limited by harsh
25 plodllclion m~thndQlogy. Most hydroxy~aLile implants are produced under
eAll~llle physical and c1~ r~l con~ition~ res--ltin~ in hard implants co"")lisedof large, fused, macro-crystals with random pores and gaps, unlike material
formed naturally in bone or other C~ t~l structures e~counl~,lcd in nature.
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Second, typical hydro~y~alil~ fonnulations are characterized by limited tissue
con~alil)ility and resorbability. For i~ nr~, the hal~less and the nature of thecrystal structure frequently make them inro~p~l;hle for applir~tion in l~,pailing
soft tissues. These shortcoming~ are caused not only by the physical
5 characteristics of the formulations, but are also due to the body's inability to
completely resorb the material over an acceptable time period. Third, known
formulations also suffer from limited cofo~ lq-tion capability. Biologically
active factors that can promote cell growth or biocolll~atible sub~lces that
offer a better support for cell growth can only be applied to the surface of
10 conventional hydrv~yapdlil~ implant by s~layillg, freeze drying or soaking since
the conditions used to produce the i~ generally destroy biological
activities. Fourth, known forml-lqtion~ may have undesirable release
char<q.~-k. ;~I;rs. Biocompqtihle or bioactive materials applied to coat a
l~dlu~ya~ile implant surface may be rapidly washed away in a physiological
15 milieu res~lltin~ in a rapid rise in local col~r~ aLion of the substance with little
or no material 1~ ;n;l,~ available for the long term action required for most ofthese ~u~L~ces. Other shOl~oll~illgs of current hydro~y~alile technology
exist.
SUMl~AR.Y OF T~F INVF.NTION
The bioco.. ~ibility of l~-llv~y~ile and the ability to
precipitate hydro~y~alile crystals under alllbi~ conditions leads to the
pro~ tion of novel biomat,lials wll~le~ biologically active sub~lces may be
hlcolpolated. These biolllat~,lials are also amenable to cofollllulation with orcoating of other polymeric (~SL- dacron, nylon, vinyl, teflon, acrylic), clf ...f .~
25 (~.g~, carbon, ~ ..), or alloy (~, steel) i...~ . Novel biomaterials
s-f lfrtf~ based on "~ n;r~l and biological plopellies have utility in providingstructural su~poll for tissue filling or co~"hlg, or the stabilization of other
l"ft~ iral devices; in promoting or su~ollillg tissue formation for repair,
- - -
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egel~ldlion or allg..~P~ tion of tissues; in deli~lhlg or releasing antigenic
alelials for vaccinqtion or genetic materials for gene therapy; in facilhqting
local, eYt~n~ or controlled delivery of phqrmqreuti~qlly active agents such as
antibiotics or cll~nlo~ ,ra~ulics or hormonal substances for e~-h~ ed
5 ~ la~ ic effect; and in providing a piezoelectric field to deliver or amplify
direct or pulsed elecllu...agn~tic stim~ q-tion to Cnh~nre healing or to gen~,dle
controlled ...~rh~n;r~l motion. These are merely examples of applications of a
bioco~alil~le hydro~y~alile formlllqtion.
Based on the con~ n~-~ and the desired site of application, the
10 forrnl-lqtions and ...~ of this hl~,llliull will be amenable to dirr~lelll means
of 1~I;n;~1. alion (~, inje~tion, i"l~ lion, ~uoscopic application,
p~q~clrin~, etc.) in dirre-elll m~irql applications. As di~c~ssed above, the
molecular composition of h~dfu~a~alile makes it highly biocolll~a~ible. A
.~ig,~;rr~ portion of an adult ~el~bldtt; animal's body weight is comprised of
15 cq1cinm ph~sphqte. Since bone is an actively rrmodeling tissue, there is a
slJbs~ l turnover of calcium ~hu~haLe in a contin-lQus m~~l. Except for
con-lition~ of sign;r~ nl hypercvlr~-mi-q- blouglll about under certain pathological
conditions, the body's major organs are capable of W;l1.~ physiological
C~1rinm PhO~h~t IU11kJ~'~1, and the excretory system, primarily the kidney, is
20 capable of proces~in~ the leleased calciu~ phosphate. ~. ..PS~;~.g the
bioc ...q ~I;bility of c~lr~ n pho~pl;~l~ in C~di11g i~ (or cqlrillm pho~l.h~e
collll~osilions that can form implants in sitU!, which are formed through t~e
pleci~i~lion of hydro~y~a~ under ~llbicinl or physiological conditions and
are lesoll,ed at dirr~,ent rates wi~in dle body, has a broad range of mr~lir~l
25 applir~tion.~.
Form~ tion~ based on such bioco...l.~l;ble materials are
pl.,f~lable over syl~ ic polymers such as polylll~yl...- Ih~ ylale, polyglycolicacid, polylactic acid and copolymers of t_ese su~sl~ces t_at are not found in
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nature. Although many of these polymers are available in a range of
consi~tenriP~, the processes of polyllR~ ion and depoly~ àtion of these
materials are frequently acco.l-~ d by lm~cceptable changes in pH or
lempcralul~, may require the use of toxic organic solvents, or lead to
5 ~desirable infl~ .ly reactions in Animsl~ and hnmqn~.
Hydro~apa~ crystals also possess unique piezoelectric
chdlac~li~lics w_ich can be exploited in mo~--lq-ting the growth and metabolic
activities of cells. Thelcrure, the use of electrical and ele~ ,.n~gnrtir
stim~ q-tion approaches for tissue healing or the gelle,alion of controlled
10 mrchqnic~l motion may be ~ignifir,cntly enhqnred through the use of implants
made of h~dlo~L~a~atile crystals. Such implants may also be ~nh~nred by
incorporation of other bioco...l~l;hle or bioactive s~bsl;~l-res involved in cell
mqnirllqtion .
It is an object of the present invention, Ill~,lerole, to provide
15 con~rolled pl~s~ on of a wide variety of biologically active or biocul~a~ibler~ at select~l locations and gradual delivery of such su~ ces into the
local milieu or sy~l-n,;rally over de~ dble time periods that may be ap~l~li~tf~to derive improved clinical bell~ril~ ccLu~ ,d to other means of ~l.~.ini~l.alion
of such ~ ,-res.
It is anot_er object of t_e present invention to provide a
formulation of a versatile delivery matrix that is composed of a bioco.n~ ihle
additive, which is amenable to Lql~ .~1;. l-~ in setting time under physiological and
ambient co~ilions, which is Lqm~nq-ble to alterations in the rate of
biodc~,adalion or lcsol~lion in an animal or human body, which is capable of
25 prodneing materials with a wide range of col.c;~t~ ~ri~s (~, slurry, paste, hard
granule, block, powder, etc.), and which is able to harden as well as dissolve
willlvul si~ irlr~ ch~ges in ~m~lalule or pH.
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It is a further object of the present invention to provide a
formulation of an implant or implantable composition which displays an
alJplvplidk co~llbiu~lion of resorbability, cell growth ~.u~olLing capability, and
pie70electric con~ ct~nre capacily for use in the enh~nrr~llpnl of electrical or
S elecllu...agn~ti~ stimlll~tion.
To achieve these and other objects, the present invention provides
formlll~tiQns of certain s~alill~ly soluble c~lcillm phosphdte salt coll~hlations,
having the ability to p~ iktle hydlo~yal)alile under physiological and/or
alnbicllL colldiliolls, ~L~lchl a biocompatible or a bioactive material is
10 hlcol~uldkd during the p~ ilalion process such that the iulcol~vl~d materhl
is retained at the site of application of the formlll~tion for eYtP-n~lPd time periods
or gradually released into the sullvulldh~ physiological milieu. The
composition of the salt colll~h~ation used in a formulation of the present
invention is chosen such that the physical propcllies (poro~.ily, tensile and
15 flexural sllc~ll, co--~;~t~ y, etc.) and the les(,l~lioll/biodegl~datioll ~,lu~llies
of the reSlllting l~dlo~yd~alik pl~;i~i~Le enable the fonnlll~tion to enh~nre the
function of the incorporated bioc~ l ihle or bioactive material.
In an embodiment of the present invention, a biocompatible
hydl~ lile forrnlll~tion is provided in which l~dl~.xyapatite ~;r~illi~ks
20 under physiological con~l;l;on~ (;n~ u~ pH, k~ Cla~ul~, ionic ~ l, etc.)
or under con~lition~ that (i) ple;,.,l~e the activity of an added biocom~alible or
bioactive sllhst~nr~ and (ii) allow the s~lbst~nre to be illl~rcgl-~tecl re~con~hly
ullirul~ly throa)gh-~llt the plec;~ilaled colll~sik. One aspect of this embodiment
C~ . "c formlll~tir,nc co. ~ g biologically c~. . .p~l ihle materials that SU ~ O1l
25 the growth or formation of dirr. lcnt cells or tissues and biologically active
~..b~S-..rec that cause or induce the formation of such cells or tissues. Another
~ aspect co.Yt~ ~.. l.lates formlll~tions col.l~ biologically active s~lb~l~.-rec. that
kill ~lice~ced or undesirable cells or tissues, or aclivate certain cells or tissues
-
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wherein the activity may produce a thel~cuLic benefit. The biocompatible
hylllo~ya~)alilc formulations may be used in products and structures with other
tec_nologies to create novel combined mP-licql devices.
In another embodimPnt, a method is provided for p~Cp~illg a
S biocoll~alible hydro~y~alile formlllqtion. A base combination of calcium
phosphqte salts is pr~ d. A liquid phase is plcpal~d. A biocomratihle
additive is provided. These components are then combined to form a mLX.IUlc.
Then, â bioco~npatihle hydro~yap~lile formulation is precil,iLa~d from this
~lule. To form the ll~luie, any two of the colllpollcll~ may be colllbilled
10 first by suitable mPth~s and then coll-bill~d with the third colll~ollelll.
Alternately, the three illgl~ien~ may be ~ nF~)..sly colllbhled. Accof~mg
to various aspects of this embo~lim~nt the biocollll,atible additive may be any
of several additives inrhl~in~, without limitation, growth factors, adhesive
agents, immllnl)gens, vaccines, genes, recombil~l cells, antibiotics,
15 ph~rm~celltir~l agents, hormones, fibers, gels, space oc-;u~ing particles, and
electrical stim~lh~ e ~-h~ . A form ~l~tion llr~alcd accordillg to this method
may âlsO incorporate a ph~ c~ll;r~lly acceptable carrier.
In another cnlbod;..~- ~1, a method is provided for treating a
patient. In this emWim~nt, a bioco...~ ible hydro~yapalile formulation is
20 prc~ d as before. Then, the form--1~tion is 1mini~tered to the patient.
Purther objects, f~alul~s and advantages of the present invention
will readily a~pa~e~t to those having o~ al ~ skill in the ~el Li~ t art from the
det~ilPd description of the pl.,f~llcd embo~ with l~,fclcnce to the
â~plOpliàle figures.
E~I2TFF nF.. ~C~TPTION OF T~F. nRAwING
Fig. 1 is a plot of the solubility iSOI .llllS of Ca4(PO4)20;
CaHPO4-2H20; CaHPO4; Ca8H2(PO4)6-5H20; ~--Ca3(PO4)2; and Ca5(PO4)30H
at 25~C in the ternary system of Ca(OH)2; H3PO4; and H20.
.
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nF.~CR~PTION OF THF p~FFFRRFn Fl\~RODTl~F~TS
The present invention is generally directed to selected ~ ules
of calcium phosphate salts formed as powders, slurries or pastes and having
various ",PC~ ir~l propl,lies, porousness, pie7oP!ectric r~lules and
5 bio~sol~)lion characte~ ics. These materials serve as novel delivery vehicles
for various ch~ al agents used as traditional phz..~ ;r~l drugs and for
biological agents including glycoproteills, polymeric hydrocalbolls, lipids,
glycolipids, carbohydrates, ploleills and nucleic acids. Additionally, these
mq-tPriql~ Prlhqnr~ the effects of electrical or ele~ ~Ptic stim~ tion in tissue10 healing.
The reclllti~ novel colllposi~ions have utility in a broad range of
mP~ir~l applications such as ;~ ion or vaccination, gene Ih~.apy,
Ih,~à~lliC m tAsllitiPc "~PA;~ ~1 tbrough selective el;n-;.~-l;ol- or mo~lifir~tion of
cells or removal of ~vbslAnr~~ from a physiological milieu, repair or
r~e~ on of biological ~IlUl;lulal material (~,~, bone, muscle, skin, tendon,
and lig,qmPnt) a~hOlillg of tooth to bone, anchoring of prosthPtir devices, or
v~ l ;on of soft tissues such as breast tissue. Other uses are envisioned and
certain s~cirlc applirationc co.l1r~.rlated for the various compositions are
(~iccllcsed in detail below.
The major CO~pOIl~ for the biocomp~tible hydro~yapalile
formlll~tir,n coll~ ise s~ ly soluble c~lrillm ph~ h~ salts. Preferably, two
salts are colllbi~d, one of which is preferably tetrac~lri~lm phosphate. The
other salt may be selPcted from the group col.~;cl;.~ of CaHPO4-2H20;
CaHPO4; Ca8H2(PO4)6-5H20; ~-Ca3(PO~,)2; a-Ca3(PO4h; and modified
Ca3(PO4)2, e.~., tri-ca1cillm phosphqte modified by plolons or up to
appl~k;ll~ttoly 10wt% mq~,.P~;...~,
The r~ f ~ l plill~iples ul~dellyi~g the selection of the second
sp~i~ly soluble cqlrillm phnsphqte salt that is to be mixed with tetracalcium
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14
phnsrh~te are described by Brown and Chow. ~ç~, ~ U.S. Patent Nos. Re.
33,221 and Re. 33,161; W.E. Brown and L.C. Chow, "J. of Dental Research,"
vol. 63, p. 672 (1983); W.E. Brown and L.C. Chow, "C~n ~ontc Research
Progress - 1986, Alllelican Cerarnic Society," p. 352 (1986); and Y. Fukase,
5 E.D. Eanes, S. Takagi, L.C. Chow and W.E. Brown, "J. of Dental Research,"
vol. 69, p. 1852 (1990). These materials are incorporated by lel;,lellce.
Racirally, each of these calcium phosph~te salts has a
ch~ac~lislic solubility behavior that may be r~l~,sellled by a plot of the totalconcentration of calcium ions at the point of saturation versus the pH of the
10 solution at a co~l~lt t~.n~ldtule. A plot of the total collce.l~lalion of ~hos~k~te
ions versus pH would be equivalent for the purposes of the present invention
be~ ce the collce.lLIdtions of phosph~t~ and c~lrilln ions in solution are linked.
The reslll~in~ curve is called an isotherm.
When the iSCllh~ S for various calcium phosphale salts are
15 plotted on the same axes, their solubility behavior relative to each other may be
~~e~,- ..-inl~d. Specifically, a c~lcillm pho~h~t~ whose iso~ l lies above the
icolll. . ll. of another C~lri~lrn pht s~k ~. at a given pH is m~ot~ct~hle with respect
to the latter. The point where the i~!kr~ of two c~lrillm pho~_a~s il~ e~;
is known as a cin~ r point. In a solution that is salwdled with respect to the
20 two c~lri~lm phos~ c~ both c~lrillm L.hos~ s will be in equilibriurn with thes&lwated solution at the singular point. This means that neither calcium
~h~ .k~' will pleci~ out of solution, but another calcium phosphate whose
icc,ll -. . - -- lies below the sin~ r point can preCi~ildlt~. The present invention in
one acpect relates to c~ alions of c~ lm ph~ . salts that form sin~ r
25 point solutions that are ~u~l~aluldtcd with respect to hydro~y~atile.
Fig. 1 is a plot of the solubility iso~ s for six c~lri~lm
phosphate salts in the ternary system CO lliShlg Ca(OH)2, H3PO4 and H20 at
25~C. The y-axis of Fig. 1 ,e~lescllt~ the total Col~c~ dtioll of c~lrillm ions in
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solution in moles per liter, while the x-axis represents pH. The isotherms for
CaHPO4-2H20, CaHPO4, ~--Ca3(PO4)2 and Ca5(PO4)30H are based,
resl ec~ ly, on the following articles: Gregory et al., "Solubility of
CaHPO4-2H2O in the System Ca(OH) 2--H 3PO 4--H ~0 at 5 ~, 15 ~, 25 ~ and
5 37.5~C.," J. Res. Nat. Bur. Stand. 74A: 461 475 (1970); McDowell et al.,
"Solubility Study of C'-q-lrillm Hydrogen Pho~,hale Ion Pari Formation," Inorg.
Chem. 10: 1638-1643 (1971); Gregory et al., "Solubility of ~--Ca3(PO4)2 in the
System Ca(OH)2--H3PO4--H2O at 5~, 15~, 25~ and 37~C.," J. Res Nat. Bur.
Stand. 78A; 667-674 (1974); and McDowell et al., "Solubility of Cas(PO4)30H
10 in the System Ca(OH)2--H3PO4--H2O at 5~, 15~, 25~ and 37.5~C.," J. Res.
Nat. Bur. Stand. 81A: 273-281 (1977). The is~ lll for Ca8H2(PO4)6-5H20 is
based on the solubility product disclosed in Moreno et al., "Stability of
Dic-qlrillm PhosphAt~ Dihydrate in Aqueous Solutions and Solubility of
Octacalcium Pho~l,hale," Soil Sci. Soc. Am. Proc. 21: 99-102 (1960). The
15 isollle~ of Ca4(PO4)20 is based on the appru~ e value of the solubility
product c-qlclllqte~ by Brown and Chow as referred to, for example, in Re.
33,221 and Re. 33,161.
In the plef~ d embof~ of this invention, the ratio of
~tr~cqlrillrn pho~ to the other salt as well as the relative particle sizes are
20 varied in the diff,le~ co~lll~lated fonnlllqtion~ in order to obtain the ones that
meet the ",~ha~ al criteria and l~,sol~lion and degradation profiles desired forthe particular bio~;live or bioc()...~ .le ~b~l~nfe incol~ula~d as an additive.
itionqlly, the relative amount of a liquid phase (preferably chosen from the
group col~c;~l;u~ of water, saline, a weakly acidic solution, a biocGlll~alilJle25 buffer solution, serum, plasma, and other bodily fluids) to the amount of the salt
coll~ iûll may be varied in order to alter setting time and the con~i~t~ y ofthe res~llting pre~ ilal~d biocc...p-~;l.le l~o~y ~dlil~ fonmllqtion. The
biocomp~ihle or the bioactive additive is preferably dissolved or essc .l;~lly
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16
ullifollllly mixed in the liquid phase prior to adding the liquid phase to the dry
salts colll~ ion and il.il;5~ the hydro~y~alil~ plccil,i~lion reaction.
~Alt ~ ;vely, the liquid phase may be first colllbill~d with the calrillm pho~,hd~
salts, followed by i~col~ dlion of the additive into the ll~Ll~lul'e. In another5 ,ql~f . ~a~ive, the bioactive additive is first colll~ined with the salts, and then the
liquid phase is added. In still another ~ql~ J ~a~ ivt;, the salts, additives and liquids
may be simlllt-qnPously colll~hled. The liquid phase may additionally contain
setting reaction or hydroxyapatite crystal growth modifiers such as a
proteoglycan (~,, hyaluronic acid), a protein (~,~, serum albumin), a
10 carbohydrate (~.&, granular sugar), a ~yll~ ,lic material (~,~, polyethylene
glycol), certain other ionic agents, and the like. Many ~l(litionql l.,plesç ,~ ive
examples of such materials may be found in U.S. Patent Nos. Re. 33,161 and
Re. 33,221.
The tetr~qlrillm ph~.~phq~ col~(Jlklll may be produced by any
15 of various suitable "l- ll,~c. In one such m~~fh~, the tetracalcium pho~phale is
produced by a solid state reaction catalyzed by high ~m~lalulc; ~l~0~ n (Ç,~L,
1500~C to 1700~C) of an equimolar ~e of ~ir-q-lrillm phosl~h~e and cqlrillm
carbonate. The tetr~eqlri~lm phosphqte thus produced is then mixed with a
desired amount of another s~. ;.¢ly soluble c-q-lrillm ph~ ~k~ salt selfvc~l from
20 the group recited above. Al~ l ;vely, a ll~ lule of a-Ca3(PO4)2 and
tetracalcium phosphqt~ may be produced by calcining a hydluxy~âtite
pl~paldlion having a molar ratio of Ca/P of ~lw~n 1.5 and 1.8 at 1150~C to
1450~C under l~luced ~ ~e. This ~ is further 1 t~ by the ~ litir.,n
of a :iui~ble ~ ply soluble cql~ium phsspk~le salt in order to provide the
25 desired setting pr~,llies and con~ y of the l~dl.~apatite formlllq-tion
produced by preci~ildlion upon mixing wit_ the qu~ Pd liquid p_ase
co.l~ P a biOâcliv~ or a b:oc(...ll, ~ible addiliv~. The c-qlrillm ~hns~h~te salts
and their colll~i~lions are preferably produced and stored under substqnti-q-lly
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anhydrous conditions in order to plccipilatc hydro~ya~alilc forml-lqtiQns of
concicte~t and superior quality upon addition of the liquid phase.
The following ~"llbo~ include methods of producing formlllqtions
colllplising the c-q-l~ illm phosphqt~ salt co"lbil~alions mentioned above and
5 various bioactive or bioco...p-lible additives, formlllqtion~ thus obtained, and
uses I~ efol. In the tliccllCcion of each embol;-.-~--l rcp~cscl~livc bioactive
or bioco---p~tible sub~ ces int~n~l~ for certain m~ al applications are
described. A person of o~.nal ~ skill in the art will be able to produce such
materials as described in these emb~i...~ and will also be able (where
10 ap~lo~liale or desirable) to ~b~lill.lr or add bioactive or biocompatible
~..ks~ es with prope,~s similar to the ones cited in any particular
clllbo~ .1 Also, while the fo,egoillg ~liccl~ssiQn relates to cqlri -m phosphatesalt co~bin~iollc such as those desclibcd by Brown and Chow, the technology
herein is not so limi~yl The embo~ , fealul.,s and aspects of the present
15 invention can be applied to other c~lril~rn phosphate salt co~ iQnc Further,
the ch~a~;t~ lics of the bioco",pa~le hydro~yapalile formulations allow
p,~;i~ilalion in vivo, ~ vivo, or partially ÇZ~ vivo and partially in vivo. Also,
the hydroxyapatite may be ,~,so,l,ed or degraded in vivo.
1. Incolpoldlion of Growth Factors For Wound T~lin~ And Soft Tissue
20 Repair
A first e~bodi",c.ll is directed to wound healing and soft tissue
repair, and addl~sses a major need which exists for materials that can be applied
to wuu~ded soft tissues (~, skin, muscle, fascia, gum, periodontium, etc.) in
order to cause or plulllo~ (as distinct from support) healing of the damaged
25 tissue. Various biological factors have the ~otel~lial for inducing growth ofdesired cells or formqtinn of such cells from ~ o....--;ll~ precursor cells in the
area of the ~ .aged site. Sy~t~"l~ic ~l ~lir~ion of such materials would requiresu~s~ y~ es~ malcing such an ap~roach both cost prohibitive and
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18
in~lacLical due to limitAtion~ on the availability of the materials. Even in cases
where such problems have been solved through the advent of reco~ lL DNA
teclmr.logy, other a~;Li\/iLies often displayed by such factors and COI~IAI~I;IIAI~I~ in
~lepaldlions of these materials may produce undesirable side effects when such
S high amounts are ~minictered ~,y~,~e llirAlly in order to achieve a~leqll~te
col.cellLlalion at a particular site. Local applications of such materials to date
involve either coating of a biodegradable lll~.l,bldne covering type material ormultiple applications over extended time periods. Such approaches are
in~ffirient, fraught with risks of infection, and cumbersome.
A first c.llbodi~llelll of the present invention, which is directed to
this m~tlirAl application of ~ ",1 nlAlion or guided tissue ,~,genefdlion, relates
to a bioco..-l~AIihle hydro~y~aliLe formulation that incorporates one or more
growth factors. The growth factor may be selected from the group consisting
of epidermal growth factor (EGF), lla~rOlll~illg growth factor alpha (TGF-a),
15 lla~rolll~ing growth factor beta (TGF-~ dccil~ia growth factor (VGF), acidic
or basic rll.lobl&~l growth factor (FGF), insulin-like growth factor (IGF) (ç,~,IGF-I and IGF-II), platelet-derived growth factor (PDGF), cartilage-derived
growth factor (CDGF), int~rlellkin-2, nerve cell growth factor (NCGF),
h~ol,oi_lic cell growth factor (HCGF), lymphocyte growth factor (LGF), bone
20 morphogenic protein (13MP) and other ..~ hc.~ of the glowing family of wound
healing factors. Further, the hemopoietic cell growth factor may be selectecl
from the group conci.ctin~ of interleukin-3, granulocyte-macrophage colony
stimlllAting factor, angiogenesis factors, macrophage colony stimlllAting &ctor,~Amlllxyte colony stim~ ti~ factor and el ~o~oit;lill. Also, the lymphocyte
25 growth factor may be sel~t~ from the group co. .~ of B cell growth factor,
T cell growth factor, interleukin~, interlellkin-5, and interleu-k-in-6. The
formlll~inn pl~,fc.ably displays ~ rh~,~ir~l plo~c.lies of a paste, a hydrogel, a
film, or the like. The formlllAtinn plef,.dbly ~ledses less than about 20% of the
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19
growth factor in about 1 day and greater than about 90% of the growth factor
in about 30 days. More preferably, the release rate equals less than 20% in
- about 2 days and greater than 90% in about 5 days. The amount of the growth
factor is p~fclably from about 0.1 ,ug to about 10 ,ug per cubic ce..li,~ter
S volume of the formulation, and is more preferably in the range of from about
3~g to about 6~g per cubic Ce~ 1 volume.
The bioco...l--lible hydro~yàpàlile formnl~tion may be plcpal~d
accor~illg to the following stecps. In a first step, a base combination of c~leium
pho~,l h~tto salts is pl~alcd. This step is preferably accomplished by ~,Çollnillg
10 the following plVCedUlC. One or more c~lrillm phns~h~lP salt co,nbil~lions are
plf~ d as desclil)cd above. Then, a liquid phase is formed by suppl~..F~
a liquid, such as sterile watcr~ saline or a physiologically co...l.~ e buffer with
a crystal growth mcKlifi~r, such as hyaluronic acid, non-cross-linked collagen,
ethylene glycol, polyethylene glycol, glyc~ , polylysine, or the like. The
lS crystal growth m~ifier pe, i~, çh~ es to the setting time and allows
achie~.,.l~.lt of a desirable col~c;~t~ ry~ which depends on the method of
~l.";n~ ;nl~ Next, the res~llti~ liquid phase is added to the one or more salt
CU111~ I;OI~C, ~ y7 pl~f~,lably at a weight-to-weight solid-to-liquid ratio
of bel~,l about 1:1 and about S:1. The solid-to-liquid ratio is more preferably
bG~eell about 1.5:1 and about 4:1, and most preferably bel~,. n about 2.5:1
and about 3.5:1. Respective hy~O~ya~alil~ samples are plccipi~ltd from these
Ul~S~ which are each evaluated for its setting P10~C.IY as well as its
cc .~ t~ ~ y. Each l~,"~cli~e sample then, is allowed to harden to a sheet, eachsheet preferably having a ll-i~L llf ,CC l~lllgillg bGIwcell about 1 mm and about 7
- 25 mm. More pl~rtlably, the ~hirL l~f-SS ranges from about 3mm to about Smm.
Pieces of the l~,S~Cli\~e h~dened materials are implanted s~lbe~ uusly and
rly into test ~nim~ ~, rats, guinea pigs, rabbits or pigs. A base
hydroAy~alile form~ tiQn~ which permits the desired release rates, is then
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s~PlPrtP~l The base co~ hlalion of calcium phosphate salts is that colllbhlationwhich can pleci~ te this base hydroxyapatite formulation.
In a second step, a liquid phase is a~g.~.r~ d by the addition of
a selPr~ growth factor. This step is prertrdbly accomplished according to the
5 following ~locelule. First, a growth factor is se!Pctçd, preferably from those already described. EGF, TGF-a, TGF-~ or VGF, for example, may be
produced by lecolllbh~ll DNA technology from a llal~Çolllled host cell
substrate (~, I~ qliqn, llliclobidl, or insect cell culture), by chr~llir~l
~yl~llesis, or from cultured cell sources that elaborate these materials. Purifled
10 p~ alions are assayed in cell culture ~y~ltllls to del~....i..~ the potency to
pr~lllolt cell growth and the ~ir.~ ;ly for binding to the recc~lol (EGFR), and
are stored as lyophili7~d powders. Other wound healing factors may be
produced by l~colll~ l or sy~ ic means, and are cha acl~liGed for polcll.;y
and s~ec;r~ y using cell culture stim llqtion assays and specific l~l)lc,l binding
15 assays, l~,s~eclivcly. One or more ~alyillg ~-".)~ of the sel~t~l growth factor
then are added to the supplf~ f .~ liquid phase desc~ibed in the above first step
to provide one or more ~llglll ~ d liquid phases, les~c~;Livcly, having a range
of final coll~e.~alions of the selectçd growth factor. Next, the lejpeclive
~1g..~f .~ed liquid phases are each mixed with the above-described base salt
cGlllbh~alion to p,ccipi~tc lei,~ecti~e ~ "lr~ 1 hydro~yal,ati~ samples.
Phf -ably, this mixing results in a solid-to-liquid weight ratio ~Iwten about 1:1
and about 5:1 (more pl.,f~,lably bclwwll 1.5:1 and 4:1; most ~lef.,lably ~elwwu~2.5:1 and 3.5:1). Theseles~;li~c5~ "~ hydro~ya~ali~samplesthenare
allowed to harden in sheets having IhirL ~.f s~es preferably l~ illg from about 1
25 mm to about 7 mm (more preferably belwwll 3mm and 5mm). Alternatively,
the samples may be allowed to harden into test blocks. Test portions of the
e~e.,live ha.dened sa~ )les are then il~ sed in water, saline, or other
physiological fluids, such as serum or plasma, under sterile con~litiQ~ at a
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t~lllpe~alule from about 25~C to about 42~C and the rc~ ive test portions are
gently qgjtqted. The test portions from the liquid mPAjllm are then tested at
- various times, by immllnological ~echni~ues, for ~ u~ of growth factor
relfeq~ecl A release kinf~tir~ diagra n may be plotted from these testing results.
5 Forrnulations which display about 30% or less release within about 5 days are
chosen for sl~hseq~ent in vivo testing. The chosen formulations are implanted
sllh;~ f~uusly and inL~ qrly at vasculanzcd sites in test animals. Release
rates of the growth factor in the ~ ~;livc samples are ~if~ te~ inf~A by collecting
serum samples as well as by collecting, via aspiration, local fluid ~ull~u~ g
10 the imrlqnt Then a final ~q~uglll~n~A~ hydroxyapatite formulation is selected,
which permits the afol~,ll-f nliol~ed desired release rates. The al~&...f ~.IPd liquid
phase is that which permits ~lf~;i~i~tion of this final q.~g...~ d hydro~y~l,alik
formlllqtion.
In a third step, the ~ d liquid phase is mixed with the base
15 combination of crqlri~lm pho~ph t salts. And, in a fourth step, the
bioco...l -I;ble hyd o~ya~alilc form~lqtion is ~lcci~i~led from this ll~lule.
2. IncGl~olalion of T.n.. ogens
The ability to i~COll~ulalc an i-~ -ogcn, ~L a ~ led protein,
~ ;lycoplolcill, lipo~lolcin or t_e like, into a biocollll)alil,lc implant t_at resorbs
and releases the ;.. ~.-ogell over several weeks has several advantages overknown technology. These include, will.oul limh~tion, avoiding the need for
multiple booster iniection~ that require multiple visits to the clinic by the subject,
and enabling the elici~lion of a more potent imm--nf response with less
;.~.. ~.og~,n material than is l~ull~d when such i.. ~.. ogens are ndmiYed with
25 various adju~a,lts and ? ~ t~ cd by various conventional methods.
A~1(1itionqlly, the innrlqnt may be form--l~t.od to produce low grade local
infl~ o.y re~unse, Il-~l~y P.~h~ the po~;y of the imml-m~gen. A
slow release apprûâch may also enable ;.. ~ ;on against mqteriqlc that are
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22
moderately toxic and thus not ~mPn~ble to the development of an effective
e;.pollse by tr~ tion~ ionprotocols.
The second embotlim~-nt of the present invention thus relates to
a bioco-~ le h~.,~L,alile form~ tion which il~Ol~)oldl~s an immnnogen.
S For i.~ re, the i.. ~nogen may be sele.-le~l from the group coli~ of viral
antigens, bact~ial antigens, fungal antigens, and parasitic ~ntigenc. The
immlmogen may also be any m~lign~nry-specific marker including without
limh~tion, tumor ~ntiglonc, peptide Ll~...~."~ of tumor ~ igens, and mrt~ct~tir-specific ~ntigrnc. The ;------~-mg~,n may be a subunit vaccine. The immlmogen
10 may be ill~ol~olàted in a vaccine, which can be active or passive. Also, the
vaccine may be a synthetic vaccine which can be org~nir~lly made or
recombil~ lly m~ade. Some specific examples of envisioned immllnrJgens
include HBV envelope ~ntigPn; HIV gpl20/gpl60/p41; lecolllbil~ or purified
protein immllnngens for v~cci.~lion against mumps, mr~cles, rubella or small
pOX (~,accil~ia); and the like. Preferably, the fnrmlll~tion displays mrch~nir~lope.lies of a granule or plug. ~. fi lably, the formlll~tion leleases about 20%
or less of the immlmogen in about 1 day and 90% or greater in about 30 days.
More p~e~lably, the release rates are 20% or less in about 2 days and 90% or
more in about 5 days. The amount of the immllnogen in the formlll~tion is
20 ~iefelably bel~.~l about 50 ~g to about 500,ug per cubic ce~ packed
volume. This amount is more preferably b~lw~n about 100 ~g to about 400 ,ug
per cubic cç~.l;...~t~ ~, and most preferably ~tween about 150 ~g to about 300
~g per c~ r.
A formlll~tion accoldillg to this embodiment is preferably
25 pl~al~,d accoldillg to the following steps. In a first step, a base coll~ dlion of
c~lrillm pho~)h-l~ salts is prepaLed. T_is first step is preferably accomplishedaccold~g to the following ploce~ e. First, one or more colllbi~lions of
spalingl~l soluble c~lrillm phosyh-l~ salts is pr~,d as previously desclibed.
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WO 96/39202 PCT/US96/08652
Next, a liquid phase is formed as previously described to have a solid-to-liquidratio as previously desclibed. In several following sub-steps, the liquid phases- are mixed with the salt c~ hlalions to plecipiL~le hydro~yapatile formulations
which are each tested as desclibed in co..l-~clion with the first embodim.ont
S Form~ tions that provide the desired ~sol~lion rates in~lir~ted above are
select~d. These formulations should permit the desired release rates of the
immlmogen.
In a second step, a liquid phase is ~ug...~ ed by the addition of
a sellocted ;.. nng~"1. This step is preferably accolll~lished accordillg to the10 following procedure. First, an ;.. ~ogell is selected. The immllnogenmay
be any of those already desclil~d. Next, one or more varying ~ of the
selected immllm~gen are ~dded to the liquid phase des~;libed in the above first
step to provide one or more ~llgm~nted liquid phases, l~ ,ecLi~ely, having a
range of final de~iti~s of the sel~ct~~ g"n. The l~i,~clive augmP-nt~l
15 liquid phases then are each mixed with the above-described base salt
colllbinalion to pl~,~,ipilaLe ~ eclive ~a lgm~ntrd hydroxyapatite s~l~les. In
subsequ~nt steps, these s~l-s are tested as desclibed in connection with the
first embodimpnt A final ~u~...f~ d ~dl~yapdtile formulation is select~d
which pelmiL~ the desired release rates. The augm~nt~d liquid phase for the
20 second embodiment is that which permits plccipiLaLion of this selected final
~ugl.. llr(l hydlOxyd~aLiLe form-ll~tion.
In a third step the ~l1gJIIr~ Iiquid phase is mixed with the base
combinaLi~ of c~lrillm l~ho~h~ salts. And, in a fourth step, the
biOCOm~a~le hydko~a~dli~ fonn~ tionis pl~Ci~ from ~s ~
25 3. Incol~,Glalion of Genes
A third embodiment of the present invention relates to gene
therapy. Recent ad~ances in the ll.l~lf ' ~ of genetic bases for di~e~P c and
the ability to m~nirll~te functional genes are revolutionizing the field of
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m,tliein,. The genetic material of hllm~n~ and other Anim_lc, which is
com~lised of deoxyribonucleic acid (DNA), is amenable to direct llalLsr~r into
~ cil~icll~ cells across species ballic~. As early as twenty years ago, a methodof ll~sr~r of DNA molecules to ~ .--.n~liAn (including human) cells in tissue
5 culture used a method of col~'~Yin~ DNA with c~lrillm phosphate and applying
this complex to the int~n-l~ r~il, c.ll cells. This resulted in the recipient cells,
albeit at a very low frequency, taking up the calcium phosphate-DNA complex
and allowing some of the DNA to be hlcol~ol~ted into the chromosomal
material, ~ ,~y allowing the product encoded by the DNA to be produced in
10 the ~ ie.ll cells.
A major obstacle in deli~e.ing genes in the form of naked DNA
into intact ol~ ;c-l-c (-AnimAlc or hllmAnc) is the susceptibility of DNA to
degrading e~yl~s in bodily fluids and the propel~sily of DNA delivered at
target tissue sites, such as muscle tissue, to be washed away prior to the ability
15 of the eYposed cells to take up a l~ aso~ble amount. Some all~ s at solving
these pJubl-~c have involved the ~ of genetic sequenrrs derived from
certain viruses ~Ivhc.~ by the desired piece of DNA, even if taken up by only a
few cells, would be s~lrrl~ .l since the Att,r~c~ viral seqllenr-es would allow
repro~llrtion of the piece of DNA in the lecip;~,nl cells. It was e~l,ec~d that
20 progenies of the ~;ipi~nl cells and cell-to-cell ll~f~. would amplify the
delivered DNA dose in the animal (or human) in order to lead to a desired
clinical u~ll( O~ . A major safety coll~e.n, how~., arises from the use of
virus-derived scquenr~s to render the desired genes into virus-like self
reproducing elem_nt.~. The conr~rn is si~nifir-Ant since most of the effective
25 viral sequenr~s that can provide the desired reproductive capability originate
from highly infectious viruses including certain viruses known to cause deadly
.lic~ces, such as AIDS and cancer.
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In the third embodiment of the present invention, a bioc-....p~ihle
l,ydroAyapalile formulation is provided for gene therapy, wh~ the
- form111qtion illcOl~ldt~s a genetic material. The genetic material may collll~lise
nucleic acids such as DNA or RNA (~, ,q~nti~Pn~e)~ ploteills or modified
ploleil~s( ~ ,~ylll~S,LIanSClil)liOn factors or trqn~1qtion factors), or cells that
express a desired protein or nucleic acid. The genetic material (~ DNA) rnay
be co r'eY~d in an ordered or random fashion with the hydroxyapatite. Cells
physically ~ r~ "l to such complexes are allowed to take up the complex and
hence the gene. Preferably, the form1-1qtion of the third embodiment
10 incol~Gldles purified DNA molecules (~, co~ q-f~ d, circular or linear
forrn) leples~ , a coding Sf~ re operably linked to regulatory and plOlllOt,
sequences that allow favorable eA~lession of a desired gene product in one or
more types of ,..~ 1iqn cells. The form~ ti-n preferably displays ~1u~irA1
pl~opellies of a soft paste or â slurry, and should deliver 20% or less of the
genetic l~atc.ial about l day and 90% or greater in about 30 days. More
pleL,ably, the delivery rates are 20% or less in about 2 days and 90% or
greater in about 5 days. The level of DNA is prer.,.àbly in the range of from
about l0 ~g to about l00 ~g per cubic c~ r of the delivery vehicle
plo~luced accc.r~i~g to this emboAimfnt More pl~Llàbly, the level of DNA is
20 plef~,lably ~t~n l0 ~g and 50 ~g.
The bioco~ le hy~LuA~a~dlil~ form111~tion of this embodiment
is preferably plepa~ed according to the following steps. In a first step, a baseCOlll~ a~iOIl of c~1ri11m ph~ h~1~ salts is pl~aled. This may be accomplished
r~col-ling to the following pl-;)CedUl~" which is similar to that of previous
25 embo~ f ~1 ~i. First, one or more colll~inalions of calciurn phosphate salts are
p~ ,alcd as previously descril~d. Then, a liquid phase is pr~)al~,d, also as
previously A. e s c il~l MiAtures of these colll~o~~ ,f._lably having solid-to-
liquid ratios as .~ .ced in col-nf~ n with previously deswil,ed embo~
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liquid ratios as fliC~ seA. in co.~n~lion with previously described embo~
are tested as ~licc~sse~ above.
In a second step, a liquid phase is ~ ~ by addition of the
desired genetic material. This step may be accomplished according to the
S following procedure. First, a desired gene ("coding sequP-nre") may be select~oA
which ~-rodec the product int~nfl~A to be eA~l~ssed in the host. The gene may
be cloned, propagated in a llliclobial host, and edited for e*.lcssion either in a
broad variety of rcci~;ent host cells or in a tissue-sl,e~,irlc ll,a~ . Requiredregulatory c~ -rces~. y for e~res~ion include general or tissue-specific
10 promoter se-lue-res, splice signals, polyadenylation signals and enh~nreJ
seqYenres. The genetic CfJ~IuCl to be i~cûl~ulaled in the hydro~ya~alile
formulation may be derived by cûlllbin.,lg the coding sequenre with an
dpl,lo~liat~ set of regulatory cl~ n~"1~ in an operable linkage. The construct
may then be plop~gsl~ d in a llliclobial host in ûrder to avoid the risks of
15 coll~nil~ating ~ule~ lly ha7~rdous genetic seq~l~nres from a ..~...n~ n cell
or a human cell-directed virus. E'~.,f~lably, all parts of the genetic construct are
cloned from normal human cellular genetic material and then mf~ifi~ or edited
and propagated in liclobial host systems. ~llr.~ ely, where a viral or
onrog~nr se.~ r~ is desired to be c A~l~ssed (~,~, for ;.. ~ . lherapy purposes20 against certain cancers, AIDS, and the like), the coding s~~ .re may be derived
from the applul)liàl~ virus or tumor cells. In certain cases, the coding seql-enre
may be created by ~eleting "introns" from the original chromosome-derived
material or through the cl~atiun of a DNA copy of the functional ~..Psse~er
RNA by a process called reverse ~ -.c- - ;l-lin.. In other cases, if the amino acid
25 seqUPnre of a desired polypeptide or protein product is known, the coding
sequence for such a protein or altered forms of such a protein with
pl~et~ ...;n~l c~ g~s may be produced by ch~ r~l gene ~ylllhesis. In these
sil~tinnc~ where the coding sequence is m~ified or ~y~ l;r~11y produced,
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editing of the coding seq~nre may involve prede1r. .,.llrA addition of new splice
signals, Ll~c.i~ /tr-q-n~1qtion start sites, or the like, in order to facilitate the
~ pro~ur,ti-)n of the product elroded by the coding se~ P~.re in the l~ci~ cells.
Purified DNA for the bioc~ mpqtihle hydro~ya~alil~ form11lq-tion of this
- 5 embodiment preferably com~lises the functional gene construct produced as
d~~r~ herein in a linear or circular form, in a single copy or tqnrl~rnly linkedmultiple copy form, or in a co~ ?osile of these forms.
Various amuull~ of the desired gene, e ~., purified DNA, are
then dissolved in a liquid phases of the type described in connection with
lO previous emb~in.f l1~ It is envisioned that the desired gene may be
e,lco..~csed in a cell, which may be a r~colllbil~l cell. The cell may be a
myeloid-derived cell or a Iymphoid-derived cell. The cell may express a
ecum~il~ll product in~ i~, wi~ ul ~ ;Qn, those sel~ct~l from the group
con~ ;~ of insulin, nucleic acid, viral ~lligens, bacterial antigens, fungal
15 antigens, p~asilic antigens, cytokines, growth factors, ho,l~ cs, cell surface
pl~te~ns, and e~yll~s. It is also envisioned that a protein or a nucleic acid may
be dil~;lly added to the liquid phase.
Mixing of the genetic material with the liquid phase preferably
results in the previously des_~ibed solid-to-liquid weight ratios. These
20 ~ug~ --l~ liquid phases are mixed with the base salt colllbi.~alion to provide
'nl~Jllrll~ Ay~ali~ Sa~ es which are tested as desclibed previously. A
final ~11gn~ rd l~dro~y~ali~e form11~tion is sel~ct~l, which permits the
desired delivery rates. The ~vg.n~.l~ liquid phase is that which leads to this
fiDal a~ nt~d hydroAy~)alile testformn1ztion
In a third step, the ~ -lr~l liquid phase is miAed with the base
cc,lllbinalion of c~lr~ n pho~h-l~ salts. And, in a fourth step, the
bioco...pq~;ble l~dloAyapdtile forTn111~tit)n is ~l~,ci~i~d from this llliAIulC.4. PYt.onAed Or Controlled Delivery Of ~1.z. ".~ 1 Agents
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Traditional phs. "~Acelltir~l agents typically col~ll,lise small
ch~mir~l molecules that are cleared from the circulatory system relatively
rapidly. An ul~desilable con~e~ çnre of this trait is the resllltin~ need to
<j.l..,;,.;~l-, a colll~uulld Col~ the desired agent to the patient multiple times
5 over several days or weeks. Also, each .~ alion involves the use of a
large amount of the colll~uulld so that, after the initial dilution, enough
compound is present in the circulatory system to cause a desired th~lapculic
effect. Current approaches to address these problems have primarily involved
m.och~l-ir~l meth-)A~, e.~., continuous intravenous infusion or the use of
10 implantable pumps. An implantable ~ul~ ce that contains the desired agent
and degrades over dirr~ ll time periods, thereby relçAcing the agent and
biocq---l-AIih~e ions such as cAlri~m and ~.hos~ , would improve upon known
a~ oaclles. Among other advantages, such an a~ploach reduces the col~lJleAily
of conventirnAl A-~ alion of ph-.,.,~ elltir~l agents, which is more
15 cA~cnsive~ fraught with risks of illfeclion, and subject to ~luced patient
compliance. An ;..~ls..l~ble ~ul.~lA~ e accor~ g to this el~ odilllclll would also
lower the total amount of the ~JhA. ",Ar~.ll;r~l agent needed over the total course
of ll..,~ -l, and would avoid ~ulclllial side effects caused by high
co~cclltl~tions of the ph- "~ ;r~l agent following each ~l...;n;~l.àlion.
Acco-di~;ly, the fourth embodiment relates to a biocompatihle
hydro~ya~alilc formlll~tiQn that inco~o~atcs a ph~ "l;~al agent.
Preferably, the agent is a biocide select~d from the group con~i~ting of anti-
neoplastic agents, anti-b~ ;Al agents, and anti-p~silic agents. An anti-
neoplastic agent may he sel~clcd from the group co.~ l;..g of
cyclopho~l,h~-.-;Aec, aLkylati-lg agents, purine ,InAl~s, py~;"~;A;"P analogs, vinca
and vinca-like aLl~aloids, etoposides and etoposide-like drugs, antibiotics,
co-lico~t~.u-ds, l il~osuu eas, A..l;...rlAholites, plAtinllm-based cylolo~iC drugs,
h~ nAl antagonists, ~l~iesLIogens, tAmoxifen, doxorubicin, L-asparaginase,
.
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dacalbazil e, ;~l~acri~e, procall,azine, hexamethylmP1~min~ and mitoxantrone.
An anti-bacterial agent may colllplise a heavy metal, an antibiotic, or another
anti-b~lclial agent. The ~h~ eutical agent may also be an infl~ at~ly
agent, an analgesic, or a cl-~mothfl~c.l~ic substance. Other ph~ r,eutir~l
S agents envisioned are known to those having O~ al~ skill in the art. The
bioco...l atible hydro~y~atile fonn~ tion should display the .llpch~nir~l
~ropcllies of granules or a soft paste. The formulation should be capable of
~!~ci.~ 20% or less of the agent within 1 day and 90% or greater with about
30 days. More p~,f~,lably, the release rates are 20% or less within about 2 days10 and 90% or greater within about 10 days. The level of the agent preferably
col~ ises from about 10% to about 50% of the total dosage conventionally
p~esclil,ed for a full course of a sl,ecirc ~eOIlllPl~l
The bioco...~alillc hy~uxy~lile formulation accor~ing to the
fourth embodiment is pler~ably produced by the following steps. In a first step,15 a base cu~ inalion of c~l~;lm ~hos~e salts is ~r~dlcd by following a
procedure similar to that in the previous embo l;...~ .~t~.
In a second step, a liquid phase is ?Ug..~ ~11ed by adding a desired
ph~ csu~ l agent. This second step is preferably accomplished by the
following procedure. The desired agent is sel~cted from among the agents
20 already described. Then, one or more liquid phases, which are l,n pared as in previous embo~ , are ~ ~ nt~d by adding ~al,ying amounts of the
~,h~....~c~"~ l agent. Each of the angJ.~r~ 1 liquid phases then are l~,spe~;livcly
mixed with the base salt co~ alion preferably at a solid-to-liquid ratio as
previously described, to fo~ .~ for~ tiQn Test samples of the
25 rcspcctive allg..-~ ~l formlll~tio~ (having varying amounts of the
ph~ 1 compound) are implanted sub.;~ Pously~ i~ sclll~rly or
tla~lilon~ally into test ~nim~l~ and ch~act~ Lics such as serum levels of the
~h-....A~ ";.~al agent co~u-ld and resorption rate of the ~l~ed forrnlll~tion
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are llloriilored. A final augmented formulation is selected which displays
rh~ 1 properties of granules or a paste, which permits the desired release
rates of the agent, and which has a level of the agent of about 10% to about 50%of the total dosage ~;ull_nlly pl~sclil)ed for a full course of Ll~ The
5 q.1~gmlontrd liquid phase is that which leads to this final ~llgm~ ted test
formulation.
In a third step, the base colllbh~tion of cq1ri~m phosphate salts
is mixed with the IqllgmPntPd liquid phase. And, in a fourth step, the
biocc---.p~1il,1e hy~O~ydl)aliL~ form111qtion is pl~cil,iL~led from the ll~Ll~lul'e of the
10 third step.
Accor~ing to one example of this embodim~ont, antibiotics and
anti-infl~.n...~1c,ly collll)ounds are il~col~o~aLed into a hydro~yal,dliLe
formlllqtion, whcl~,in a final a11gJ..~ d formlllqtion is selected that provides a
suitable dosage for about 7 to about 14 days. In another exarnple, a
15 ch~n~ a~c;ulic s~b~ re (or a colllbil alion thereof with other s11bsl;.nr~s) is
incorporated into a hydlo~yapalil~ formulation to produce an ,q~ngrnpntlod
form111qtion capable of delivering the chcllw~ la~ulic s11bstqnre over a period
of about 20 to about 30 days.
5. FYtPntled Or Controlled Delivery Of Holll~l~es
Small peptide ho. ",,~I~r.~ or their derivatives are believed to exert
their physiological action on specific target cells by binding to specific
mem~lalle ror~plo. ~ on such cells. As in the case of small çk~ 1 molecules
that are hlcolpoldted in coll~enlional ph~ re~lirql agents, the small peptides
have only a limited circlll ~tin~ half-life. This cl~a~ islic can be si~nifirantly
i~u~loved by h~collJoraling such molecules in a biodegradable hydro~ya~alile
material.
Thus, according to a fifth embodiment of the present hl~e.,lion,
a bioc~ ;blP ~ y~ali~ fonnlllqtion is provided as a delivery vehicle for
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holuwlles inclu~ing peptide hûl~lulRs and hormone-like agents. Preferably, the
fonnul~tion comprises a hormone or peptide factor such as a regulatory-type
hormone S~PI~CtPd from the group COl~ ing of insulin, atrial naLliu,~tic factor(ANF), c~lcit~nin, ~aSOpl i,sin, relaxin and the like. The hormone may also be
S a sex hormone selected from the group co~ ;n~ of estrogenic hormones,
progestational ho-~-oll~s, al~lrog~ c holmolles and any active delivati~/e of
these. The form~lqtion should display ~,Pl~ha.~ l plu~llies of a paste suitable
for SUbw~ f'-OuS pl~~emPnt and pel~;uL~eous application. Preferably, the
formlllq-tion is capable of delivering the hormone at a rate of about 20% or less
10 in about 1 day and about 90% or greater in about 30 days. More preferably, the
delivery rates are 20% or less in about 2 days and 90% or greater in about 7
days. The corlcf-lltlalion of the active material in the formulation is preferably
from about 10% to about 50% (per cubic C~ f~l of the ~l...;..i.~te.cd
formlll~qtior~) of the coll~f-lllional l~cn---..-- n~led cl~m~ qtive dosage for a
15 conventional L~ .l period of about S to about 30 days.
The bioco...p~lible hydro~yal,alil~ fonm~l~tion may be
accomplished by the following steps. In a first step, a base cc.nlbinalion of
calcium pho~ e salts is l,l.,pared preferably by following the procedure
described in the fourth embo~im~nt (i~, the embodiment illcol~olaling a
20 ph-.l..q~ ~..l;r~l agent) or steps similar thereto. In a second step, a liquid phase,
such as that des.ilil)ed in the previous embo~l;...P ~!i, is qu~ ~1 by adding the
desired horrnonP (or peptide factor or hol,llolle-like agent). This is preferably
accomplished by following the plùcelult describe in the fourth embo~1im~n
In a third step, the base salt c~n~ ion is n~ixed with the au ~ r~l liquid
25 phase. And, in a fourth step, the bioc~ l al ;hle hydlo~ya?alile formlll qtion is
pleci~iL~Led from the ll~i~Lul'e of the third step.
- 6. Bone R~pl~q~ . Repair and Re~n~lalion
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Bone is unique in its ability to fully legellelaLe among the
complex tissues in higher ol~a~ such as velleblalcs. Due to the complex
nature of bone tissue and the varied functions pelrolllled by the skeleton at
dirr.,renl locations within the body, dirr.,l~lll approaches for hllcl vclllion may
5 provide greater Ill~,,a~culic b~ f fil~ in dirr~ inl locations. The respective roles
played by the cellular compollf nL, the matrix component, and the interaction
bcl~n these two phases of bone, colltlil,.lle to the ~gell~,.alive process and the
functioning of the tissue. Certain plolcills that are unique to bone have been
i.l- ., iriP~l with respect to their ~lluc~al and mineral binding propellies. Thus,
10 a sixth embo(limpnt of the present invention relates to bone replqrernf nt repair
and ,egel~.alion.
One aspect of this embodiment deals with osteoconductive
h~ les. A biocc~ ihle hydro~y~alilc form~llqtion which leples~ the
major mineral colllpo~enl of bone in the plesence of the major structural and
15 mineral binding plOt~5 found in bone, may be provided which has utility as a
filler of bone voids with a matrix resembling that of normal bone. Such a
form~ q-tion is c~ ' ;l .lc with bone . nd capable of u~ ;uing resGl~lion to be
replaced by new bone that ren ~lelc along natural ~rocesses. This novel
formlllqtion is useful in filling various ~ g~1 sites and is also useful in filling
20 gaps b,lw~n ~lo~ ir devices and sullu~.Ai..g bony tissue, thus providing a
better ~ mpnt for orthopedic, periodontal and dental applications. The
formlll~tion is also useful as an e~rtton~llor of autografts or allografts where the
........ ~I'i normally available are not sufficient.
Thus, according to this aspect, a bioco...~alible hydro~y~alile
25 formnl~tion is provided which is capable of su~pol~ g bone growth.
Preferably, a formlll~tion accoldhlg to this aspect co.~ one or more adhesive
agents. The adhesive agent may be chosen from the group consi~ g of
hlt~glil,s, extracellular matrix protcills~ leukocyte adhesion plote,ns, collagen,
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albumins, bone ~rul~i,ls, osteol.ectins, cell surface receptor ~)roteins, bone gla
protein, and rnatrix gla protein. The bioco...~ hle hydroxyapatite formulation
should display mPc~laniral ~iop~llies of a paste that hardens into an implant.
The implant ~le~lably has a co.llyl~ssi~e strength greater than 10 MPa, which
S is more preferably greater than about 50 MPa, and most preferably greater thanabout 100 MPa. The implant should be about 45 % porous with an average pore
size of from about 15 ,l,i~;rons to about 30 ~icrolls. More preferably the
average pore size is from about 20 llliClOl~ to about 25 lllicr~ns. Most
preferably, the average pore size equals about 23 lllicrolls. The mqximllm pore
10 size should be less than 100 microns, preferably less than 50 microns. The
formlll-qtion is preferably resorbable within about 60 days to about 2 years.
More pl~f~l~.bly, the formlllqtion is resorbable within about 60 to about 90 days.
The amount of the osteogenic protein should be from about 10% to about 40%
of the total weight of the fnrmlllqtion, and the relative amount of the proteil,s to
15 each other (if more than one is incorporated) should be similar to that found in
the bony tissues at the site of application. The biocomp-q-tible hydroxyapatite
formlllqtion may ad~1itionqlly contain (a) an antibiotic s~lbst-qnre that is locally
ele~cd from the formlll-qtiQn during the repair process and pl'~:vellls infections
that could hinder regell. ~aliO"; or (b) a holmoll~, e.g., calcitonin, that may be
20 locally released during the repair process to inhibit bone loss due to an
~d~llyi~ metabolic disease such as osteoporosis.
The q~vg~ ..led hydloxyal)alile formlllqtion of this aspect of the
sixth embo~lim~nt is preferably formed accofdil,g to the following method. In
a first step, a base colllbi~d1ion of c-q-lri-lm phos~hate salts is ple~ d. This25 may be accomplished accor~ing to a procedure similar to that of previous
emb~;..~
In a second step, a liquid phase is ~ ~g~"l ~ecl by adding an
osteogenic adhesive-type protein. This may be accoll,~lished by the following
-
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procedure. Firstl one or more ~roteils are selected from those already
described. For P~A...l~le, human bone gla protein, human matrix gla protein and
human osL~o,~liu may be produced by ,econ.~ means and ~irled. Then,
a liquid phase is formed as described previously. The liquid phase may be
5 supplf mf~n~~~ (or additionally sllrplf~ ~llf '1'~) with granular sugar, such that upon
mixing with the base salt colll~ ation the final formulation will contain up to
about 20~ by weight of sugar. The select~d ploleills are then ullirollllly
dissolved in the supplf mf nte~ liquid phase to form an ~11g1.~ t d liquid phase.
In a third step, the base salt co"lbi,lalion is mixed with the ~llg1nf ntf d liquid
10 phase. In a fourth step, the biocolll~alil)le hydro~yd~dlile formulation is
plt~ iL~led from the Il~uL~ule of the third step.
The paste thus formed is preferably allowed to harden under
p~i~ule (~,~, from about 40,000 psi to about 80,000 psi)in vitro in desirable
mold conro....A~;ol-~ for a period of about 12 hours to about 48 hours. The
15 hardened for~ ti-)n is then subjected to llC~III.f.~l with warm water at
telllpc.àlul~,s in the range from about 50~C to about 70~C, more ~rer~,.dbly
about 50~-55~C. This L1~A1~....t should last for about 4 to about lO hours, morepreferably from 4 to 5 hours, to remove most of the granular sugar, thus
Cl~,alillg a highly porous material. The mqtf ri~l may then be implanted into a
20 body, for e~A..1l~le, at a selçcted bone void.
Another aspect of the sixth embodiment deals with osteoin(l~lctir~n.
Hy~lO~a~)aliLe ;~lp~ i, inrl~ i~ the porous forms, f~e~u~ y allow iu~ lowLIl
of newly developed bony tissue at bone defect sites. As a result, the
hydro~y~aLile imrl~nt is incorporated into the newly formed bone which
25 remo~lç1~ naturally. A hydro~yapali~ implant that can actively effect the
dirr~ Lialion process that causes the plu~ otellt stem cells at the site of a
trauma to follow the path of bone folllldlion - "osteogenesis" - will provide
iu~loved Lll~,la~eulic ~n~rlL~. A group of factors, generally called osteogenic
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factors, bone morphogenic protein, or chondrogenic factors, have been shown
to m~rliq~e such lJhel1o",~non in ~nim~lc and h.ln.;.-~c,
Thus, this aspect of the sixth embodiment of the present invention
provides a bioco...p~ihle hydro~y~paliLe form~ tion, which may cause active
5 hlgl~JWIll of bone into implants. Preferably, the formulation is capable of
canCing in~ ction of new bone formation and co,ll~,ises one or more growth
factors sel~ct~d from the group CO~ -g of osteogenic factors, bone
morpho~rlir, pr~t~s, and chol~oge~c factors. These growth factors may be
inrlllded either alone or in COl~illaliOll with one or more adhesive agents, which
10 may be se1ected from the group concicl;~-_ of integrins, extracellular matrix ~ol~h~s, leukocyte ~hPcion prol~hls, collagen, albumins, bone ~role~s,
o~t~u.~ -c, cell surface rc~ptol ploleins, bone gla protein, and matrix gla
protein. The formulation should display ~ h~nir~ lies of a paste that
hardens into an implant having a tensile s~ of at least 20 MPa. More
15 preferably, the tensile sll~ lh is greater than 60 MPa, and most preferably
greater than about 70MPa. The fonnlll~tion is preferably porous with average
and .~.zx;.. pore sizes as described above. The level of the active growth
factor should be in the range of from about 10 ~g to about 100 ~g for every
cubic c~n~ Pte~ of a delivery vehicle Co~ ,g the sele~ted growth factor
20 alone, or of a delivery vehicle co.~ p up to about 40 wt% of the one or more
adhesive agents.
The final formlll~tion may ~ itionqlly contain a h~lase.
This term is genf~ ly j"t .~ to mean an enzyme that either degrades heparin
sulphate or causes the release of molecules in vivo that bind to heparin or
25 heparin slllph~te. The final formulation may also contain an antibiotic
Sl~ nrl~.
The fonmll~tion of this aspect of the sixth embo~limPnt may be
p,~ ~ acco,~i~g to the following m~th~xl. In a first step, a base combination
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36
of ~p--l ;n~ly soluble c~ m phosphate salts is ~rcpdrcd as before. In a second
step, a liquid phase is all m~nt~ by adding the al,plo~liate one or more growth
factors and the desired adhesive agents. Various suitable factors, including those
already mentioned as examples, have been described in the art along with
5 reco~bi~l means of production either in ~e,~f I irally Pnginpered ".z."",~ n
cells or microbial host cells. In a third step, the ~-g"~ l liquid phase is
mixed with the base salt colll~h~alion. Preferably, this l~ lw~, has a solid-to-liquid ratio as described previously. In a fourth step, the biocompatible
hydro~y~alilc fonnlll~tion is pleci~ilated from the mi~lulc of the third step.
10 The formulation preferably has the above described characteristics. The finalform~ tio~ may be hdr~ll~d ex vivo or rapidly in vivo when applied in an open
rla~;lw~ as an implant or a gl~JUlillg material in co"j~ l;on with other implants.
Still another aspect of the sixth embodiment focuses on bone
lcgelle.alion. Bone defects at dirr~,lelll skeletal sites may require dirr~ t
15 b~l~nrps beLwccl, bone l~gell~lalion and short term bi~ lprll~nir-al support. The
fo~ tions described in the previous aspect of the sixth embodiment can
provide modest ...~ ha.~ al support and allow efficient bone iLIglCWlll such that
the implant is rapidly hlcOlpolalcd into newly formed bone for long term
mPch-~ir~l support. Yet, these fonn~ tions also provide reasonable stress
20 bearing capability within a short tine pOst~ alivcly by virtue of their
bi~~lP~ha~ alS~c~l. In other appl;ra~ ;nn~, where short term load bearing is
not an illpo~L~-l factor (~, spinal fixation, radial and ulnar non-union
rla.;Lule, and the like), there is a need to be able to cause rapid fc,ll"alion of new
bone for long term benefit with short term support being provided by other
25 fLlcation devices.
Thus, according to this aspect of the sixth embodiment of the
present i"v~"lion, a bioco..~ ble l~dro~yal)alilc form~ tion is provided which
is capâble of actively in.l.Jc-i.,p. bone repair in small defects. The form~ tion
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preferably comprises a growth factor se!PctP-d from the group conci~ting of
osteogenic factors, bone morphogenic pro~ins, and chondrogenic factors. The
form~ qtiQn may also col~ ise one or more adhesive agents, which may be
selected from the group con~;~l;,lg of illl~glins, extracellular matrix ploleills,
5 leukocyte adhesion prol~ins, collagen, albumins, bone p~oteins, osteonectins,
cell surface receptor lJiot~ s, bone gla protein, and matrix gla protein. The
formlllqtion should be in the form of a soft paste or slurry that can be applied~ ;u~leously. ~c~lably, the formlllqtioll is char~t~li7~ by the lcsoll~&bilitydescribed in co.~l~fc1ion with the previous aspects and preferably conlains
10 ~lwt;ell about 50 ~g and about 500 ,ug of the selectPd growth factor for every
cubic c~ t~ of the fornllllqtion If illcol~ulaled, the adhesive agent should
be at a level of up to 40 wt% of the final formulation.
The formlllqtion additionally may contain he~a~se, which is
gel~ ically ;..~ A to mean an enzyme that either degrades heparin slllph-qt~ or
15 in-lur,es the release of molecules in vivo which bind to heparin or heparin
slllph~te. The formlllqtion may additionally contain an antibiotic substance
Compared to t_e formulation described in the previous aspects of this
embo~limPnt, this formlllqtion is not intpn~led to have signifirqnt "~rrhAnira
sL~ and is ;.~t. .~1~ to provide a more rapid, local delivery of growth factors
20 for a shorter duration.
The formlllqtion of this aspect of the sixth embodiment may be
pl~.,d accor~illg to the following m~th~d In a first step, a base cc.lllb~ation
of sl,~ingly soluble c-q-lrillm ph~.h~t~ salts is plepaled as before. In a second
step, a liquid phase is ~ -1 by adding the a~rop~ one or more grow~
25 factors and the desired a&esive agents. In a third step, the nugm~ntç~l liquid
phase is mixed with the base salt combin ion. In a fourth step, the
bioco---l- 1;l.1e ~O~apalit~, fnrmlll~tinn is pl~ it.~d from the ll~i~lur~ of the
third step. The forrnlll~tinn preferably _as the above~ ;..nPd Ch~aCLIis~ics
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38
Also, this ~ lule should have a solid-to-liquid ratio as previously described.
P~,f~,lably, the final fnrm~ tio~ is applied pel~;u~ eously and hardens in vivo.Examples of this and the previous aspects of this embodiment
include: (a) a form~ tinn that hlcc.l~âles a hom~im~r of BMP-2 produced in
5 Chinese h-...ct~, ovary (CHO) cells; (b) a formulation that uses molecules
design~tecl COP5 or COP7 or Vgl produced in E coli cells; and (c) a
form~ tion that incorporates a heterodimer of BMP~ and BMP-5 produced in
a g~l;.. t;r~lly engi.. ~ ~red .. -.. ~li~n host cell.
7 F.nh~nrf~ r.~l Of Electrical Or Ele~ u~ gnrtir Stimlll~tiQn Of Bone
10 Growth
A seventh embodiment of the present invention relates to the
el-h~n~e~..r..l of osteogenesis around and into hydro~yapàLile implants in
.,sponse to cle~;L,u~ lir stimlll~tion. ElecLlull~gnrtir stimlll~tiQn has been
reported to be ~rreclive in stimlll~tin~ bone healing and bone inglowlll into
15 l~dro~ya~alile i~ t~ when the ;"~ "l~ have an average pore size preferably
in the range of from about 15 microns to about 30 microns (more preferably
~Iwwn 20 microns and 25 llliclons; most preferably equal to about 23
~lClOnS). The incorporation of pa~ gn~tir, ~ magn~tir, conductive, and
inclll~ting material into an implant can hl~l siry or Z~ ; the electrical and
20 ",~g".1;r fields proximal to the implant when it is subjected to ele~ u-~l~n~tir
stimlll~tion This illll)loved formulatiûn can h~ edse the rate of bone healing
and reduce the field h~ sily required for elecll~,...~nrtir stimnl~tion. This
embodiment of the present invention relates to an ~llg... ~.l~ hydlu~yapàli~
formnl~inn with altered ma~r~tir and con~lllrtive p~ ~ies which enh~nre the
25 effects of ele~ o...agn~tir field-in~l~lr~cl bone repair and bone follllalion.
Thus, the seventh embodiment of the present invention provides
a biocompatible l~oAya~dli~ formlll~ti-)n, which hlcOl~olates an cle~tlical
stimlllllc ~..h~n~.. The stimlllllc enh~l-re. may be one or more of a
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39
parqm,qgn-PtiC material, a ~liqma~nPtic material, a conductive material, or an
in~llqtor. The l,~;..n~nPtir material may be selecteA. from the roup consi~ling
of iron, iron ammonium alum, UlaniWll, pl,qtim-m, and al~ . The
diqmagnPtic material may be selçctP(l from the group co..~ g of bislllulll,
5 lllel-;wy, silver, carbon (diamond), lead, and copper. The conductive materialmay be selçcted from the group CO~ of silver, copper, ql.~,..;,.~.." and
And, the in~lllqtor may be selPrtP~ from the group co~ of glass,
lucite, mica, quartz, and poly~L,~fluoroe~ylene (PI~:E). Preferably, the
f~rmll1qtion is capable of causing new bone illgl~w~l and l,lolllo~illg os~ogenesis
10 in the tissue ~ulloullding the il~l~ll. The formlllqtion should display
mPchqnirql plo~,lies of a paste that hald~ns into an illlpl~l having a tensile
SL~ greater than 20 MPa (more preferably greater than 60 MPa; most
preferably greater than 70 MPa). The formll1qtion should be porous with
average and mq~timllm pore sizes as described above. The formulation
15 preferably has the resorbability already desclibed (i.~, 60 days to 2 years;
~f~ldbly 60-90 days).
The l~l~'o~yà~ali~ formulation of the seventh embodiment may
be ~l~dlCd accordillg to the following mPth~ In a first step, a base
combina~ of ~la~ ~ Iy soluble c~lrillm phos~ e salts is ~ d as before.
20 In a second step, a liquid phase is :ug~ n~eA by adding one or more
eleetlu...agn~tir, e-~h~.r~ls select~A from those already described. In the cases
where the _aterial is toxic, it can be enc~ed in glass or ErrFE. In a third step,
the qvgn.~ 11PCl liquid phase is mixed with the base salt colllbma~ioll. And, in a
fourth step, the biocon.p~ih~ ydlo~LyalJali~ formlll~tion is ~l~,ci~i~ttd from
25 the ~lw~, of the third step.
The hydro~yapaLi~ formlll~tion may he hardened in vitro and
used as a bone repl~e~ In a l~ t example, an implant formed from
the for~mllqtion may be implanted in a human hody. Next, the implant is
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stim~ electrically or mAgn~AtirAlly to induce bone growth within and around
the implant.
8. F.nh~......... re,~ t Of Electrical Or ElecL,v".~gn~tic Pulse In Local Release
Of Materials
5Several embo~limPnt~ of this invention generally relate to the use
of a hydroxydpalile formulation for the delivery of a bioreactive substance.
Application of an elecL,n..~n~tic field can stiml-l~te bone and tissue growth and
vascularization in and around a hydro~y~d~ implant. ElecLlolllAgn~tically
stim~lAtin~ a hydro~yapatiL~ implant or im~l~.l~d h~dlv~ydpali~ formulation
10 can also regulate the delivery of bioreactive subs~lces. Electric fields havebeen shown to cause desol~lioll of factors from a matrix which is exposed to thefield. In ~ ition to causing direct release, elecllv...agn~ti~ stim~ tion will also
stim--l~te tissue lnglowlll into the implant, thus increasing contact ~lweell the
bioreactive ~u~s~lce and the tissue.
15Thus, the eighth embodiment of the present invention relates to
a biocolll~aLible hydro~Ly~dLile formulation which is subjected to electrical orelecLl. ..~n~ti~ stim~lAtion after being inserted into a human or animal. The
hydro~al~dtile forml~l~tion is preferably plepale~ accoldillg to the following
m~ thod. In a first step, a base colll~ ion of calcium phosphate salts is
20 lJl~af~ as before. In a second step, a liquid phase is 2~ 1llr~l~ by adding one
or more bioreactive subsL~ces. The biol~,a~;liv~ s~bstAnce can be any of the
bi~ additives ~l~sc~ herein. In a third step, the All~ 'nled Iiquid
phase is mixed with the base salt colllbh,àlion. In a fourth step, the
biocompatible hydro~-yal)aLile forml~l~ti- n is preei~ a~d from ~e ,~Iu,e of ~e
25 third step. The p~cil~i~lion can be in vivo. Alternately, the pl.,cil.iL~lion can
be ç~ vivo. Accor~ing to this Alt~ 1 iv~, an a~ itionAl step is required in which
the pl~i~iLdted l~dloAyd~aLiLe f~ tion is implanted into a human or animal.
In a fifth step, the imp1Ant is stimlllAt~d electrically or ele~;Llo,..~gn~tir~11y to
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induce bone and tissue growth and vascularization within and around the
hydroAya~dliLG implant and to induce release of the bioreactive ~u~s~lce from
the hydro~ya~alile formnlqtion
9. Multico~ ollGlll, Layered Devices For Dual Tissue Interfacing
The use of implantable devices in guided tissue legellG~alion
processes would be signifir-q-ntly more ~llGficial if such devices could be
col~llu~;lGd to allow one surface to be compatible with one tissue type while
another surface is co~ )alible with a second tissue type. In periodontal defects,
for çYqmplf., the devices hllelrace with both the soft tissue of the gum and theulld llyil~ bony :illllCIUlG. Similarly, in articular joints such as hip, knee, elbow
and alilde joints, where .1.f ~ toid a,lhlilis or osteoa~ ilis has caused cartilage
and bone ~lqmage, a desirable device should have one surface colll~d~il,le with
bone and allulh~l colllpàli~le with cartilage. It will be readily appa~fll~ to one
of ordin~y skill that other examples could be easily iclentifif d with application
throughout the human body, as well as ,qnimql~.
Thus, the ninth embo~imf nt, which may be used for such dual
tissue interface applicalions, provides a layered device having a first layer
Colllplisillg a ~ylllllclic polymer (~.~, a glycolide/lactide/acrylic material
sPlectfd for c~ p~ ility with soft tissues or cartilage) and a second layer of a20 biOc~r.alil,lc l~ ya~alilc formlllqtion which is colllyalible with bone. The
second layer may be created by the application of â paste formed from â
spalill~;ly calri~lm ph-)sphqte salt ll~ixlulc, such as those previously described.
This embodiment further contemplates the illcolyolalion of one class of
l.h~ tirqlly active or biocc,~ alible material in the first layer to fa~ilit~te
the r~ge~flàlioll of soft tissue or cartilage while the hydro~yapalilc layer
iulCOl~ulat~S one or more biologically active or bioco...~ ;hlf s..lL,sl ~ s, which
facilitate bone repair as described in previous e lllbo~l;...f .~
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42
A si~lifir.~nt advantage of the layered devices of this embodiment
is that biologically or ph~ lly active sub~ces that negate the
formation of selected tissues may be incorporated into one layer while other
~b~ nres which ellcoulage such formation can be incol~olal~d into the other
5 layer. This allows clear dP!inP~tion of the dual tissue interface created in the
guided tissue l~gellelalioll process. For ~x~ll~le, in the case of articular joints,
the ~ylllllc;lic polymer layer that is made to be compatible with cartilage may
contain an anti-angiogenic factor to prevent migration of bone tissue into this
layer from the bony tissue being legel~cla~d at the hydroxyapatite layer.
10 Accordillg to an example, the ~yllll~Lic polymer layer is illll)legnàled with Type
II collagen and hyaluronic acid to allow better cartilage co"-l,a~ibility, while the
hydroxyal,ali~ layer is hll~)leg~ P-d with Type I collagen, bone gla protein,
osteonectin and the like, for the facilitation of bone l~ge.~.alion.
10. Other rcalul.,s and Aspects
The embo~lim~Pntc thus desclibed are merely int~n-led as examples
of the present invention, which is not limited thereto. For example, as
previously ~ s~l many of the embo(1;...-P~t~ described herein involve a
process whelelll a liquid phase is ,~ m,ntPd with a bioc~)."p~lible additive.
Then, the ~u~nPn~-d liquid phase is mixed with a base coll~h~ation of calcium
20 phosph-tP salts. However, it is envisioned that the col~ollel~ts of the ln~lul~
which ~re.,i~ les hydroxyapalil~, can be co~illed in dirr~l~,nl orders or
.cim~ P(Jusly. Also, ~1th~-gh various release rates are desclil,cd, those havingordh~aly skill will recognize that the CO111~O1Y~nIS of the hydroxy~a~ile
form~ -ti- ns may be marupulated to achieve dirr~.cllt release rates which may
25 be more suitable for a given application.
Accor.li~g to a feature of the various emho~lim-ntc of the present
invention, the colll~ull~ s of the ~ixlule which pl~cil,it~s a biocom~ le
hydro~patite formlll~ti~n may be provided in a kit form. For exarnple, a
.
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43
package co~ ises a kit having t_ree vials. A first vial preferably contains a
.nca~uled amount of the base c~ m l~h~sph~le salt culllbhlalion. A second vial
preferably contains the selected additive, which may be any of the additives
already described (~.g." a growth factor). Depel~ding upon the additive and
5 desired ll~ criteria, the additive may be in any of several forms. For
el~mple, a growth factor might be provided in a lyophilized state. The third
vial colllaills the liquid phase. Alternately, the kit could contain two vials. In
a first t~,vo-vial options, one vial would contain the calcium phosphate salts and
the other would contain a ll~i~lul~ of the liquid phase and the additive. In a
10 second two-vial option, one vial contains a co,llbilld~ion of the additive and the
salts, w_ile the other vial colllalns the liquid phase.
Accordi~g to an aspect of the kit feature, the colll~ of the vials
_ay be con~ d to produce a paste of the bioco...l.alihle hydro~yal~a~
m~t~n~l. The resllltin~ paste may be n~n;.. ~t~ r~d by itself in a surgical site or
15 may be used to ~ugment sutures, staples, lll~,lll~lal~S (resorbable or
ol~e;,oll,able) or the like that are in wides~lead use for wound stabilization and
closure. The paste may also be used to ~ m.ont artificial skin and n~enl~al~e
used to cover major burn woullds.
ACC(Jr~1~ to ano~ . feature of the previous embo~ , pre-
20 i~ u~ed halde~d sheets of a precipildt~d bioco...l)~lihle hydro~yd~aLilforml~l~tion incorporating an additive may be provided in sterile form
Preferably, these sheets are capable of being cut into desired shapes and applied
to cover wound sites or to pack deep wuul~ds. Also, the sheets thus applied may
be stabilized using Imown surgical closure techniques. Various pluce~ses of
25 pr~ the sheets or similar ~ ,s are co..lr~..plated in this embo~im~n
The embo~1;...r..l~i described above may also incorporate an
antibiotic substance that ple~ellls infection of the ~llg~ 1~ hydro~a~dti
l~,lial. The antibiotic ~ub~l~ce may also be released at the wound site in
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44
order to prevent infection of the wound site while the material is in place. Some
of the types of antibiotics which may be used include, without limitation,
aminoglycosides, ~ )h~.-icols (~,~, chlo~ henicol), ~-~ qrtqm~ antibiotics,
penicillins (~,g,, ampicillin), peptide antibiotics, and tetracycline antibiotics
5 (~ cllacycline).
According to another feature, two or more formulations are
in-l~pçnrl~ntly pl~paled. For example, an implant may be plepalcd ex vivo by
mixing the salts, additives and liquid phase as described above to produce a
paste. The paste may be allowed to harden into a component which is more
10 slowly resorbable than a sepalalcly plcpaled formulation. This paste may be
extruded through an applul)lidlc d~llulc to produce granules of such a size that
may be applied through a large gauge hypodermic needle or other acceptable
injector ~.r~hA";,~n. A second formlllqtion may be freshly pl~aled at the time
of arl~;n;~llalion (~,~, by adding a liquid phase co..li~;n;..g approximately one
15 third of the total inl~ led dosage of the additive to the base salt colll~illation at
a ratio that pçùduces a softer paste, which can harden i~iL into a more rapidly
biodegradable implant. ~d~qllqtP An~O~ of the granules can be mixed
unirullllly with the base salt col~h~lion prior to adding the ~qll~n~nt~ liquid
phase, such that the granules co~lliblllc about three times as much of the
20 ;.n..n...-g~n as co.~ u~d by the softer paste. Such a system colll~ g a paste
with ~mix~l granules and a freshly pl~al.,d paste provides a two-colllpollel
system W~ the freshly ~lc,pal~,d paste l~leases a first dose of the selecte~
additive relatively rapidly and the ~ .e of paste and granules delivers
booster ~....,~..l~ more slowly over an ç~le~ d time period. According to an
25 aspect of this feature, a layered composite may be produced wh~.eu~ the central
core co.~ ;..c a more slowly l~,soll,able fonmllqtion that is ~ùulvul~ded by a more
rapidly resorbable formlllqtion
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According to still other fealllres of these emboAim~ntc~ the
form~ tion may be a~ el~d ~L~ ..cclll~rly, intravenously, ~ u~sly
or p~l~;u~leously (de~llding on the desired target tissue) where the additive
~,~, DNA) is to be delivered. The solid-to-liquid ratio and the amounts of
5 supplements recited herein for the liquid phase are selected based on results of
animal evaluations where various test formlll~tionc are ~ l.llin;~ ed, and the
release of the additive and the biodegradation of hydroxyapatite are monitored.
It will be recognized by those having oldil~aly skill in the
~cllimlll art that other variations and moAifir~tions to the specific e~mples
10 disclosed herein can be easily ~c~-,m~lished without departing from the scopeand spirit of the present hl~lllion. Accordingly, the present invention is only
limited by the following claims.
