CA2195957C - Biocompatible hydrogel - Google Patents

Abstract

A BIOCOMPATIBLE HYDROGEL. is provided for use in the treatment of humans for cosmetic and functional defects (e.g. in mammas, vocal cords, penis etc. as endoprostheses). in the provision of intratissue storage sites the prolonged-action medicinal preparations, in various applications as electroconductive immersion media, and in the life-long tamponing of caverns. Its contains an acrylamide-based polymer produced in the presence of an initiator of radical polymerization in apyrogenic water as the dispersion medium. An increase in elasticity, shape retention capability, and stability of bulky implants, as well as corresponding therapeutic and cosmetic efficacy, mainly in the endoprosthetic applications is achieved due to the hydrogel containing cross-linked polyacrylamide produced by using a biocompatible cross-linking agent, such as methylene-bis-acrylamide, and preferably a mixture of ammonium persulfate and tetramethylenediamine as the initiator of polymerzation. A preferred concentration of the novel polymer in the hydrogel is from 3.5 to 9% by mass.

Description

B IOCOMi'AT IBL~: HYDROGEL
t:ield of invention The invention rel::ztes to fr)rmrsl~~tions of biocompatible 05 hydrogels for rnedic<~l apio ic;at ~.car~s and which ~~an be used:
in endaprasthesis laract:ic~,~ kay way of' purposeful injections to remedy prveferably Chase defects in humans which are due to traumatic, congerti Cal or agF~ distorsions of the shape and dimensaonv~ ar due to lass of form stability of ~>ame organs consisting of soft t:is~~ues, e.g.:
- plastic surwgervy far' c_~orrectlng the farm and dimensian:.s of tile. face acrd other par°ts of the bady and, :.~pecifically> mamniop ~~.st;y (preferably in the case of marnmary apiasia or hypamastirz) , - otolaryngology pract~ic:cra for the treatment of the larynx fey cc:~rr°f~~t:irig tt~f~ t~c~cal cord shape and dimensions.
- male serology ( in tire cases of feeble erection) for improving paterrcy thr ough inject,in~; an elastic medium into span~;y vascular tissue of the penis;
in endoprosthesis prar;tic;e combined wi th skin surgery, by premolding er~dopro;~theses intermediate products and implantating of the :.game unto the operation si te;
in long-term therapy ~e.g., abscesses or tumor) to provide an internal :;forage =>itfe far a prolonged-action drug both inside or' near tin a of f~~c: fed organ:
far filling c=~.vitic>s resultirug from various diseases (e. g., tuberculosis caverns) or icnjuries:
as a physiologically roeut~ral electraconducting immersion medirzm between the patv.ent':~ skin anti electrodes i n the case:) o f - long--term moni Coring of tale systemic electrophysi.ologic var:iablE~s (e. g. , cardiac and cerebral activity>, - percutaneous elei;t~rophoret:,ic drug injections;
a:> a base far° prirnan°il.y me:~c:iic~~irual aintrnents, water being a disper;aion mE;din~r~a.
Demands fcor° an irnf>r°avemerrt in bodily shape and ~

N..
functioning as mentioned abovc.~ and other the similar cases have become widespread and i s I'r°ecluen t 1 y reap.>oned by the mere patiF~nt's vesir~~. Aksc~ widespread are demands for electrophysialogica~ diagno~tic~s,> drug tre<~trn~.ent;, including 05 drug storage :sites, arnci ccFnserv<~tive plty:>iolagically efficient tarnponir~g of various ca.vernrs.
That is why t~ioi;arnpar,ibka m~.aterials i'or the above mentioned applications :~h~:~l.slcl satisfy varying requirements. Among true noost important requirements are long-term (preferably 1 a t~~-lcang) retention of the shape and dimension; of ari or~;~r~ wtu~re cvclopr°osthe:>is has been p 1 aced i rr°ta;~pec (; i v a o f tkue age the pa t i en t was operated;
the maximum possit;le biocampatibility, i. e. absence of carcinogenicity, allergic; roactiorn~ (including short-term apes imrnediately t'allowiry; the injection of :>eiected material into the body or" applic~:~tic~n thereof on the skin and, particularly, mucosa.l membr<:~nr:) , absence of hard capsules or the rejectic~rl ~' f ~~r.rc~c~pr°o:~these s, or drug ' a storage sites, to mention I~ut a f'ow, provision of unhampered rnetabol is prac~:rses ra', ~~k~e si to of ~~lacement of a biocompatible material;
minimal trauma occurvance ~~rul the short~:esi~ possible introduction of a bic-cornpatible material, :specially in large-dosage (up to 1000 ml ) ap~~n u;at~.ons Thus, glicerol--based fluoroplastic (teflon) paste as a ~0 biocompatible material i"or t,reatrrrent of the larynx produces a considerably stable clinical effect (see: Beck Ch. L.
Unsere Erfahrungen mi t cler irrtt°alaryngealon Tef'loninjektion // Laryngol. ktninol. i_~tr~l. - 1!80. -- f3d. 5~i, _. Na 11. -S. ?15 '71g; Lewy t~. B. 'fef can irr;jr:c,tion of the vocal cord:
Complications, errors <~rrr~:k prec<_~utiorrs ;'/ Ann.Otol. -- 1983, V. 5~> No 5, pp. 1, 4'I,3--41'u'~: her"grraru:~ A. ~erfahren cur Unterf i tterung von Stimrr~l i pperu : i H. rJ. U. i 98 ~' , Bd. 3 >, No 6, S. X27-3) .

~~-Ulicerol is known to have higher viscosity than water, therefore, the paste proves to be rather stable in storage. But when injected, glicerol is an effective lubricant.
Being readily soluble in water and aqueous fluids found in an organism, glicerol, however, is quite readily (a few hours to one day) removed from the operation site.
When teflon microparticles are used as implants, it is not impossible that teflon microparticles are progressively removed by the lymph- and blood flow. Such removal results in ~ decrease in the prosthesis volume and in substantially low therapeutic efficacy. Therefore, though teflon is biochemically inactive, operations t~:~ place endoprostheses made of teflon must be repeated at clinically ascertained intervals.
Also, teflon particles, being hard, me~;har~i~ally damage the tissues in contact with the endoprostlaesis to initially cause, actually in all cases, a manifest aseptic inflammatory reaction an rl, occasionally, laryngostenosis with a necessity for the urgent tracheotorru,~~.
That is why to meet the abcove applic~rtion requirements it is practical to use gelling biocompatible materials.
Minimal trauma occurenc~3 anal the shortest possible introduction of a biocompatible material, absenc~a ref' carcinogenicity and minimal allergic reactions being the objective, it has been ~;r~ggested to use aL water solution of bovine collagen, which is a highly refir2ed and partially depolimerized product, turns into an elastic and mecl~ani~;ally stable hydrogel at a temperature below ~~7 deg.C', is in ,jectable into the organ that has been treated as to shape and dimensions (see ( l;'orri t'h., Martin D.. M., Warner Th.F. Lnjectable collagen ir1 la~-yngval ~-eabilitation// L.~IRYr~TGOSCOPE, 1984, 94, pp.513-518).
Being protein, collagen, however, would completely be resorbed in the patient's body in a consideraL~ly short pericod of time (less than half a year).
It is, therefore, suitable for use in endoprc>sthetic practice primarily in the cases when a complete substitution of an endoprosthesis for connective tissues is acceptable or when a p~~ti~r~t, ~~ccko~ding to medical indications. needs a precisely tempor~<~.ry endoprosthesis.
It should be also} r~otec~l tk~at the bovine collagen solution, c.~ue to it.. rfe:aorbt,ior~ ability and tcl~ intra-tissue and inter-tissue rnigraticir~, i~p irnefficient for storage of 05 medicinal preparations and. ~rlmr~ea~~ it is su;~ceptive to an enzyme at tack and ha,:> :~. I cv~w a lec; tr'c>corrd~cc t i vi ty> is practically unsuitable fc~r a~~~plicatiorr as an immersion med ium.
Conv;iderirrg thc-: rwrt~;ov~e, gellirrg~ biocompatible materials based- ors synt.hetic ~>olyrner~s are more preferable.
Thus, the biocompatil.~le gk~~lling material in the form of hydrophilic esters c~I' polygl;y~~:ol~ and of metacrylic acid is known to be appl i ed i r~ enc~,.~I~z~c>~; tt~e t i c pr°ac t ice ( Kresa L. > Rems T. , Wichterle t). Hycl.i~orn gel implantat in vocal cord // U to laryngal . 1~Ic.aci Neck '~~~~r~;. - 1988, V. 98, No 3, pp . 242-245 ) .
A rec:Iuirecl. dose of :u.mtn dr~s,~ rruaterial is implanted via a section in tine region of cc>srnetic or~ functional treatment and then the operative wc:o.cnc~ i.:~ :_~atured. The~~eafter, the material swells by ak~sor~bin~wat,c~r~ frorr~ adjacent tissues.
to - thereby pr~ovicie fc~r~ a. loc;t_t~ increase in the volume of the corrected organ, e. g. , a vc:~cal cord.
This biocompatible materi<rl is characterized by a high biochemical stability.
In application, however, r~ durt~ble therapeutic effect is achieved at the expense of traumatic surgical interventions associated with edemas and aseptic inflammations; its use as a mater°ial of intro-tissue drug storage si tes involves considerable cli f f icul ties. and i t is impracticable to u:~e as r_i b~rsi: for conductive immersion media.
Therefore> the most promising for encloprosthetic practice and the other abav~! mentioned applications are commercial in,jecta.ble l i.~~~.ric~ triocompatible gelling materials.
The biocompatible gelling material as a-i solution containing w~~ter--insc~llrb.le ~:~olymers, awnong them non-cross._.linkecl ac°;r~ylc:~r-iii,rilc~~ L~c>lyrners or their ._ copolymers, polyvinylac:etate, a 1 inear or low--branched polymer or copolymer c:~f ~?--tlyctroxyethyl-acrylate and methyl-acrylate, poly--n--vinyliminocarbonile and dimethylsulfoxide or other polar readily miscible with 05 water organi~~, solvents, may be ~~,xamplifir~d (Stoy V.
, Chvapil M. U,.~ Patent ~Vo. ~1>~~,~1.1~~8p 198k~). Tn obtaining copolymers, use may be made of ;add i t:ional moniamers>
such as acrylamide t inc lodin~; IV--srato~ti t,ur~ed) , ~~crylhydra2ide ( includ irlg N__sub~.>t:i tut~.c:~~ , ~~~~ry i :ic; ac id <~.~rd acrylates, glutarimide arid vinyl :_>ul font:: and the i~olar~ readily miscikale with water' solvents m<~y be glycerol and its mono-or diacetat,es, methanol, cthanro, ~~rc:>panol amI izopropanol, dinoethylformamide, glycols and c~trre~rw suitable solvents.

This material is highly Nt~iicier~rt in tree treatment of minor cosmetic or functional of~fec,ts. specifically lips and other parts of a facc:> above rnrer~tior~ed vocal cords, etc.

However, in filling volurnirrous caverns. or correcting the mammary ft~r~m and dimensions with endoprostheses. up tc:~ 1 l i ter ~:af the material can be required. Tn such cases, arr amount of an organic :>olvent, injected together with the gelling polymer, sustantially exceeds the physiologically permissible minimum to result in erythema and, ir1 some ease:.:,, arr allergic shock- Also, due to a linear structure of the gelling polymer applied, endoprostheses are observed to have a low forym-stability, the greater in volume, the lower irl quality..

That is why, the most l:rr~el'er~able arc; commercial hydrogels that corrtain rrco <~llergen:..

Among them, tl~e r~r~ost prefer red is a, l~iocornpatible hydrogel containing ~.(~'ro by wei~;lot of; a polymer based on acrylamide pr~odticed by the use of a free-radical polymerization initiator (specifically, ammonium persulfate) in a dispersion mediurrr such as ahyrcogenic bidistilled water 85 (USSR Inventor's certificate l,b~'7,'~~6).

This hydrogel is ire fact c;cprrapLet~:ly hioc;orr~patible with human tissu~.:s and fluids xn all the above aspects and, therefore, c;an be appl ~eci in c;i:~rrfie~erable ~,crp t.o 1 li ter) amounts, causing no perceptible negative biochemical and biological after effects. In the region of injection (placing of endoprosthe~;es, filli~~g, etc. ), it forms a structure readily permeable riot ~~~nly to water, ions, oxygen 05 but by low-molecular metabolites as well. The hydrogel implants, are invaded <~t, ~j. r:~on:~icler~ably high rate (by the 5-6th month) with a yc~c~r~g fito~o~t; tissue of a recipient.
This result is especially valuat~Le lrr (micro)alloplasty of the larynx.
This hydrogel, however, teas low vis.,osity and, therefore, low elasticity arnd High mobility. Water contained in the hydrcagrel i_> lc.~o~ely bound with the macromolecules of polyac;rylarnicle arZc~ is rE.ardi.ly removed from the implants to re:;trl t; in r~~~.rn fey>t ,~hrinl~;age thereof and a con.~iderable dcecrease ~.rr cosmetic oo~ t,eurapeutic effect. That is wl~y, ire t,l~tf.:: c;a~r:~ of plar:ln~; voluminous (e. g. , irctramarnmar) em:lc~~nr~~o:.>tiu~ ~~~..~, filling caverns, and in providing long-term ir~t;r,~t,i:>~~uF~ dr~~g storagE~s si tes, the implants shrwv low resistance t~:~ external detorrrvation loads and shrinkage a:> i.ar°~~v~ i.> t r~~eir irli trial volume.
Due to i is tiigrn f 1 ~.ndi t,y, ti~i;~ lyclrogel h<~s low efficiency a.s an E,lectt~~~c:;arrclue_;tivre immersion medium for external applications.
Therefore, the invention has for its object to provide ~5 a biacornpatible hydrogel wtrich, by improving the polyacrylamide compositior~r, would ensure elasticity, shape retention, and stability of balk implarrt~~ and offer greater therapeutic and cosmetic rA~~sult:~, preferably in cases of endoprosth~;tic~ uses.
The above problem has t~eero resolved by providing a biocompatible hydroge:l containing a polymer based on acrylamide produced by using :gin ini Bator of radical polymerization in apyrogenic wager as a dispersion medium in which according tca the invention said polymer is cross-linked polyacrylamide produced by using a biocompatible cross--lini;.ing agent.
Being permeable to watF:,r~, ions, oxygeru and low-molecular metabol i. tes ar~;l racing suitable for applications by irijec;tion, the hydrogel of the invention has a more regular arur~ more advantageous water--binding structure to thereby pr~cavide fc~r balky, highly elastic and forrn-retaining implant:; (e. ~;. . inc:.r~:irnammary endoprostheses.
05 supporting rod: iti the >pongy vas~e.zlar tissue c~f the penis, filling in lung cavern:_>) t.l,at <-rr~e irrvadect with a soft highly-vascularizc:~d connectives ~,is,~ue at an extremely slow rate (months to years). Urge tc~ st:rwc;tural, b:iocYremical.
anatomical and phys i c~ l off; i c;al -~cj~~~an Cages, ~~.s described above, there is a sui:~st,arrt7.onal e:cos~netic andior~ ti~erapeutic effect as well, ir1 eraclcy;rwcastin,etic~ prvactice and filling applications, and <rr~ ewc~rea:~f~ try c~ltzr,~.bi l i t,y of such effects.
According to Lhe f it st, furtY~er characterizing feature of the invention t;hc, ltiu>corr~patibl.k: hydrogel contains cros s-linked polyac;ryl<~roicie l~r~ocl~rce~l by using methylene--bis--acr~ylannid~~, :~~~~ a c:r~uss--linking agerut, and a mixture of ammonium per:,ulphat~~ r_rnd tet;ramet,hyleneiliamine as an ini Bator of polyme~wizatioru. Methylene-bis-acrylamide is ananalogous to the baste o~oncamc.~r (~.c;rylamide~ both by its composi lion arrd bioc.:c~~rnl><~t,a l:01 i ty, whi l.e use of the abovementioned mixture c'f pcilymerization initiators is favorable in the f'airl.y regular cross-linking of polyacrylamide chain mac:rorz~olec;ult::~ +~o provide an elastic space network suitable l~or~ i.n,ject,ing the hydrog~el.
According to the second further charact~eri~ing feature of the invention the r~iocc~ml.~atible hydrogel contains from 3. 5 to 9. 0 °~ by weight; cif saica c:rc:~5s--1 inked polyacrylamide.
This range of corrcerzt;ratic~rr 1.laroviding the maximum therapeutic or c~o:~met,ac:, ef ~'~~c~t, in the injection endoprosthetic use or fillirug t~s~. Ooncentrations below 3. 5~ make the hydroge 1 rzn:~ gala 1 a c ~n 1 y t o be app 1 i ed as a base for medicinal oirrtmer~ts car electroconductirng immersion media for cardio- or f~ncelW~lc~grafpliy> while concentrations above 9. 0~ decrease f luicli I:~y cif t~hie liyc~r~ogel pr<~c~t.ieally to zero to be used, in Borne c~~rr.,e>, ire manufacturyin~, relatively firm, form-retaining, i~rler;a:t ~ekcaollrosthf~ses that require an incision access C,o tru_e rTc~~T;lc~ra for placir:g such an , endoprosthesis.
According to the third further characterizing feature of the invention tile biocompatible hydrogel contains a physiologically neutral watery solub,e salt to be used the 05 most advantageously as :err eiec~r~oconductirng irrrmersiom medium.
Accord ing to the f our~tfl ftzr~k~n~-ar characterizing feature of the invention the i.ooc,oml~<:~tikolf~ hydrogel contains commercially av~.l:i.ablc~ s~:oa i_urrl chloride as said physiological ly neu tral. wr.~ter~ sc.~ l l.ik~l a sal t.
The invention i:; thc~rc~aftc~r~ di:vriosed by:
a description of t;tre ini ~; i.al reagents, method of preparing the nove:t k~ioc;ornpat~kole hydrogel, examples of carrying out the method, and tire resin is o.f laboratory tests on said trydrogel;
examples c~f ttue f orvrulatir~rus of the t~iGCOmpatible hydroge l a description of t>'rc=a metrrocls and the results of cherrrical, biochemical aruc:i mecli~;al studie.~ of the novel biocompatible hydrcagel~
a description of tYrc' ways of cc}rr~ecLing cosmetic and functional defects of a ru.~marr body by means of injections with the novel biocompatible llyc~r-ogc~l, anc~
information on its practical applications.
To prepare the ncavel bic~compatible hydrogel use was made of the reagents as shown irr 'fable 1.
Table 1 REAGENTS F'OR PkEPARI NC~ NOUEL i3I0C:OMPATIBLE 11YDROGEL
Reagent and (Consumption I Controlables, empirical formula (per 100 g ofI r~niis and limiis hydrof;e 1, g 1 d,.
Akrylamide CI~HjNO Is3.5 - ~.O IMel~;ing point i ~lf~g t' , 84. 5+/-_0. 5 j I 1)err.:;i ty, g/c;ub. cm, 1. 122 I I t><:i:~ c. ingredient, wt. l, - g -Table 1 continued 1 I 2 ( 3 05 I (not less than 98 . Methylene-bis- 10.01-1.00 (Melting point acrylamide I ( deg. C, 184+/-1.

C5 Hl o Nz 02 I I Basic ingredient, wt.
~, (not less than 96 TMED - tetramethyl- I 0. 001-1.I Density, g/cub. 0.
00 cm, 78 ethylenediamine ( (Basic ingredient, wt.~, C6 Hl 6 N2 I I no t 1 ess than Ammonium persul fate I 0. 001-1. I Densi ty, g/cub. 1.
00 cm, 98 (NH4)25208 I (Decomposition poin t, I I deg. C, 120 Basic ingredient, wt.
~, (not less khan 98 Bidistilled apyro- (balance (Refraction index, genic water I I 1.3329 Apart from bidistilled water, reagents commercially avaliable under the tradename REANAL (Hungary) were used in the experiments, namely: acrylamide and methylene-bis-acrylamide in the form of white crystals, tetramethylethylenediamine as a white oily liquid and ammonium persulfate in the form of colorless crystals.
Conventionally, the novel biocompatible gel is prepared by the following method:
Under aseptic laboratory conditions, calculated amounts of acrylamide and diluted water solutions of the cross-linking agent (methyl-bis-acrylamide) and initiators of polymerization (ammonuim persulfate and TMED), are placed into a sterile glass vessel. These reagents are thoroughly stirred, then diluted with water (alternatively with the physiological solution, alternatively other diluted water solution of a physiologically neutral salt, e.g. sodium acetate) the mixture is then filtrated and the filtrate is allowed to stand until the hydrogel of cross-linked polyacrylamide (hereinafter CL PAA) is obtained.
The prepared CL PAA hydrogel is controlled for the following characteristics:
05 appearance by sight (the hydrogel should be transparent, colorless, free of impurities);
refraction index (to be within the range of 1.334 to 1. 350) ;
pH (to be within the range of 7.0-9.0);
heavy metal contents (to be no less than 0.001 by we i gh t ) , and sterility.
The invention will be readily understood by reading the following examples.
Example 1. Preparation of a low-concentration biocompatible hydrogel 20. 3 g of acrylamide, 8. '7 ml of a 2~
methyl-bis-acrylamide aqueous solution, 7.5 ml of a 1~ TMED
aqueous solution, and 15 ml of a 4~ ammonium persulfate aqueous solution were mixed in a 1 liter capacity glass vessel. Water was then added to obtain a total volume of 580 ml, the mixture was filtered through a glass filter and the filtrate was allowed to stand for at least 20 minutes until 3.5 ~ CL PAA hydrogel was formed.
Example 2. Preparation of a high-concentration biocompatible hydrogel 34.2 g of acrylamide, 60 ml of a 1~
methyl-bis-acrylamide aqueous solution, 6 ml of a 1~ TMED
aqueous solution, and 25 ml of a 0.48 ammonium persulfate aqueous solution were mixed in a 1 liter capacity glass vessel. Water was then added to obtain a total volume of 380 ml, the mixture was filtered through a glass filter and the filtrate was allowed to stand for at least 20 minutes until 9~ CL PAA hydrogel was formed.
Example 3. Preparation of a medium-concentration biocompatible hydrogel 24 g of acrylamide, 50 ml of a 1~
methyl-bis-acrylamide aqueous solution, 25 ml of a 1~ TMED

aqueous solution, and 50 ml of a 1,3~ ammonium persulfate aqueous solution were mixed in a 1 liter capacity glass vessel. Water was then added to obtain a total volume of 350 m1, the mixture was filtered through a glass filter and 05 the filtrate was allowed to stand for at least 20 minutes until 5 ~ CL PAA hydrogel was formed.
Example 4. Preparation of a low-concentration electroconductive biocompatible hydrogel The CL PAA hydrogel was prepared as in Example 1, except the physiological solution was used instead of water.
Example 5. Preparation of a high-concentration electroconductive biocompatible hydrogel The CL PAA hydrogel was prepared as in Example 2, except the 9%-sodium acetate aqueous solution was used instead of water.
In experiments, formulations of the CL PAA
biocompatible hydrogel (hereinafter BCH) were used as shown in Table 2.
Table 2 EXAMPLES OF SPECIFIC FORMULATIONS OF NOVEL CL PAA
BIOCOMPATIBLE HYDROGEL
IngredientslFormulations and concentrations, ~ by weightl I_______________-------------__________------I

CL PAA I 3. 0 I 3. 5 I 6. 0 I 9. 0 I 9. 5 I 4. 0 I 7. 0 I 5. 0 I 8. O
Na chloride I - I - I - I - I - I - I - I 0. 9 I 0. 9 Na acetate I - I - I - I - I - 10.9 10.9 I - I -Water I b a 1 a n c a As can be seen in Table 2, the BCH2, BCH3 and BCH4, BCH6, BCH7, BCH8 and BCH9 formulations have the preferred concentrations of CL PAA in the hydrogel, BCH2 and BCH4 being in conformity with the preferable concentration limits of CL PAA in the hydrogel, while all the other formulations reflect intermediate concentrations, and the most preferable ones. Tn corrtr<~.st, the F3CH1 anal 8CH5 formulations are rvepr~e:~erltati.ve cif tyre CL PAA
concentrations in the hydrc~gW whic;Yr arf: uc>eful ir~~ a very limited number of appl.icatio~l::,.
05 Laboratory studies oL the novel hydro~;el were conducted in terms of cherrlicai, biochemical, arrd medical and biological properties. These st~~iltk=:~> were not, strictly delimited and virtually wcerc based pan conventional methods and techniques.
Thus, a dry residue was studied to conve.ntially determine a precise concentration of a ~aubstance in a true or colloidal solution.
Then, a dry residue was studied ira conformance with the USSR State Standard COS T 15. 0~.~.3---r~FP "Medical devices"
following the technique set-for~ttr in ,~;r practical guidance enti_lled "MeTO,~r~l aria.rrrr;3a a~:i.m~~a~rc~a rr Memarcpu.naz~os", M.: XYI-MI~H. 1t~'72 ( "Methods o f ~.n~ 1. ys i::~ c~ f acryla tes and methacrylates", KHLM1A publ i~f~ers, Mcu>cc~w) , 1~~37~.
A precise concentration of CL I7AA in tYl2 hydro~;el is usually determined from <~, dry t°e:;idrze. 'this method involves weighing a hydrogel sample ,~nct dryirythereof to a constant weight (for about 20 ho~~r~ at ~bf~> deg. C and residual pressure of 12 to 15 mm Hg i'ollowc-'d toy a c:,onverltional calculation of CL PAA perceuta~;e in the Yryclrof;el.
This method was emplcuyed fi:ov evaluatzn~, chemical stability of the novel hydrc~gel.
To this end, a Ylydrogr:l wars prepared to contain relatively loo:~ely cro~.~-lir~~eci (by introducing (~.~~5~ of methylene-bis-acrylamic.fe by wei.gYut cPf <rcr~ylarnide> ('L, PAA of about 5~ calculated conc;entrvai:~ic.~rr.
Five samples of such hydr~ogel c~f about 20 nrl each, were subjected to four suce:.-a~>ivc:~ t;e:~ts <~.~; follows:
Test 1. 'rhe samples werv~ weighed and dried, at 35 deg. C
and residual pressure 1~-15 rrrrra ~lg, urlt,il a cc~n~t,ant weight was reached ( for about ~(~ h. > ;
Test ~. The sam~~le,:~ were wei~,hed, immersed in bidistilled water, boiled ~'i:n~ i,'~ n~irmt~?;>, anal cried as above;

Test 3. The samples were weighed, immersed in bidistilled water, bringing up the level to 200 ml in each case, soaked in water for ? days, water being changed each day, and dried as above;
05 Test 4. The samples were weighed, soaked in water for 7 days as in Test 3, boiled for 15 minutes as in Test 2 and then dried as above.
Percentage of the polymer in the gross mass of the hydrogel was calculated by conventional methods for all the samples. The results are shown in Table 3.
Table 3 CHEMICAL STABILITY EVALUATION BASED ON DRY RESIDUE
OF NOVEL CL PAA BIOCOMPATIBLE HYDROGEL
I Sample average weight, g, (mean +/- SD) Testsl Prior to treatment I After treatment 1 I 20. 84+/-0. 96 I 0. 983+/-0. 0048 ' 2 I 20.15+/-0.87 I 0. 951+/-0.0076 3 I 20. 65+/-0. 83 I 0: 923+/-0. 0065 4 I 20. 41+/-0. 63 I 0. 913+/-0. 0095 As can be seen from Table 3, even soaking followed by boiling causes no destruction to CL PAA in the hydrogel to indicate that it can be thermally sterilized (whenever, need arises) and features stability though loosely cross-linked.
Next, acrylamide was examined for its capability to migrate into biotissues from stability characteristics of the base of the (CL PAA) hydrogel of the invention in an aqueous medium in conformity with "Pyxoso,~~u~~e MeTO,~~~ec~~e MaTepHa.n~ no Toxc~~co~toro-r~rueH~~ecx~M ucc.ne,~osaH~~M no,nn-MepH~x MaTep~a,noB a H3Ae,~~i~ A,~~ oHAonpoTes~poBaH~~" M~-HHCTepcTSa a,~pasooxpaHeH~~ CCCP, 1987, c.18-25 ("Guidance on toxicological and hygienic examination of polymeric materials and devices for endoprostheses", Ministry of ~ 19~~5 l Public Health of USSR, 198'l, p.18-25) This capability was determined by the HPLC (highly performance liquid chromatography) involving detecting UV
radiation absorption in the range of 190-210 nm that is 05 typical of the monomer, using a LIQUOCHROM chromatograph (Hungary).
To this end, extracts from the novel hydrogel were obtained by soaking the samples thereof for 14 and 30 days at a temperature of 40 deg.C and a ratio of 100 ml extractant (bidistilled water) to 1 ml hydrogel. Samples for the HPLC were prepared by drying aliquots of the extracts of 5 ml at room temperature and a residual pressure of 12-15 mm Hg and eluating once a residue at a rate of 0.2 ml/min .with a 2 ml 1:1 mixture of water and methanol in a column 150 mm long and 4 mm in diameter filled with the Separon C18 sorbent and by feeding 20 microliters of the eluate into an injector loop.
A minimum of detected concentration of acrylamide by the HPLC was 0.000001 mg/l, whereas its maximum permissible concentration in aqueous extracts from the material from implaY~ts was 0.02 mg/1.
Acrylamide was not detected by the HPLC in aqueous extracts from the hydrogel prepared by the method here described to indicate that on the whole both CL PAA and the biocompatible hydrogel of the invention are chemically stable.
In terms of medical and biological properties, the samples of the CL PAA hydrogels prepared by the method here described were tested under laboratory conditions for:
biochemical and hemolitic activity, embriotoxic activity, mutagenic activity, and carcinogenic activity.
Biochemical and hemolitic activity of the CL PAA
hydrogels was evaluated from variations of chemical composition of the plasma and blood cell composition in males of albino rats of Wistar line of 300-350 g body weight from test and control groups of 16 animals each.

Prior to experiment, a dose of 5 ml 5~ hydrogel of the invention was injected intraperitoneally into each of narcotized rats from the test group.
The rats were fed on a regular basis.
05 Two weeks thereafter, blood samples were drawn from the rats and were examined for the contents of the ions of Na, I~, Ca, a Cl; urea, blood urea nitrogen and uric acid;
creatinine and enzymes (amylase, alkaline phosphatase, alanine- and aspartate-aminotransferase, hereinafter AlAT
and AsAT respectively, lactatedehydrogenase, hereinafter LDG, and creatinine phosphokinase) using a MORNING
biochemical analyzer (Sweden). In this case, contents of potassium and urea were determined by a LACHEMA bio-test (Czech republic). The results are shown in Table 4.
Table 4 EFFECT OF IMPLANTS FROM BIOCOMPATIBLE CL PAA HYDROGEL
ON BIOCHEMICAL COMPOSITION OF BLOOD PLASMA IN RATS
Biochemical~characteristics and (:Examination results units therefor I control I test Sodium, mmol/1 I 151 I 148 Potassium, mmol/1 I 8.20 I 6.82 Calcium, mmol/1 ( 0.97 I 0.90 Chlorides, mmol/1 I 97.5 I 102.1 Urea, mmol/1 I 4.8 I 4.8 Blood urea nitrogen, mmol/1 I 2.2 I 2.2 Creatinine, mmol/1 I 0.05 I 0.05 Amylase, mgt I 89.1 I 83.33 Alkaline phosphatase, mmol/1 I 84.5 I 55.9 AsAT mmol/1 I 133 I 130 AlAt, mmal/1 I 41 I 51.7 LDG (total), mmol/1 ( 217 I 189 Creatinine phosphokinase, units15960 5685 I

Uric acid, mmol/1 I 0.14 I 0.10 2 ~ 95957 As can be seen from Table 4, the main characteristics _ of the ion exchange demonstrate that there is no manifest damage to the cell membranes. ATPase activity is normal as well.
05 Stability of characteristics of nitrogen exchange demonstrate normal metabolism including purine exchange and, togheter with creatinine stability, functional stability of the urogenital system in the presents of CL
PAA in human body.
Normal activity of AlAT and AsAT represents stability of hepatocytes and a proper state of the miocard which, judging from the activity of creatinine phosphokinase being normal, are not subjected to appreciable overloads.
Sufficient activity of alkaline phosphatase is an evidence that there is no inflammation in the endothelium of the biliary ducts.
Also, a blood cell count in the same rats was performed to be shown in Table 5.
Table 5 EFFECT OF IMPLANTS FROM BIOCOMPATIBLE CL PAA
' HYDROGEL ON BLOOD COMPOSITION IN RATS
Characteristics of blood cells I Measurement resalts composition and units therefor I control I test Leukocytes, thsd/mcl I 3.5+0.2 I 5.4 Erythrocytes, mln/mcl I 6.86+0.431 "1.02+0.31 Hemoglobin, g/1 1125+12 1139+9 Hematocrit, ~ I 35.0+1.5 I 36.5+1.3 Erythrocyte mean diameter, nm I 51.0+0.2 I 52.0+1.5 Hemoglobin average content per erythrocyte, pg I 35.7+0.3 I 38.1+0.5 Thrombocytes, thsd/mcl 1992+12 1694+50 Thrombocyte mean diameter, nm I 8+1.5 I 14.25+1.6 As can be seen from Table 5, leukocytes, in the experiment, unessentially exceed the normal content of 4.5*1000 per cub. mm, while erythrocytes and hemoglobin in erythrocytes indicate normal blood oxigenation. In terms of hematocrit, it can be claimed that a fluid-and-electrolyte 05 balance is approximating normal.
The data avaliable indirectly indicate that biochemical stability and biocompatibility of CL PAA per se, fairly acceptable.
Embriotoxic activity of the CL PAA hydrogels was determined according to "Pyxoso,~~Il~e MeTORI'I~ec~,I~e MaTepna.nbl IIO 3KCIIepIdM2HTaJIbHOMy I~I KJI~IHH~ieC~COMy H3y~ieHI~IIO HOBbIX JIe ~apcTSeHHbIx cpe,~cTS", M.: M3 CCCP, 1975, c.42-48 (Guidance on experimental and clinical studies of novel drugs, Moscow, Ministry of Public Health of USSR, 1975, p.42-48) and "Pyxoso,~~u~e MeTOA~~ecl~ue MaTep>ila,nbl no Toxcl~xo~toro-rl~
rHeHH~ecx»M Hcc~te,~osaHl~~M no,nnMepHbIx MaTepl~a~IOS n I~a,~e,>Ini~
A,n~ oH,~ortpoTe3l~posaHH~", M.: M3 CCCP, 1987 (Guidance on toxicological and hygienic examination of polymerical and devices for endoprostheses, Moscow, Ministry of Public Health of USSR, 1987).
Three groups of mongrel albino female rats of 180-200 g body weight were used in the experiment, each group containing 16 animals.
The rats in the first group were injected intraperitoneally with 2 ml of the novel 5~-hydrogel to be coupled in a week.
The rats in the second group were injected intraperitoneally as well with 2 ml of the novel 5~-hydrogel on the third day of pregnancy.
Pregnant intact rats constituted the third group.
Two rats in the first group showed no pregnancy. 14 rats in the first group and all of 16 rats in the second and third groups gave birth to normal healthy cubs to prove that the novel hydrogel is not embriotoxic.
Mutagenic activity of the CL PAA hydrogels was examined, according to a guidance of the USSR Public Health Ministry "OUeH~a MyTareHHOI~ axTI~BHOCTI_~ x~M~~ecl~ux seluecTs MI~IIS.pOA,~,epHbIM MeT0A0M" ( "Evaluation of Moscow, mutagenic ~1959~7 activity of chemicals by micronucleous method" 1984, 14 pages.), on reticulocites of the bone marrow from the C3H1-line mice (of both sexes) at the two-month age in two groups of 10 animals each.
05 Experimental animals were injected with 0.01 by body _weight of a 30-day aqueous extract obtained at the temperature of 40 deg.C and a ratio of 100 ml extractant per 1 g gel from the 9~-CL PAA hydrogel.
In 24 hour period the experimental and intact mice were killed by shifting the spinal marrow. Further, smears of femoral marrow diluted with a serum of fresh non-stabilized human blood of group AB (IV) were conventionally prepared to be Pappenheim stained thereaf ter.
Reticulocytes with the micronuclei in the smears were counted under the microscope. Variations in the reticulocytes counts in the marrow smears of both the experimental and intact mice in 20 visual fields containing 1000 cells each were not found to exceed 2.3~. It is an evidence that the CL PAA hydrogel does have any mutagenic effect.
Carcinogenic activity of the CL PAA hydrogel was evaluated by immunodetection of inaccessible tumor-associated antigens.
This type of evaluation involves determination of electrophoretic mobility (hereinafter EPM) of stabilized and tannin-treated erythrocytes which are sensitized to a tumor-associated antigen of rhabdomiosarcoma and, additionally, to an inaccessible embryonal antigen, the latter being an indicator of a tumor progressive growth when the EPM-test is positive. Usually, the EPM-tests are positive, if the electrophoretic mobility of cells-indicators is decreased by 20~ or more.
12 non-linear albino male rats of 180-200 g body weight were used in the experiment, constituting a test group and a control group, each containing 6 animals.
The rats in the test group were injected with 4 ml 6~
of the CL PAA hydrogel into the femoral muscle under local . 2195957 anesthesia. Then, the rats of both the groups were kept on their usual diet for 18 months. Afterwards, in all the animals blood samples were let from the tail vein, erythrocytes were isolated from the samples and sensitized 05 with the abovementioned antigens to carry out EPM-tests.
_ A decrease in the EPM of the sensitized erythrocytes compared with those that were not sensitized was observed as follows:
4.17+/-1.58 for rhabdomiosarcoma antigen and 1.67+/-0.95 for inaccessible embrional antigen in test animals and 1.50+/-0.62 for rhabdomioblastoma antigen and 1.83+/-1.28 for inaccessible embrional antigen in controls.
Therefore, the EPM-test appeared to be negative for the rats of both the groups, which is indicative of the fact that the novel CL PAA hydrogel does not display carcinogenic activity.
More detailed medical and biological examination of the novel CL PAA biocompatible hydrogel applicability in endoprosthetic practice and tamponing was conducted on mongrel male dogs of 25 to 30 kg body weight at the age of three to four years. The dogs were subjected to a test placing of endoprostheses, under sterile conditions, following disinfection of the skin covering of the penis with 10~-iodine tincture and local anesthesia, among which:
6 dogs, subcutaneously, were once injected with 5 ml of the 3.5~ CL PAA hydrogel;
also 6 dogs, endofascially, excluding penetration under the tunica albuginea, were injected with the 9~-CL
PAA hydrogel into three segments along the penis on opposite sides thereof in the amount of up to 1.5,m1 per segment to make the total 8.0 ml, and other 6 dogs, intracavernously, including penetration under the tunica albuginea and mainly into trabecula corporum cavernosum but excluding injury of the urethra, were injected with 6~-CL PAA hydrogel into three segments, along the penis on opposite sides thereof in the amount of up to 1.5 ml per segment to make the total 8.0 ml.
The fourth group of three dogs was used as control.
The dogs were killed one by one by an intravenous nembutal injection, among them:
05 the test animals in 1, 7, and 14 days and 1, 3, and 6 months after the CL PAA hydrogel was implanted;
the controls in 1, 3, and 6 months.
Excised pieces of full cross-section slices of the penis, regional lymph nodes, and lungs of the dogs, together with control slices, were fixed in 10 and 6~
neutral formaline and in the Carnoit liquid, dehydrated in alcohols of increasing strength and were covered with paraf in.
Mounts were stained with hematoxylin and eosin, van Gison-stained with pyrofuchsin, Weigert-stained for elastica, and with toluidine blue at various pH of the staining solution to thereafter detect glycosaminoglycanes by chemical and enzymatic methods.
Glycoproteins and glycogen were detected by the McManus-periodic acid Schiff reaction (hereiafter PAS-reaction), calcium salts were detected by the von Doss method, RNA was detected by the Brachet method (with ribonuclease).
Activity of the following enzymes was studied, namely:
malate dehydrogenase (hereinafter MDG);
succinate dehydrogenase (hereinafter SDG) by the Nachlass method;
lactate dehydrogenase (hereinafter LDG);
glucose-6-phosphate dehydrogenase (hereinafter G-6-PDG), NAD- and NADP-diaphorase by the Hess, by the Scarpelli and by the Pearce methods respectively;
alkaline phosphatase (hereinafter AP) by the Gomori me thod, and adenosintriphosphatase (hereinafter ATPase) by the Wachstein- Meisel method.
Nervous tissues were impregnated with the silver nitrate by the Bielschowsky-Gross method.
Histochemical reactions were conducted and controlled as recommended in the Manual by E.Pearce "HISTOCHEMISTRY"
(rus. edition "rYICTOXI~MYI~I"/transl. from the English/2nd Ed.- Moscow, 1962).
The studies revealed that:
05 A. In the case of subcutaneous injection of the CL PAA
hydrogel:
the following day, a sleeve-like swelling of a soft-elastic consistency with some thinning of the skin was observed at the injection site (one dog developed inconsiderable edema and hyperemia of tissues surrounding the implant with a small focal hemorrhage which had resolved as of the 7th day to be considered as a manipulation injury);
7 days thereafter there were no visually perceptible hemodynamic, alterative, and inflammation reactions. On histological examination, the implant became a large light-blue vacuole surrounded by a thin connective-tissue capsule separating the CL PAA hydrogel and the fascia of the penis and the skin covering. The capsule consisted of one or two layers of young fibroblasts with gentle collagen and elastic fibers to be seen around. Pyroninophilia and enchanced activity of the redox enzymes (SDG, MDG, NAD- and NADP-diaphorases, LDG) and AP are typical of cytoplasma in fibroblasts. An increase in activity of G-6-PDG revealed the activation of pentose=way metabolism. A loose infiltration of leukocytes and macrophages was observed in the layer near the surface of the hydrogel. The capsule had a periphery of granulation tissue of moderate number of newly grown vessels covered with swelled epithelial cells, the lumens of which were locally enlarged and filled with blood formed elements. Proliferating fibroblasts, histiocytes and solitary plasmatic cells were detected in the adventitia of thr vessels. In all cases, no giant-cell reaction was observed. Upon staining with toluidine blue, no metachromatic foci were detected at all pH values of the solutions used. A small number of nerve fibers, being impregnated with silver nitrate, showed various changes such as local swelling of axons, ~ loss of their fibrous ~~?
structure, vaCU011Lat10n. Vcir'1C(~s~. ty, hypo- Or' hyperimpregnation. At times accumr.u.ation of oxoplasma along the nerve f i tiers or a t the i r' erTd:~, i:r~rr° t i al unevenness=~
o f the myeline membrane and 1. t;s dec:;c;;mpci,i tion int4:~ .>hort and 05 long fragment s were ob::~erved. These changes are typical of the nerve fibers in thei r' c~c3mpensatory--adaptive restructurfing in response to compression from tfTe vacuola on the CL PAA hydrogel"
of ter 1~ days, tine Grracr~ophage- leu)";ocyte rr::ac:tion in tissues ad~lacent t.hc~ (L~ t-'AA hydroge l implant insignificantly increaasec ; a ~9r~c:~r~oo.rrlcecl fibroblastic reaction, including c:crntinuc~L~~ formation of the connective-tissue capsule aroc.znd t:he v<~cuole, wa:.~ observed, the capsule locally appearing as r°a.rrdoml.y arrangE~d collagen and elastic fibers having young fibroblasts and newly formed capillaries irTbetweer~. wherveas at otrrer locations as more mature connec t i ve t i ssue cons i s t i ng o f same rows o f collagen and el.<~st:i.c; fitar~s arranged ride by ~;ide as well as proliferating f'ibrobla~~~:~. Tt~e E~.NA--content irx cytoplasm and nucleoli increased <~s well as ~~ctivlty of redox and hydrolytic enzymes. Fibrol~last cytoplasm has been enriched in metachromatic granules which ar'e reac~i ly detec;table with toluidine blue at pH ~.3 to eviderTCe an irTCrea:>e in the synthesis of glycosaminoglycanes. The number af° the newly grown vessels srTar~ply decr°easc;d in tissues surrounding the capsule and hystiogenous-type cells producing glycosaminoglycanes and collagen were ~jrevailing i:n t;heir stead. Giant cells were extremely rrn~rw.quent. Changes in the nerve fibers were as above.
one month after injection, a mature connective-tissue capsule develored around the vacuole of the CL PAA hydrogel to consist of circularly arrang~Jd collagen and elastic fibers with mature fibroblasts ir~bE:tween containing a moderate amount of F~NA and high--sulf't~tt: glycosaminaglycanes detectE;d with toluidirre bl.uo~ at pH ~~. Vii. Activity of redox and hydrolitic enzymes lrr fil~rocytp:~~ c;ytoplasm was normal.
A cell reaction that apl,~eared ~r.:~ a loose diffuse infiltration of mac_ropha.~;es a.rTd pl<~smic cell: was, at times, observed on the hydrogel surface. The structure of tissues surrounding the implant was completely normalized and was not different from that of like tissues of the intact animals. Reactive changes in sensitive nerve fibers 05 began to decline and to appear mainly as irregular enlargement or thinning of the axons and their focal hypo-or hyperimpregnation.

3 months later, same thickening of the CL PAA hydrogel was observed along with an increase in basophilia and a well defined separation of the implant from neighboring tissues by the connective capsule consisting of collagen and elastic fibers and fibrocyte-type cells. Structural and histological changes in the neighboring tissues were not observed, whereas nerve fibers developed their normal shape.
6 months later, the shape and dimensions of the implant remained practically as during the first 24 hours upon injection of the CL PAA hydrogel. Gistologically, the implant appeared as an integral well encapsulated dark blue vacuole. The capsule consisted of one or two arrays of fibrocytes and orderly arranged collagen and elastic fibers in which no salts of calcium were detected by the von Doss method. The tissues enclosing the implant displayed no reactive, hemodynamical, dystrophic, necrotic, inflammatory and other changes including tissue and cell irregularities.
Upon impregnation with silver nitrate, nerve fibers appeared normal.
B. In the case of endofascial injection of the CL PAA
hydrogel:
the following day and after 7 days, the penis appeared evenly swelled and showed an increased resiliency. Body temperature of the dogs was normal, skin coloring at the injection sites was as usual, local inflammations were not observed. Histologically, the implants at the injection sites appeared as light blue vacuoles. After seven days, the CL PAA hydrogel vacuoles were observed to be enclosed with thin-walled capsules mainly consisting of one or two layers of young fibroblasts, and gentle connective fibers and same newly formed capillaries surrounding the fibroblast layers, wYiereas leukocytes and macrophages were obserwved on true hydroge 1 surf ac~~. 'l'he RNA-con ten t in cytoplasm and nrzcleoli irncrea~>ed its well as activities of 05 SDG, MSG, NAD-- and NADP-ciiaphorasE:is. LDG and G--6--PDG in cytoplasm. The grc~nulataorr t.i:~si_te enclosing the capsule had newly formed capillaries w:i ztlr t~ll~ihtly widcaned and blood-f i l led lumens and swel l.ce~i eruic~ t;herl ium. Pro ~ i f'erat i on fibrobLasts and sarne pl~:~sma~t~.co cel.~.~> were foun~_~ irn the adventitia of the blond ve>sels. Hi~;tochemical reactions have confirmed that dyst;rophi.canci, mc~rEaaver, n~~crobiotic changes were not detec;tecl in the I~ascial tissue enclosing the implants and forced ap<~rrC, by ttat~ in~plant;~. Thus, upon staining the mounts witty toluidir~e bane solutions at any pH, metachromasy foci wrrictr would suggest of destruction of the CL PAA hydrogel were rrat found. Permeabili ty of the vessels remained to be nc.7rrnal :since tine PA;~-positive material was not detected wr~ictr is stable to amyl<~se and in perivascular spaces as well as in the walls of small and middle-sized vessels, wherve<~s AC' <~ncl ATP-ase activities in the walls of microcirculatc:~ry i:~ed Remained to be low. In some cases, the nerve f fibres i.mpre~:;nated with silver nitrate and exmined by the 5pielmeyer method, were observed to be wave- or spiralshaped. ,:end, in or~her cases, they had swelis at the ends. Demyei inat,inn si tea were rare as was rare local spreading of nerve fibers developing loop-like structures. Infrequent proliferation of hypertrophic Schwann's cells was observed. 'I'he ~..:hanges are to b~e treated as a response of nerve fibers to compreasian by implants, i.e.;
after 14 days, the macrophage rveaction near the implants was somewhat more intensive tut giant cells were not observed. A distinctive flbroblt~.st reaction and the growth of connective tissue c~apsul~~s around vacuoles were observed some capsules corrsi:~tin~c.~f' randomized collagen and elastic fiber.,s wi th yc~ur~g f ~x~z~co~>~~:r:~ts thereioetween a high RNA content in the ~-°yr or:~l~~:,>rrr <~rnd enhanc~ced redox <~ctivity of en~,ymes. Mc_~r~cl m<~trrn~e connectivfe tissue consisting of several arrays of collagen and elastic fibers and fibroblast-type cell~> was found at other sites. An increased number of histiogeraous cells and a decreased number of newly--grown ve;~~~c=:~1::> were ob:~erved in granulation 05 tissue adjacent the cap~~ules. Endothelium arid middle membrane structures of the adventitia of the vessels, and hemodynamic factors clid not change while change:> of nerve cells were as described in conjunction w~ th the previous term:
in a month's time, the implant capsules consisted of cell elements of r~ fibrob:lastic: series, fibroc;ytes with a mederately pyroninophilous cytoi3lasm being prevail out.
Staining with methylene blue at. p1-1 ~.8 showed a moderate number of high--sulfated l.lycosamin9~glycans in fi.broblasts.
Enzyme activity in the fibrocyte cytoplasm was in accordance with the corutr~c~l. UndenAsur~f~ace layer of the CL
PAA hydrogel was to same extent infiltrated by the macrophages and plasmatic cells. 'There were either blood-flow disorders, inflammatiaru, degeneration nor .necrosis observed in the t issues ae~;jac~ent; the implants. The changer in nerve fibers, mentione:,ed Y>efore, were :till observed;
3 months after inject.ic:~:n, an i;r~crc:ase in ba~~op:hilia of the CL PAA hydrogel was observed. 'The gel vacuoles were well defined from the fir::>r;i~~. by ttnrl connective-tissue capsules of the collagen arrd elastic fibers with fibrocytes therebetween. Blood vessels wf~re norm<~~ . Na response of the penis tissues were observed (fascia, as in controls.
appeared as circurlarly arranged well defined collagen and elastic fibers without a lass ~of ~ritegrity a.nd without calcipexis at both the micro- and macrcflevels, nerve fibers being normal);
after 6.months, the penis as to form and dimensions in the dogs was by visual ins>pect,ion, ~~imt lar to that observed on the second-seventlu d<~,y.;. llistologically, the implants appeared as integral wcell.-inc.;<~i:~su1<~.t,ecj dark-blue vacuoles.
The capsules cons 1 s ted o f c ~rre or tw~~ a~°rays o f f ibrocy tes and orderly arranged thin collagen a~r~cj elastic fibers, and 21~~951 no salts of calcium were found either by the macroscopic.or microscopic von Doss method. None of the reactive, hemodynamic, degenerative, necrotic, inflammation or other changes including tissue and cell irregularities were found 05 in the implant-adjacent tissues. Nervous tissues impregnated with silver nitrate, both in the experimental and control animals, were virtually indentical. In the regional lymphonodes, intra-trabecular and trabecular spaces of the corpus cavernosum penis, penis veins and in the lungs, the hydrogel particles were not found;
C.In the case of intracavernous injection of the CL
PAA hydrogel:
in 1 day and 7 days, staining with hematoxylin and eosin, revealed the CL PAA hydrogel appearing as homogenous light-blue vacuoles which, in seven days, were surrounded by thin connective-tissue capsules that caused a shift and slight compression of the trabeculas of the corporum cavernosum penis and tunica albuginea. The capsules were consisted of thin, mainly collagen, fibers and one or two arrays of fibroblasts. Connective-tissue of the trabeculas of the corporum cavernosum penis adjacent the capsules were of the usual structure, having clearly defined smooth muscles with a small number of elastic fibers without any treits of degeneration and necrosis on histochemical or histological examination. Minor affluxes of leukocytes and macrophages were observed on the surface of the CL PAA
hydrogel implants. Intra-trabecular spaces were filled with minor amounts of blood and the endothelium was slightly swelled. Lesser arteries and veins were moderately filled with blood and having slightly thickened walls (primarilly, due to the swelling of endothelium and proliferation of fibroblasts, histiocytes and plasmatic cells in the adventitial membranes). Capsules were observed to be surrounded by granulation tissue consisting of a small number of the thin-walled newly-grown vessels and different cells, mainly of a histiogenous origin such as fibroblasts, histiocytes. Some nerve fibers, when impregnated and examined by Spielmeier method, showed changes in axon, the _.
myelin membrane, and Sc:,hwanro';> cells. 'there were no giant--cells c9bserved as ~~ r~e~>u1 t ~,7f ttue re:>olution of foreign matter. There were ob served twist°ir~g, local swelling and irregular ttlicker~ings in nervous tissues as 05 well as varicosity and a lr3ss ui' ('fibrous structure in axons, spherical and c l.ub-~srraped enlargement:, at their ends, and lokal demielin:i~~~tiorn. uceactive proliferation of Schwann's cells, some oT wrni cl~ tiyfner trophied, was found, l.. e. ;
in 14 days, the lt~ukocyte and macrophagve reaction around the CL PAA hydr~ogel i~nplants became somewhat more intensive but the giant c:~ell:~; c::af~ablca of resolving foreign matter were, <~s mEantiorrecl befog°t~, not found. 'rhe connective-tissue capsul~:;a:a around the implants were, in some cases, sl ightly porous arid consisted of randomly scattered collagen and elastic; fibers and young f ibroblasts, and some of therr~ appearwd more mvture and consisted mainly cf l:~~rrv~11e1 c;c~llag~en fibers including elastic fibers and filirvc>f~la~>ti~:~ e-lements. Perifocal granulation tissue was t_:onr~istin~of a small number of flatened newly-grown vessel: Kind fibroblasts conl~ai.ning a moderate amount of RNA and granules of highl;~-sulfated glycosaminoglycans. Changes in rrervc~us tissues occured as a local swelling of axons, loss cal' fil>rous str~.zctur~~~ in them.
vacuolization, varicosity, tnypo- arc liyperimprf.gn<~tion and, occasionally, in Local accum~~lation of the oxopla;~m, either along the nerve f fibers orT at their ends, <~, partial roughness of the myeline membrane and its disintegration into short and long f ragment:.~, what, should be can:>idered as a compensatory-adaptive r~c~si~onse to compression. In the region<~l lymphonodes, intc.~rvt;rx<~t~ecc.~l~~r :~>paces of corporum cavernosum, the viens of the penis; end in the lungs, CL PAA
hydrogel particles were not, detEact~~cl;
in a month, the t'L, hAA hydrogel implants were surrounded by the thin rna~,ure c~c~r3rn:ctlve-tissue capsules consisting of circlularly ar°ranl;ec:1 ~~allagen arid elastic fibers witYr mature 1'i.t~rololast,ic ~~.lc;mc~r~ts found between their arrays. Irac;an~>iciera:~.lrlcy dll~f~~.~ivr~ ir~filtrvation of _...
macrophages and plasmatic c~Eyls wa:> observed in the layers of the implants adjacent ttreir~ sur~f'ace. Connective-tissue trabeculas of the corpor~um c.;~rvrr°rrc~.-~um were not str7uc;turally differrent from the controls arrd were covered with the 05 normal endothel.iurn. A >mal.l <~rnc:~urrt; of blood coulc:l be found in the inter-trabecular spaces. The walls of the corporum cavernosum veins ~rnd artervir~e~ :~kxoweci no visible ~~~tructural changes. Reac t i ve charnge:~ i r~ r~er~vr~ f i hers, com;~~ared wi th the previous periad, were le:~~=> clist.inct and appeared mainly both as irregul<~r thic~~kerning:, c~r° triinnings in axons and local hypo- or hyperimpregrlatic~rr:
in 3 montri s the hyi~lrwo~;~al w~:r~ getting tkoic~ker and became basoph i 1 i ~~ . The i rni~ 1 an t s were separa ted from adjacent tissues thin-walli:el c;~rp:;ul~;~5 0l.' parallel collagen and elastic fibers with <i small number of fibrocytes between them. Cell elerriernt~t> werr.~ riot observed on the surface of the implants. ~djacarerot, tissues with blood vessels were of the usual str~ucl:urt;. (~lycasaminoglycans in the ground irltE~rsti tial subst~:in~;e, f ihraus formations and cell elements of connective tissue werE.~ virtually identical to those in the control. Changes irn r~e:rve fibers were not observed:
in G mon ths, the pen i s in dag:~ was o f the shape and dimensions by visual abservation, similar to those observed on the second-seventh days. ili~tologit,ally, the implants appeared as integral well - inc;aps~~latec~ dark-blue vacuoles.
The capsules consisted of one or~ twu ar=rays of fibrocytes and regularly arranged tkrirm;oll;~gerr and elastis fibers, and no salts of calcium were found either by the macroscopical or micrascopicai. vorr ltos:> method. There were observed no reactive, hemadynamic, degenerative, necrotic, inflammatory and otrrer clrarugE~s, inclircli.rrg tissue and cell irregularities, i.n the ti slue adtjacent the implant. Upon impregnation with silveu nits°ate, nerve fiber°s appeared and were virtually identical irr tile oxperim~;nta( acrd control animals. In the regional lymphonodes, in inter--trabecular spaces of the carporum cavEe°r-xo~~urri ~rrrd tale veins of the pen i s as wel l as i n t.k~e l ure,:, the CL PAA hydrogel _ ~c~ _ particles were not found. .
Similar morphologic data werv~ obtained b:y c;linical experiment. Used as a test m<~c;ex~ial. wa;. <r bioptic sample of hypodermic cel lular ti slue t3kE~rr in a healthy male 05 volunteer, age 4,'>, wt~co. E'> years t_~r~ior to biopsy, was intradermally in,jectc:cl wirra i() nvl ~>~' the novel h;ydrogel at 8~ concentration t'L PAA.
The bioptic sample was fixed in 10~ formaline, dehydrated in alkohols c~f 'tr~c;r'easiry; s-rength and E=mbedded in paraffin. Mounts were st-~irnad wit;~u lnym<~toxylin a~.nd eosin;
the collagen fibers were determined by the van Gison method and the elastic f fibers wi tic resorc i~~-fuc:hsin by ttze Weigert method; glycosamino~Iycar~es were ~l~,~ter'mimed with toluidine blue solutions at various pii vale<~t <rpplying the required chemical and enzymatic corut,rol.; glycoyroteide and glycogen concentratiorns were deterrnirzerl by rn~.ean> of the PA~:~-reaction by the McManus rc~e thoc:l .
Macroscopically, the bioptic sarrrple was oval in shape, soft elastic, light-pink in color without any visible changes which would c~ist;irigui:r~ i 1. t torn adjacent tissues.
On microscopic examination, all preparations showed the CL PAA hydrogel. stained with hematoxylin and eosin to acquire blue color of' va~'ious degree of intensity. The hydrogel implant was t;r~r~eaded t~~r~oughout; with well vascularized gentle connective tissue mainly con;>isting of orderly-arranged collagen and elastic fiber:> and the ground substance which included ~~r~ irmigrni fi.c.~.r~t number' of cell elements (as a rule, such as imact,ive fibroblast:>, because, on staining with toluidine iOue at pH ~.8, no traits of metachromasy evidenced by glycosamir~oglycarns were detected in the cytoplasm of these f vi l~rvc~b l ar t~, and }such as sol i tary mononuclears-macrophagces).
This connective-tissue had tt~e vessels located as a groups and having walls of it~regular thickness with the 3'> flattened endothel iurr~.
Signs of acute <~nd chronic; irrflammation, such as polymorphonuclear leukocytes, epitiu~lioid cells, giant cells capable of r'esolva.rlg for'k:aigrn rr~<.~tter and both lymph _, v -and histiocyte infiltrates, were en~;ir~E~ly absent. a:> absent were signs of allergic reactiora~~, such as lymphocytes, macrophages and h i s t i o~;y te:~ ~a:~ we l ~ as s igns o f hramodynam i c disorders such a.s plet;rmrw<~ ;:~f the v=essels, pre--stasis, 05 hemost<~sis, ttlrombo;~is, arac~ mtzl igxri~ation> e. g. cel l or tissue irregularities arid cell prop feration. Cal~~:ium salts were not detected in the rnour~ta> ei ther m<~cro- or microscopically. Alterative> i. r~. cfyA;trophic or necrotic, changes were no t found.
A f fibrous capsul a ~,czrrol.zrrd i rng the implan f was no t observed.
The basic method of c;orrectirrg c~crsrnetir; or functional defects in a human body icy cz:>vrrg l,rre novel CL PAA
biocompatlble hydrogel c~nn>i>t,s in I_,he fcnllc~wing:
based on arr<:inrne~> i:~. c~xazrn i na ~, ~ r7ri t_rrrd. i f required>
laboratory studies genera.ll;~ ,.~~;cep~able for~-patir~nts to be surgically treated (partlc~zl<~r~ly f,~rw individual rre spouse to antibiotics) , a tentative c~;onc~ fcr~>ion is drawn up as f of lows:
~0 - first, i;he organ to be trvet~tf-acl either as to i.ts form and dimensions or func:, tai onal ef f is<rcay, is def i ned, and - second, an amount, tactics arid a form (r~utpatient or patient) of the future treatment;
prior to i.n~jectican of tfre novae! r~ydrogel> <~nesthesia (as rule, local infiltratlon~ is irrca.uc~4d;
Sterile CL, PAA hydrog~el, adds t,~fc:~rral ly satur<ated with antibacterial pre:paration_;. i.s ~>yringed <~t a low rate (usually, in t;wo or trrree stages) info the si lie to be treated a temperature approxirrratiry normal bod;~ hceat (30-3'~
deg. C ) .
This method is tyre most apY~licable in m~~mmaplasty (preferably> in tyre caA>e of~ <~plasfcr. and hypoma:~tia) and alloplasty> in the case r7f' impr:~t,ency manifestcad as poor erection due to age or priczx~ zn,jur>iex:~.
TYrus> in mammopla,~fy. the c', L. PAA hydroge:l having a preferable concentratiarn wit,Irin tyre range of 3.5--6.0~ and the mast preferable concer~tr~atic~rr wi t,hin the range of 5. 0-6. 0~, i s i n;jec ted rca trornamrrrari 1 y> intracapsularry and/or subfascially, in two or three stages depending on the individual anatomic mamrn<:~ peco l,~,rr~i ~,i cs, usual ly in the amours t o f 40-160 ml ( bra t rnc:~ t exceed i ng 200 rnl 7 per mamma per stage.
05 In phalloplasty, tyre CI~ t'AA hydrogel h<iving a preferable concentration wi thin the range of 4. 5--6. 0~ and the most preferable concentr<~.t;ion cU' '>.(~, is, ,-vs a rule, intracavernously injected irutr~ trrrc:ye trabecular segments along each side of the penis. A total amount of the CL PAA
hydrogel required for cn~e tolallc:>pl~~slaic; oper;~ti.on is preferably within the range of 40 to 60 ml. The: specific amount of the hydr~ogel to t~f~ in,jecJ teed i.> calcul,-cted from the criteria of acceptablEe volume arrcl resilieruce of the penis, possible compression of t;he irrethr~a exclE~cl~.ed.
Tl~e novel t iocarn~~at iloLce ~~u~c~r7o~~el was c;l inically tested.
Specifically, it was ar~:ipliec:i fc~r cosmetic; correction of fac ial corrgerri t,al defer t:z a:rrrd in rraarrEm~>plr~.:~ty ir~r cases of aplasia and hypomastia in wornc.~n.
Pa t i en t reco rds, toy w<ry c:~ f' exrrm~a 1 e, are pr~f::esen ted as follows:
( 1 ) Pa t i en t, M. ( ca;.~e r~cecc_>rcl Nc:~. 1 aLi) , b. 1965.
Diagnosis: Caongenital rvigt~t--,ide m<~ndibula-neuro-muscular craniafacial microsomia.
Treatment (general anesl;toesi~r: ir~tr<~venc~u:~ and NLA):
- the first stage (Noverotver, l~l~ay - two injfjctions of 10 ml of the 3. 5°& Cf., f-'AA hydr~oge l intramuscularly;
- the second stage (,Ir.rrma, l~~t~4) - the injec:,tions and the hydrogc~l (15 ml) as abav~:.
Improvement s of r~ec~.~rcj: ttuc~ r~i~;ht and lef t sides of the face appearved symrnetri~~al.
Patient 1,. (ca;~e record No. 1~r) , b. 196'l, porous.
Diagnosis: Symmetrical rnarr~rna apl.asia..
Treatment in three stages ( local anesthe;~ia: 0. 5~
novocain solution, 80 ml):
- the first stage (Jarruary. 1001 ) - intramuscular, retramammar, and suY~c;<~~~;_,r~lar~° injections of 740 ml of the 6.0~ CLy PAA tnydrwogcy~l int,c4 kett~ breasts;

- the second stage (March, 10t~1) -- the injections and the hydrogel (40 ml) as above=:~;
- the third :stage (Ma~y> 1991) - tree injections and the hydrogel (f~0 ml ) as <~bovc~.
05 Impravemerrt is of recur°rl: the patient's breasts, in shape and dimensions are consi~~terut,, wi th her physique;
their resilience is similar° t,o natur°al soft tissur:~s.
(3) Patient N. (c ase record Na. '7i3) , b. 11369, rnonparous.
Diagnosis: Symmetrical rrypoma~~~ria.
Treatment (local anesthe;;ia: Co.S~ novocain solution, 80 ml):
- the first stage (F ebruary, 1903) - intr amuscular>
retromammar°, arid subcapsular° irijectiorns of 130 ml of the 6. O1 CI, PAA rrydrogel intro heath breasts;
- the second stage (March, 10~t3) -- tile injec:,tions and the hydragel ( 100 ml ) a.s above.
Improvement is of record as ire the above cage.

(4) Patient I~u. (~;ase r~ec~card Ncl. 1'7L) , b. 1967, porous.
Diagnosis: Symmetr°icai rryporna:~tia.
Treatment (local anest,he.:>i~r: C-. ~>~ novocain solution, 80 m1):
- the first stage (January, 1904) - intramuscular>
retromarrrmar> and subcapsular~° injections of 130 ml of the 6.0~ CL PAA nydrc~gel into both breasts;
- the second stage (July, 1004) -- the injections and the hydrogel (60 ml) as above.
Improvement is of r°ecord as in the above case.
The effect of the tr°e<~tment, in trris case, was additionally evaluated frarrr tree data obtained by computerized axial tomogrjaprry c:af t;h~J crres~t on a SIEMENS
"SONATRON CR" tomograph CGermaruy) by scanning each 8 mm deep section of the trweatecf nramrna irr a sr.zpine pass. tion. Two tomograms, of the many that were taken are shown here as follow,:
Fig. 1 i l lustrates the lef t breast of ter treatment for malformation anc:i for° :=~ize correction:
Fig.2 illustrates similar form and size correction of tile right breasi.

~~j As can be seen ire the i llustrat;ions> both the mammas, as a resin t of" the treatnuerat;> < rw found in a normal situation arid have a regularw form. ~>kir~ thickness does not exceed ~. 0 mm, tLre te<~.ts and at~eo la:> acre normal (nei then 05 deformed nor drawn lrr). Ilypopl~r:atlc~ glandular tissue of both the mammas i.~ ventrally ;hif t,f:ct lay t;lre ~;L PAA hydrogel (dif ferring in clen,:~i t;~ camparve~~ do that of tile tissue) injected into the retr>arnarrrmar>y ~>pac~E~ (cFer~sity of glandular tissue is +;3. 0 to +4. 0> d~ansi ty q~~'' tlrf=~ hydrogel is +4. 6 to +7. 2, and densi ty of hypoclcar>mi~, ad) pa,e cellular tissue is --73 to -95 urri t:~ Hu) .
Glandula rnamrrraria ditnE~rn~:ions off~er~ treatment are as follows:
transvers<~.l taf 7.4 cm dextra efi 8.0 cm sinistr<~.;
anteroposterior about, F~.O c;m df:~xtna E:t sinistra.
Regional lymphonodes are not en:lar>gfad, ossear~s tissues of breast borre:~ arnd ribs are r~armally ~trTuctured.
Laboratory, experitnent<~1 arrd ~~linical date, allow to conclude that the navel CL 1'AA hydt~a'gel is chemically and bioligically stable> inert, bioccawnt.~a~,iL:~le, and perfectly suitable for implantation as encLofrr~~~~stl~eses> for filling of caverns and for formation of z~-rtr~~~tissue storage sites for prolonged-action medic;iraal preparations.
Tyre novel biocampatible hydrsage:l was tested as a medium for long--term cardi.o-- and encepnalography an samples having the CL PAA corrcentr~atlcarl w~ t;hin the range of 4_ 0--8. 0% and prepared on thc: 0. 9;~ ,rciueous solution of sodium chloride arid sodium acetat;cD.
The test included the followinf:
measurement of elecl~ricfal z~~,°s~istivity of the hydrogel arranged as 1 mm layer l:aetweerr rli:>k-type E~lectrographic electrodes ( type EI~:MI~-6;r ~~ rrirrt ir~t diamE3ter arid 3 mm in thickness, with <~ tin-, c:oppc:~r- ar al~.~rnirrum-plated contact surface, measurement of tyre electrical resistivity as to its 24-hour s tab i 1 i ty" acrd determinatioln of abil.itiE~s to bear prolonged (1, '7>
and 15 day observation) app:l i c:;at i or~:,~ ta:~ a Corearnr skin area near the elbow of medical stuff volunteers including two men and two women.
The electrical resistivity was from 8.0 to 9.0 kOhm/cm _ of the BCH6 and BCH? samples and from 10.0 to 20.0 kOhm/cm 05 of the BCH8 and BCH9 samples; and it remained unchanged for each sample during a 24-hour period, measurements being repeated an electrode paste commercially available from SIEMENS AG has an electrical resistivity of about 8.0 kOhm/cm.
In all tests, the polarizability of . tin-plated electrodes was about 450 mV, of copper-plated electrodes was 150 mV, and of aluminum-plated electrodes was about '~00 mV. In measuring the electrical resistivity no parasitic polarization was felt.
On visual observation, none of the skin application sites, during the above mentioned periods, showed visible irritations (reddening or pruritus) and, what is more, skin injuries (maceration). On day 15 in one case, the skin around the plaster covering the hydrogel application turned pink in one of the women-volunteers.
Spontaneous flow of the CL PAA hydrogel having viscosity of 10-11 poise, out of the space between the horizontally arranged measuring electrodes or from underneath the plasters, was not observed.
These data indicated that the novel CL PAA hydrogel is applicable as an immersion medium for monitoring the electrophysiologic parameters of a human organism and for electrophoretic drug injection through the skin.

Claims (15)

1. Biocompatible hydrogel for placing endoprosthesis by injection, containing cross-linked polyacrylamide produced by radical polymerization and pyrogen-free water, said cross-linked polyacrylamide constituting from 3.5 to 6.0% by weight based on the total weight of the hydrogel.

2. An injectable biocompatible hydrogel prepared from a hydrogel comprising pyrogen-free water and about 3.5% to about 6% polyacrylamide by weight, wherein the resulting biocompatible hydrogel is used for the preparation of a prostheses.

3. The use of the hydrogel of claims 1 or 2 for addressing a cosmetic or functional defect.

4. The use of the hydrogel of claims 1 or 2 in the manufacture of an endoprosthesis injectable for in-situ treatment of a cosmetic or functional defect.

5. The use of claims 3 or 4, wherein the biocompatible hydrogel is prepared from a hydrogel comprising about 3.5 to about 6.0% by weight polyacrylamide and pyrogen-free water.

6. The use of claims 3 or 4, wherein the defect is any one of mammaplasty, phalloplasty, or cosmetic defects.

7. The use of claims 3 or 4, wherein the defect is aplasia or hypomastia.

8. The use of claims 3 or 4, wherein the cosmetic defect is a facial congenital or facial cosmetic defect.

9. The use of claims 3 or 4, wherein the defect is mandibuloneuromuscular craniofacial microsomia.

10. The use of claim 4, wherein the endoprosthesis is intramuscular.

11. The use of claim 4, wherein the endoprosthesis is subcapsular.

12. The use of claim 4, wherein the endoprosthesis is subfacial.

13. The use of claim 4, wherein the endoprosthesis is intracavernous.

14. The use of claim 4, wherein the amount of biocompatible hydrogel injected is from about 1.5 mL to about 200 mL.

15. An injectable biocompatible hydrogel prepared from a solution of about 3.5% to about 6% polyacrylamide by weight, wherein the resulting hydrogel is used for the preparation of injectable prostheses.