3-Methoxybutylcyanoacrylate: evaluation of biocompatibility and bioresorption

1077 %Methoxybutylcyanoacrylate: evaluation of biocompatibility bioresorption and Alex M. Henderson and Martin Stephenson* Ethicon Lfd (a Johnson & Johnson Company), 1421 Lansdowne St W., Peterborough, Ontario KSJ-769, Canada The biocompatibility and bioresorption of 3-methoxybutylcyanoacrylate (MBCA) was evaluated in vivo using female Wistar albino rats. MBCA was found to elicit slight to moderate tissue reaction similar to isobutylcyanoacrylate (IBCA) which has been sold commercially as a surgical adhesive (Bucrylate@, Ethicon). MBCA was judged less reactive to tissue than ethylcyanoacrylate (ECA). The MBCA implants in rat gluteal muscles also resorbed within approx. 16 wk while iBCA implants remained essentially unchanged at 36 wk in vivo. In vitro resorption in phosphate buffer (pH 7.2) at 37°C showed the same trend. The MBCA performed similarly to iBCA as a haemostat on excised rat livers and as an adhesive on rat skin incisions and had comparable adhesive bond strength. Keywords: Received Cyanoacrylate, 30 October biocompatibility, 1991; revised 6 December Various means of haemostasis have been used by the medical profession, including ligation, application of pressure, application of absorbent pads and haemostatic agents such as thrombin and alkyl-2-cyanoacrylate adhesives. Surgical wound closure is typically effected by sutures, staples and clamps. Alkyl-Z-cyanoacrylate adhesives are used as adjuncts to these traditional methods and also in procedures where they are considered the superior technique. The isobutyl and n-butyl-2cyanoacrylates have been sold commercially as surgical adhesives under the trade names Bucrylate@ (Ethicon Inc., Somerville, NJ, USA) and HistoacrylTM (B. Braun Melsungen, AG, Germany). Matsumoto has reviewed the use of these and other adhesives in surgical applications1 and Coover has reviewed cyanoacrylate development’. Cyanoacrylate monomers are not universally bacteriostatic or bacteriocidal and need to be sterilized before use as surgical adhesives31 ‘, Acidic stabilizers such as SO, added to the cyanoacrylate allow them to be heatsterilized at temperatures up to 150°C (Refs 5, 6). Increasing the level of stabilizers has been reported to reduce the strength of the tissue adhesive bond and increase polymerization time7. SO, inhibitor forms a protonic acid in contact with body fluids. Proton transfer can increase the termination of anionic polymerization, lengthen conversion time of monomer to polymer and lower the molecular weight of the polymer’. Correspondence to Dr A.M. Henderson at present address: AT Plastics Inc., Technical Centre, 142 Kennedy Rd. S., Brampton, Ontario L6W-3G4, Canada *Deceased. 0 1992 Butterworth-Helnemann Ltd 0142-9612/92/151077-08 bioresorption 1991; accepted 20 January 1992 Isobutylcyanoacrylate (iBCA) and n-butylcyanoacrylate (BCA) have low toxicity toward tissue and elicit only slight tissue reaction but these monomers produce polymeric material which is not significantly resorbed in viva. In many applications the adhesive remains in the soft tissue as a hard foreign material after it has outlived its usefulnessgs’O. Methylcyanoacrylate (MCA) was found to degrade much faster in vivo than the homologues with longer chain alkyl groups”. The methyl, ethyl and isobutyl homologues have been reported to be cytotoxic and MCA is mutagenic to bacteria’2-‘5. A lack of mutagenic potential was observed in ECA, BCA and iBCA13. Tseng et al. reported that iBCA did not inhibit 3T3 cell growth”j. In general, it has been found that as the alkyl group on the cyanoacrylate becomes longer, the tissue reaction elicited by the cyanoacrylate decreases, the wetting of the tissue by the adhesive increases and the rate of in vivo degradation decreases7, 17-‘l. This has led to the search for cyanoacrylate tissue adhesives which resorb in vivo and which spread well on tissue but elicit only minimal tissue reaction. Two primary mechanisms of bioresorption of cyanoacrylates have been proposed: a retro-Knoevenagel condensation to yield formaldehyde and cyanoacetate (Leonard et al.“] and hydrolysis of the ester group to produce polycyanoacrylic acid and alcohol (Lenaerts et al. 23). The in vivo resorption of the alkoxyalkyl-z-cyanoacrylates was first described by Banitt and Nelsonz4 who studied ethoxyethylcyanoacrylate (EECA), methoxyethylcyanoacrylate (MECA), butoxyethylcyanoacrylate (BECA), 2-methoxypropylcyanoacrylate (MPCA), Z-isopropoxyBiomaterials 1992, Vol. 13 No. 15 1078 Biocomnatibility of 3-methoxybutylcyanoacrylate: ethylcyanoacrylate (iPECA) and l-methoxyisopropylcyanoacrylate (MIPCA). The alkoxyalkyl cyanoacrylates were found to resorb much faster than the alkylcyanoacrylate homologues, Recently, Tseng et al. studied the in vitro resorption of EECA, iBCA and ECA. The more rapid resorption of EECA was attributed to the increased hydrophilicity of the alkoxyalkyl cyanoacrylate due to its pendant ether moiety25*Z6. The degree of tissue reaction was inversely correlated with the rate of cyanoacrylate degradationZ7, Jaffe et al. describe the synthesis and in viva evaluation of 1,2-isopropylideneglyceryl Z-cyanoacrylate (iPGCA). Resorption of this material in vivo was attributed to incorporation of the inherently hydrolytically labile ketal group”. Synthesis and evaluation of alkyl Z-cyanoacryloyl glycolates as bioresorbable tissue adhesives has also been described”’ 30. Acceptably low tissue reaction was only achieved with polymer pendant groups containing alkyl groups of four or more carbons. The present work was carried out to synthesize and evaluate the in vivo compatibility and resorption of 3methoxybutyl cyanoacrylate (MBCA) and determine its efficacy as a tissue adhesive. MBCA was chosen as a target candidate because to our knowledge it had not been studied previously, the &carbon butyl pendant group was predicted to produce minimal tissue reaction comparable to Bucrylate (iBCA) and Histoacryl (BCA), and the alkoxy moiety was expected to increase resorption. A.M. Henderson and M. Stephenson vacuum overnight in a 100 ml round bottomed flask to remove volatiles. Hydroquinone (0.2 g) and P,O, (0.5 g) was added and the resin heated in an oil bath under vacuum with the 100 ml flask attached to a Bantamware still. The resin started to crack when the oil bath reached approx. 17O’C and the cyanoacrylate distilled at 85”C, 400 PmHg. The distillate was yellow so P,O, was added and the product re~stilled in a Bantamware apparatus at 68”C, 456 FmHg (yield - 40%). Anhydrous SO, was added to a known weight of cyanoacrylate using a gas-tight syringe. For example, 100 p 1 SO,/g cyanoacrylate corresponds to approximately 0.02% w/w. The MBCA was analysed bylH n.m.r. in CDCl, using a Varian 300 MHz spectrometer and found to be greater than 99% pure. A typical ‘H n.m.r. spectrum is shown in Figure 2. The cyanoacrylate purities are summarized in Table 1. Analysis of SO2 content The cyanoacrylates were analysed for SOZ content using a Hewlett-Packard 5890A gas chromatograph with a thermal conductivity detector and a Hewlett-Packard 3390A integrating recorder, A Poropak-T column was used with a glass liner in the injection port containing glass wool to prevent polymer from depositing on the column, Standards were prepared in dried 7 ml septum vials. The results of the analyses of the MBCA, iBCA and ECA are summarized in Table 2. Synthesis of Zkmethoxybutylcyanoacetate The 3-methoxybutylcyanoacetate precursor to the cyanoacrylate was synthesized by methods similar to those previously described3’% 32, Cyanoacetic acid (ZOOg) was refluxed with 244 g 3-methoxybutanol in 800 ml Ccl, containing 0.5 g ~-toluenesulphonic acid (PTSA) with stirring. The water of reaction was removed over 3 d with a Dean-Stark trap. The reaction mixture was washed twice with 400 ml distilled water containing 35 g sodium bicarbonate. The organic layer was dried with anhydrous calcium chloride, the solvent removed with a rotary evaporator and the product distilled twice (8Z°C, 500 FmHg). The product was a water white liquid (yield >70%). ----A 10 a Synthesis of 3-methoxybutylcyanoacrylate The 3-methoxybutylcyanoacetate (171 g), paraformaldehyde (6.7 g) and piperidene catalyst (50~1) were refluxed with stirring in toluene (75 ml) for 8-12 h while removing the water of reaction with a Dean-Stark trap. Once no more water collected in the trap the reaction was stopped. The reaction mixture was washed first with 100 ml 4% PTSA in distilled water and then with 100 ml distilled water. The organic layer was dried with anhydrous sodium sulphate and filtered through a 10 pm Millipore Teflon* filter and the solvent removed on a rotary evaporator. The resulting product was a hard tacky resin the colour of honey. This resin was heated at 90°C under Biomaterials 1992. Vol. 13 No. 15 6 4 2 0 ppm Figure1 ‘H n.m.r. spectrum of 3-methoxybu~icyanoac~iate. Table1 Purity of experimental cyanoacrylates and cyanoacetate as determined by ‘H n.m.r. spectroscopy Compound Purity 3-methoxybutylcyanoacetate 3-methoxybutylcyanoac~late (MBCA) isobutylcyanoacrylate (iBCA) ethylcyanoacrylate (ECA) >99.8% w/v 99.6 rt 0.3% wan 99.5% W/Vb >99.0% WIVC of five lots. bBucrylate” supplied by Ethicon plc, Edinburgh, *Average ‘Supplied by Permabond International Co. UK. Biocompatibility Table 2 Sulphur dioxide content (measured by gas chromatography, Cyanoacrylate of the cyanoacrylates TCD detector) SO, content isobutylcyanoacrylate (IBCA) 3-methoxybutylcyanoacrylate ethylcyanoacrylate (ECA) Heat treatment A.M. Henderson of 3-methoxybutylcyanoacrylate: (MBCA) (O/ow/w) 0.020 0.022 0.024 of MBCA and sterility testing MBCA (1 g) containing 0.02% SO, was pipetted into an amber 2 ml glass ampoule and pure MBCA (1 g) was loaded into a second ampoule. The MBCA was blanketed with prepurified nitrogen and then flame-sealed in the ampoules. The ampoules were heated in a forced air oven for 6 h at 100°C. At the end of the heat treatment the cyanoacrylate containing SO, did not appear macroscopically to have increased in viscosity, while the and M. Stephenson 1079 cyanoacrylate without SO, had solidified. Heat-treated cyanoacrylates containing SO2 were used in all further studies. Two flame-sealed ampoules containing 1 g each of MBCA which had been heat-treated for 6 h at 100°C were tested for sterility. The MBCA samples were each pipetted under aseptic conditions into 25 ml aliquots of tryptic soya broth (TSB) and incubated at 32 f 2°C for 14 d. The aliquots were examined macroscopically for growth. There was no evidence of growth in the two samples. The bioburden appeared to be very low or nonexistent in these two samples. In viva biocompatibility and bioresorption Female Wistar albino rats were used as the animnal model in the in viva evaluations of adhesive effectiveness, ?tidti&t+eaction and absorption. All rats were held in the animal colony for at least 1 wk before being used in the Figure3 Histological sections of adhesive implants in rat gluteal muscle (original magnification X40): a ethylcyanoacrylate, b isobutylcyanoacrylate, and c 3-methoxybutylcyanoacrylate implants recovered after 1 wk; d isobutylcyanoacrylate recovered after 36 wk; e 3-methoxybutylcyanoacrylate recovered after 16 wk. Biomaterials 1992, Vol. 13 No. 15 1080 procedures. guidelines Laboratory _. Biocompatibility of 3-methoxybutylcvanoacrvlate: __ _-____ _ They were maintained according to the set down by the Canadian Association of Animal Sciences and had food and water ad libitum. All procedures were carried out under general anaesthesia (Metofane:methoxyflurane@, Pitman-Moore Inc., Washington Crossing, NJ, USA) using aseptic techniques. The cyanoacrylates were injected into the gluteal muscles of rats using a 16 gauge Teflon Cathlon I.V.@ catheter (Critikon Inc., Tampa, FL, USA). A 2 cm incision was made parallel to the spinal column in the dorsal skin to expose the gluteal muscle groups. The muscles were freed from the overlying fascia by blunt dissection. Once the catheters were placed in the muscles, the needle portions were withdrawn leaving the Teflon tube in place. Syringes (1 ml) containing the experimental adhesives were attached to the metal luer locks of the catheters and the adhesives introduced into the muscles. The catheter was slowly withdrawn from the muscle as the adhesive was injected to allow polymerization in situ. Each injection was of 0.050.07 ml. The adhesive did not adhere to the Teflon to allow catheter removal. This technique produced rodlike implants of fairly consistent cross-section. The skin incisions were closed with 11 mm Michel clips. For ECA and iBCA implants, two rats were killed by CO2 inhalation post-operatively at 1, 4, 8, 12, 16 and 36 wk. For MBCA implants, six rats were killed by CO, inhalation post-operatively at 1,4,8,12,16 and 24 wk. At 1 wk the implants averaged 2 mm in diameter although they were irregular in shape. For all in viva absorption/tissue reaction evaluations of the cyanoacrylate tissue adhesives, the implanted gluteal muscles were recovered at designated times, fixed in 10% buffered formalin and processed in methacrylate. The plastic sections were cut at 3.5 iurn on a rotary microtome and were stained with Oil Red 0 and then haematoxylin and eosin in a manner similar to that evaluation described by Galil et al, 33 Histopathological ‘ of overall tissue inflammatory response was conducted using the method of Sewell et a1.34. Absorption was estimated semiquantitatively as the approximate percentage of tissue adhesive remaining at designated intervals after implantation. Until absorption was marked, an ocular micrometer was used at low power to approximate the cross-sectional diameters of the implanted tissue adhesive in the gluteal muscle. Polymerization chromatography and size exclusion (SEC) analysis S~~uijo~ ~u~~erizaijon MBCA was polymerized in acetone solution [l g in 10 ml dry (0.04% H,O) HPLC grade acetone) by addition of 1OOpl 10% sodium bicarbonate solution. Heat was evolved after the addition of the bicarbonate solution to the mixture. The mixture was allowed to stand overnight in a sealed flask. The polymer was recovered as a fluffy white powder by precipitation in distilled water and filtration. Polymerization in rat glu teal m uscle MBCA was introduced into the left and right gluteal muscle groups respectively in two female Wistar albino Biomaterials 1992, Vol. 13 No. 15 A.M. Henderson and M. Stephenson rats. Cathlon I.V. Teflon catheters were used to guide the adhesives. The catheters were placed in the muscle group and syringes containing the tissue adhesives were attached to the catheters. In each case a large bolus of adhesive (0.5-0.6 ml) was introduced into the muscles by simultaneously injecting the adhesive and pulling the catheter out of the muscle. The process took approximately 2 min to ensure complete polymerization of the adhesive, After 1 d the animals were killed with CO, and the polymerized glue recovered as a 5-6 mm diameter lump from the muscles. The muscle surrounding the adhesive was peeled away before analysis. SEC analysis The molecular weights of the polymers were measured using size exclusion chromatography (SEC) in hexafluoroisopropanol (HFIP) solvent with micro-Styragel columns. The results are summarized in Table 3. In vitro resorption The in vitro solubilization of cyanoacrylate polymers which were produced by solution polymerization in acetone were studied. Weight loss The fluffy polymer powders (‘I g) were stored in 25 ml phosphate buffer (pH 7.2) at 37°C. The phosphate buffer was prepared by dissolving 1.135 g potassium phosphate [monobasic) and 3.552 g sodium phosphate (dibasic) in 500 ml distilled H,O. At specified times the polymer was collected by filtration, dried and weighed to determine weight loss with time, Two samples of each MBCA and iBCA were studied. One of the MBCA samples was recovered at 3, 7, 21 and 35 d. After each weighing, the remaining polymer was returned to fresh buffer. The other MBCA sample was recovered after 56 d. One iBCA sample was recovered at 3,14,28,42 and 84 d. After each weighing, the remaining polymer was returned to fresh buffer. The other iBCA sample was recovered after 777 d. ‘H n.m.r. analysis of resorption products In vitro hydrolysis studies were also carried out at Ethicon (Somerville, NJ, USA) using D,O. ‘H n.m.r. analysis of the D,O soluble fraction was carried out using a Varian 300 MHz spectrometer (D.B. Johns and C. Ace, personal communication). Skin-adhesive bond strength The bond strengths of cyanoacrylates were evaluated by adhesion to rat skin which had been freshly harvested. Strips of rat skin with the subcutaneous fat and fascia Table 3 Site exclusion chromatography (HFIP solvent, Waters Styragel Columns): Molecular weights of cyanoacrylate polymers Polymer Mol wt (~“/~~~ PoI~(MBCA)~ PoI~(MBCA)~ 10000/11000 aPolymerized in vitro(acetone solution). bPolymerized in viva(rat gluteal muscle). MBCA. 3-methoxybutylcyanoacrylate. 18 000179 000 Biocompatibility of 3-methoxybutylcyanoacrylate: A.M. Henderson removed (2 cm X 4.8 cm] were positioned on a moist towel so that the strips overlapped an area of 1 cm’. The upper strip was folded back as a flap to expose the lower overlap area poised for gluing. With one hand positioned to lower the skin flap quickly after the glue was applied, three drops of cyanoacrylate were applied to the lower piece of skin overlap area. The upper flap was then immediately folded on to the glue. After a prescribed time the bonded strips were positioned in the jaws of an Instron tensile tester and the tensile strength of the bond determined under the following conditions: 2.5 cm gauge 2.5 cm/ length, - 140 000 Pa (20 p.s.i.) gauge pressure, min crosshead speed. An estimation of long-term effectiveness of the adhesives in closing incisions was made by evaluating the approximation of full-depth dorsal skin incisions in rats 7 d post-operation. The complete dorsal skin of female Wistar rats was cleanly shaved, wiped free of all loose hair, washed with an antiseptic and bactericide and painted with Ioprep@ (Surgikos Inc., Arlington, TX, USA) presurgical solution. Incisions of approx. 1.5-2.0 cm in length were made through the full depth of the dorsal skin using a scalpel or very sharp surgical scissors. Incisions were placed one each in the left and right gluteal region or the left and right scapular region. Excess blood was sponged away from the wound surface. The edges of the incisions were everted with the aid of mosquito forceps. The adhesive under test was painted into either one or both of the everted edges using heat-sterilized artist’s brushes and the incisions re-approximated very quickly by manipulation of the mosquito forceps. Topical applications of the experimental adhesives were made to seal the incision. When the procedures were completed each rat was placed in a separate clean cage and allowed to withdraw slowly from the effects of anaesthesia. Haemostasis of excised rat liver An estimation of the effectiveness of the cyanoacrylates as haemostats was obtained by applying the adhesive to excised rat livers and comparing the ease of application, speed of haemostasis. and presence/absence of bleeding leaks. Two rats were used for each adhesive. The left lateral lobes of the livers were exposed and externalized (using plastic drapes as barriers] through a ventral incision in the skin, pectoralis and underlying muscle groups. The incision was caudal to the diaphragm and slightly to the right of the midline. The tip of each liver lobe (15-30 mm) was excised. Gentle finger pressure was used to control and reduce the bleeding from the excision. Excess blood present on the wound surface was sponged away just before application of the adhesive. The tissue adhesives were then painted quickly on to the excised liver surfaces using heat-sterilized artist’s brushes. The adhesives were allowed to polymerize before releasing finger pressure on the lobes. In most cases the rats were not allowed to regain consciousness and were killed immediately after the procedure by a Metofane overdose. To allow longer term ev’aluation of the wound, the muscle layers were sutured in a continuous manner and the skin incision closed using a subcuticular stitch. The rats were observed closely for a week after the operation. 1081 and M. Stephenson After 1 yr the rats were killed by CO, inhalation and the livers exposed and externalized for observation as described earlier. RESULTS AND DISCUSSION Molecular weights of poly(3-methoxyhutylcyanoacrylate) The SEC results in Table 3 indicate that the molecular weight distribution (MWD) of the polymer produced by applying cyanoacrylate to tissue was wider than the polymer produced in solution under controlled conditions. Therefore it is important to study resorption in vim. Since it has been reported35 that renal glomerular tubules are relatively impermeable to mol wt above approx. 60 000, the mode of resorption of the high mol wt fraction of the polycyanoacrylate produced in vivo is important. If the resorption mechanisms proceeds primarily via the retro-Knoevenagel route as proposed by Cameron et al.” (found to be the case in vitro for poly(EECA) by Tseng et aLz5, then excretion by the kidneys should occur. If the resorption mechanism occurs primarily via hydrolysis of the ester pendant group (as found in vivo for poly(iBCA) nanoparticles by Lenaerts et al.‘” then excretion of the high mol wt portion may be inhibited. The in vitro resorption mechanism for poly(MBCA) was examined in a preliminary fashion by lH n.m.r. analysis. In vitro resorption Figure 2 compares the in vitro resorption in phosphate buffer (pH 7.2 at 37°C) of polymeric powders of iBCA and MBCA which were prepared by solution polymerization in acetone followed by precipitation. The mol wt of the poly(MBCA) are given in Table 3. The mol wt of the solution polymerized polymer of MBCA is lower and the MWD narrower than that of the polymer produced in vivo. The poly(MBCA) is resorbed substantially in 3 months, while the poly(iBCA) was essentially unchanged after 26 months. The poly(MBCA) wetted faster than the poly(iBCA) and sank sooner in the buffer solution. Both polymers stayed as powders and did not clump noticeably. If the poly(MBCA) was left to sit several days and the jar gently swirled so as not to disturb the settled polymer particles, then swirls of dissolved polymer could be seen leaving the solid as the concentration gradient was 100 ,+-+,+\ k +-+ Time (days) Figure 2 In vitro resorption of cyanoacrylates (pH 7.2, 37°C). 0, 3-methoxybutylcyanoacrylate; i-, isobutylcyanoacrylate. Polymer produced by solution polymerization in acetone. Biomaterials 1992, Vol. 13 No. 15 1082 Biocomoatibilitv Figure Skin-adhesive the results of skin adhesive bond strengths for MBCA compared with iBCA and ECA. MBCA was equivalent to the other adhesives in strength and was not noticeably different in set-up time (i.e. within seconds of application). Table 6 shows that, in this preliminary testing, the MBCA adhesive was comparable to ECA and only slightly less effective than iBCA in the approximation of full-depth dorsal skin incisions in rats at 7 d postoperation. and bioresorption Haemostasis sections from gluteal muscle implants of the adhesives. All photomicrographs were taken at magnification X46 for comparison. Table 4 summarizes the in viva absorption and tissue reaction of the cyanoacrylates. ECA showed more initial tissue reaction at 1 wk [moderate] compared with iBCA [slight] and MBCA [slight to moderate) as expected from its shorter alkyl pendant group. Significantly, iBCA was inert as an implant and was essentially unchanged even after 36 wk in viva. However, MBCA was 95-100% resorbed after 16-24 wk in viva. The photo shows the remains of an implant. In some cases no evidence of the MBCA implant could be found in the gluteal muscle. This In viva absorption Cyanoacrylateb Time and tissue period (wk) reaction Absorption 1 rat liver Table 7 summarizes the preliminary tests of effectiveness of the adhesives iBCA, ECA and MBCA as haemostats applied to excised rat livers. MBCA compared favourably with the other adhesives and provided effective rapid haemostasis of the excised liver. After 1 yr implantation in one rat, gross observation of the externalized liver revealed that iBCA remained on the surface of the liver. There was no apparent absorption, After 1 yr implantation in two rats, gross observation of the externalized liver revealed that MBCA was present in a small amount in one liver and not at all on the other liver. ECA iBCAC 36 1 55 0 iBCA 36 0 1 0 16 24 Tissue (%) 0 95 100 MBCA MBCA of excised of cyanoacrylatesa ECAC MBCAd bond strength Table 5 summarizes 3 shows histological Table 4 and M. Stephenson is further evidence that introduction of the alkoxy moiety to the alkyl pendant group increasesin viva resorption, disturbed. The polar alkoxy group increased the wettability of the poly(MBCA) compared with the poly(iBCA) which increased potential for resorption via retroKnoevenagel or hydrolysis. ‘H n.m.r. analysis of the D,O soluble fraction from in vitro resorption studies of poly(MBCA) in D,O indicated that the major species present in the D,O were 3-methoxybutanol and a polymer of the poly(cyanoacrylic acid) type. A small resonance at 4.8 p.p.m. was also observed which could indicate trace formaldehyde (D.B. Johns and C. Ace, personal communication). These results suggest that the hydrolysis resorption mechanism predominates in vitro for poly(MBCA). Biocompatibility A.M. Henderson of 3-methoxybutylcyanoacrylate: reaction - moderate - no significant necrosis - macrophages and lymphocytes were predominant, polymorphs evident -slight with macrophages predominant -slight - no significant necrosis evident - macrophages were the greatest in number at the implant sites -slight - no necrosis evident - slight to moderate - no significant necrosis evident - macrophages were predominant, however a few polymorphs were present - slight with macrophages being greatest in number - slight with a few macrophages almplanted in rat gluteal muscle. bContain -0.2% w/w SO,, heated 6 h at 100°C in flame-sealed ampoules. ‘Two rats (four muscles) examined per time period. dSix rats (12 muscles) examined per time period. ECA. ethylcyanoacrylate; IBCA, isobutylcyanoacrylate; MBCA, 3-methoxybutylcyanoacrylate Table 5 Rat skin-adhesive bond tensile strengthsa Adhesiveb Glue cure time (min) Tensile ethylcyanoacrylate (ECA) isobutylcyanoacrylate (IBCA) 3-methoxybutylcyanoacrylate 15 15 15 0.90 * 0.2 1.07 * 0.2 1.05 + 0.2 (MBCA) aApproximately 1 cm* bond overlap area, average of five pulls. bHeated 6 h at 100°C in flame-sealed glass ampoules, contain -0.2% Biomaterials 1992, Vol. 13 No. 15 w/w SO,. strength (kg) Comments Glue peeled Glue peeled Glue peeled from from from skin skin skin Biocompatibility Table 6 A.M. Henderson of 3-methoxybutylcyanoacrylate: Soundness of rat skin incision9 closed with cyanoacrylate Adhesiveb Time ECAC 7 iBCAC 7 MBCAd 2 MBCA 7 adhesive Comments 6 of 6 incisions remained closed 4 of 6 incisions remained closed period Incision 1083 and M. Stephenson (d post-op) soundness 5 of 8 incisions remained closed 8 of 8 incisions remained closed Tended to polymerize too accurate approximation tended to run over the control Exhibited good handling over the surgical site as contain rapidly to allow neat and of skin incisions. Also surgical site, difficult to properties. It did not run much as iBCA, easier to al 52.0 cm dorsal skin incisions in rats. bHeated 6 h at 100°C in flame-sealed glass ampoules, contain -0.2% w/w SO,. ‘Two rats each with two scapular Incisions, two rats each with two gluteal incisions. dTwo rats each with two gluteal incisions, one rat with two scapular incisions, one rat did not revive from anaesthesia ECA. ethylcyanoacrylate; IBCA. isobutylcyanoacrylate; MBCA, 3-methoxybutylcyanoacrylate Table 7 Adhesive haemostasis of excised rat liver Adhesivea Ease of applicationb Polymerization ECA - good - one brush stroke no repeat application necessary - good - one brush stroke no repeat application necessary - good - one brush stroke no repeat application necessary - slightly iBCA MBCA %eated 6 h at 100°C In flame-sealed glass ampoule. bT~o rats used for each adhesive. ECA. ethylcyanoacrylate: IBCA. isobutylcyanoacrylate; Haemostasis - immediate contain -0.2% after 1 yr - 2 rats tested. - after 1 yr MBCA was found in a small amount on one liver and not at all on the other liver on MBCA. 3.methoxybutylcyanoacrylate. Introduction of a polar methoxy moiety into the butyl pendant group of an alkyl cyanoacrylate (iBCA or Bucrylate) produced 3-methoxybutylcyanoacrylate which had similar tissue reaction to isobutylcyanoacrylate but which resorbed more readily. Also MBCA performed similarly to iBCA as a haemostat on excised rat liver and as an adhesive on rat skin incisions and had similar skin adhesion strength. ACKNOWLEDGEMENT A. Henderson gratefully acknowledges the assistance of Dr D.B. Johns and Dr C. Ace at Ethicon USA for the lH n.m.r. and SEC analyses, MS L.M. McKey of Ethicon Canada for the in vivo and histological work, and Mr M. Mathews for assistance in synthesis. 3 4 5 6 7 6 REFERENCES 2 status w/w SO,. CONCLUSIONS 1 - 1 rat tested. - after 1 yr iBCA remained the liver surface - good, no leaks - some flexibility - difficult to peel - immediate - not tested - good, no leaks - hard coating - difficult to peel delayed Adhesive - good, no leaks - hard coating - difficult to peel time Ulin, A. and Matsumoto, T.: Bucrylate tissue adhesive, in Tissue Adhesives in Surgery (Ed. T. Matsumoto), Medical Examination Publishing Co., Flushing, NY, USA, 1972, pp 226-237 Coover. H.W., Cyanoacrylate adhesive, a day of 9 10 serendipity, a decade of hard work, ACS Organic Coatings and Applied Polymer Science Proceedings 1983, 48, 243-247 Fenzl, T.C., Fenzl, R.E. and Harris, L.. Antimicrobial properties of alkyl-2-cyanoacrylate tissue adhesives invitro, Am. J. Ophthalmol 1983, 95(l),125-126 Matsumoto, T., Bacteriological study of cyanoacrylate tissue adhesives, in Tissue Adhesives in Surgery (Ed. T. Matsumoto), Medical Examination Publishing Co., Flushing, NY, USA, 1972. pp 106-113 Rice, T.B. and Hawkins. G.F.. Method for stabilizing a-cyanoacrylate, Canadian Patent 876.131, July 20, 1971, granted to Eastman Kodak Co.. Rochester. NY, USA Wicker, Jr, T.H. and McIntire, J.M., tx-Cyanoacrylate adhesive composition, lJS Patent 3,527,841, Sept. 8.1970, assigned to Eastman Kodak Co., Rochester. NY, lJSA Matsumoto. T., Aron alpha A ‘Sankyo’ Japanese tissue adhesive in surgery of internal organs, in Tissue Adhesives in Surgery (Ed. 7’. Matsumoto), Medical Examination Publishing Co., Flushing, NY, IJSA. 1972, pp 5-8 Odian, G., Principles of Poolymerization. McGraw Hill, NY. USA, 1970, pp 313-341 Matsumoto, T., Aerosol tissue adhesive spray in the repair of injured kidney, in Tissue Adhesives in Surgery (Ed. T. Matsumoto), Medical Examination Publishing Co., Flushing, NY, USA. 1972, pp 40.-41 Matsumoto, T., in Tissue Adhesives in Surgery (Ed. T. Matsumoto), Medical Examination Publishing Co., Flushing, NY, [JSA, 1972, pp 112 Biomaterials 11192. Vol. 13 No. 15 1084 11 12 13 14 15 16 17 18 19 20 21 22 23 Biocompatibility of 3-methoxybutylcyanoacrylate: Leonard, F., Kulkami, R.K., Brandes, G., Nelson, J, and Cameron, J.J., Synthesis and degradation of poly(alkylacyanoacrylates), 1, Appt. Potym. Sci. 1960, 10, 259272 Zumpano, B.J., Jacobs, L.R., Hall, J.B., Margolis, G. and Sachs Jr, E., Bioadhesive and histotoxic properties of ethyl-2-cyanoacrylate, Surg. Neurol. 1982, 18(8), 45% 457 Rietveld, EC., Garnaat, M.A. and Seutter-Berlage, F., Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates, Mutat. Res. 1987, 188, 97-104 Anderson, M., Binderup, M.L., Kiel, P., Larsen, H., Maxild, J. and Hansen, S.H., Mutagenic action of methyl 2-cyanoacrylate vapor, Mutat. Res. 1982, 102, 373381 Papatheofanis, F.J., Cytotoxicity of alkyl-2-cyanoacrylate adhesives, J. Biomed. Mater. Res. 1989, 23, 661-668 Tseng, Y.C., Hyon, S.H. and Ikada, Y., Effect of poly(~,~lactide) addition to 2-cyanoacrylates on their physical properties and toxicity, I. Bioactive Biocompat. Polym. 1989, 4,101-109 Matsumoto, T., Aerosol tissue adhesive spray in the repair of injured kidney, in Tissue Adhesives in Surgery (Ed. T. Matsumoto), Medical Examination Publishing Co., Flushing, NY, USA, 1972, pp 37-40 Ulin, A. and Gollub, S., Hemostasis: isobutyl-Z-cyanoacrylate in surgical bleeding, in Tissue Adhesives in Surgery (Ed. T. Matsumoto), Medical Examination Publishing Co,, Flushing, NY, USA, 1972, pp 238-247 James, P., Clinical use of the alkyl-alpha cyanoacrylate monomers: indications, techniques of application, results and complications, in Tissue Adhesives in Surgery (Ed. T. Matsumoto), Medical Examination Publishing Co., Flushing, NY, USA, 1972, pp 248-269 Gipps, E.M., Groscurth, P., Kreuter, J. and Speiser, P.P., The effects of polyalkylcyanoacrylate nanoparticles on human normal and malignant mesenchymal cells invitro, Int. J. Pharm. 1987, 40, 23-31 Leonard, F., Collins, J.A. and Porter, H.J., Interfacial polymerization of n-alkyl a-cyanoacrylate homologs, /, Appt. PoJym. Sci. 1966,10, 1617-1623 Cameron, J.L., Woodward, SC., Pulaski, E. J., Sleeman, H.K., Brandes, G,, Kulkarni, R. and Leonard, F., The degradation of cyanoac~late tissue adhesive. I, Surgery 1965, 58(Z), 424-430 Lenaerts, V., Couvreur, P,, Christiaens-Leyh, D., Joiris, E., Roland, M., Rollman, B. and Speiser, P., Degradation Biomaterials 1992, Vol. 13 No. 15 24 25 26 27 28 29 30 31 32 33 34 35 A.M. Henderson and M. Stephenson -- of poly(isobuty1 cyanoacrylate] nanoparticles, Biomaterials 1984, 5, 65-68 Banitt, E.H. and Nelson, R.A., Method of adhesively repairing body tissue with alkoxyalkyl 2-cyanoac~late, US patent 3,559,652, Feb. 21971. assigned to 3M Co., St Paul, MI, USA Tseng, Y.C., Hyon, S.H. and Ikada, Y., Modification of synthesis and investigation of properties for 2-cyanoacrylates, Biomaterja~s 1990, 11, 73-79 Tseng, Y.C., Hyon, S.H., Ikada, Y., Shimizu, Y., Tamura, K., Kawarasaki, S. and Hitomi, S., Medical application of cyanoacrylates as surgical adhesives, effects of thickened cyanoacrylates on healing of skin wounds, fpn. 1. Artif. Org. [Jinko ZokiJ 1989, 18(l), 409-413 Tamura, K., Hitomi, S., Kawarasaki, S., Tseng, Y.C., Hyon, S.H., Ikada, Y. and Shimizu, Y., Application of ethoxyethylcyanoacrylate as surgical adhesive, Jpn, 1. Artif. org. ~Jinko ZokiJ 1988, 17(Z), 739-742 Jaffe, H., Wade, C.W.R., Hegyeli, A.F., Rice, R.M. and Hodge, J., Synthesis and bioevaluation of a rapidly biodegradable tissue adhesive: 1,2-isopropylidene glyceryl2-cyanoacrylate,]. Biomed. Mater. Res. 1986,20, 213-217 Kronenthal, R.L. and Schipper, E., Surgical adhesive, US Patent 3,955,641, Dec. 7, 1976, assigned to Ethicon Inc., NJ, USA Jaffe, H., Wade, C.W.R., Hegyeli, A.F., Rice, R, and and bioevaluation of alkyl Hedge, J., Synthesis 2-cyanoacryloyl glycolates as potential soft tissue adhesives, J. Biomed. Mater. Res. 1986, 20, 205-212 Joyner, F.B. and Hawkins, G.F., Method of making a-cyanoacrylates, US patent 2,721,858, Oct. 25, 1955, assigned to Eastman Kodak Co., Rochester, NY, USA Kimura, K. and Sakabe, K., Novel 2-cyanoacrylate, process for producing same and curable composition comprising same, US Patent 4,364,876, Dec. 21, 1982, assigned to Toagosei Chemical Industry Co. Ltd Galil, K.A., Schofield, I.D. and Wright, G.Z., Detection of cyanoacrylate tissue adhesive in histological sections, 1. Biomed. Mater. Res. 1984, 18, 609-616 Sewell, W.R., Wiland, J. and Craver, B.N., A new method of comparing sutures of ovine catgut with sutures of bovine catgut in three species, Surg. GynecoJ. Obstet. 1955, 100, 483-493 Blecher, L., Lorenz, D.H., Lowd, H.L., Wood, AS, and Wyman, D.P., Poiyvinylpyrollidone, in Handbook Of Water-Soluble Gums and Resins (Ed, R.L. Davidson), McGraw Hill, NY, USA, 1980, pp 21.6