text: in accordance with the present invention , it is contemplated that various types of implantable biological tissue derived from numerous animal sources and parts of the anatomy can be made resistant to calcification . thus , the tissue can be derived from various sources such as but not limited to bovine , porcine , horse , sheep , kangaroo , or rabbit ; and can include tendons , ligaments , heart valves , or tissue used to construct heart valves such as dura mater and pericardium . it is further contemplated that tissue used for augmentation such as skin patches , pericardial patches , aortic patches , and tympanic membranes is suitable in the present invention . in accordance with a preferred embodiment of the present invention , porcine heart valves or pericardial tissue which was fixed in glutaraldehyde and subsequently treated with a surfactant was implanted subcutaneously in rabbits . this treated tissue unexpectedly and beneficially effected a sustained mitigation or reduction of calcification after implantation . this sustained mitigation of calcification provides a method of increasing the durability of implanted tissue , particularly of heart valve bioprostheses . in accordance with the present invention , the tissue may be stored and processed in conventional well - known conditions and may be fixed ( tanned ) conventionally in from about 0 . 2 to about 0 . 6 weight percent and preferably from about 0 . 5 to about 0 . 7 weight percent glutaraldehyde in either phosphate - buffered solutions , or phosphate - free buffers as described hereinafter . the tissue handling conditions as conventionally known are not considered part of out present invention unless otherwise stated . likewise , tissue may be sterilized in 0 . 625 percent glutaraldehyde or from about 4 to about 5 percent formaldehyde . organic surfactants within the scope of the present invention include anionic , cationic , and nonionic surfactants and their salts . preferred salts of the surfactants in the present invention include sodium , potassium , ammonium , and halide . anionic surfactants of the present invention are those having a relatively large hydrophobic region of hydrocarbon residues including both aliphatic groups , aromatic groups and combinations thereof bonded to a negatively charged ionic group . the aliphatic residues may be branched chains , straight chains , cyclic , heterocyclic , saturated or unsaturated . these hydrophobic residues may either be connected directly to an anionic group such as carboxylate , sulfate , or sulfonate ; or connected thereto through an intermediate linkage such as an ester , amide , sulfonamide , ether , or aryl group . anionic surfactants in one embodiment of the present invention are those having carboxylates bonded to the alkyl side chain of a steroid or through amino acids in the side chain ; such as in the bile acids . illustrative bile acids in accordance with the present invention include but are not limited to deoxycholic acid , cholic acid , lithocholic acid , taurocholic acid , and glycocholic acid , and their salts . a preferred bile acid and its salt which we have found effective in mitigation of calcification of implanted tissue is sodium deoxycholate . anionic surfactants in accordance with the present invention further include those having a carboxylate group bonded to a straight - chained aliphatic group preferably having from about 8 to about 20 carbon atoms ; such as the sodium salts of fatty acids . anionic surfactants containing carboxylate groups in accordance with the present invention further include those having the carboxylate group coupled to a hydrophobic portion through an amide , sulfonamide , or ester linkage such as in the n - alkanoyl amino acids and n - acylated amino acids . illustrative of n - alkanoyl amino acids are those including but not limited to surfactants having the formula r 1 conr 2 chr 3 co 2 -- where r 1 is an aliphatic residue preferably having from about 8 to about 18 carbon atoms , r 2 is hydrogen or methyl , and r 3 is a conventional amino acid side chain . illustrative side chains include the non - polar aliphatic side chains of alanine , leucine , isoleucine , valine , and proline ; the aromatic rings of phenylalanine and tryptophan ; the polar side chains of glycine , serine , threonine , cystine , and the like ; and the charged polar groups of aspartic acid , glutamic acid , lysine , and the like . preferred carboxlate containing surfactants in accordance with this embodiment of the present invention are those containing an amide linkage such as n - lauroylsarcosine . anionic surfactants in accordance with an alternate embodiment of the present invention include ethylene oxide modified sulfates of aliphatic alcohols , sulfated ethanol amides , or alkyl phenols such as the sulfonated alkylphenyl ethers . further anionic surfactants include alkane sulfonic acids and alkylaryl sulfonic acids . alkane sulfonic acids in accordance with the present invention include those having the sulfur directly attached to the hydrophobic residue , such as 1 - decanesulfonic acid ; or coupled through an ester , amide , or ether ; such as n - methyltaurine . alkylaryl sulfonates are those having the sulfur directly attached to an aromatic ring such as phenyl or napthyl which is , in turn , coupled to the hydrophobic residue preferably having from about 8 to about 18 carbon atoms . illustrative of this latter type of surfactant is dodecylbenzenesulfonic acid . cationic surfactants in accordance with the present invention include alkyl quaternary amines and their halide salts . preferably surfactants in the present invention include the chloride and bromide salts of tertiary amines connected directly to a hydrophobic residue or connected thereto through an amide linkage . preferably the amines are directly connected to a relatively large hydrophobic portion having an aromatic residue such as benzene , pyridine or napthylene ; aliphatic chain which is branched , unbranched , cyclic , saturated , or unsaturated ; or a combination of both aromatic and aliphatic residues . illustrative alkyl quaternary ammonium surfactants include but are not limited to cetylpyridinium chloride , cetyltrimethylammonium bromide , trimethylphenylammonium chloride , decyltrimethylammonium bromide , hexdecyltrimethylammonium bromide , and the like . nonionic surfactants in accordance with the present invention include polyoxyalkylene ethers , polyoxyalkylene alkylaryl ethers , aliphatic esters , polyethers , polyoxyalkylene ester derivatives , saccharide ester derivatives , and combinations thereof . nonionic polyoxyalkylene , and preferably polyoxyethylene , ethers are those having a relatively long hydrophobic residue and a hydroxyl end connected by one or more alkylene oxide residues . examples of polyoxyalkylene ethers are polyoxyethylene lauryl ether ( brij ), polyoxyethylene oleyl ether , polyoxyethylene cetyl ether , and the like . nonionic polyoxyalkylene , and preferably polyoxyethylene , alkylaryl ethers are those having a relatively large hydrophobic residue and a hydroxyl end connected thereto by an aryl , such as benzene or napthaline and one or more alkylene oxide residues . examples of polyoxyalkylene alkylaryl ethers include polyethylene glycol p - isooctyl phenyl ethers such as triton x - 100 and the like . nonionic polyethers are those having the formula ch 3 ( ch 2 ) n -- o --( c 2 h 4 o ) m where n is about 11 , and m is about 23 . nonionic aliphatic esters include aliphatic fatty acid esters , polypropyleneglycol fatty acid esters such as propyleneglycol monostearate , and glycerol fatty acid esters such as glycerol monostearate . aliphatic fatty acid esters are those having the formula r 4 coor 5 where r 4 is an alkyl preferably having from about 8 to about 20 carbon atoms , and r 5 is an aliphatic residue having from 1 to about 5 carbon atoms . saccharide and polyoxyalkylene ester derivatives are those having either a 5 or 6 carbon sugar in the former or a polyoxyalkylene chain in the latter coupled to a relatively long hydrophobic residue through an ester bond . illustrative saccharide derivatives include sorbitol coupled to fatty acids to form surfactants such as sorbitan trioleate , sorbitan strearate , sorbitan monooleate , and the like . polyoxyalkylene ester derivatives include polyoxyethylene monooleate , polyoxyethylene monostearate , and the like . combinations of polyoxyalkylene ether derivatives and sorbitol ester derivatives found to be useful in the present invention include polyoxyethylene sorbitan fatty acid derivatives such as polyoxyethylene ( 20 ) sorbitan monooleate ( polysorbate - 80 , tween - 80 manufactured by difco ). in accordance with the present invention , the effective concentration of surfactant will vary somewhat depending on the molecular weight thereof ; and is preferably from about 0 . 1 to about 10 percent ( w / v ) and more preferably from about 0 . 5 to about 5 percent . most preferably , the surfactant concentration is from about 0 . 5 to about 1 . 5 percent . moreover , the treatment of the tissue with surfactant can be performed during the fixation ( tanning ) process , during the sterilization process , or in a separate step after fixation and prior to sterilization ; and from about 2 to about 30 hours and preferably from about 6 to about 24 hours . in accordance with a preferred embodiment of the present invention , the tissue is treated with surfactant at temperatures of from about 20 ° c . to about 40 ° c . in one embodiment , the surfactant is included in the sterilization step whose effectiveness has been found to be enhanced at temperatures above room temperature ( 20 ° c .) to a range of from about 30 ° to 40 ° c . in accordance with the present invention , it is preferable to store , fix , and sterilize the tissue within a tissue - stabilizing ph range ; that is , within a ph range that is not deleterious to the tissue components . a preferred ph range is from about 7 . 0 to about 7 . 6 , and a more preferred ph range is from about 7 . 1 to about 7 . 4 . the most preferred ph in accordance with the present invention is 7 . 3 . buffers used in accordance with one embodiment of the present invention are preferably stable , non - interacting with the stabilization process , and have a buffering capacity sufficient to maintain an acceptable ph , particularly during the fixation of the tissue . the choice of the appropriate buffer , and its concentration will depend upon specific tissue preparation conditions ; variations of which have been introduced by several manufacturers . the buffers can be either conventional 0 . 01 - 0 . 02m phosphate - buffered saline ( pbs ) or phosphate - deficient solutions such as those containing less phosphate than these 0 . 01 to 0 . 02m pbs solutions , and preferably less than about 0 . 001 to about 0 . 002m phosphate . preferred buffers in accordance with the present invention include borate , carbonate , bicarbonate , cacodylate ( found to be nontoxic in animals ), and other synthetic , artificial , or organic buffers such as hepes , n - 2 - hydroxyethylpiperazine - n &# 39 ;- 2 - ethanesulphonic acid ; mops , 2 -( n - morpholino ) propane - sulfonic acid ; and pipes , 1 , 4 - piperazinediethanesulphonic acid . preferably , the buffered or unbuffered solutions , used in accordance with the present invention should not interfere with the tissue stabilizing process afforded by fixing agents such as glutaraldehyde . that is , they should not react with the fixing agent or prevent the fixing agent from achieving proper fixation of the tissue . illustrative of this are buffers containing primary and secondary amines such as tris ( hydroxymethyl ) aminomethane ( tris ), which are known to react with the aldehyde groups of glutaraldehyde or formaldehyde and thus interfere with the normal tissue stabilization process . the present invention is further illustrated by the following examples which are not intended to be limiting : extracted porcine aortic heart valve tissue was throroughly rinsed and shipped in an isotonic ( 285 ± 15 milliosmols ) solution of 0 . 02m phosphate - buffered saline ( 0 . 885 weight percent sodium chloride ) at ph 7 . 3 and at about 4 ° c . ; and fixed in 0 . 625 weight percent glutaraldehyde in an isotonic phosphate - buffered solution at ph 7 . 4 and at room temperature . extracted porcine aortic heart valve tissue was thoroughly rinsed and shipped in an isotonic ( 285 ± 15 milliosmols ) solution containing 0 . 54 grams / liter of the sodium salt of n - 2 - hydroxyethylpiperazine - n - 2 - ethanesulphonic acid ( hepes ) and 0 . 885 weight percent sodium chloride at ph 7 . 3 at about 4 ° c . ; and fixed with 0 . 625 weight percent glutaraldehyde in an isotonic solution containing 5 . 39 grams / liter of the sodium salt of hepes ( 0 . 02m ), 0 . 440 weight percent sodium chloride and 2 . 6 grams / liter of mgcl 2 . 6h 2 o at room temperature . the extracted tissue of example i was further sterilized in a 0 . 02m phosphate - buffered saline ( 0 . 885 weight percent sodium chloride ) solution ( 3 square inches of tissue in 70 ml ) containing 4 ± 0 . 4 percent sorbitan monooleate polyoxyethylene ( tween - 80 ), ph 7 . 3 at 35 ° c . the tissue was removed from the solution after 24 hours , rinsed 4 times with 0 . 625 percent glutaraldehyde in 0 . 02m phosphate - buffered saline for 10 minutes each and implanted subcutaneously in growing rabbits . the value tissue was retrieved up to six weeks later at regular one - week intervals . after retrieval , the extent of tissue calcification was assessed by quantitatively monitoring the weight percent calcium in dried tissue using atomic absorption analysis ; and histologically by visually monitoring the degree of calcification in von kossa - stained tissue sections . both the histologic and quantitative results indicate that the implanted valve tissue effected a significant reduction in calcification compared to a valve tissue treated identically to that described herein in all essential details with the exception that no tween - 80 was added . the extracted tissue of example ii was further sterilized in a 0 . 02m hepes ( 5 . 39 gram / liter of the sodium salt ) buffered saline solution ( 3 square inches of tissue in 70 ml ) containing 4 ± 0 . 4 percent formaldehyde , 22 . 5 percent ethanol , 0 . 26 grams / liter mgcl 2 . 6h 2 o , at ph 7 . 3 and 35 ° c . the tissue was removed from the solution after 24 hours , rinsed 4 times with 0 . 625 percent glutaraldehyde in 0 . 02m hepes - buffered saline for 10 minutes each , and implanted subcutaneously in growing rabbits . the valve tissue was retrieved up to six weeks later at regular one - week intervals . after retrieval , the extent of tissue calcification was assessed by quantitatively monitoring the weight percent calcium in dried tissue using atomic absorption analysis ; and histologically by visually monitoring the degree of calcification in von kossa - stained tissue sections . the results of the histologic and quantitative analyses were used for comparison with results obtained for tissue treated with various surfactants . the extracted tissue of example ii was further sterilized in a 0 . 02m hepes ( 5 . 39 gram / liter of the sodium salt ) buffered saline solution ( 3 square inches if tissue in 70 ml ) containing 4 ± 0 . 4 percent formaldehyde , 22 . 5 percent ethanol , 11 . 3 mm ( 1 . 5 weight percent ) sorbitan monooleate polyoxyethylene ( tween - 80 ), 0 . 26 grams / liter mgcl 2 . 6h 2 o , at ph 7 . 3 and 35 ° c . the tissue was removed from the solution after 24 hours , rinsed 4 times with 0 . 625 percent glutaraldehyde in 0 . 02m hepes - buffered saline for 10 minutes each , and implanted subcutaneously in growing rabbits . the valve tissue was retrieved up to six weeks later at regular one - week intervals . after retrieval , the extent of tissue calcification was assessed by quantitatively monitoring the weight percent calcium in dried tissue using atomic absorption analysis ; and histologically by visually monitoring the degree of calcification in von kossa - stained tissue sections . both the histologic and quantative results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated identically to that of example v in all essential details with the exception that no ethanol was added . after sterilization and rinsing the tissue was implanted in growing rabbits and retrieved up to six weeks later at regular one - week intervals . after retrieval , the extent of tissue calcification was assessed by quantitatively monitoring the weight percent calcium in dried tissue using atomic absorption analysis ; and histologically by visually monitoring the degree of calcification in von kossa - stained tissue sections . both the histologic and quantitative results indicate that there was no effect of the presence of ethanol in the surfactant solution on mitigating calcification . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 24 . 0 mm triton x - 100 ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 57 . 2 mm 1 - decanesulfonic acid ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 45 . 9 mm dodecylbenzenesulfonic acid ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 42 mm potassium coconut fatty acid hydrolyzed protein ( maypon - 4c ) ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 55 . 3 mm n - lauroylsarcosine ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 36 . 2 mm deoxycholic acid ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 53 . 5 mm decyltrimethylammonium bromide ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 41 . 2 hexadecyltrimethylammonium bromide ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the extracted tissue of example ii was treated , implanted in growing rabbits , and analyzed identically to that of example v in all essential details with the exception that 87 . 4 trimethylphenylammonium chloride ( 1 . 5 weight percent ) was used in place of tween - 80 . the results indicate that the implanted valve tissue effected a significant reduction in calcification compared to the valve tissue treated in accordance with example iv which did not include surfactant . the tissue treated in accordance with the process of example iii was further analyzed to assess the integrity of the tissue after exposure to surfactant . the results of our analysis show that there was no significant difference in the cross - link stability as indicated by shrinkage temperature , tissue stability as indicated by pronase digestion ; amino acid analysis , ninhydrin analysis ; uronic acid content , histologic examination as indicated by staining with hematoxylin eosin , aldehyde fuschin , pas / alcian blue , and trichrome ; and surface morphology as determined by scanning electron microscopy and transmission electron microscopy . the present invention has been described in specific detail and in reference to its preferred embodiments ; however , it is to be understood by those skilled in the art that modifications and changes can be made thereto without departing from the spirit and scope thereof .