traditionally , tissue engineering scaffolds have been made by the solvent casting ( mikos , a . g ., et al ., “ preparation and characterization of poly ( l - lactic acid ) foams ” polymer 35 : 1068 , 1994 ) and gas foaming ( harris , l . d ., et al ., “ open pore biodegradable matrices formed with gas foaming ” j biomed mater res 42 : 396 , 1998 ) processes . in the solvent casting process ( mikos , a . g ., et al ., “ preparation and characterization of poly ( l - lactic acid ) foams ” polymer 35 : 1068 , 1994 ), a polymer is dissolved in a suitable solvent , added to a porogen - containing mold and the porogen is dispersed . the solvent is then allowed to evaporate from the mixture under ambient conditions leaving a polymer matrix containing a porogen which can then be leached out in an appropriate solvent . in the gas foaming process ( harris , l . d ., et al ., “ open pore biodegradable matrices formed with gas foaming ” j biomed mater res 42 : 396 , 1998 ), polymer particles are mixed with porogen particles and the mixture is compressed into a solid mixture of a discontinuous polymer with an interspersed porogen . the resulting pellet is then exposed to high pressure co 2 gas , and after the pressure is allowed to equilibrate over a period of time the pressure is rapidly released causing a thermodynamic instability in the polymer component of the mixture . the instability causes the polymer to foam , and the originally discontinuous polymer particles fuse together to form a continuous polymer matrix around interspersed porogen particles , which are then leached out in a solvent . in each of these processes the degree of interconnection between pores in the resulting scaffold is determined by the interconnection of porogen particles during the solvent evaporation or polymer foaming steps , respectively . because the porogen is dispersed within the polymer before fusion , the degree of interconnection between porogen particles is not actively controlled and the interconnectivity of pores in the final scaffold product is uncontrolled . enhancement in and control over porogen interconnectivity may be an important concern in various tissue engineering strategies in view of the substantial advantages of pore interconnectivity within scaffolds . certain embodiments of the present invention solve these problems in control over scaffold porosity and interconnectivity . in a preferred embodiment of the present invention , the porogen is not dispersed . rather , the progen is fused to create a framework ( or lattice ) and a polymer solution is introduced over and into this framework . it is not intended that the present invention be limited by the source of the cells . in one embodiment , the cells used for tissue engineering are from a biopsy . cells from the biopsy are then cultured from explants or a collagenase digestion to create a “ cell bank ”. these cells are then further cultured on substrates and scaffolds , under the correct physiological conditions , to form tissue - engineered constructs for implantation . the process is carried out in a tissue culture facility to maintain a sterile environment . cellular biochemical and physical activity can be enhanced by the addition of growth factors or cytokines and also by the use of physical stimulation . in some applications , a device that applies minute physical loads stimulates the resident cell population in the scaffold into biochemical and bio - physical activity normally associated with organogenesis and tissue repair . after further tissue culture under the correct conditions , the construct can then be implanted back into the patient from whom the cells were originally removed . this technology will eliminate the need for anti - rejection drugs because the tissue engineered tissue has been grown from the patients own cells and , therefore , will be accepted as a natural part of the patients body . in an embodiment of the present invention , salt crystals can be fused via exposure to certain conditions ( e . g ., approximately 95 % humidity ), resulting in enhanced pore interconnectivity within both solvent cast and gas foamed plg scaffolds . fusion of a salt matrix prior to solvent casting results in formation of holes in pore walls , and the diameter and sphericity of these holes increases with increasing salt fusion treatment . salt fusion treatment causes an increase in the compressive modulus of solvent cast scaffolds , possibly due to the formation of thick annular struts adjacent to holes in pore walls . enhanced pore interconnectivity may be useful in a variety of tissue engineering applications , particularly those requiring intimate cell - cell contact ( i . e ., neural and muscular applications ). also , because the salt fusion method imparts improved pore interconnectivity in both the solvent casting and gas foaming processes , the concept may be applicable to other solid porogen - based methods for producing macro - or micro - porous material systems with high interconnectivity . utilization of a fused salt mold in a solvent casting , particulate leaching method results in the formation of holes between pore walls in the scaffold . with increased salt fusion time the pore structure within the scaffold cross sections became less organized . the apparent lack of an organized pore structure is due to the excellent interconnectivity of the salt fused samples , which reduces the presence of well - organized , largely closed - off pores . upon scaffold bisection many of the pores have flattened out due to their lack of a continuous pore wall . in effect , the increased continuity of the fused salt matrix creates corresponding discontinuity in the polymer matrix , leading to large openings between pores and superior interconnectivity . additionally , intact samples could not be fabricated using salt fusion time periods of 48 hours or more . this further supports the inverse relationship between salt matrix continuity and tissue engineering scaffold continuity . previous studies using solvent casting , particulate leaching processes in which salt is dispersed allow no control over pore interconnectivity in accord with the holes in pore walls displayed in the present study ( mikos , a . g ., et al ., “ preparation and characterization of poly ( l - lactic acid ) foams ” polymer 35 : 1068 , 1994 ; kaufmann , p . m ., et al ., “ highly porous polymer matrices as a three - dimensional culture system for hepatocytes ” cell transplant 6 : 463 , 1997 ; murphy , w . l ., et al ., “ growth of continuous bone - like mineral within porous poly ( lactide - co - glycolide ) scaffolds in vitro ” j biomed mater res 50 : 50 , 2000 ). in a recent study , investigators utilized heat to fuse polymeric porogen particles together prior to solvent casting ( ma , p . x . and choi , j . “ biodegradable polymer scaffolds with well - defined interconnected spherical pore network ” tissue eng 7 : 23 , 2001 ). although the use of heat may prove useful in several tissue engineering applications , the localized dissolution approach described herein may hold more broad applicability due to its potential for room temperature fusion of several types of porogen particle ( both organic and inorganic ), and its potential addition to processing techniques that include bioactive inductive factors ( i . e ., gas foaming / particulate leaching ). the fusion of nacl crystals within plg / nacl mixtures prior to gas foaming also has a pronounced effect on pore structure . the pores within 24 hr of salt fusion ( sf ), gas foamed scaffolds appear to feed directly into one another , implying a very high interconnectivity without a large decrease in scaffold compressive moduli ( see , for example , fig6 b ). the gas foaming process has previously been used to process scaffolds containing biologically active vascular endothelial growth factor ( sheridan , m ., et al ., “ bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery ” j control rel 64 : 91 , 2000 ; murphy , w . l ., et al ., “ sustained release of vascular endothelial growth factor from mineralized poly ( lactide - co - glycolide ) scaffolds for tissue engineering ” biomaterials 21 : 2521 , 2000 ) and plasmid dna encoding for platelet - derived growth factor ( shea , l . d ., et al ., “ dna delivery from polymer matrices for tissue engineering ” nat biotech 17 : 551 , 1999 ) to promote ingrowth of vascular tissue . adding the novel salt fusion method of the present invention to the gas foaming and solvent casting methods has lead to the formation of a highly interconnected vascular supply throughout the interior of a tissue engineering scaffold . achieving vascular ingrowth to maximum depths within a scaffold system is a substantial goal in bulk tissue engineering strategies , and the highly interconnected pore structure of the present invention is advantageous for optimal vascular tissue ingrowth . when exposed to humid environments , adjacent salt crystals fuse in a process called ‘ caking ’, which often results in the formation of large agglomerations of rock salt or improperly stored table salt ( anti - caking agents , such as calcium silicate , are added to table salt to prevent caking , essentially by absorbing moisture inside the package that otherwise would be absorbed into the surface of the salt particles ). in a preferred embodiment , the present invention does not contemplate the use of anti - caking agents . the rate of diffusion of atoms within the solid salt crystal lattice is increased by the presence of absorbed water . the increased diffusion allows the surfaces of the contacting salt particles to coalesce , forming bridges between particles in a process similar to that used for solid sintering of non - vitreous ceramic materials . the individual particles begin to coalesce because , in the process , the total surface area of the salt particles is reduced , thus reducing the surface energy ( van vlack , l . h . “ elements of materials science and engineering ,” 4ed . addison - wesley publishing company , reading , mass ., pp . 120 & amp ; 316 , 1980 ). the increased sphericity of each particle of salt is also thermodynamically favored , since this also reduces the total surface energy of each particle . the solvent cast scaffolds had a significantly increased compressive modulus after 24 hours of salt fusion , whereas the gas foamed scaffolds did not . the solvent cast scaffolds the thicker struts of plg material that were permitted to form in the space vacated by rounded corners and edges of the salt particles formed a stiffer structure , without an increase in the volume fraction of plg in the scaffold . a similar increase in the modulus of the gas foamed scaffolds did not occur with increased nacl fusion time . this may be due to the presence of plg particles during the salt fusion in the gas foaming process . undoubtedly there is some void space for interaction between adjacent nacl crystals , even in the presence of both types of particles ( nacl and plg ). the displacement of the salt surface resulting from diffusion was restricted to movement within the available void space . evidence in support of this is clear in fig5 ( b & amp ; d ), in which the scaffold is composed of micro perforated sheets , not present in the solvent cast scaffold , suggesting that during the nacl fusion process the moving salt crystal surface was obstructed by , and perhaps flowed around , the smaller plg particles . thus , although bridges were formed between adjacent salt particles , the movement of the crystal surfaces was constrained by the presence of the plg particles . this may have prevented the growth of void spaces in the salt structure that would lead to the formation of thick - section struts in the plg scaffold , explaining why there was increased pore interconnectivity , but not increased compressive modulus , in the gas foamed scaffolds . although not limited to any particular application , the salt fusion method of embodiments of the present invention will be applicable to the engineering of neural and muscular tissues due to their dependence on pore interconnectivity . regenerative processes in the bridging of neural tissue defects ( axonal elongation ) and the development of functional skeletal muscle tissue ( myoblast fusion ) are examples of physiological processes requiring intimate cell - cell interaction . strategies to bridge nerve gaps using a variety of natural and synthetic scaffolding materials have been only moderately successful even in gaps less than 10 mm in length ( valentini , r . f ., et al ., “ collagen - and laminin - containing gels impede peripheral nerve regeneration through semipermeable nerve guidance channels ” exp neurol 98 : 350 , 1987 ; aldini , n . n ., et al ., “ effectiveness of a bioabsorbable conduit in the repair of peripheral nerves ” biomaterials 17 : 959 , 1996 ), and reasons for failure in many cases include lack of adequate pore interconnectivity and inadequate mechanical integrity of the conduit . recent studies using porous poly ( lactic - co - glycolic acid ) ( evans , g . r . d ., et al ., “ tissue engineered conduits : the use of biodegradable poly ( d , l - lactic - co - glycolic acid ) scaffolds in peripheral nerve regeneration ” in : stark , g . e ., horch , r ., tanczos , e ., eds . biological matrices and tissue reconstruction . berlin : springer , 1998 , pp . 225 - 235 ) and poly ( l - lactic acid ) ( evans , g . r . d ., et al ., “ in vivo evaluation of poly ( l - lactic acid ) porous conduits for peripheral nerve regeneration ” biomaterials 20 : 1109 , 1999 ) scaffolds for neural regeneration have shown promise in 12 mm nerve defects in a rat sciatic nerve model . extension of this basic concept to larger , critical nerve defects requires controlled pore interconnectivity to allow vascular ingrowth , avoid pruning of regenerating fibers during axonal elongation and ensure that elongating axons reach their target organs . enhanced and controlled pore interconnectivity are necessary scaffold characteristics to promote successful myoblast fusion . further , the survival of cells within a functioning muscle organoid is diffusion limited ( dennis , r . g . and kosnik , p . e ., “ excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro ” in vitro cell dev biol — animal 36 : 327 , 2000 ) and thus ingrowth of vascular tissue is essential to increase the maximum diameter of functional muscle constructs in order to amplify contractile properties . although not limited to any particular application , certain embodiments of the present invention ( e . g ., the salt fusion process ) are particularly applicable in preparing highly interconnected scaffolds for neural and muscular applications . although the present invention is not limited to any particular theory , the substantial advantages of pore interconnectivity in promoting three dimensional cell - cell interaction are believed to aid in the growth of neural and muscular tissue in tissue engineering scaffolds . the following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof . in the experimental disclosure which follows , the following abbreviations apply : eq ( equivalents ); m ( molar ); μm ( micromolar ); n ( normal ); mol ( moles ); mmol ( millimoles ); μmol ( micromoles ); nmol ( nanomoles ); g ( grams ); mg ( milligrams ); μg ( micrograms ); l ( liters ); dl ( decalitters ); ml ( milliliters ); μl ( microliters ); cm ( centimeters ); mm ( millimeters ); μm ( micrometers ); nm ( nanometers ); ° c . ( degrees centigrade ); rda ( representational difference analysis ); nts ( nucleotides ); kv ( kilovolts ). in this example , nacl frames and biodegradable polymer scaffolds are produced . the salt particles ( mallinkrodt , paris , ky .) were sieved to yield a range of sizes . nacl crystals of a diameter of about 250 - 425 μm were used . porous scaffolds were prepared either by solvent casting / particulate leaching , or gas foaming / particulate leaching processes using nacl as the particulate porogen . the solvent cast scaffolds were prepared essentially as described by mikos , a . g ., et al . (“ preparation and characterization of poly ( l - lactic acid ) foams ” polymer 35 : 1068 , 1994 ; which is incorporated herein by reference ). nacl molds were made by subjecting nacl crystals ( diameter of about 250 - 425 μm ) to 95 % humidity for periods from 0 - 24 hr to achieve fusion of nacl crystals prior to solvent casting . a closed , water - jacketed cell culture incubator ( forma scientific , inc .) held at 37 ° c . was used to create a 95 % humidity environment for fusion of nacl crystals . poly ( lactide - co - glycolide ) ( plg ) pellets with a lactide : glycolide ratio of 85 : 15 were obtained from medisorb , inc . ( intrinsic viscosity ( i . v . )= 0 . 78 dl / g ) and boehringer - ingelheim inc . ( i . v .= 1 . 5 dl / g ). high inherent viscosity plg was used in the solvent casting process to ensure that the scaffolds would retain adequate mechanical integrity despite their relatively high porosity (˜ 97 %). plg pellets were dissolved in chloroform ( mallinkrodt , paris , ky .) to yield a solution of 10 % weight / volume ( w / v ). the polymer solution was then poured into an nacl - containing mold wherein the salt crystals had been fused , as described above . following solvent evaporation , the salt was removed by immersion in distilled water for about 48 hours . the gas foamed scaffolds were essentially prepared as described by harris , l . d ., et al . (“ open pore biodegradable matrices formed with gas foaming ” j biomed mater res 42 : 396 , 1998 ; which is incorporated herein by reference ). nacl molds were made by subjecting nacl crystals ( diameter of about 250 - 425 μm ) to 95 % humidity for periods from 0 - 24 hr to achieve fusion of nacl crystals prior to solvent casting . following treatment in 95 % humidity samples were dried in a vacuum desiccator for 48 hr before further processing . a closed , water jacketed cell culture incubator ( forma scientific , inc .) held at 37 ° c . was used to create a 95 % humidity environment for fusion of nacl crystals . plg pellets ( prepared as above ) were dissolved in chloroform . frames of fused nacl were mixed with plg were loaded into an aluminum die ( 1 . 35 cm diameter ; aldrich chemical co ., milwaukee , wis .) and was compressed at 1500 psi for 1 minute using a carver laboratory press ( fred s . carver , inc ., menominee falls , wis .) to yield solid disks ( thickness of about 3 . 4 mm ). the samples were then exposed to high pressure co 2 gas ( 800 psi ) for 24 hours to saturate the polymer with gas . a theromodynamic instability then was created by decreasing the gas pressure to ambient pressure . this lead to the nucleation and growth of co 2 pores within the polymer matrices . the nacl particles subsequently were removed from the matrices by leaching the matrices in distilled water for 48 hours . all processing steps were performed at ambient temperature . scaffolds were circular disks with a diameter of about 12 mm and a thickness of about 3 mm . the pore size range was controlled by using nacl particles with a diameter of about 250 - 425 μm in the processing . the total porosity of scaffolds was calculated using the known density of the solid polymer , the measured polymer mass of the scaffold , and the measured external volume of the scaffold . in this example , the scaffolds are characterized . incubation of nacl crystals in 95 % humidity resulted in fusion of the crystals , creating a highly interconnected nacl matrix ( fig1 a - b ). fused salt molds were bisected and imaged prior to solvent casting to observe the extent of nacl crystal fusion . in addition , polymer scaffolds were bisected after preparation via freeze fracture . a carbon coating was evaporated onto the surface of each bisected salt mold and polymer scaffold , and samples were imaged under high vacuum using a hitachi s - 3200n sem operating at 20 - 30 kv . fusion of salt crystals prior to addition of plg in chloroform ( solvent casting ) resulted in enhanced pore interconnectivity within the scaffold . the pore structure within the scaffolds ( fig2 ) appears similar to the structure of the fused salt matrix ( fig1 a - b ), as expected . pores within the cross section of 1 hr salt fusion ( sf ) samples display a defined pore structure with intermittent holes in pore walls , ( fig2 a , 2 c ), while the cross section of scaffolds created from 24 hr sf samples display a much less organized pore structure and a very large density of holes in pore walls ( fig2 b , 2 d ). the hole size increased significantly with fusion time , from an average diameter of 31 ± 10 μm after 1 hour of fusion to 78 ± 21 μm after 24 hours of fusion ( p & lt ; 0 . 05 ). in addition , the pore walls in the 24 hr sf scaffolds display thickness contours such that the walls appear thicker in the area adjacent to the holes in pore walls and along the outer diameter of the walls ( fig2 d ). a higher magnification view of a pore wall within a 24 hr sf scaffold further displays the contoured structure of the pore walls ( fig2 e ). the salt fusion process had no effect on the porosity of the scaffolds , and the calculated total porosities of the solvent cast scaffolds for each salt fusion time period were 97 ± 1 %. a close examination of the electron micrographs of the solvent cast scaffolds formed after 1 and 24 hours of nacl fusion indicate that the exposure to 95 % humidity has caused several important changes in the structure of the salt particles . in addition to the formation of bridges between particles at the points of contact , the radius of curvature of edges and corners in individual particles of salt has increased ( fig1 a and b ). these changes are shown schematically ( fig3 ). the radius of curvature of salt crystals was calculated from electron micrographs using microsoft ™ paint ™ software . the pixel size for each image was calibrated , and the pencil tool was used to mark tangent points on crystal edges . the calibration values and pixel coordinates were then used to calculate the cord length between tangent points , which was multiplied by ( 2 / 2 to obtain the crystal radius of curvature . the diameter of holes in pore walls was determined by measuring the major and minor diametral axes of each hole using microsoft paint and taking the average . the increased radius of curvature at the edges and corners of each particle of salt results in an increased sphericity of each particle ( fig4 ), and thus in each resulting pore in the scaffold . the mean radius of curvature of the crystal edges increased from 19 ± 10 μm , to 32 ± 15 μm after 12 hours of exposure to 95 % humidity , then to 62 ± 18 μm after a full 24 hours of exposure ( fig4 ). as a result , many of the smaller crystals became nearly spherical in shape after 24 hours of fusion . one additional consequence is that thicker polymeric struts may be formed in the space vacated by the corners and edges of each salt crystal , which may result in the thickness contours in pore walls described above and in varied mechanical properties . fusion of salt crystals in plg / nacl pellets prior to gas foaming also resulted in a pronounced variation in pore structure . the cross section of 1 hr sf samples ( fig5 a , 5 c ) shows small holes in pore walls similar to those in the solvent cast 1 hr sf samples . the 24 hr salt fusion samples lack a defined pore structure and pores appear to simply feed into each other ( fig5 b , 5 d ). the gas foamed sf scaffolds do not display any of the contours in pore walls observed in the solvent cast sf samples . again , the salt fusion process had no effect on the total scaffold porosity . the total porosities of the gas foamed scaffolds for each salt fusion time period were 94 ± 1 %. fusion of salt crystals for 24 hr resulted in a 2 - fold increase in the compressive modulus of the solvent cast scaffolds ( fig6 a ). compressive moduli of scaffolds were determined using an mts bionix 100 mechanical testing system . samples were compressed between platens with a constant deformation rate of 1 mm / min . compression plates had a diameter of 45 mm , and thus covered the entire 12 mm diameter surface of the scaffold . a small pre - load was applied to each sample to ensure that the entire scaffold surface was in contact with the compression plates prior to testing , and the distance between plates prior to each test was equal to the measured thickness of the scaffold being tested . compressive moduli were determined for scaffolds without salt fusion and for each of four samples for each salt fusion time . values on graphs represent means and standard deviations . statistical analysis was performed using instat ™ software , version 2 . 01 . at each time point , experimental moduli were compared to control moduli via a student &# 39 ; s t - test to reveal significant differences in compressive modulus . no significant modulus change is observed after 1 hr , or 12 hr of salt fusion . alternatively , there was a statistically significant decrease in the compressive modulus of gas foamed scaffolds processed using salt fusion when compared with control scaffolds ( fig6 b ). in this example , the scaffolds of the present invention are use for the culture of cells . the scaffold is sterilized by gamma radiation , ethylene oxide or cold sterilants . cells are seeded on to the scaffold in an appropriate culture medium with any necessary growth factors , if needed . cell culture media may be replaced by batch feeding or by perfusion methods . sterility is maintained at all times . in this example , scaffolds of the present invention are used for the implantation of tissue into the body of test subjects . in other examples , scaffolds are used for direct implantation without tissue or cells to allow the conductive and inductive migration of cells from the body . biopsies are taken form 16 mice . biopsies are taken from any non - hemopoietic or hemopoietic tissue . biopsies are taken from the same tissues source from all test animals . this example may be preformed on as many tissue types as desired . cells are disassociated from basement membranes and syncytia by collagenase treatment . cells from 8 of the mice are cultured in petri dishes . cells from the other 8 mice are cultured on the scaffolds of the present invention . after culture of about 7 to 21 days , the cells or scaffolds with cells are implanted back into the respective mice . follow up observation shows that the cell / scaffold implants of the present invention become invaginated with blood vessels and maintain a three dimensional structure . follow up observation shows that the cells cultured without the scaffolds of the present invention dissipate into the host animal and do not become invaginated nor maintain a recognizable three dimensional structure . in examples where cells migrate into the scaffold from the surrounding tissues , follow up observation shows establishment of cells in the scaffolds with invagination of circulatory vessels . as is evident from the foregoing , the present invention contemplates novel compositions and methods for the production of tissue engineering scaffolds . these novel compositions and methods allow for the efficient production of biocompatable structures of consistent pore size and interconnectivity .
the present invention relates to methods and compositions for the production of scaffolds , such scaffolds to be used for a variety of purposes , including tissue engineering . more specifically , the present invention relates to the use of fused crystals , such as fused salt crystals to form a framework . the methods for producing the scaffolds of this invention improve the porosity , interconnectivity and ease of manufacture as compared to prior art methods .