Patent Application: US-87768297-A

Abstract:
disclosed and claimed are : a composition including at least one glycosaminoglycan , e . g ., cis , and at least one alginate , e . g ., sodium alginate , wherein : the at least one glycosaminoglycan and / or the algninate are cross - linked or polymerized , e . g ., the alginate is cross - linked or polymerized , for instance by addition of an inorganic salt , such as a calcium salt ; or the at least one glycosaminoglycan and the alginate are covalently bound , e . g ., by means of a coupling reaction involving a linker molecule such as dvs ; or the at least one glycosaminoglycan and / or the algninate are cross - linked or polymerized , e . g ., the alginate is cross - linked or polymerized , for instance by addition of an inorganic salt , such as a calcium salt , and the at least one glycosaminoglycan and the alginate are covalently bound , e . g ., by means of a coupling reaction involving a linker molecule such as dvs , and the covalent binding can have been performed prior to cross - linking or polymerizing or vice versa ; and , gels comprising the composition ; mixtures of such gels or of at least one such gel and at least one such composition ; and , methods for making and using such compositions and gels , including products therefrom such as “ paints ”, sprays , matrices , beads , microcapsules .

Description:
without wishing to necessarily limit the foregoing , the following shall discuss the invention with respect to certain preferred embodiments . it has now been found that the chemical polymerization of an algal polysaccharide , alginic acid , under the form of sodium alginate , with a specific group of animal heteropolysaccharides , e . g ., chondroitin sulfate - 4 and - 6 , cis , results in new materials , e . g ., biodritins , that can be classified as neo - heteropolysaccharide conjugates having novel properties in relation to the parent molecules , while preserving most of the chemical characteristics of both parent molecules . the general properties of this new class of compounds include any or all , and preferably all of biodritins are polyanions , carrying both carboxylate and sulfate groups ( alginate contains carboxylates , while chondroitin sulfate contains both carboxylates and sulfates ); biodritins form infinite gels when treated with calcium ion , a property imparted by the alginate moiety . biodritins dissolve readily in water . additionally , the viscosity of the solutions can be regulated by adjusting the concentration of biodritin , as well as the concentration of calcium . biodritins can also be prepared by polymerization of cis and alginate , in mixed solutions , by addition of calcium ions , thereby resulting in the formation of stable gels of semi - interpenetrating polymer network , in which the cis component is entrapped in the gel network formed by calcium alginate . by adding calcium in sufficient concentration , biodritins form stable gels , as the synthesis process of the invention was designed to preserve , with extreme care , the calcium binding sites in the alginate component . the presence of the chondroitin sulfate component , on the other hand , imparts a stronger negative charge of sulfates and similarity to an extracellular matrix to biodritins , rendering them entirely biocompatible for applications in transplantation of cells and tissues , among others . biodritin - derived materials can have their permeability barrier controlled by ionic complexation with positively charged polymers such as poly - l - lysine or poly - l ornitine , which form a thin external capsule to the gel . the pore size of this capsule can be adjusted as desired , by changing the concentration and molecular weight of the positively charged polymer during capsule formation . in the synthesis of the invention compositions , biodritin preferably has a gag content ranging from 2 to 200 % of the alginate composition by weight , depending on the specific reaction conditions . the final composition can be adjusted by combining biodritin that has been cross - linked with biodritin formed by the gelling of alginate and cis , in the same solution . within this range , the compounds have the desired properties for use as transplantation materials . insoluble materials can also be prepared by controlling the polymerization conditions . these may have utility as fillers in tissue repair , or as surfaces to allow cell growth in culture . the surface of biodritin beads exposed to an aqueous solvent carries the functionalities of both alginate and chondroitin sulfate ( gag ) carboxylates , sulfates , hydroxyls , as well as glycosidic links and the sulfone group of the cross - linker moiety ; the negatively charged sulfate and carboxylate groups are able to form strong ion - ion complexes with polycations such as poly - lysine or poly - ornithine , thereby providing additional external structure and protection to the beads , which has been observed for alginate capsules . these complexes formed at the outer surface of the microcapsules also help to define pore sizes . the viscous solutions obtained by dissolving biodritin in water or saline or other desired aqueous medium are easily handled , especially if the concentration is kept under 4 % ( w / v ) and that they are not exposed to calcium ions . such solutions can be transformed into infinite gels by complexation with calcium ions ; e . g ., by dripping them from syringes or capillaries into solutions of calcium chloride one obtains microbeads of desired spherical size . further treatment with positively charged molecules such as poly - lysine results in a thin external capsule that may , again , be protected by layering biodritin on top of it from a dilute solution . a biodritin solution can be extruded , e . g ., from a thin tubing directly into a calcium chloride solution , whereby a gel in the form of a rod or cylinder of uniform diameter is obtained . cells or tissues can be held within these cylinders for the purposes of culturing or transplantation . the biodritin gel may also be conformed , e . g ., as a slab of desired size and shape where cells or tissues may become entrapped , for purposes of culturing or implantation . another use of biodritin is in the protection of recent sutures : a biodritin sol may be “ painted ” or applied directly with a sterile brush or sprayed around the suture area , followed by spraying with an appropriate calcium chloride solution . a soft , protecting gel forms immediately around the suture area , protecting it from invading cells by blocking cell adhesion . biodritin gel itself can be sprayed , painted or applied on a suture to protect it from adherences . the skilled artisan can determine the suitable viscosity of the gel and the amount thereof to employ to spray , paint or apply , without undue experimentation , from the knowledge in the art , the disclosure herein , and typical factors such as the wound or suture , and the condition of the patient . the chemical coupling of the two major biodritin components , preferably , sodium alginate and chondroitin sulfate - 4 and / or - 6 is carried out by reaction with a linker molecule , e . g ., divinyl sulfone , abbreviated dvs . a linker molecule is a small molecule that is very susceptible to nucleophilic attack ( see u . s . ser . no . 08 / 417 , 652 , incorporated herein by reference , with respect to other types of linker molecules and types of coupling reactions ). the reaction is carried out at alkaline ph , under conditions which allow the calcium binding sites in alginate to be protected from reaction . the final product of the cross - linking reaction is then rendered soluble and can be separated from the reagents through purification by repeated alcohol precipitation . the concentration of linker molecule , e . g ., dvs , used in the reaction can define the final solubility of the glycopolymer complex ; beyond a certain range insoluble materials are formed . an alternative to the chemical coupling of cis and alginate is the preparation of biodritin by formation of a physical gel from a solution containing both polymers , in desired concentrations . this gel forms when calcium ions are added , and the resulting gel is of the semi - interpenetrating polymer network type , s - ipn . in this type of polymeric gel , the cross - linked structure is derived from only one component , e . g . alginate , by the formation of the “ egg - box ” structures resulting from the binding of calcium ions to poly - guluronic blocks in alginate . the resulting gel network entraps and holds the cis chains that are intertwined with alginate , such that cis molecules remain as components of the gel structure , imparting to that gel structure the properties of cis . an element of the invention is the ability of the final product , biodritin , to form gels with calcium ions . this is achieved by protecting the calcium binding sites in alginate , also called “ egg - box ” sites , from reacting with the cross - linking reagent during the synthesis of the chemically coupled variant of biodritin . this is accomplished by adding the appropriate amount of calcium ions to the alginate solution prior to the coupling reaction in such a way that the calcium -“ egg - box ” complexes are pre - formed , while avoiding strong gelling , and the solution co - polymerization reaction is not blocked . early in the purification steps of the final product the protecting calcium ions are removed from the “ egg - box ” complexes , allowing for the dissolution of the particles to form a viscous solution that can , then , be treated as desired . the ability of the inventive compositions to easily and rapidly go from solution to gel and from gel to solution , by manipulation of the calcium ion concentration , is another significant property of the invention , as it leads to additional applications of this new material outside the transplantation field . in another embodiment of the present invention , the chemical combination of alginate , e . g ., sodium alginate and gag , e . g ., chondroitin sulfate - 4 and / or - 6 is made by dehydrothermal reaction , at temperatures of about 100 ° c . in this case , dvs is not used and ester bonds are formed between the alcoholic hydroxyls and acidic groups in alginate and chondroitin sulfate . also using dht , protection of the “ egg - box ” sites is obtained , and this is useful for preservation of the gelling properties of biodritin . it is emphasized that , given the purification steps after reaction , commercially available reactants have been used in all tests herein without preliminary purification , and with no negative effects on the properties of the final product , biodritin . a schematic representation of the complex chemical structure of biodritin is shown in fig1 and 4 , respectively , for the chemically coupled and the physical gel . the following non - limiting examples are given by way of illustration only and are not to be considered a limitation of this invention , many apparent variations of which are possible w / o departing from the spirit or scope thereof . chondroitin sulfate : the commercial cis used contains 70 % of 4 - sulfate and 30 % of 6 - sulfate ; 2 . 5 g was allowed to dissolve in 25 ml 0 . 1 m sodium carbonate solution , to give a 100 mg / ml final concentration . in other formulations , higher concentrations of cis are used , to give final concentrations of 150 , 200 or 250 mg / ml . sodium alginate ( 5 . 0 g ) was suspended in 45 ml of water , heated to 40 ° c ., after which another 30 ml water were added in 10 ml portions to facilitate dissolution and decrease viscosity of the final solution . the amount of sodium alginate used corresponds to 28 . 3 mmoles of the unit disaccharide residue of alginate ( f . wt .= 176 . 2 ). the total blockage of the calcium sites in the alginate solution would require 6 . 5 ml of a 4 . 5 m cacl 2 solution , which is not practical to use . a 4 . 5 m calcium chloride stock solution was diluted 1 : 5 and 200 μl portions were added at intervals to the sodium alginate solution . during additions , and thereafter , the solution was vigorously mixed with the aid of a power mixer to completely avoid formation of gel clumps . eight such additions , plus one , were made , in a total of 1800 μl , corresponding to 1 . 5 mmol of calcium ions added . biodritin preparations can also be made in which the calcium binding sites are blocked to a lesser extent , from 25 % to 75 % of that described above . this also results in soluble materials , provided the coupling with dvs is not extensive . in applications where the s - ipn gel is prepared , protection of the calcium binding sites is not necessary , as there is no chemical reaction with the alginate component of biodritin prior to the gel - forming step . keeping the alginate gel with protected calcium binding sites under continuous , vigorous mixing , 25 ml of the cis solution were added , at which time the solution viscosity decreased . the solution was rapidly homogenized by the vigorous mixing and the coupling reagent , dvs , was added in 10 portions of 0 . 3 ml each , under continuous mixing at 2 - 3 min intervals . when dvs addition was complete the mixture was kept for 2 hours in a water bath at 40 ° c . at the end of this interval the reaction mixture was slightly purple in color ; it was removed from the water bath and left overnight at room temperature . during the reaction steps , continuous mixing is required , which can be easily achieved with help of an electrical mixer with blades or paddles . after reaction is complete , the mixture resembled a mousse . to each 100 ml portion of this material was added 15 ml of 1m nacl and 5 × 1 ml portions of a 0 . 63 m edta solution , to remove calcium ions from the “ egg - box ” sites . mixing was maintained to facilitate solubilization . a very viscous and brownish solution was finally obtained . the solution was cooled in an ice - water bath , after which 3 × 100 ml portions of ice - cold ethanol were slowly added with strong mixing with spatula , to precipitate the product . a white , thread - like , copious precipitate formed ; the mixture was left for one hour in the ice - water bath , after which it was centrifuged at 4 ° c ., at 6000 rpm for a minimum of 20 minutes , in 250 ml plastic bottles . after discarding the supernatant , the centrifuge bottle was inverted for drainage of excess liquid , the precipitate was taken and pressed between filter papers , using gloved hands . it was , then , suspended in 50 ml 1 m ethanolamine at ph 9 . 4 , to which 1 ml 0 . 63 m edta was also added . as dissolution started , an additional 50 ml ethanolamine solution was added in two equal portions ; small clumps were broken with a spatula . the solution was then stirred while heated to about 40 ° c ., when complete solution occurred . the container was closed with parafilm and left overnight at room temperature or , alternatively , kept in a water bath at 40 ° c . for four hours . a golden - yellow , transparent solution resulted that was transferred to a 2 liter beaker for further treatment . the solution of the preceding step was cooled in an ice - water bath and , after 30 min , 3 × 100 ml portions of ice - cold ethanol were added with hefty mixing with spatula . a white , fibrous precipitate formed that sedimented easily by gravity . the suspension was kept for 1 hour in an ice - water bath , after which it was centrifuged at 6000 rpm for 20 min at 4 ° c ., as before . the clear supernatant was discarded and the precipitate collected as in example 4 above and dried between filter papers . this precipitate was re - dissolved in 2 × 50 ml portions of water for a third ethanol precipitation . the solution was transferred to a 2 liter beaker and cooled to ice - water bath temperature . after slow addition of 3 × 100 ml portions of ice - cold ethanol a white , fibrous precipitate formed and the mixture was left for one hour in the ice - water bath . the precipitate was collected after centrifugation carried out exactly as described in this example ; it was drained , pressed between filter papers and dissolved , with help of a spatula , in 100 ml water . the final solution was clear , very viscous . it was poured into petri dishes , frozen and freeze - dried . the dry material obtained after freeze - drying was white , weighing , on the average , 5 . 9 g for this batch size . it was recovered as brilliant , fibrous sheets that break into flakes . the final material is completely soluble in water or 0 . 15 m nacl solution . solutions in both solvents at the levels of 10 to 30 mg / ml form gel spheres when dripped into an 1 % calcium chloride solution . sodium alginate and cis solutions were prepared as in example 1 with the important modification that the solvent for each one was now water . “ egg - box ” sites were combined with calcium ions as in example 2 . cis solution was , then , added to alginate ; cis solution can also be added before the calcium ions addition . a power mixer was used to break any clumps that could have formed at the calcium chloride addition steps . the very viscous final mixture was transferred to petri dishes , frozen and freeze - dried . the hard , dry cake in the petri dishes was then submitted to the dehydrothermal treatment as follows : the dishes containing the cakes were placed in an electric hood maintained at 102 - 106 ° c . and kept there for 24 hours . after this time allowed for reaction , the cakes were left to cool to room temperature and further processed . at this point an average 6 . 7 g of cake is obtained ; it is reduced to powder and suspended into ˜ 75 ml water to which 5 - 6 ml 0 . 63 m edta is added to remove calcium from the “ egg - box ” sites . to 100 ml of the mixture is added 10 ml of 1 m nacl , to render it 0 . 1 m in nacl , and the mixture is gently heated . coarse lumps that would not dissolve with a spatula are removed and the mixture is centrifuged to remove insolubles . the clear supernatant is purified by ethanol precipitation as described in example 6 ; a total of 3 re - precipitations are carried out , the first two after addition of 2 ml edta to assure complete calcium removal . after the third precipitation the cake is suspended in ˜ 75 ml water , gently warmed to facilitate solution . the clear solution obtained is transferred to petri dishes , frozen and freeze - dried . a typical yield for such a batch is 5 . 6 g of final product . the resulting powder is easily water soluble at concentrations from 10 to 40 mg / ml water or 0 . 1 m nacl ; the viscosity is a little less than that of equivalent concentrations of biodritin prepared by chemical co - polymerization with dvs but microcapsules are formed when the solution is dripped into calcium chloride . stock solutions of biodritin heteropolysaccharide were prepared at 10 , 15 , 20 mg / ml ( dvs polymerized biodritin ) or 30 mg / ml ( dht biodritin ) in 0 . 15 m nacl , at room temperature . dissolution is rapid and easy . the biodritin heteropolysaccharide solution was dripped from a syringe into a 1 . 1 % cacl 2 solution , with mild agitation , from a height of about 15 cm , forming beads . the beads were left to mature for different time intervals in the calcium chloride solution , from 5 to 40 min and were , then , processed as described by sun et al ( 1984 ). i - briefly , beads are washed , successively in 0 . 1 % ches ( cyclohexyl - ethane sulfonic acid ) and 1 . 1 % calcium chloride ; they are then treated for 6 min with a 0 . 5 % poly - llysine solution , in order to form a capsule of biodritin - polylysine complexes at the bead surface . ii - the beads are , then , washed in 0 . 1 % ches , 1 . 1 % calcium chloride and 0 . 15 m nacl , in this order ; each washing lasts 2 minutes . iii - an outer coating of biodritin is now applied by incubation of the capsules with a 0 . 03 % biodritin heteropolysaccharide solution for 4 minutes , after which the capsules are extensively washed in 0 . 15 m nacl . they are stored in 0 . 15 m nacl until used . in this example no cells were encapsulated ; the microcapsules contained only biodritin - calcium gel and the outside coatings . pure microcapsules are used to investigate their biocompatibility in animal experiments . the external diameter of the microcapsules prepared in this example was 4 - 5 mm but , using devices where a coaxial air stream flows around the syringe needle tip , much smaller microcapsules can be prepared , as is known to those versed in this field . in this example solutions were prepared having the following concentrations of components : solvent was 0 . 15 m sodium chloride ; the alginate concentration was fixed at 3 . 0 % and the cis concentrations used were 1 . 5 %, 3 % and 6 %, respectively , giving cis / alginate ratios by weight of 0 . 5 : 1 , 1 : 1 and 2 : 1 . the semi - interpenetrating polymer network was prepared in bead form by dropping each of these solutions into a solution of calcium chloride at 1 . 1 %, as described above . beads were formed from a 20 gauge syringe needle , at a rate of 1 ml / min ( peristaltic pump ); this needle was inserted into a larger one linked to an air pump so that air could be blown around the dropping needle in order to form microbeads , by fractionation of the out - flowing liquid column , that dropped into a calcium chloride solution . a typical preparation consisted of forming beads from 3 ml of each solution , received in 30 ml 0 . 15 m nacl in a small beaker . immediately thereafter the beads were transferred to a 50 ml centrifuge tube , where they were gently mixed with the calcium solution for 10 minutes . in this step the periods can vary from a few minutes to much longer ones , as 40 min , for example , depending on the extent of gelling that is desired . after this period the beads are centrifuged off at low force ( 1000 rpm , 3 min ), transferred to a 15 ml centrifuge tube and washed with 0 . 15 m nacl . a washing sequence as described in example 8 was applied . a poly - lysine capsule can be applied to the beads , exactly as described above . biodritin beads of the s - ipn type were tested for stability , without the poly - lysine capsule , by incubation for 140 hours at room temperature , in 0 . 15 m nacl . analysis of cis and alginate leakage , made by ultra - violet spectrophotometry at 204 and 215 nm , revealed that only 6 % of the total alginate in the beads leaked within the first 50 hours for the beads containing 6 % cis , about 4 % alginate leaked from the beads with 3 % cis , and ca . 1 % leaked from beads with 1 . 5 % cis , approximately the same leakage observed with alginate control beads . the leakage of cis , on the other hand , was minimal , in all cases , less than 1 %. after this period , the beads were further washed by daily exchanges of 0 . 15 m nacl . the leaked amounts decreased and reached zero for all beads by the third exchange , i . e ., after 72 hours . these data demonstrate that beads formed by s - ipn are stable and keep their composition after gelling . the beads formed in this example had an average diameter of 550 μm . example 10 — s - ipn gel formed from a mixture of dvs - cis - cross - linked biodritin and added cis a biodritin solution at 2 %( w / v ) was prepared in 0 . 15 m nacl . the biodritin powder preparation used had a dvs - crosslinked cis content of ca 2 % by weight , which was raised to 15 % by addition of cis . the final solution , thus , had a composition of 2 . 0 % alginate and 0 . 3 % cis , on a w / v basis . beads were prepared by dropping this solution into 1 . 1 % calcium chloride solution , followed by 40 min incubation in the same solution . additional treatment included formation of a poly - l - lysine capsule , further layered with biodritin , and washing as described in example 8 . beads and capsules prepared in this way have identical properties to beads of similar composition prepared from alginate and cis as in example 9 . they can be treated exactly as the beads described in the next examples . in addition to the possibility of sterilizing the biodritin heteropolysaccharide solution , as well all other solutions used in the capsule preparation by membrane filtration before the gelation step , biodritin microcapsules can also be sterilized by treatment with 70 % ( v / v ) isopropanol in 0 . 15 m nacl . thus , biodritin microcapsules were successively treated by isopropanol / 0 . 15 m nacl solution of increasing isopropanol concentrations , as follows : 25 %, 50 % and 70 %, all by volume . the first two treatments were applied for 30 min each , to allow slow dehydration of the capsules by the increasing concentrations of isopropanol . when submitted to 70 % isopropanol , the microcapsules were left for up to 4 days at room temperature . under these conditions , they shrunk to about one - half the initial diameter , presenting wrinkles on the surface . when re - hydrated 4 days later , by decreasing concentrations of isopropanol , in the reverse order , as above , until 0 . 15 m nacl was attained , they recovered the original spherical form , without shape deformations , breaks or peeling of the outer layer . the surface examination was done with help of a loupe with varying degrees of magnification . differently from this , microcapsules prepared with alginate alone treated the same way and in parallel experiments , shrunk to ⅓ the original volume and did not recover the initial spherical shape . most became ellipsoids of revolution , with intact surfaces showing wrinkles that did not disappear with time . microcapsules treated as in examples 8 and 9 were also implanted into mice to test their biocompatibility . the preservation of the outer membrane after sterilization and re - hydration was tested by dissolving a considerable part of the gel in the interior of the microcapsules by treatment with 0 . 050 m sodium citrate . microcapsules were incubated with sodium citrate at room temperature for varying times while observed under a loupe at high magnification . depending on the capsule size , the time course of events changes but , for a group of larger microcapsules the following results were obtained : after ˜ 10 min , the central core was surrounded by a narrow , very transparent zone , the latter limited by the external membrane . at 30 min observation , the solubilization of the biodritin gel by calcium removal by citrate reached about 50 % of the capsule diameter ; the outer membrane was intact . mixing the microcapsule suspension by rotation of the flask makes possible to observe the undulating movement of the intact external membrane , also indicating that it is permeable , allowing fluid to move in and out . lacking the support of the subjacent gel structure , dissolved by action of citrate , the membrane can be broken by strong aspiration with a pipette . alcian blue is a well known stain for chondroitin sulfate ( turnbull , 1993 ); we found that it also stains alginate , although with less intensity . microcapsules were stained in 0 . 5 % alcian blue in 2 % acetic acid , for 20 min ; de - staining was in 2 % acetic acid , in repeated washings . biodritin microcapsules give a deeper blue than alginate capsules , as expected . when biodritin microcapsules prepared as in example 8 , are cut in half and stained , the interior of the capsules stains more intensively than the outside , indicating that the external membrane has an effect on the stain diffusion to the capsule interior . example 14 — preparation of structured “ spaghetti ”- like cylinders of biodritin for cell and tissue implantation a new use of the biodritin invention is in the preparation of structured spaghetti - like cylinders to contain cells or tissues for implantation , itself an invention to be described for the first time herein , called biodritin spaghetti . these biodritin spaghetti are called “ structured ” because the thin gel cylinder that receives the name spaghetti has an interior structure formed by a string of sewing cotton line or , alternatively , of surgical suture line . the cotton line has a multitude of very fine lateral extensions , as short branches in a long caulis , that spring from the line surface and are embedded into the gel . the surgical suture does not have such branches , but they can be created by scratching the surface of the line with a knife blade , before sterilizing . these lateral extensions of the central line core provide additional area for adhesion between the gel matrix and the central core , reinforcing the structure in a way similar to the iron wires in concrete . 1 ) a sterilized cotton sewing line , size 50 or thinner is inserted into a 10 to 20 cm sterile polyethylene catheter of the desired diameter — usually size 60 , but size 90 can be used with thinner lines — up to the point where the line reaches the needle , itself connected to a syringe . a surgical suture line can be used in place of the cotton line , with equivalent results . the line should extend beyond the free end of the catheter tubing for 4 to 5 cm , so that in can be handled , anchored or restrained later on in the process ( see fig2 a ). 2 ) a biodritin heteropolysaccharide solution of desired concentration , between 0 . 5 and 3 . 0 %, for example , in the required solvent ( saline , culture medium , or hank &# 39 ; s medium ), and containing the cell or tissue preparation desired , is introduced into the catheter tubing by syringe aspiration through the catheter , with the line in place . after a given length of the catheter is filled with the solution the catheter tip and the line extension are briefly rinsed with solvent and the set up is ready for spaghetti formation . 3 ) a structured spaghetti is formed as follows : the catheter filled with biodritin heteropolysaccharide solution containing cells and connected to the syringe is quickly placed into a calcium chloride solution in a large petri dish or shallow tray and the syringe plunger is pressed , as the catheter is continuously moved forward in the calcium chloride solution . as the biodritin heteropolysaccharide solution exits the catheter , it enters into immediate contact with calcium ions and instantly forms a cylindrical gel around the cotton line or surgical line . when all the biodritin heteropolysaccharide solution in the catheter is expelled by the syringe , one is left with a cylindrical gel formed around the line , whose extension is dictated by the amount of biodritin heteropolysaccharide solution originally in the catheter . at each end of the biodritin gel there is a continuous piece of line that is now used to hold the gel cylinder during further processing . 4 ) the biodritin spaghetti is now left in the calcium chloride solution for a given period of time to strengthen the gel ; this period varies according to the type of gel desired , and ranges from 5 to 40 minutes . after the incubation with calcium ions the biodritin spaghetti are treated exactly as under example 8 , to form a biodritin - poly - l - lysine membrane covering the spaghetti surface area . this external biodritin / polylysine / biodritin membrane helps to support the gel and controls permeability according to molecular size . 5 ) when islets of langerhans or other cells or tissue are suspended in the biodritin spaghetti , they distribute themselves in the double cylindrical space contained within the limits of the cotton or surgical line surface , in the inside , and the biodritin / poly - lysine / biodritin membrane , in the outside , as shown in fig2 a . the number of islets contained per unit cylindrical volume can be controlled by the islet suspension prepared originally in the biodritin solution . the biocompatible biodritin gel structure that forms the bulk of the spaghetti supports the islets away from each other and prevents that they clump together , thereby avoiding central necrosis in islets aggregates . 6 ) the biodritin spaghetti , with the line extensions at each end , may be implanted individually or can be tied together by the line ends before inplantation , as shown in fig2 b . in this way , a much larger mass of cells may be implanted , in a rather limited space , but under conditions that preserve the individual spaghetti . 7 ) another embodiment of the present invention is that a group of biodritin spaghetti strings with cells , tied together as in fig3 b , can be placed inside a porous , protective and biocompatible external cylinder initially prepared from veins or arteries of an animal or from the recipient himself . once attached within such a biocompatible container , to form a sort of living tissue cartridge , the biodritin spaghetti strings can be implanted into a patient . a possible and interesting alternative proposed in this invention is through the umbelical scar in such a way that , eventually , replacement cartridges may be exchanged using rather simple surgical technique . 8 ) under the preparation conditions described above , a biodritin spaghetti of gel length equal to 10 cm would have the following approximate dimensions ( fig4 ): a ) gel length — 100 mm ; b ) total internal diameter — 0 . 76 mm ; c ) cotton line diameter — 0 . 20 mm ; d ) thickness of biodritin gel cylinder ( b - c )— 0 . 50 mm ; volume of biodritin gel — 42 . 2 mm 3 , or 4 . 22 mm 3 per linear centimeter length . biodritin microcapsules of 4 - 5 mm diameter were implanted intra - peritoneally in mice , 3 capsules per mouse , 2 mice per group , following standard surgical procedures and approved protocols . after one week , one month and three months post - implantation periods the capsules were removed from the animals . the week - long implanted capsules were found to be free in the peritoneal cavity , clean and with no adherences ; no inflammatory reaction could be observed in or around the site where they were found . similar results were found with the microcapsules that stayed for one month or three months intra - peritoneally in mice . the capsules showed a clean , brilliant surface , free of any adherences . as with the week - long experiments , no inflammatory reaction was noticed . fig4 shows a capsule removed from a mouse after three months intraperitoneal implantation . histological study of the preparations fully supported the conclusion : no cells were found on the beads surface after intraperitoneal implantation for eight days , one month and three months . thus , the biocompatibility of this novel material was established , as expected from the ubiquitousness of chondroitin sulfate in the animal kingdom and from its demonstrated inherent non - immunogenicity . in another embodiment of the present invention , a 0 . 8 to 1 . 5 % ( w / v ) biodritin heteropolysaccharide solution in saline is painted on and around a surgical suture ; immediately thereafter a 1 % ( w / v ) calcium chloride solution is briefly sprayed as a mist over the painted area . there immediately forms a biodritin gel over the painted area that protects it from invading cells or from adhering to adjoining tissue . this has important applications in abdominal surgeries , where undesired adherences may form after surgical procedures . as an alternative mode of application , the biodritin heteropolysaccharide solution can be sprayed over and around the suture , followed by spraying of the calcium chloride solution to form the biodritin gel . a third manner of biodritin application is to mix the biodritin heteropolysaccharide solution with an insoluble calcium salt , such as tricalcium citrate , forming an uniform suspension of the calcium salt and , immediately before application , mixing this with an aqueous solution of the δ - lactone of gluconic acid , which slowly hydrolyzes to gluconic acid , which dissolves the calcium ions from the citrate , thereby forming the biodritin gel in situ . this formulation is called “ internal gelation ” and has been employed to encapsulate microbial cells by johansen and fink ( 1988 ). to perform this formulation the following proportions are recommended : to each 8 ml of biodritin heteropolysaccharide solution of adequate concentration ( 2 . 5 %, for example ) add 1 ml of a tricalcium citrate solution ( 11 . 4 mg / ml ) and , finally , 1 ml of glucono - lactone ( 10 mg / ml ). upon addition of the gluconolactone the solubilization of calcium oind takes place in minutes , when the gel starts forming . this is the preferred embodiment of this application . having thus described in detail the preferred embodiments of the present invention , it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above descriptions as many apparent variations thereof are possible without departing from the spirit or scope of the present invention . 1 . devos , p ., de haan , b . j . and van schilfgaarde , r . 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