Patent Application: US-63968303-A

Abstract:
hydrogel - bacterial cellulose nano - composite materials are created using a hydrogel and never dried bacterial cellulose fibers . such materials are suitable for a broad range of soft tissue replacement applications . in addition controlled release of bioactive agents properties can be designed into medical devices fabricated from such composite materials .

Description:
a composite material based on a combination of a hydrogel in combination with bacterially produced cellulose having cross sectional dimensions on the nanometer scale is disclosed herein along with a method of making the composite . the bacterial cellulose is produced in its original as produced state and is not dried but used directly to produce the composite . the preferred bacterial cellulose is produced using a microbial fermentation process using the bacteria acetobactor xylinum in either a static , shaken or agitated culture as disclosed in u . s . pat . no . 5 , 846 , 213 ( which is incorporated herein by reference ). the hydrogel can be chosen from the following list including polyvinyl alcohol ( pva ), poly ( vinyl pyrrolidone ) ( pvp ), poly ( ethylene glycol ) ( peg ), poly ( hydroxyethyl methacrylate ) ( phema ) and polyacrylamide . polyvinyl alcohol is the preferred choice for the purpose of this invention . the hydrogel can be dissolved in a hydroxylic solvent including water , alcohol , ketone and aldehyde or carboxylic acid , or any other aprotic solvent capable of forming effective hydrogen bonding to dissolve pva . examples of dipolar aprotic solvents which may be used include dimtheyl sulfoxide ( dmso ), dimethyl formamide ( dmf ), dimethyl acetamide ( dmac ) and n - methyl pyrrolidone ( nmp ). if the solvent is not water , the solvent would have to be removed by solvent exchange with water by immersion in water before use . as described above , the composite material can either be prepared using water as the solvent or solvent systems consisting of combinations of water and other solvents . the final product consists of microbial cellulose , hydrogel and the solvent used . in the case when either water is used in combination with other solvents or when solvent systems not containing water are used in the fabrication process , an additional step of solvent exchange with water will be necessary to replace the non - water solvent before the resulting product can be used for biomedical applications . bacterial cellulose is blended into the hydrogel solution and the composite material is solidified into the desired shape of the intended medical device . in the case of polyvinyl alcohol - bacterial cellulose nanocomposite , properties of the composite is a function of polyvinyl alcohol concentration , bacterial cellulose concentration and the processing conditions used to generate the composite material . the polyvinyl alcohol concentration may be in the range of 5 to 20wt %, and the bacterial cellulose concentration in the range of 0 . 05 to 5 wt % may be used with the balance being the solvent system used in preparing the hydrogel solution . the low temperature thermal cycling method is preferred in creating the pva - bacterial cellulose composite material . in this case , material properties are a function of the number of thermal cycles , the freezing and thawing rate . another method that can be used is fast cooling followed by cold soaking and controlled thawing . the composite material produced according to the method disclosed herein may be formed into various pre - selected shapes for use as medical devices . non - limiting examples include forming the composite material in the shape of a substantially planar sheet for a wound dressing , dental implant , vascular grafts , catheter covering dressing , dialysis membrane , coating for cardiovascular stents , coating for cranial stents , and membrane for tissue guided regeneration . the pva solution prepared was 5 - 15 % by weight . this concentration was chosen as reference for the purpose of comparison against previous results and among different pva composite materials . the procedure for pva preparation was consistent with the protocol implemented by wan [ 36 ]. the pva used in all the experiments was purchased from aldrich chemical company ( catalogue no . 36 , 306 - 5 ). a preferred pva average molecular weight range ( mw ) was 124 , 000 to about 186 , 000 , 99 +% hydrolysed and was received in powder form . the pva solution in distilled water was prepared in a pyrex resin flask combined with a reflux column to prevent excess vapor pressure build - up and water loss . the solution was heated between 2 - 3 hours at a temperature of around 80 ° c . when all the pva had gone into a clear jelly - like solution , the flask was removed from the heating mantle . more broadly , the polyvinyl alcohol ( pva ) may have a molecular weight in a range from about 100 , 000 to about 200 , 000 . if the molecular weight of pva changes , in order to achieve the same mechanical properties , the corresponding cellulose concentration range will be adjusted accordingly . a 1 . 5 l stirred tank bioreactor equipped with a disk flat blade turbine and temperature and ph control was used for bacterial cellulose production . an incoulum was prepared using the bacteria acetobacter xylinum ( atcc # 700178 ). it was added to the sterile media and the mixture was allowed to mix at 28oc , ph of 5 , air flow rate of 1 l / min and a mixing speed of 700 rpm for 72 hours . the media used has the following composition . fructose 4 % w / v , corn steep liquor 4 % v / v , ammonium sulphate 0 . 33 % w / v , potassium dihydrogen phosphate 0 . 1 % w / v , magnesium sulphate heptahydrate 0 . 025 % w / v , tri - sodium citrate 0 . 42 % w / v and citric acid 0 . 88 % w / v . after 72 hours , the reactor was shut down and its contents were centrifuged to remove the bacterial cellulose fibres from the spent broth . the crude bacterial cellulose was treated with 1 n sodium hydroxide at 90oc for 30 minutes to remove any bacteria that still may be clinging to the fibres . the mixture was then centrifuged to recover the bacterial cellulose . the treated bacterial cellulose was washed three times with distilled water to remove any excess sodium hydroxide . the purified bacterial cellulose was stored in distilled , purified water in the refrigerator at around 6 ° c . two different methods of making up the pva - cellulose solution may be used depending on the composition of pva - cellulose solution . when preparing a low concentration cellulose and pva solution , the preferred method is to start with the cellulose in suspension and add solid pva to it , while when making up a higher concentration of cellulose and pva solution , the preferred method is to mix pva already in a solution with a cellulose suspension of known concentration . it will be understood that the difference between these two methods of making up the pva - cellulose solution is more for convenience than being critical to the solution preparation procedure . suspensions of microbial cellulose nanofibres in distilled water in the range of 0 . 3 - 0 . 5 wt % are prepared . the suspension is added to pva solution with mechanical stirring such that the final concentration of pva is in the range of about 5 to about 15 % and microbial cellulose concentration is between about 0 . 15 to about 0 . 5 %. depending on the viscosity of the resulting solution , extra care must be taken to prevent air bubble introduction in the mixer process . table 1 contains a summary of the pva - bacterial cellulose samples prepared . as seen on table 1 , the concentrations were widely varied to observe the effects of both components on the material properties . first , a 10 % pva concentration was kept constant and the concentration of bacterial cellulose was varied from 0 . 15 to 0 . 61 %, which was the highest concentration of cellulose obtained . then , a ˜ 0 . 31 % bacterial cellulose concentration was kept constant , varying the pva concentration from 7 . 5 to 15 %. the two extremes were also investigated , a low pva and low bacterial cellulose ( 5 % pva — 0 . 15 % bacterial cellulose ), as well as a high pva and high bacterial cellulose concentration ( 15 % pva — 0 . 5 % bacterial cellulose ). after preparing pva or the different pva composites , the solution was poured or injected using large syringes onto stainless steel molds , with rubber spacers of either 1 . 6 or 3 mm thickness . these moulds were placed vertically into a temperature controlled bath . the freezing and thawing rate were kept constant at 0 . 1 ° c ./ min and the samples were cycled between about + 20 ° c . to about − 20 ° c . for 6 cycles . tensile properties ( stress - strain ) and relaxation properties ( stress remaining vs . time ) of the pva - bacterial cellulose composite were determined using a mts tensile tester . mechanical properties for the pva - bacterial cellulose composite were determine for the composition of 10 % pva and 0 . 61 % bacterial cellulose and compared to that of pva reference . fig1 shows the stress - strain curves for samples undergoing low temperature thermal cycling of cycles 1 through 6 . the curve for the 10 % pva reference ( cycle 6 ) was also included for comparison purposes . there are significant differences of the stress - strain relationship and the pva - bacterial cellulose composite . referring to fig1 , the stress - strain curve of the 10 % pva cycle 6 is similar to that of 10 % pva with 0 . 61 % bacterial cellulose cycle 2 up to a strain of 45 %. at this point , the stiffness of the material greatly increases and deviates from the curve of the pva reference . this difference can be attributed to the presence of the bacterial cellulose in the composite . the effect of the two components can be seen when comparing the moduli as a function of the strain while keeping one component constant . fig2 shows the moduli of 5 composites with 10 % pva and various bacterial cellulose concentrations ( 0 , 0 . 15 , 0 . 23 , 0 . 31 , 0 . 61 %) for cycle 6 . it can be clearly seen the increase in modulus by adding extremely small amounts of bacterial cellulose , with an increase in modulus of almost 3 times at 30 % strain and more than 6 times for 60 % strain by adding 0 . 61 % bacterial cellulose . in addition to 10 % pva , two extreme concentrations were also examined , including a low concentration of 5 % pva with 0 . 15 % bacterial cellulose , and a high concentration of 15 % pva with 0 . 5 % bacterial cellulose . these two compositions gave results that defined the limits of mechanical properties of the pva - bacterial cellulose composites studied . fig3 shows the stress - strain curves for cycle 6 of all the 9 pva - bacterial cellulose composites , including these two extremes , and the reference 10 % pva . the moduli up to 40 % strain for all the 9 composite compositions and the reference pva can be seen on fig4 . fig3 and 4 illustrates that any tissue with mechanical properties that fall between this range of stress - strain curves can be matched by a pva - bacterial cellulose composite with an appropriate composition of components . the stress - strain curves presented in fig3 are only for cycle 6 . it is therefore clear that the range of mechanical property control is very broad . thus , the stress - strain curve of any target tissue falling within this range can be matched by altering and controlling a combination of variables , including pva and bacterial cellulose concentrations , number of freeze / thaw cycles , thawing rate , and freezing holding time , among other parameters . a large increase in modulus and stiffness was obtained by the high concentration composite ( 15 % pva with 0 . 5 % bacterial cellulose ) and for the first time a very stiff pva material at low strains was obtained . [ heading - 0066 ] matching of pva - bacterial cellulose composite properties to that of the aortic root the stress - strain curves for porcine aortic root in both directions were similar to the stress - strain curves of various types of pva - bacterial cellulose composites . fig5 shows the comparison of the stress - strain curves of various concentrations and cycles of pva - bacterial cellulose composites and circumferential and radial aortic root . as seen in fig5 , there are various parameters that can be altered to obtain similar mechanical properties to the targeted aortic root tissue in any direction . the stress - strain curve of aortic root in the circumferential direction was similar to three different bacterial cellulose composites , including the 10 % pva with 0 . 61 % bacterial cellulose cycled 2 times , the 15 % pva with 0 . 31 % bacterial cellulose cycled 2 times , and the 10 % pva with 0 . 23 % bacterial cellulose cycled 6 times . the stress - strain curve of aortic root in the radial direction was similar to four different bacterial cellulose composites , including the 10 % pva cycled 4 times , the 10 % pva with 0 . 31 % bacterial cellulose cycled 2 times , the 10 % pva with 0 . 15 % bacterial cellulose cycled 3 times , and the 10 % pva with 0 . 23 % bacterial cellulose cycled 2 times . pva had been reported as an ideal cell entrapment material for cell immobilization carriers , due to their physico - chemical , thermal , mechanical , and biological stability and highly porous structure that facilitates the nonhindered diffusion of solutes and dissolved gases [ 13 ]. these characteristics are important for drug delivery applications . the bacterial cellulose - pva composite with a cellulose concentration in the range of 0 . 05 - 0 . 5 % is expected to retain all these useful characteristics of pva . as used herein , the terms “ comprises ”, “ comprising ”, “ including ” and “ includes ” are to be construed as being inclusive and open ended , and not exclusive . specifically , when used in this specification including claims , the terms “ comprises ”, “ comprising ”, “ including ” and “ includes ” and variations thereof mean the specified features , steps or components are included . these terms are not to be interpreted to exclude the presence of other features , steps or components . the foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated . it is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents . 1 . popma , j . j ., et al . “ lipid - lowering therapy after coronary revascularization .” american journal of cardiology 86 . 4b ( 2000 ): 18h - 28h . 2 . korossis , s . a ., j . fisher , and e . ingham . “ cardiac valve replacement : a bioengineering approach .” bio - medical materials and engineering 10 . 2 ( 2000 ): 83 - 124 . 3 . schoen , f . j ., and r . j . levy . “ founder &# 39 ; 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