Patent Application: US-66416405-A

Abstract:
the invention relates to a composite comprising a semi - permeable barrier layer that is permeable to oxygen and impermeable to moisture ; and a scaffold fiber layer formed by electrospinning fibers on one side of the barrier layer .

Description:
fig1 is a schematic diagram of an electrospinning apparatus used to produce a nanofiber scaffold layer . the electrospinning system 300 comprises a syringe pump 10 , a high voltage power supply 20 , a movable multiple spinneret system 30 and a nanofiber collector 25 . the syringe pump 10 feeds a conducting fluid used for forming nanofibers to the multiple spinneret system 30 through a series of tubes ( 12 a , 12 b , 12 c ). a plurality of spinnerets comprising three syringe needles ( 31 a , 31 b , 31 c ) is mounted on the multiple spinneret system 30 . each of the three syringe needles ( 31 a , 31 b , 31 c ) is mounted on to a spinneret holder 38 by means of respective plugs ( 32 a , 32 b , 32 c ). in use , the conducting fluid flows from the pump 10 through the series of tubes ( 12 a , 12 b , 12 c ), into each of the three syringe needles ( 31 a , 31 b , 31 c ) via the plugs ( 32 a , 32 b , 32 c ). the multiple spinneret system 30 is operable to move in a reciprocating manner , for example , from left to right , as indicated by arrows ( 35 a and 35 b ). a grounded collector 25 is positioned below the syringe needles ( 31 a , 31 b , 31 c ) to create an electric field between the charged syringe needless ( 31 a , 31 b , 31 c ) and the collector 25 . the electric field causes tiny jets 34 of the conducting fluid to be ejected from the tip of each syringe needle ( 31 a , 31 b , 31 c ). the jets 34 are deposited onto the collector 25 , and form a scaffold of nanofibers on the collector 25 . a semi - permeable barrier layer 50 may be located on the collector 25 to form the scaffold fiber layer onto the semi - permeable barrier layer 50 . the structure and working of apparatus of fig1 is described in further detail in singapore patent application no . sg 200403355 - 1 . fig2 represents another embodiment of the syringe needle 31 . fig2 shows schematic diagram of a syringe needle 431 that can be used in place of the syringe needle 31 in the electrospinning apparatus of fig1 . the syringe needle 431 comprises two capillary tubes 406 and 408 arranged concentrically . the capillary tubes ( 406 , 408 ) are made up of stainless steel . the syringe needle 431 is provided with two inlets 412 and 414 for supplying a first conducting fluid and a second conducting fluid to the syringe needle 431 . the concentric arrangement of the capillary tubes ( 406 , 408 ) forms two separate channels 402 and 404 for the passage of the first conducting fluid and the second conducting fluid from the respective inlets ( 412 , 414 ) to the tip of the syringe 431 . the inlets ( 412 , 414 ) are connected to two different syringe pumps ( not shown ) by means of teflon tubes ( not shown ). the syringe pumps used in this embodiment are identical to the syringe pump 10 of fig1 and the tubes used in this embodiment are identical to tube 12 of fig1 . the syringe 431 is connected to the power supply 20 by means of a copper wire 420 . in use , two different conducting fluids are pumped into the syringe needle 431 by the respective pumps . when electric field is applied to the syringe needle 431 , jets of conducting fluids are ejected from the needle and are deposited on to the grounded collector in the form of nanofibers . fig3 shows a schematic diagram of a nanofiber 430 produced by using the syringe needle 431 of fig2 . the nanofibers produced by using syringe needle 431 are composed of two fiber - forming materials . the first material corresponds to the first conducting fluid and the second material corresponds to the second conducting fluid . the first material forms core 420 of the nanofiber 430 and the second material form the shell 422 of the nanofiber 430 . a composite manufactured as described above is able to be used for treating a dermal condition . referring to fig1 there is shown steps of an autologous layered dermal reconstitution ( aldr ) method . in step 1 a first tg - nf composite 10 comprising scaffold 14 of poly - ε - caprolactone ( pcl ) that has been electrospun onto a layer of tegaderm ™ would dressing 12 is placed onto a patient &# 39 ; s burnt skin wound 8 . the scaffold layer 14 of the tg - nf composite 10 is seeded with human dermal fibroblast ( hdf ) cells . in step 2 the tg - nf composite 10 seeded with hdf cells , is left on the skin 8 for a period of time to allow skin healing assisted by the hdf cells as they proliferate within the scaffold 14 in situ . in step 3 the layer of tegaderm ™ 12 is peeled off the scaffold layer 14 . in step 4 , directly after step 3 , a second tg - nf composite 10 a comprising scaffold 14 a of poly - ε - caprolactone ( pcl ) that has been electrospun onto a layer of tegaderm ™ would dressing 12 a is placed onto the first scaffold layer 14 so that the second scaffold 14 a is in direct contact with the in - situ scaffold 14 . additional layers may be placed on the skin by repeating steps 3 and 4 until 70 - 80 % of the original dermal thickness is reconstituted on the patient &# 39 ; s skin 8 . finally , in optional step 8 , a skin graft 16 is placed on top of the last remaining scaffold 14 a . non - limiting examples of the invention , and a comparative example will be now be described in greater detail by reference to specific examples , which should not be construed as in any way limiting the scope of the invention . it has long been assumed that the adhesive in polyurethane materials , such as tegaderm ™, would lead to cell death . however , the inventors have surprisingly found that keatinocyte cells can not only survive but can in fact grow directly on tegaderm ™. human epidermal keatinocyte cells were seeded directly onto a layer of tegaderm ™ and cultured in an assay as described in t . t . phan [ 33 ]. fig5 shows a sem image of the human epidermal keratinocyte cells cultured direct on tegaderm ™ wound dressing material . the image was taken at a resolution of 1311 ×. fig7 is a bar graph illustrating the difference between growth of human epidermal keratinocytes cultured on tegaderm ™ wound dressing material and on tissue culture plastics as disclosed in t . t . phan et al . the growth was assessed by means of an mts assay . fig7 shows that cell proliferation on tegaderm ™ wound dressing material is comparable to cell culturing in tissue culture . accordingly , the inventors have found that tegaderm ™ membrane material can be used to support cell proliferation and is therefore a suitable material for use in a composite for treating dermal conditions . example 1 was repeated only in this example , human dermal fibroblast ( hdf ) cells were seeded directly onto a layer of tegaderm ™ rather than keratinocyte cells . fig6 shows a sem image of human dermal fibroblast cells cultured on the tegaderm ™ wound dressing material . the image was taken at a resolution of 1106 ×. fig8 is a bar graph illustrating the difference between growth of the hdf cells cultured on tegaderm ™ wound dressing material and on tissue culture plastics . the growth was assessed by means of a mts assay . again , the inventors have found that tegaderm ™ membrane material can be used to support hdf cell proliferation and is therefore a suitable material for use in a composite for treating dermal conditions . a composite was prepared according a disclosed embodiment . in this composite , tegaderm ™ wound dressing material was employed as a semi - permeable barrier layer . using the apparatus of fig1 , a nanofiber scaffold layer was electrospun onto the tegaderm ™ material . an electric field strength of 10 kv was used , a needle radius of 0 . 21 mm , a spinning solution feed rate of 0 . 8 ml / hr and an electric field distance of 12 cm . poly - ε - caprolactone ( pcl ) was used as the fiber forming material . the conducting solution was prepared by dissolving poly - ε - caprolactone ( pcl ) in a mixed solvent of chloroform and methanol ( 3 volume of chloroform : 1 volume of methanol ) to form a 10 wt % pcl solution . the conducting solution was used to form nanofibers onto the tegarderm ™ material . the composite thus prepared is called as tg - nf composite . using a sem ( scanning electron microscope ), images of the scaffold fiber layer of the composite were taken at various resolutions . fig4 a is a sem ( scanning electron microscope ) image of the scaffold fiber layer of the formed composite . the image was taken at a resolution of 3600 ×. fig4 b is another sem image of the scaffold fiber layer of the composite . the image was taken at a resolution of 14400 ×. human dermal fibroblast ( hdf ) cells were seeded onto the scaffold fiber layer of the tg - nf composite of example 3 . the cells were allowed to proliferate for a period of 21 days . the hdf cells were obtained from an 8 - month - old chinese infant ( cell research corporation ). the hdfs were plated as a monolayer and cultured to confluence in dmem containing 10 % fbs ( fetal bovine serum ) and 1 % antibiotic solution ( penicillin - streptomycin ). media was replaced every 3 days and the cultures were maintained in a humidified incubator at 37 ° c . with 5 % co 2 . all culture media and reagents were purchased from research biolabs ( sigma , st louis , mo ., usa ). the growth of cells on the scaffold layer was assessed using standard mts ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carbomet and also by hoxyphenyl )- 2 -( 4 - sulfophenyl - tetrazolium innersalt ) assay standard cell count method . to study cell proliferation on the substrates , viable cells was determined by using the calorimetric mts assay ( celltiter 96 ® a queous assay , madison , wis ., usa ). the mechanism behind this assay is that metabolically active cells will react with the tetrazolium salt in the mts reagent to produce a soluble formazan dye that can be absorbed at 492 nm . the substrates were rinsed with pbs , followed by incubation with 20 % mts reagent in serum - free culture medium for 3 hours . thereafter , aliquots were pipetted into a 96 - well plate . the 96 - well plate was then placed into a spectrophotometric plate reader ( fluostar optima , bmg lab technologies , germany ) and the absorbance at 492 nm of the content of each well was measured . in order to count the number of viable cells attached to the pcl scaffolds and tg - nf constructs , the substrates were harvested , washed with pbs to remove non - adherent cells , and then incubated in 0 . 5 ml of 1 × trypsin at 37 ° c . for 5 min . the trypsinization process was stopped by adding 0 . 5 ml of dmem to each sample . the cell numbers were then counted using a hematocytometer and microscope . fig9 shows a comparison between growth of hdf cells cultured on a tegaderm ™- nanofiber (“ tg - nf ”) composite of example 1 and growth of hdf cells cultured on pcl scaffold without tegaderm ™ (“ pcl nanofiber ”). the growth was assessed by means of a mts assay as outlined in example 2 above . hdf proliferation on pcl nanofiber scaffolds and tg - nf constructs was studied at days 1 , 3 , 5 , 7 , 9 , 11 , 14 , 16 , 18 and 21 , with the results shown in fig9 and 10 . both the optical density and number of cells were noted to have increased significantly through the 21 - day span , demonstrating that cell proliferation occurred successfully on both types of substrates and that cell proliferation was not adversely affected by the presence of the tegaderm ™ would dressing material . in fig9 , it was observed that optical density of hdfs within pcl nanofiber and tg - nf kept increasing until day 16 when it started decreasing . this was because hdfs had proliferated till confluence at day 16 when there were no more available spaces on the constructs for further proliferation . the results were substantiated by the observations made in fig1 , which shows a comparison between growth of hdf cells cultured on a tg - nf composite of example 1 and growth of cells cultured on the pcl nanofiber scaffold . the growth was assessed by cell counting . fig1 shows that the cell counts were noted to have reached a plateau from day 16 . the cell counting results are in agreement with the assessment by mts assay . at every time point , the numbers of cells present on the pcl nanofiber scaffold and tg - nf construct were comparable , demonstrating that cell growth was comparable on both the substrates . therefore , it can be concluded that the tg - nf construct is a suitable non - toxic substrate for cell growth and proliferation . for characterization , the pcl nanofiber and tg - nf constructs were sputter coated with gold ( bal - tec ; scd 005 sputter coater ; germany ). morphological imaging of the constructs was performed using field emission scanning electron microscopy ( fei co . ; xl30 feg sem ; usa ) at an accelerating voltage of 15 kv . cell morphology on the pcl scaffolds and tg - constructs was studied by fesem at days 3 , 7 , and 21 . it was noted that at day 3 ( refer to fig1 a and 11 b ) hdfs only reached approximately 10 % confluence . this was due to the low hdf seeding density used in this study in order to observe proliferation over the 21 day span . the cells were seen to be characteristically spindle shaped and stretched across the nanofibrous substrates during the course of proliferation . subsequently at day 7 ( fig1 c and 11 d ), it was observed that the hdfs had increased in number and reached about 30 % confluence on both substrates . hdf proliferation and growth continued progressively and at day 21 ( fig1 e and 11 f ), hdf proliferation had almost reached 100 % confluence . both the pcl scaffold and tg - nf construct were almost completely covered with a continuous hdf monolayer . cracks were observed from the micrographs at this point . this could have been due to the contractile force exerted by the hdf monolayer during the sample preparation phase for fesem , where the fibroblasts populated substrates were dehydrated with increasing grades of ethanol solutions and air - dried overnight . from the fesem micrographs of fig1 , it was established that there was successful hdf proliferation and adherence on both nanofibrous substrates . comparing the fesem micrographs taken for both the pcl scaffolds and tg - nf constructs at each time point , it was observed that the density of hdfs on both substrates was always comparable and that the cells reached almost 100 % confluence on each at an near equivalent rate . from this experimental data , it can be seen that the tg - nf construct can be considered a suitable host substrate for fibroblast population , with results of cell proliferation was much comparable to that of the pcl nanofibrous scaffold . the pcl nanofiber scaffolds and tg - nf constructs were fixed in 4 % formalin and stained with hematoxyline and eosin ( h & amp ; e ). thereafter , the individually stained substrates were embedded between two layers of oct embedding medium ( leica ; germany ) by immersing the whole structure in liquid nitrogen . serial sections ( 5 μm ) were sliced using a cryostat ( leica cm3050s ; germany ) and examined under an inverted optical microscope ( leica dm irb ; germany ). since fibroblasts are anchorage - dependent cells , it was speculated that high specific surface properties and porosity of the electrospun nanofibrous constructs may aid in the attachment and migration of the cells and its extracellularmatrix ( ecm ) within the substrate . histological examination revealed ecm integration ( light orange stains ) inside the pcl scaffolds and tg - nf constructs , which were h & amp ; e - stained at days 1 , 7 , and 21 ( refer to fig1 ). for the individual substrates , it was observed that the density of the hdfs formed had increased over the four time points . this illustrated that not only did the hdfs attach themselves to the surface of the substrates , their ecm have also managed to successfully migrate into its substance as well . this verifies that both the pcl nanofiber scaffold and the tg - nf construct can be further looked into as effective three - dimensional scaffolds for hdf growth and population . however , the scaffolds and constructs produced by the electrospinning techniques were non - woven . therefore , the porosity and pore sizes were generally irregular and randomly distributed throughout the entire substrate . this provided a better understanding as to why ecm migration and integration was predominantly observed only in certain areas of the substrate and not throughout . nevertheless , this shortcoming can be rectified with recent developments using aligned nanofibers to fabricate scaffolds , where porosity and pore sizes can be readily controlled . another interesting point to note was that at any time point , the densities of hdf in both substrates were relatively comparable . this demonstrates that hdf ecm growth and migration within the tg - nf construct is comparable to the pcl nanofiber scaffold , which can been regarded as a suitable biodegradable scaffold for a skin substitute . with reference to y . z . zhang et al [ 26 ], where bone marrow stromal cells ( bmsc ) are noted to be able to proliferate within a gelatin / pcl composite scaffold to a depth of 114 μm . the inventors investigated the use of a gelatin and pcl polymer as a suitable scaffold material for cell proliferation and penetration . gelatin has numerous merits which include biological origin , biodegradability , biocompatibility and commercial availability at relatively low cost . gelatin has also established itself to be used widely in the pharmaceutical and biomedical field as wound dressings , carrier for drug delivery and sealants [ 26 - 27 ]. the composite material was fabricated using gelatin type a ( 300 bloom , sigma , st . louis , mo .) from porcine skin in powder form , pcl ( mn 80 , 000 , aldrich ) with solvent 2 , 2 , 2 - trifluoroethanol ( tfe ) ( purity ≧ 99 . 0 %, fluka , buchs , switzerland ). solutions of gelatin / tfe and pcl / tfe mixtures were prepared in 10 % wt concentration and subsequently mixed together in 50 : 50 ratio under gentle stirring to obtain the gelatin / tfe / pcl solution to be used for fabricating the composite fibrous scaffold using the apparatus of fig1 under the same operating parameters as example 3 above to produce the gelatin / tfe and pcl / tfe tegaderm ™ composite (“ gelatin / tfe / pcl tegaderm ™ composite ”). the diameter of the gelatin / tfe / pcl tegaderm ™ composite nanofibers was noted in the range of 300 - 700 nm ( 80 % of nanofibers ) with a mean diameter of 500 ± 120 nm using an image analysis software ( imagej , national institute of health , usa ). through 3 hours of electrospinning , a nanofibrous mat with approximately 30 μm thickness was obtained . with a known bulk density of ( 1 . 34 g / cm 3 ), the porosity of the gelatin / tfe / pcl tegaderm ™ composite nanofiber scaffold can be obtained from the equation : where d and d represents the apparent density and bulk density respectively [ 28 ]. fig1 shows the fesem morphological images of the gelatin / tfe / pcl tegaderm ™ composite at a magnification of ( a ) 3 , 000 × ( b ) 8 , 000 × ( c ) 12 , 000 × and ( b ) 20 , 000 ×. fig1 shows fesem cross - sectional views of gelatin / tfe / pcl tegaderm ™ composite through freeze fracturing at a magnification of ( a ) 750 × and ( b ) 1 , 500 ×. mechanical properties of the electrospun gelatin / tfe / pcl tegaderm ™ composite were measured using the tabletop uniaxial testing machine ( instron 3345 ). this was done using a 10 - n load cell with a cross - head speed of 10 mm / min under ambient conditions . all samples were prepared in the form of rectangular shape with dimensions of 20 × 10 mm from the scaffold construct , with an average thickness of 120 μm measured from the digital micrometer . four samples were tested for this characterization procedure . specific sample preparation methodology is as mentioned in z . m . huang et al . [ 27 ] fig1 shows the stress - strain behavior of the gelatin / tfe / pcl scaffold before and after detachment from the tegaderm ™ wound dressing . interestingly , two different phases were noticed from the tensile loading graph . the first phase of the tensile loading graph is achieved with the gelatin / pcl scaffold still intact with the tegaderm ™ wound dressing . the second phase occurs after the scaffold has broken and tensile loading continues purely with tegaderm ™ wound dressing alone . this result has been extrapolated and shown in fig1 . it is known that the combined polymer of gelatin / pcl has lower tensile and elongation properties similarly , with an understanding that the tegaderm ™ wound dressing , has an almost elastic rubber - like tensile property , it is expected that the nanofibrous scaffold will break before the wound dressing does . however , the combined construct of the nanofibrous scaffold and tegaderm ™ wound dressing has given rise to an improvement in the tensile properties of a construct based solely on gelatin / pcl alone . this tegaderm - gelatin / pcl composite construct has shown to offer much better tensile strength , deformability and flexibility which is particularly important in skin rehabilitation procedures . as shown from the optical density of cellular activities from the cell viability test from fig1 , it can be noticed that hdf cell viability increased greatly over the 7 days period on gelatin / pcl nanofiber scaffold as compared with the pcl scaffold . this result can be verified with cell counting test from fig1 . it is observed that cell counts on gelatin / pcl composite scaffold are approximately an 80 % fold increase as compared with that of the normal pcl scaffold . it is interesting to note from fig1 that by day 7 of cell culture , optical density has begun to stagnate for the gelatin / pcl composite scaffold . this does not mean that cell growth has come to a stop , but rather at a slower rate . a probable reason will be at this phase the cells are working on penetrating into the composite scaffold through cell proliferation or growth in extracellularmatrix . results from fig1 shows that the hdf cells , being anchorage cells , tend to attach better to the gelatin / pcl composite scaffold , achieving results that are almost comparable with that of tcps . it can be concluded that the inclusion of the biopolymer gelatin into the pcl polymer solution has greatly enhanced the hdf affinity onto the scaffold structure . fig2 shows the cell count of hdfs on tcps , pcl nfm and pcl - gelatin nfm . cells were seeded at density of 1 . 5 × 10 4 cells / well and cultured for a period of 7 days . fig2 shows fesem morphological views of hdf proliferation on gelatin / pcl composite scaffold : ( a ) day 1 , ( b ) day 3 , ( c ) day 5 , ( d ) initial hdf penetration into scaffold structure . it will be appreciated that the disclosed composite is highly useful in the treatment of dermal conditions such as skin burns . it will be appreciated that the skin cells seeded into the scaffold layer assists in the healing of the skin . furthermore , the composite forms a protective barrier on the skin and prevents infection of the dermal , sub - dermal and epidermal tissue , particularly in situations where the body is unable to repair itself . it will also be appreciated that the skin cells seeded into the scaffold layer provide a useful alternative , or compliment , to skin grafting . additionally , as the inventors have surprisingly found that adhesives in commercially available polymer plasters , such as tegaderm , do not kill or inhibit skin cell growth , the composite is able to be used in dermis and epidermis repair . the composite additionally provides a useful alternative to the use of permanent skin replacement products . furthermore , electrospinning the fibers onto the semi - permeable barrier provides a relatively low cost means to manufacture the composite . furthermore , the composite can be used in ulcers that have signs of clinical infection or sinus tracts . 1 . s . t . boyce , g . d . warden . principles and practices for treatment of cutaneous wounds with cultured skin substitutes . the american journal of surgery . 2002 ; 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