the present invention will be described further with reference to various embodiments in the drawings . according to a preferred embodiment of the present invention , such a guided bone regeneration membrane can be manufactured through the steps of electrospinning and soaking in a simulated body fluid ( sbf ). in the electrospinning step , a positive high voltage is applied to a polymer solution , and the resulting polymer solution is sprayed as fibers to a negatively charged collector . a spinning solution for the formation of the pla layer ( pla spinning solution ) is prepared by dissolving a poly ( lactic acid ) in chloroform ( chcl 3 ) or dichloromethane ( dcm ). the pla spinning solution preferably has a poly ( lactic acid ) concentration of from 4 to 12 percent by weight for easy spinning . in this connection , the poly ( lactic acid ) generally has a molecular weight of from about 20 × 10 4 to about 30 × 10 4 . for maintaining conditions for satisfactory spinning , the pla spinning solution may further contain dimethylformamide ( dmf ) and / or methanol ( ch 3 oh ) in an amount up to about 50 percent by weight relative to the amount of chcl 3 or dcm . another spinning solution for the formation of the si — caco 3 / pla layer ( si — caco 3 / pla spinning solution ) is prepared by adding si — caco 3 to the pla spinning solution . the si — caco 3 is preferably added to the solution so that the si — caco 3 / pla layer has a si — caco 3 content of from 40 to 60 percent by weight . this allows an apatite to deposit efficiently on si — caco 3 / pla fibers in the sbf soaking step . alternatively , a si — caco 3 / pla spinning solution can be prepared by kneading a poly ( lactic acid ) and si — caco 3 in predetermined proportions using a heating kneader to give a composite , and dissolving the composite in a solvent . the si — caco 3 may be prepared , for example , by the method described in japanese patent application no . 2006 - 285429 ( corresponding to japanese unexamined patent application publication ( jp - a ) no . 2008 - 100878 ). the pla layer preferably contains a poly ( lactic acid ) ( pla ) alone or a copolymer between a poly ( lactic acid ) and a poly ( glycolic acid ) ( pga ) ( copoly ( lactic acid / glycolic acid )) exemplary other biodegradable resins usable herein include synthetic polymers such as polyethylene glycols ( pegs ), polycaprolactones ( pcls ), as well as copolymers among lactic acid , glycolic acid , ethylene glycol , and / or caprolactone ; and natural polymers such as fibrin , collagens , alginic acids , hyaluronic acids , chitins , and chitosans . each of these can be used instead of the pla component in the si — caco 3 / pla layer . the si — caco 3 / pla layer and the pla layer may further contain inorganic substances that are usable without biological problems . examples of such inorganic substances include tricalcium phosphate , calcium sulfate , sodium phosphate , sodium hydrogenphosphate , calcium hydrogenphosphate , octacalcium phosphate , tetracalcium phosphate , calcium pyrophosphate , and calcium chloride . using an electrospinning apparatus , each of the pla layer spinning solution and the si — caco 3 / pla spinning solution is charged and sprayed from a nozzle , converted into fibers in an electric field while evaporating the solvent , the charged fibers are jetted toward a collector on a negative electrode and form a thin layer of fibers on the collector . a desired guided bone regeneration membrane can be prepared by changing spinning conditions such as the concentration , solvent type , and supply speed ( feed rate ) of the spinning solution ; spinning time ; applied voltage ; and distance between the nozzle and the collector . the prepared nonwoven fabrics may be pressed so as to be compacted or to have a desired thickness . a guided bone regeneration membrane having a bi - layered structure is configured by spraying the pla spinning solution to form a pla layer , and thereafter spraying the si — caco 3 / pla spinning solution to form a si — caco 3 / pla layer on the pla layer ; or by preparing a pla nonwoven fabric and a si — caco 3 / pla nonwoven fabric independently , and combining the two nonwoven fabrics . the guided bone regeneration membrane having a bi - layered structure is cut to a desired size and soaked in a simulated body fluid ( sbf ) or a solution with 1 . 5 times higher concentration of inorganic ions compared to sbf ( 1 . 5sbf ) at about 37 ° c . for a predetermined time to precipitate an apatite on the si — caco 3 / pla layer . this gives a bioresorbable guided bone regeneration membrane including a novel mechanism that effectively induces the bone reconstruction ability . the sbf soaking can be performed even after the combining ( or laminating ) the two layers . even in this case , the apatite deposits substantially not on the pla layer but selectively on the si — caco 3 / pla layer . this is because silicon contained in the si — caco 3 / pla layer induces nucleation of apatite , and the calcium component dissolves out to abruptly increase the degree of supersaturation of apatite , and the apatite selectively deposits on the surface of the si — caco 3 / pla layer ; but the surface of the pla layer is hydrophobic to avoid the deposition of apatite substantially . manufacturing methods of guided bone regeneration membranes according to embodiments of the present invention will be illustrated with reference to several examples below . it should be noted , however , that these examples are included merely to aid in the understanding of the present invention and are not to be construed to limit the scope of the present invention . silicon - releasable calcium carbonate ( si — caco 3 ): vaterite having a silicon content of 2 . 9 percent by weight and prepared by using slaked lime ( microstar t ; purity 96 % or more ; yabashi industries co ., ltd ., japan ), methanol ( analytical grade reagent ; purity 99 . 8 % or more ; kishida chemical co ., ltd ., japan ), γ - aminopropyltriethoxysilane ( tsl 8331 ; purity 98 % or more ; ge toshiba silicones co ., ltd ., japan ), and carbon dioxide gas ( high - purity liquefied carbon dioxide gas ; purity 99 . 9 %; taiyo kagaku kogyo k . k .) poly ( lactic acid ) ( pla ): purasorb pl poly ( l - lactide ), molecular weight of 20 × 10 4 to 30 × 10 4 , purac biochem chloroform ( chcl 3 ): analytical grade reagent , purity 99 . 0 % or more , kishida chemical co ., ltd ., japan n , n - dimethylformamide ( dmf ): analytical grade reagent , purity 99 . 5 % or more , kishida chemical co ., ltd ., japan example 1 a pla spinning solution having a pla concentration of 6 . 8 percent by weight was prepared by blending 10 g of pla , 110 g of chcl 3 , and 27 . 5 g of dmf . independently , a si — caco 3 / pla spinning solution having a si — caco 3 concentration of 7 . 5 percent by weight and a pla concentration of 5 . 0 percent by weight was prepared by blending 15 g of si — caco 3 , 10 g of pla , 140 g of chcl 3 , and 35 g of dmf . using the prepared spinning solutions , a guided bone regeneration membrane having a bi - layered structure of nonwoven fabrics was manufactured through electrospinning . spinning solution feed rate : about 0 . 1 ml / min ., applied voltage : 15 kv , distance between the nozzle and collector : 10 cm , nozzle : laterally moves in a width of 3 to 4 cm at a rate of 15 cm / min , conveyor - type collector ( conveyor speed : 5 to 6 m / min ), spinning time : about 170 minutes spinning solution feed rate : about 0 . 16 ml / min , applied voltage : 20 kv , distance between the nozzle and collector : 10 cm , nozzle : laterally moves in a width of 3 to 4 cm at a rate of 15 cm / min , conveyor - type collector ( conveyor speed : 5 to 6 m / min ), spinning time : about 130 minutes the microstructure of the prepared pla layer ( side for preventing soft tissue invasion ) is shown in the scanning electron microscope ( sem ) photograph of fig1 . the microstructure of the si — caco 3 / pla layer ( bone regeneration side ) is shown in the scanning electronmicrograph of fig2 , demonstrating that si — caco 3 particles are attached to pla fibers . a spinning solution having a pla concentration of 9 . 0 percent by weight was prepared by blending 9 g of pla and 91 g of chcl 3 , and using this spinning solution , a pla layer was prepared through electrospinning . spinning solution feed rate : 0 . 05 ml / min , applied voltage : 20 kv , distance between the nozzle and collector : 15 cm , nozzle : fixed , plate collector : fixed , spinning time : 60 minutes independently , pla and si — caco 3 were kneaded in a heating kneader at 200 ° c . for 15 minutes to give a si — caco 3 / pla composite containing 60 percent by weight of si — caco 3 . a spinning solution having a si — caco 3 concentration of 13 . 0 percent by weight and a pla concentration of 8 . 7 percent by weight was prepared by blending 25 g of the si — caco 3 / pla composite and 90 g of chcl 3 , and using this spinning solution , a si — caco 3 / pla layer was prepared through electrospinning . spinning solution feed rate : 0 . 05 ml / min , applied voltage : is 20 kv , distance between the nozzle and collector : 15 cm , nozzle : fixed , plate collector : fixed , spinning time : 30 minutes the two nonwoven fabrics prepared by the above procedures were each cut to a desired size and affixed or combined with each other to give one membrane . specifically , the pla layer was laid over the si — caco 3 / pla layer , and a stainless steel mesh ( 40 - mesh ) was laid over the pla layer . a plate heated at 150 ° c . to 160 ° c . was placed on the stainless steel mesh and pressed under a suitable pressure for about 10 seconds to give the combined membrane ( composite membrane ). the scanning electron micrographs of the pla layer surface and of the si — caco 3 / pla layer surface are shown in fig3 and fig4 , respectively . the scanning electron micrograph of fibers configuring the si — caco 3 / pla layer is shown in fig5 , demonstrating that si — caco 3 particles are attached to pla fibers . the si — caco 3 / pla layer surface of the resulting composite membrane was brought into contact with 1 . 5sbf at 37 ° c . for one day . the scanning electron micrograph of fibers on the side in contact with 1 . 5sbf is shown in fig6 , demonstrating that a substance quite different from si — caco 3 covers the surface of fibers , as compared to fig5 . the x - ray diffraction patterns before and after soaking in 1 . 5sbf are shown in fig7 , indicating that peaks of apatite appear after the soaking . these results demonstrate that the si — caco 3 / pla layer surface is coated with apatite . fig8 shows how cellular numbers ( in terms of cellular numbers per 1 cm 2 ) vary after the inoculation of osteoblastic cells on the apatite - coated si — caco 3 / pla layer surface ( si - composite ), on the pla layer surface ( pla ), and on a control ( thermanox : plastic disc for cell culture which has been treated on its surface ). the data in fig8 demonstrate that the layer including pla in combination with a novel mechanism gives higher growth capability to osteoblasts , and the resulting guided bone regeneration membrane is expected as a bioresorbable guided bone regeneration membrane that excels in bone reconstruction ability . cell type ; murine osteoblastic cells ( mc3t3 - e1 cells : riken institute of physical and chemical research ) cell counting method : the measurement was performed using the cell counting kit - 8 ( cellular growth / cellular toxicity analytical reagent ; dojindo laboratories ) in accordance with the protocol attached to the reagent . while the above description is of the preferred embodiments of the present invention , it should be appreciated that the invention may be modified , altered , or varied without deviating from the scope and fair meaning of the following claims .
disclosed is a guided bone regeneration membrane including a novel mechanism that effectively induces a bone reconstruction ability . the mechanism is provided by forming a bi - layered structure of a first nonwoven fabric layer containing a silicon - releasable calcium carbonate and a poly as principal components and a second nonwoven fabric layer containing a poly as a principal component ; and coating the first nonwoven fabric layer with an apatite . the guided bone regeneration membrane is available by using a nonwoven fabric manufacturing technique through electrospinning and a simulated body fluid soaking technique .