Patent Application: US-201013144074-A

Abstract:
the present invention relates to a membrane comprising at least one positively charged , synthetic , hydrophobic polymer , at least one hydrophilic polymer and at least one plasticizer ; wherein said membrane is flexible and is capable of supporting at least one of cell adherence , cell proliferation or cell differentiation . the invention further relates to use of a membrane of the invention in the preparation of an implantable devices including cell delivery systems , cell growing surfaces and scaffolds . the invention further provides methods for promoting tissue regeneration in a defected tissue region applying membranes of the invention .

Description:
the present invention provides a membrane composed of at least three elements , the first being a synthetic , hydrophobic polymer having a positive surface charge , which is non - biodegradable under physiologic conditions , at least one hydrophilic polymer which is biodegradable under physiological conditions and at least one plasticizer . without wishing to be bound by theory , the combination of these elements generates a membrane which is flexible enough to be able to generate three dimensional structures suitable for various therapeutic applications , for example , a hollow tube . moreover , upon exposure to fluid ( in vivo or ex vivo ), the hydrophilic polymer at least partially disintegrates and the membrane becomes porous , thus enabling the adhesion of cells . cells may be seeded on a membrane of the invention as will be further described below in detail . in further embodiments , a membrane of the invention may further include an active agent , as further detailed below . a membrane of the invention can serve as an infrastructure to allow guided tissue repair as well as a cell delivery system . a membrane of the invention may also serve as a barrier membrane for eliminating infiltration of unwanted cells , blood vessels and soft / scar tissue into the treated area , and for isolating the cells delivered in said membrane from the surrounding tissue , and preventing the leakage of cells and factors from the space inside the membrane to the surrounding tissue . a membrane of the invention may be used as such , for example , by covering a region into which the cells are delivered , however , in certain embodiments it may be used to form a three dimensional device ( for example a hollow tubular device ) which holds the cells to be deliver , or may coat a tissue engineering scaffold containing the cells to be delivered . in some embodiments , a membrane of the invention is folded into a desired three dimensional structure , e . g . a tubular device . the tubular device can be used as an infrastructure to allow guided tissue repair as well as to deliver cells into a tubular region of defect , such as a bone defect , and the membrane can be used to hold the delivered cells and components in the device and prevent infiltration of cells , extracellular matrix and blood vessels from the surrounding tissue into the space surrounded by the device . in other embodiments , a membrane of the invention is used for coating a tissue engineering scaffold . such a membrane - coated scaffold can hold cells to be delivered into a site in the body . the membrane coating isolates the cells delivered in the scaffold from the surrounding tissue and prevents the leakage of cells , and soluble factors from the space inside the scaffold into the surrounding tissue . coating of the scaffold with a membrane of the invention may allow better cell adhesion and higher doses of cells to be delivered to the target site . in yet other embodiments , a bone defected area can be wrapped after implantation of a scaffold with a membrane of the invention in order to prevent leakage of cells and soluble factors and to prevent growth of soft tissue into the scaffold . the term “ cell delivery ” refers to introduction of cells into a desired site in the body of an individual for therapeutic purposes . a membrane of the invention is suitable for seeding of any type of cells for example stem cells ( both adult and embryonic stem cells ). in other embodiments cell type may be selected from the following non - limiting list : mesenchymal ( stromal ) stem cells , umbilical cord blood cells , osteoblasts , chondroblasts , or cd105 + cells . the invention also encompasses seeding of pluripotent stem cells of embryonic origin as well as adult cells that have been reprogrammed to become pluripotent . the cells may be autologous , allogenic or xenogenic . in some embodiments , the cells are autologous adult stem cells , obtained , for example , from bone marrow or adipose tissue . cell seeding is performed in some embodiments ex vivo . the cells may be placed on the membranes ( for example formed as a hollow tubular device ) or placed in a tissue engineering matrix ( also termed herein “ scaffold ”) coated by the membrane of the invention . examples of tissue engineering matrix are those fabricated from either biological materials or synthetic polymers . in certain embodiments , a membrane of the invention , a tubular implant of the invention or a coated scaffold of the invention , with or without ex vivo seeded cells are placed at a desired location in the body . this location is typically a location where it is desired to generate new tissue which has been damaged by trauma , surgical interventions , genetic or disease processes . in some embodiments a desired site is a site where tissue should be generated from adult stem cells ; is some embodiments such a site is ligament , tendon , cartilage , intervertebral disc , dental tissue or bone tissue , most preferably bone tissue . generation of bone tissue is required in conditions such as non - union fractures , osteoporosis , periodontal disease or teeth implantation , osteolytic bone disease , post - plastic surgery , post - orthopedic implantation , post neurosurgical surgery that involves calvaria bone removal , in alveolar bone augmentation procedures , for spine fusion and in vertebral fractures . generation of tendon / ligament tissue is required for example following tissue tear due to trauma or inflammatory conditions . generation of cartilage tissue is required in conditions such as rheumatoid arthritis , osteoarthritis , trauma , cancer surgery or cosmetic surgery . generation of intervertebral disc tissues including nucleous pulposus and annulus fibrosus , is required in conditions such as nucleous pulposus degeneration , annulus fibrosus tears , or following nucleotomy or discectomy . typically the membrane , for example in the form of a hollow tube is placed at the desired site by implantation . in certain embodiments , the membrane of the invention comprises a synthetic , hydrophobic positively charged polymer , a hydrophilic polymer , a plasticizer and an active agent and is further seeded with cells . in a specific embodiment the membrane of the invention comprises a synthetic , hydrophobic positively charged polymer and peg and is further seeded with stem cells . as used herein the term “ cell - growing surface ” refers to any artificial surface suitable for cell growth for example a slide , vessel or cell / tissue culture dish . the membrane coated cell growing surface in accordance with the invention thereby gains properties suitable for cell adhesion , proliferation and / or differentiation . the present invention provides a flexible membrane capable of supporting msc adherence , proliferation and differentiation . such a membrane can be used as treatment for bone regeneration applications . the healing of displaced fractures and regeneration of bone defects does not result only from proliferation of the locally present osteoblasts , but involves recruitment , proliferation , and differentiation of preosteoblastic cells . the differentiation of multipotent osteoblastic precursors is the main initial event in bone healing and callus formation , although preexisting osteoblasts might also be involved . any failure in the recruitment , establishment , proliferation , and differentiation of these progenitor cells can lead to delayed union or nonunion . there are many difficulties related to the healing of critical - size bone defects . in general , these difficulties result from the fact that there is an insufficient number and / or activity of osteogenic cells of the host to allow for healing . a membrane of the invention can guide bone regeneration as well as prevent unwanted vascularization in the newly formed bone . the membrane can also protect the area of bone defect from infiltration by connective and scar tissues , guide the osteogenic cells and allow storage of osteogenic components in the space enclosed by the membrane , which may potentially be released from the bone ends and bone marrow [ 10 , 12 ]. furthermore , placing msc attached to a membrane at the site of critical size defect model will provide starting material for a new bone tissue . therefore , implanting gbr membrane with expanded ex vivo msc may greatly improve the bone repair outcome . as demonstrated in the examples provided below , several polymers were tested in conjugation with various plasticizers . in one embodiment , a membrane constituted from amca and 15 % peg 400 could support good msc adhesion , proliferation and differentiation : ( i ) msc adhered to amca membrane with 15 % peg 400 as determined by light microscopy , fluorescent microscopy and sem . ( ii ) msc maintain their proliferative activity as determined by cfse labeling and flow cytometric analysis ( iii ) msc maintained their differentiation ability as determined by alizarin red staining . amca membrane containing 15 % peg 400 supported msc differentiation to osteoblasts . ammonio methacrylate copolymer type a nf ( amca , eudragit ® rl , degussa , germany ) and ethyl cellulose ( ec , ethocel ® n 100 , hercules inc ., wilmington , del .). polyethylenglycol 400 ( peg 400 , merck , germany ), glyceryl triacetate ( triacetin , fluka , rehovot , israel ), glycerin ( frutarom , israel ), triethyl citrate ( fluka , rehovot , israel ), dibutyl sebacate ( fluka , rehovot , israel ), dibutyl phtalate ( fluka , rehovot , israel ). membranes were prepared using solvent casting technique as disclosed in friedman m . and golomb g . j . [ 13 ]. polymeric membranes were cast from solution consisting of polymer , plasticizer and ethanol ( frutarom , israel ) into teflon ® moulds ( round plates , inner diameter 9 . 6 cm ) and the solvent was allowed to evaporate over night . membranes width was : 100 ± 5 μm . prior to use in tissue culture , membranes were immersed in pbs ( biological industries , beit haemek , israel ) for 24 hours to wash out possible remains of ethanol and then sterilized by uv irradiation for 2 hr . amca membranes containing 15 % peg 400 were fixed with 2 % glutaraldehyde in cocodylate buffer ( 0 . 1 m ; ph = 7 . 2 ) for 2 hours . the specimens were then processed according to the air drying method skipping the ethanol dehydration series ( ethanol dissolves amca ; therefore it should be excluded from the specimen preparation ). the process was accomplished through 100 % freon 113 . the specimens were vigorously shaken , which allowed rapid evaporation of the freon phase . the membranes were mounted in copper stubs , coated with gold and then examined in fei quanta 200 at an accelerating voltage of 30 kv . hmscs were obtained from discarded bone tissues from patients undergoing total hip replacement surgeries , under approval of hadassah medical center helsinki ethics committee following an informed consent . the hmscs were separated from other bone marrow - residing cells by plastic adherence and were then grown under tissue culture conditions , as described in krampera m . et al . [ 14 ], and djouad f et al [ 15 ]. the cells were maintained in a low - glucose dulbecco &# 39 ; s modified eagle medium ( dmem ) supplemented with 10 % heat - inactivated fetal calf serum , 2 mm glutamine , and penicillin / streptomycin ( biological industries , beit - haemek , israel ). primary cultures were usually maintained for 12 - 16 days , and were then detached by trypsinization and subcultured ( barry f p . et al . [ 16 ]). the medium was changed every 3 - 4 days . for msc labeling , msc were re - suspended in pbs ( 10 7 cells / ml ) containing 5 - carboxyfluorescein diacetoxymethyl ester ( bcecf / am or cfse ; 5 μg / ml ; calbiochem ), incubated at 37 ° c . for 10 mm , and the cells were then washed three times . cells were cultured on a sterilized membrane wetted with pbs , 15 × 10 4 cells in 150 μl medium , and incubated for six hours at 37 ° c . afterwards 3 ml of medium were added . cells were examined 24 hours after seeding by fluorescent microscope . tissue culture polystyrene dishes were used as a positive control for membrane in cell adhesion test . for cell division studies , msc were resuspended in pbs ( 10 7 cells / ml ) containing 3 ′- o - acetyl - 2 ′, 7 ′- bis ( carboxyethyl )- 4 or 5 - carboxyfluorescein diacetoxymethyl ester ( bcecf / am or cfse ; 5 μg / ml ; calbiochem ), incubated at 37 ° c . for 10 min , and washed three times . cfse - labeled cells were then seeded on the membrane or on the tissue culture dishes as described above . at the indicated time points cells were harvested and proliferation of cells was visualized by incremental loss of cfse fluorescence as analyzed on a facscalibure flow cytometer ( becton dickinson ) using cell quest software . msc were seeded on the membranes or on the center well organ culture dishes ( falcon ) for control , as described above . as soon as msc were confluent , the culture medium was supplemented with ascorbic acid ( 50 μg / ml ), dexamethasone ( 10 − 8 m ) and β - glycerophosphate ( 10 mm ). medium was changed twice a week for 17 days , afterwards membranes and dishes were dyed with alizarin red , as described below . a stock solution of 2 % alizarin red in distilled water was adjusted to ph 4 . 2 with naoh and passed through a 0 . 22 μm filter . cultures in the center well organ culture dishes were rinsed with 150 mm nacl three times , fixed in ice cold 70 % ethanol , rinsed with distilled water and stained at room temperature for 10 min with 500 μl of alizarin red stock per well . individual wells were rinsed five times with distilled water ; a sixth and final wash with distilled water was performed for 15 min ( halvorsen y d . et al . [ 17 ]). membranes due to their positive charge had a higher affinity towards alizarin red stain than a negatively charged center well organ culture dishes , therefore rinsing with distilled water didn &# 39 ; t remove the stain from the membranes well enough . to reduce background we applied a single rinse with 0 . 02 m hcl on the membranes . photomicrographs were then obtained . various membranes were tested for their ability to support cell attachment and growth . the tested membranes varied in their polymer and plasticizer types . several plasticizers were tested , i . e . glycerin , polyethylene glycol , triethyl citrate , dibutyl sebacate , dibutyl phtalate , triacetin . the plasticizers tested were hydrophobic or hydrophilic and were added in order to contribute flexibility to membrane . msc were seeded on sterilized membranes as described hereinabove . msc cells showed little adherence to all formulations of ec membranes and cell aggregation was slight . the various plasticizers had no influence on either cell adhesion or cell shape . as control , poly - 1 - lysine coated membranes were used . poly - 1 - lysine , a highly positively charged amino acid chain , is commonly used as a coating agent to promote cell adhesion in culture . cells adhered in monolayer spindle shape to ec membranes coated with poly - 1 - lysine , hence it was concluded that ec does not support cell adhesion , as such . however ec was found to be non toxic in the presence of poly - 1 - lysin . cell adhesion test was performed with ammonio methacrylate copolymer type a ( amca , eudragit ® rl , degussa , germany ) [ 85 %], mixed with various plasticizers disclosed herein above [ 15 %]. msc adhered well to amca membranes prepared with the various plasticizers ( fig1 d - f ) in spindle monolayer shape . cell spreading on the amca membranes was similar to spreading on the polystyrene dishes which were used as a positive control for cell adhesion ( fig1 a - c ). the mode of spreading is indicative of the cells &# 39 ; well being . cell adhesion was further analyzed using sem . as shown in fig2 cells on the amca membrane , were flat and monolayer spindle shaped . furthermore , at higher magnification , cell - membrane interaction was seen , with a cellular podia attached to the membrane , ( fig2 , d - f ). moreover , the release of numerous vacuoles from the cell surface was observed , demonstrating cell functionality . similar results were obtained using both human as well as rabbit msc . proliferative capacity of msc was tested using the fluorescent marker of cell division , cfse and flow cytometric analysis . this method is based on the fluorescein related dye cfse , which is partitioned with remarkable fidelity between daughter cells allowing eight to 10 discrete generations to be identified both in vitro and in vivo . the technique allows complex information on proliferation kinetics and differentiation to be collected according to this technology ; individual cells are tagged with the fluorescent cfse dye that binds irreversibly to cell cytoplasm . as cells divide , their fluorescence halves sequentially with each generation , allowing the proliferative history of any single cell present to be monitored over time ( see lyons ab . et al [ 18 ]). msc proliferated on amca and peg 400 membrane ( fig3 b ) ( but no proliferation was detected with other plasticizers ; data not shown ) although at somewhat reduced rates as compared to their proliferative capacity on tissue culture dishes used as control ( fig3 a ). subsequently , msc proliferation rate was tested over time on membranes containing different concentrations of peg 400 ( 10 %, 15 %, 20 % and 25 % w / w ). the rate of msc proliferation inversely correlated to the mean fluorescent intensity value ( mfi ) ( fig4 ). this analysis revealed that , membranes containing 15 % peg 400 and 20 % peg 400 were fairly close to the polystyrene control , while other concentrations of peg resulted in either higher or lower proliferation rates . in addition , amca membrane with 15 % and with 5 % peg 400 was characterized using scanning electron microscopy ( sem ). membranes were observed before and after immersion in pbs ( fig5 ). it is noted that membranes were immersed in pbs for 24 hours before each msc seeding , in order to wash out residual ethanol . since peg 400 is soluble in water and thus porogenic , only after immersion in pbs , pores were observed on the membrane surface ( fig5 b - c ). in both concentrations of peg 400 , sem pictures demonstrated a porous surface , with average pore size of 0 . 18 μm . pore distribution correlated directly to different peg 400 concentrations . differentiation medium was added as described hereinabove . membranes and dishes were then dyed with alizarin red . ( fig6 a and 6b ). alizarin red binds irreversibly to bivalent positive ions and has especially high affinity towards calcium . calcium is secreted from osteoblasts and deposits on the membrane as part of the creation of an extracellular matrix . therefore presence of calcium marks the differentiation from msc that do not secrete calcium into osteoblast . fig6 demonstrates that msc cultured on both amca membrane and polystyrene controls have differentiated to osteoblast and produced extracellular matrix . this finding confirms that amca membrane with 15 % peg 400 supports msc differentiation and that msc after adhesion to membrane maintain their stem cells traits . example 4 — in vivo bone regeneration study using a membrane of the invention five male new zealand rabbits weighing 3 . 8 - 4 . 4 kg underwent bilateral midshaft resection of radial bone segment ( 1 cm in length ) in forelimbs . tubular amca membranes were implanted in the left forelimb ( treated osteotomy ) and the right limb served as a control ( untreated osteotomy ). radiographic evaluation — lateral radiographs of forelimbs were obtained 2 , 4 , 6 and 8 weeks postoperatively . to obtain standardized measurements of the bone defects during the regenerative healing process , true lateral radiographs of both forelimbs were performed in standard conditions ( 42 kv , 2 mas ). radiographs were examined using osirix medical imaging software to evaluate the area and density of the new bone . total area of regenerated bone tissue ( appearing around and within the bone gap defect ). to eliminate possible bias by variability of bone dimensions , data calibration was made using the diameter of olecranon process at its narrowest zone as a standard reference . this diameter was defined as 10 mm in each specimen . relative density of the newly regenerated bone in the gap defect . the segmented area was outlined , and the density was measured . the bone density in the center of the olecranon process was measured in each forelimb for a calibration , as a reference value . the density of olecranon process was defined as a 100 % for each specimen ( see mosheiff r . et al . [ 10 ]). fig7 shows bone regeneration expressed by mean callus area ( mm 2 ) throughout the study ( weeks 2 to 8 ). at week 2 of the study the mean callus area produced in control arm was larger then that of arm treated with amca membrane , possibly due to formation of hematome or blood clot at the surgery site . when the site was surrounded by membrane it isolated the area and thus slowed the degradation of the hematome . however from week four of the study , mean area of callus generated in the limb treated with amca membrane was slightly bigger than that of the control ( 144 . 8 mm 2 vs . 114 . 5 mm 2 ). this trend continued at weeks 6 and 8 , hand in hand with widening the difference between mean callus areas of amca membrane treated limb and control limb . at week 8 , the difference between mean callus areas produced in two limbs ( treated with amca membrane and control ) reached its peak and was 143 . 91 mm 2 ( see table 1 below ). however , this difference is not statistically significant , due to small sample size ( n = 5 ) of this preliminary study and high variability of results , as it often happens in in vivo studies . in 6 male new zealand rabbits critical size defect ( 10 mm ) was created in both forelimbs . in one forelimb ec membrane which contained simvastatin was inserted , in the contralateral limb ec membrane with no active agent was inserted . callus density and callus area were measured and calibrated using osirix software . fig8 a and 8b show the quantitative analysis of the radiographs . fig9 shows the microct of bone regeneration with ec membrane . in this experiment bone defect was left untreated . the bone defect is in non union state . arrows mark the bone defect area . fig1 shows microct of bone regeneration with ec membrane containing simvastatin . arrows mark the bone defect area . in this experiment bone defect was treated and successful bridging of the defect is evident . example 6 — in vivo bone regeneration study using an amca membrane of the invention further comprising simvastatin critical size bone defect of 1 cm in radius bone were created . 5 rabbits were treated with simvastatin controlled release amca membrane and 5 others with amca membrane without any active ingredient . simvastatin 20 % w / w — 0 . 36 g amca ( eudragit ® rl ) 70 % w / w — 1 . 26 g peg 400 10 % w / w — 0 . 18 g membrane width was 180 micrometer . amca ( eudragcit ® rl ) 90 % w / w — 1 . 26 g peg 400 10 % w / w — 0 . 18 g membrane width was 180 micrometer . fig1 shows significantly larger callus area formed at the defect site treated with simvastatin controlled release amca membrane ( wilcoxon summed ranks test ), as well as increase in callus growth rate at 2 first post operation weeks — may be important from clinical point of view . example 7 — in vitro release rate of simvastatin from different membranes of the invention the effects of various parameters on simvastatine release from membranes of the invention were measured in vitro as follows : the effect of simvastatin concentration on simvastatin release rate is shown in fig1 a ; the composition of the tested membranes was as follows : the effect of membrane width on simvastatin release rate is shown in fig1 b ; the composition of the tested membranes was as follows : the effect of plasticizer on simvastatin release rate is shown in fig1 c ; the composition of the tested membranes was as follows : the effect of plasticizer type on simvastatin release rate is shown in fig1 d . critical size bone defect of 1 cm in radius bone was created . two rabbits were treated with amca membrane carrying hmsc in one forearm and on another forearm amca membrane without hmsc . fig1 a demonstrates the development of the callus area in the effected bone . as shown in fig1 b the histological score of various parts of the defected bone area at 8 weeks post operation is higher in bones implanted with an amca membrane carrying hmsc . 1 . gerstenfeld l c , cullinane d m , barnes g l , graves d t , einhorn t a . fracture healing as a post - natal developmental process : molecular , spatial , and temporal aspects of its regulation . j cell biochem 2003 ; 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