Patent Abstract:
a biomimetic biosynthetic nerve implant casting device includes a matrix casting tube ; a matrix casting tube protective shield comprising a male coupling portion joinable to a female coupling portion , wherein the joined portions encase the matrix casting tube ; microchannel forming fibers ; a fixing point for holding one end of the microchannel forming fibers ; loading fiber guideholes for placement of the microchannel forming fibers ; one or more ports for injection of matrix material into the casting tube ; and a cell suspension loading well in fluid communication with the matrix casting tube when the device is fully assembled such that removing the fibers from the formed implant can draw fluid containing cells and / or other agents into the microchannels .

Detailed Description:
an embodiment of the disclosure is shown in fig1 , and provides a casting device useable to cast a multi - luminal scaffold a novel biosynthetic nerve implant . the device includes an outer biodegradable or non - biodegradable tube or sleeve with a plurality of perforations to allow cellular migration inside the lumen , a multi - laminal matrix with multiple microchannels , which in turn can be loaded with single or multiple selected cell types or molecules . the surface area of each microchannel can be further modified to incorporate micro - or nano - domains that are cast into the microchannels during the extraction of the conduit - casting / cell suspension loading fibers . in certain embodiments , the fibers are coated with chemically treated , cell - anchoring , nano - or micro - structures , or a combination thereof . the micro - or nano - structures are released and remain embedded in the matrix upon extraction of the fibers , which also draws cells or molecules into the lumen of each microchannel . the preferred nerve conduit provides great flexibility for custom fabrication of a cell scaffold designed for a particular nerve to be repaired . preferred casting devices allow for the reproducible production of a nerve conduit with relative ease , and within a short period time . the hydrogel - based multi - luminal scaffold is designed to allow fascicular growth of axons through the multiple microchannels . as indicated in fig1 , each microchannel of the multi - luminal matrix may incorporate cells or molecules in the lumen , and / or micro - structures or nano - domains either in the lumen or embedded in the walls of the microchannels , in order to present extracellular matrix molecules and growth factors to the regenerating nerves . furthermore , these domains , molecules , and / or cells inside each microchannel are used to evaluate and quantify cellular growth and function . the hydrogel - based multi - luminal scaffold is designed to allow compartmentalization of the regenerated nerve tissue and segregation and directed growth through the combination of physical microchannels and specific molecular cues . the external conduit is preferably a tube composed of biocompatible and / or bioresorbable material ( s ). such materials may include , but are not limited to cellulose , hydroxymethyl cellulose , hydroxyethyl cellulose , carboxymethyl cellulose , carboxymethyl chitosan , poly - 2 - hydroxyethyl - meth - acrylate , poly ( r - 3 - hydroxybutyric acid - co -( r )- 3 - hydroxyvaleric acid )- diol ( phb ), collagen , keratin , gelatin , glycinin , synthetic polymers , including polyesters such as polyhydroxyacids like polylactic acid ( pla ), polyglycolic acid ( pga ) and copolymers thereof such as poly ( lactic acid - co - caprolactone ), some polyamides and poly ( meth ) acrylates , polyanhdyrides , as well as non - degradable polymers such as polyurethane , polytrafluoroethylene , ethylenevinylacetate ( eva ), polycarbonates , and some polyamides - methyl , or silicone rubber . the perforations in the external conduit are designed to facilitate the migration of endogenous cells , such as those in the muscular fascia , which then vascularize the intra - luminal matrix , providing enhanced exchange of nutrients and gas for the cells seeded within the multi - luminal channels or the regenerated tissue . fig2 shows a graphic representation of the three - dimensional multi - luminal nerve implant matrix casting tube . hand - made prototypes of a bni matrix - casting device were built ( fig3 ) to demonstrate the principle disclosed herein . the device , made of dental cement , has plastic fibers guided through it by a series of holes cast at both ends of the device . the device has a matrix casting well to accommodate the external tubing and a loading well for the placement of cells and / or molecules that are loaded into the hydrogel matrix simply by removing the plastic fibers once the hydrogel has polymerized . the microchannels may be geometrically distributed in different shapes and sizes to maximize tissue regeneration and to better match the fascicular nature of the specific nerve to be repaired . an advanced casting device is illustrated in fig1 - 15 . the multi - luminal matrix is made by casting multiple cylindrical microchannels within a biocompatible and bioresorbable , biopolymeric material capable of forming a hydrogel , wherein the cylindrical microchannels are formed inside the external tubing and parallel to the longitudinal axis of the tube ; each cylindrical matrix has two ends . the intra - luminal matrix may include a material selected from the group consisting of agar , agarose , gellan gum , arabic gum , xanthan gum , carageenan , alginate salts , bentonite , ficoll , pluronic polyols , carbopol , polyvinylpyrollidone , polyvinyl alcohol , polyethylene glycol , methyl cellulose , hydroxymethyl cellulose , hydroxyethyl cellulose , carboxymethyl cellulose , carboxymethyl chitosan , poly - 2 - hydroxyethyl - meth - acrylate , polylactic acid , polyglycolic acid , collagen , gelatin plastics , and extracellular matrix proteins and their derivatives . by placing a solution or suspension in the loading well of the casting device , one can easily incorporate any combination of cells and bioactive compounds presented within the lumen of each microchannel . of particular interest is the combination of growth factors and extracellular matrix molecules with or without cells . in a preferred embodiment , a slow release formulation is prepared as nano - or micro - spheres in a size distribution range suitable for cell attachment and drug delivery . the spheres are embedded in the hydrogel scaffold partially exposed to the luminal surface of the multiple microchannels . the anchored intra - luminal particles function as a method for selectively restricting the delivery of cell effectors , promoters or inhibitors , and provide cellular anchoring points for cell development within the lumen of the conduits . several molecules , pharmacological agents , neurotransmitters , genes , or other agents may be entrapped in the biodegradable polymer - manufactured micro - or nano - spheres for on - demand drug and gene delivery within the microchannels . systems may be tailored to deliver a specified factor for cell attachment and growth , such as acidic and basic fibroblast growth factors , insulin - like growth factors , epidermal growth factors , bone morphogenetic proteins , nerve growth factors , neurotrophic factors , tgf - b , platelet derived growth factors , or vascular endothelial cell growth factor , as well as active fragments or analogs of any of the active molecules . the disclosed devices are also amenable for controlling the loading and subsequent maintenance dose of these factors by manipulating the concentration and percentage of molecular incorporation in the micro - or nano - sphere , and the shape or formulation of the biodegradable matrix . in certain embodiments of the invention , the controlled release material includes an artificial lipid vesicle , or liposome . the use of liposomes as drug and gene delivery systems is well known to those skilled in the art . further , the present disclosure provides for pharmaceutically acceptable delivery of neural molecules such as neuroactive steroids , neurotransmitters and their receptors . yet another aspect of the disclosure is the manipulation of factors that modulate or measure the ionic transport across cell membranes . suitable biodegradable polymers can be utilized as the controlled release material . the polymeric material may be a polylactide , a polyglycolide , a poly ( lactide - co - glycolide ), a polyanhydride , a polyorthoester , polycaprolactones , polyphosphazenes , polysaccharides , proteinaceous polymers , soluble derivatives of polysaccharides , soluble derivatives of proteinaceous polymers , polypeptides , polyesters , and polyorthoesters or mixtures or blends of any of these . the polysaccharides may be poly - 1 , 4 - glucans , e . g ., starch glycogen , amylose , amylopectin , and mixtures thereof . the biodegradable hydrophilic or hydrophobic polymer may be a water - soluble derivative of a poly - 1 , 4 - glucan , including hydrolyzed amylopectin , hydroxyalkyl derivatives of hydrolyzed amylopectin such as hydroxyethyl starch ( hes ), hydroxyethyl amylose , dialdehyde starch , and the like . other useful polymers include protein polymers such as gelatin and fibrin and polysaccharides such as hyaluronic acid . it is preferred that the biodegradable controlled release material degrade in vivo over a period of less than a year . the controlled release material should preferably degrade by hydrolysis , and most preferably by surface erosion , rather than by bulk erosion , so that release is not only sustained but also provides desirable release rates . the disclosure also provides for the use of the micro - structures or nano - domains as a means to evaluate cellular function either through a calorimetric or calorimetric molecular or physiological indicator . the present disclosure is not limited to regeneration of nerve cell connections or to nerve tissue of either the central or peripheral nerve systems . while specific alternatives to steps of the invention have been described herein , additional alternatives not specifically disclosed , but known within the art , are intended to fall within the scope of the present inventions . thus it is understood that other applications of the present disclosure will be apparent to those skilled in the art upon the reading of the described embodiments and a consideration of the claims and drawings . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . preclinical data on animal models was obtained to evaluate surgical morbidity , immunogenicity , and cellularity of the implants . using the sciatic nerve gap repair model , two separate cohorts of rats repaired with either seven or fourteen multi - luminal bnis were examined and compared to animals repaired with empty tubes , tubes filled with collagen , or autologous grafts . some of the animals were implanted with ptfe micro - renathane ® tubing that included conical perforations . as expected , the recovered implant showed a nerve cable 10 weeks after implantation ( fig4 ). the benefit of the perforations to the polyurethane micro - renathane ® tubing is also illustrated in fig4 . in sharp contrast to the single nerve cable that characterizes the autograft ( fig4 a ) and the simple tubularization repair method ( fig4 b ), multiluminal repair revealed fascicular - like nerve growth throughout the length of the multiluminal bnis 10 - 16 weeks after injury ( fig4 c - h ). in most cases , the nerve cables were similar in thickness and occupied all the available area within each microchannel ( approximately 250 μm id ) of bnis that contained 7 or 14 channels . vascularization of the bni nerve cables was observed both inside each microchannel ( fig4 e ), and along the mesenchimal layer that formed between the inside tubing and the outer agarose ( fig4 f ). no gross evidence of inflammation or tissue reaction was observed in any of the bni - implanted animals . to confirm that the gross tissue regeneration observed within the bni was filled with nerve - associated cellular structures we performed histological and morphometric analysis ., as shown in fig5 . compared to the uninjured controls ( fig5 a ), autograft repair ( fig5 b ) or collagen - filled tubing repairs ( fig5 c ), qualitative normal nerve regeneration was facilitated by the bni ( fig5 d , e ). a highly vascularized mesenchimal layer covered the outer surface of the bni hydrogel ( fig5 d ). furthermore , each channel was vascularized and filled with numerous axons , and was surrounded by a perineurium - like outer membrane that resembled the multifascicular architecture of the normal nerves ( fig5 e ). we then evaluated whether the total area occupied by the regenerative axons differed among the repair methods . the total area of tissue regeneration was determined by tracing the area of toludine - blue stained tissue containing visible nerve growth . the area occupied by the regenerated nerves was comparable among the autograft - repaired and collagen - loaded tabularized animals , and was similar to nerves of uninjured animals ( fig5 f ). in contrast , a three - fold reduction in the regenerated area was observed in animals repaired with the 14 - channel bni ( fig5 f ). to determine the efficacy of nerve growth in the bni - repaired animals , we evaluated the tissue using electron microscopy and performed morphometric analysis as shown in fig6 . as expected , compared to the uninjured controls , injured animals in all groups showed a qualitative increase in axon density and reduction in myelin thickness ( fig6 a ). myelinated and unmyelinated axons in the bni ( fig6 d ) were comparable to those in the autograft and the collagen - filled tube repairs ( fig6 b , c ). quantification of the number of axons per fixed area ( 0 . 033 mm 2 ; see methods ) revealed a significant increase in the density of both myelinated and unmyelinated axons in all the injured groups , compared to the uninjured controls ( fig6 e , f ). the apparent sprouting of myelinated axons was more pronounced in the autograft and bni groups , compared to the tube / collagen repairs ( 5 - fold and 3 - fold , respectively , compared to uninjured controls ). axonal sprouting of unmyelinated axons was also evident . a 3 - 4 fold increase was documented in all repair groups compared to the uninjured control , with the highest number present in the bni group ( fig6 f ). the increased number of axons in the autograft and the bni , together with the significant reduction in the total area available for regenerative growth in the 14 - channel bni , indicated that axonal density within the bni was increased four - fold compared to that in the autograft . to evaluate whether specific neuron subtypes are preferentially influenced by the different repair strategies , we studied the distribution axon diameters in the regenerated nerves ( fig6 g ). the number of axons ( per standardized area ) was lowest in the uninjured control for all axon groups except in the 2 - 4 μm range , where axon number was increased over that in the collagen - loaded tube - repaired group . both the autograft and the bni groups demonstrated the highest increase in axonal number for all diameter ranges . however , small - diameter axons (& lt ; 4 μm ) were more abundant in the autograft group , whereas axons at the 2 - 6 μm diameter range were more prevalent within the bni . to evaluate the “ maturity ” of the regenerative process , we measured the myelin thickness in all groups . as expected , myelination was thicker in the uninjured controls , and significantly reduced in all other groups . however , the axons in the autograft and tube collagen groups showed increased myelin thickness compared to the bni ( fig6 h ). a separate group of animals underwent fluoro - gold ( fg ) tract - tracing of the sciatic nerve distal to the graft , as shown in fig7 . numerous fg + cells were visualized in the ventral motor neuron pool of the spinal cord ( vmn ; fig7 a - c ) and in the sensory dorsal root ganglia ( drg ; fig7 d - f ) in nissl counter - stained sections , as expected from their anatomical contribution to the sciatic nerve ( fig7 g ). the number of fg + motorneurons in the bni - repair animals ( 20 - 40 % reduction ) was significantly less when compared to the uninjured , autograft and tube / collagen groups ( fig7 h ). conversely , the number of fg + sensory neurons was statistically comparable among all the groups , with a trend for reduced fg + neurons in both the tube / collagen and the bni - repaired animals ( fig7 ). this data indicates that both sensory and motor axons spontaneously regenerate in all repair strategies . the behavioral recovery of the rats was evaluated by the dynamic plantar aesthesiometer test ( fig8 a - c ) and the digit abduction assay ( fig8 d ). as expected , the normal response of the hindlimb to mechanical stimulation ( 50 g ) of the plantar surface declined after injury , reflecting the lack of force opposition caused by muscle denervation . the required force to elicit a response increased progressively over 16 weeks . such recovery reached comparable levels to baseline and to the contralateral control limb in the autograft group ( fig8 a ), and progressed , albeit less effectively , in the tube / collagen and bni repaired animals ( fig8 b , c ). these data suggest that the functional sensory regeneration of the paw plantar surface in the tube / collagen and the bni groups remains suboptimal compared to that obtained with the autograft repair method . the digit abduction score ( das ) was used to evaluate motor neuron functional regeneration ( aoki kr , 1999 ; fig8 d ). baseline measurements were normal for all treatment groups and significantly increased to the worst score ( 4 ) immediately after injury to the sciatic nerve . the recovery of animals with autografts was noted as early as 4 weeks post injury ( p . i .) and reached their best score ( 1 ) at 7 weeks p . i . conversely , those repaired with either a tube / collagen or bnis , reached their best score ( 2 . 5 ) at 8 weeks p . i ., with slight improvement to score of ( 3 ) at week 12 in the collagen / tube group ( fig8 d ). we tested the electrical conduction of the regenerated nerve by stimulating the proximal end of the sciatic nerve , and recording in the common peroneal , sural and tibial branches of the sciatic nerve distal to the implant ( fig8 e ). in the simple tubularization repair , a single compound action potential was recorded in the sural and tibial nerves , but not in the peroneal nerves ( fig8 f ). in contrast , recordings from the bni showed multiple compound potentials , which were detected in the common peroneal tibial and sural nerves ( fig8 g ). large myoelectric depolarizations were observed in all cases , indicating the capacity of the regenerated nerves to elicit muscle contraction . the tissues into which the bni may be introduced to induce nervous tissue regeneration include those associated with neurodegenerative disease or damaged neurons . non - limiting examples of neurodegenerative diseases which may be treated using the methods described herein are alzheimer &# 39 ; s disease , pick &# 39 ; s disease , huntington &# 39 ; s disease , parkinson &# 39 ; s disease , cerebral palsy , amyotrophic lateral sclerosis , muscular dystrophy , multiple sclerosis , myasthenia gravis , and binswanger &# 39 ; s disease . injury to the adult mammalian spinal cord results in extensive axonal degeneration , variable amounts of neuronal loss , and often - severe functional deficits . restoration of controlled function depends on regeneration of these axons through an injury site and the formation of functional synaptic connections . resorbable pla tubing has been studied as a possibility to bridge the injured spinal cord ( oudega , et al . biomaterials 22 , 1125 - 36 , 2001 ). clearly , the bni design can be adapted for spinal cord repair . we implanted animals that underwent dorsal hemisection injury of the spinal cord with bnis that contained channels filled with collagen only , or with collagen mixed with schwann cells that expressed the reporter green fluorescent protein ( gfp ). fig9 demonstrates the use of the bni implant in repairing the injured spinal cord , as shown in photographs of the injured spinal cord 45 days after repair , which show regenerated tissue inside each microchannel ( arrows ). photograph of the injured spinal cord 45 days after repair visualized by the nuclear staining dapi demonstrates numerous cells filling the bni microchannels . the implanted gfp - labeled schwann cells survived inside the microchannels as indicated in the gfp and merged photographic panels in fig1 . fig1 shows a higher magnification of the regenerated tissue inside a bni microchannel in the injured spinal cord , 45 days after repair . numerous cells are visualized inside the microchannel as indicated by the nuclear staining dapi . the implanted gfp - labeled schwann cells survived inside the microchannels as indicated in the gfp , and more importantly numerous regenerated axons are visualized with the specific neuronal marker b - tubulin ( arrow heads in fig1 ). thus , demonstrating the successful nerve regeneration in the adult injured spinal cord though bni bridge repair . in addition , damaged neurons caused by vascular lesions of the brain and spinal cord , trauma to the brain and spinal cord , cerebral hemorrhage , intracranial aneurysms , hypertensive encephalopathy , subarachnoid hemorrhage or developmental disorders may be treated using the methods provided by the present disclosure . examples of developmental disorders include , but are not limited to , a defect of the brain , such as congenital hydrocephalus , or a defect of the spinal cord , such as spina bifida . non - limiting examples of tissues into which the bni method may be used to foster and induce regeneration include fibrous , vesicular , cardiac , cerebrovascular , muscular , vascular , transplanted , and wounded tissues . transplanted tissues are for example , heart , kidney , lung , liver and ocular tissues . in further embodiments of the invention the bni design is used to enhance wound healing , organ regeneration and organ transplantation , including the transplantation of artificial organs . agarose , a natural polymer widely used as a biomaterial for tissue engineering with demonstrated safety and biocompatibility , was experimentally selected as matrix . multiple plastic fibers ( 0 . 25 × 17 mm ) were placed inside the custom - made casting device . ultrapure agarose was dissolved in sterile 1 × pbs , injected into a perforated micro - renathane ® tubing ( braintree scientific , inc ; od 3 mm , id 1 . 68 mm , and length of 12 mm ) previously placed into the casting device , and with various plastic fibers ( i . e . 7 or 14 ) running longitudinally through the tube for channel casting and polymerized at room temperature for 15 minutes . syngenic cultures of schwann cells were obtained from adult rat sciatic nerves and expanded in vitro according to established methods ( mathon et al ., science 291 , 872 - 5 , 2001 ). in order to enhance cellular attachment and growth , the cells are mixed with 10 % matrigel or collagen - i prior to seeding . the cell suspension is then added to the loading chamber of the casting device and by carefully removing the fibers , the cells are drawn into the microchannels of the agarose matrix by negative pressure . the cellular density inside the channels can be varied through the use of different cell titers at the time of seeding . the conduits are then seeded with several types of cells . in the preferred embodiment schwann cells obtained from rodent sciatic nerves culture in dmem / 10 % fbs , supplemented with forskolin , pituitary gland extract and herregulin , were seeded within the microchannels by placing the cell suspension into the loading well and then removing the synthetic fibers ( fig1 , panel b ( c - d )). by this method , both the channel casting and cellular loading can be done within minutes , in a simple and reproducible manner . under anesthesia induced subcutaneously ( ketamine 87 mg / kg / medetomidine 13 mg / kg ), the left sciatic nerve was exposed through a dorsolateral incision of the gluteal muscles . a 5 - 7 - mm segment was then excised proximal to the bifurcation of the sciatic nerve . in animals receiving an autograft , the excised segment of the sciatic nerve was immediately sutured back . those in the tube and bni groups were repaired using 10 - 0 sutures to co - apt the nerve stumps with the micro - renathane ® tubing . the muscle was sutured and overlying skin clipped . post - operatively the animals received atipamezole 1 mg / kg , and were allowed to recover for 16 weeks . the animals were tested for recovery of motor and sensory function . sensation was evaluated using the dynamic plantar aesthesiometer test ( ugo basile ). after a 5 min habituation period , a metal filament applied increasing pressure to the plantar surface until the rat withdrew the paw . the actual force at which the paw was withdrawn was recorded from both the injured and contralateral paws . the digit abduction score ( das ) assay semiqualitatively measures muscle weakness ( aoki kr , 1999 ), and was used to evaluate motor axon reinnervation . briefly , the animals were tail - suspended to elicit hindlimb extension and digit abduction . the extended hind limbs were photographed each week and digit abduction scored on a five - point scale ( 0 = normal to 4 = maximal reduction in digit abduction and leg extension ) by two observers blind to the treatment . a subset of animals ( n = 4 per group ) was evaluated for anatomical regeneration using a fluorescent retrograde tract - tracer from the sciatic nerve distal to the implant . fluorogold ( fg : fluorochrome , englewook colo ., usa ) crystals were placed for 10 min on the regenerated nerve transected distal to the repaired site . the nerve stump was then carefully rinsed , the skin sutured , and the animals given atipamezole ( 1 mg / kg ) during the recovery period . the animals were allowed to survive for six days prior to tissue harvesting . fg - positive cells with clear nuclei were counted in a subset of sections obtained from the dorsal root ganglion and the ventral horn of the spinal cord . cells or tissues were incubated with a combination of primary antibodies against acetylated b - tubulin ( 1 : 200 ; sigma ) and s - 100 ( 1 : 500 sigma ) to identify axons and schwann cells , respectively . visualization was achieved by tissue incubation in cy2 - and cy3 - conjugated secondary antibodies ( 1 : 400 ; jackson labs , west grove , pa .). neurotrace ( 1 : 250 : molecular probes .) was used as fluorescent nissl conterstain . the staining was evaluated using a zeiss pascal confocal microscope . animals were euthanized with pentobarbital and perfused with pbs followed by 4 % paraformaldehyde . overnight post - fixation was done by placing the tissue in 2 % glutaraldehyde / 1 % paraformaldehyde / 0 . 15m sodium cacodylate , ph 7 . 2 at 4 ° c . tissues were rinsed , stained in 2 % uranyl acetate , dehydrated , and infused in propylene oxide / durcupan ( fluka chemika - biochemika , ronkonkoma , n . y . ), in 25 / 75 ratio , for 1 hr at room temperature . sciatic nerves were flat embedded in fresh durcupan resin and polymerized 24 - 36 hours at 65 ° c . one gm thick sections were stained in toluidine blue . thin sections were viewed at 60 kv and photographed on a jeol 100 cx conventional transmission electron microscope . for quantification , twenty - one pictures were taken of each nerve cross - section at random covering 1575 μm 2 per picture , and totaling 0 . 033 mm 2 in sampling area per animal . a macro ( zeiss , co .) was written to evaluate the number of myelinated axons , axon diameter and myelin thickness in each electron micrograph , which was validated by direct comparison with measurements obtained manually . the number of unmyelinated axons was estimated manually from photographic prints . raw data was analyzed by anova followed by neuman - keuls multiple comparison post hoc test ( prism 4 ; graphpad software inc .). synthetic or metal fibers measuring 250 micrometers in diameter by 18 millimeters in length were dipped in matrigel ( ecm ) forming a five - micrometer film coating . the ecm coated fibers were allowed to polymerize at room temperature for ten minutes and then rolled across a monolayer of 10 micrometer latex beads . in this manner , the beads were partially embedded into the ecm coating of the fibers . the ecm coated , bead embedded fibers were inserted into a multi - channel matrix casting device . next , 1 . 5 % ultrapure agarose , 1 × phosphate buffered saline solution was heated to its boiling point and poured into the casting well . the agarose was allowed to polymerize at room temperature . it is contemplated that in cases in which various degrees of gel opacity are desired , various gelling agents are used with the present disclosure , including , but not limited to chitosan , collagen , fibrinogen , and other hydrogels . the beads embedded in the ecm are partially embedded and have an exposed surface . when liquid agarose is poured into the casting well , this exposed bead surface becomes embedded into the agarose matrix . since ecm is a hydrophilic gel substance and agarose is a hydrogel matrix , when the fiber is extracted , the ecm embedded beads are released from their attachment points on the fiber and remain anchored in the luminal wall of the resulting conduit , presenting a bead surface area that is now exposed to the lumen of the conduit . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . al - 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