(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2014/0030315 A1
`JOHNSON
`(43) Pub. Date:
`Jan. 30, 2014
`
`US 20140030315A1
`
`(54) NANOFIBER SCAFFOLDS FOR
`BOLOGICAL STRUCTURES
`
`(71) Applicant: Jed K. JOHNSON, Columbus, OH (US)
`
`(72) Inventor: Jed K. JOHNSON, Columbus, OH (US)
`(21) Appl. No.: 13/740.913
`(22) Filed:
`Jan. 14, 2013
`
`Related U.S. Application Data
`(60) Provisional application No. 61/585,869, filed on Jan.
`12, 2012.
`Publication Classification
`
`(51) Int. Cl.
`A 6LX 9/70
`A6 IK 47/32
`A6 IK35/12
`
`(2006.01)
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl.
`CPC. A61 K9/70 (2013.01); A61 K35/12 (2013.01);
`A61K47/32 (2013.01)
`USPC ....... 424/444; 424/400; 424/93.7: 514/772.1;
`435/396: 118/621
`s
`
`57
`(57)
`
`ABSTRACT
`
`- Y
`-
`A system for manufacturing an artificial construct suitable for
`transplantation into a biological organism that includes a two
`or three three-dimensional preform that is based on the actual
`two or three-dimensional structure of a native mammalian
`tissue; and an electrospinning apparatus, wherein the electro
`spinning apparatus is operative to depositat least one layer of
`polymer fibers on the preform to form a polymer scaffold, and
`wherein the orientation of the fibers in the scaffold relative to
`one another is Substantially parallel.
`
`NANOFIBER 1014
`Nanofiber v Acera
`IPR2021-01016
`
`

`

`Patent Application Publication
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`Jan. 30, 2014 Sheet 1 of 6
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`US 2014/0030315 A1
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`NANOFIBER 1014
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`IPR2021-01016
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`Patent Application Publication
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`Jan. 30, 2014 Sheet 2 of 6
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`US 2014/003O31S A1
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`NANOFIBER 1014
`Nanofiber v Acera
`IPR2021-01016
`
`

`

`Patent Application Publication
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`Jan. 30, 2014 Sheet 3 of 6
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`US 2014/003O31S A1
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`NANOFIBER 1014
`Nanofiber v Acera
`IPR2021-01016
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`Patent Application Publication
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`Jan. 30, 2014 Sheet 4 of 6
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`US 2014/003O31S A1
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`NANOFIBER 1014
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`IPR2021-01016
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`

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`Patent Application Publication
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`Jan. 30, 2014 Sheet 5 of 6
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`US 2014/003O31S A1
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`NANOFIBER 1014
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`Patent Application Publication
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`Jan. 30, 2014 Sheet 6 of 6
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`US 2014/003O31S A1
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`NANOFIBER 1014
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`IPR2021-01016
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`

`

`US 2014/003O315 A1
`
`Jan. 30, 2014
`
`NANOFIBER SCAFFOLDS FOR
`BOLOGICAL STRUCTURES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001. This patent application claims the benefit of U.S.
`Provisional Patent Application Ser. No. 61/585,869 filed on
`Jan. 12, 2012, and entitled “Biocompatible Nanofiber Mate
`rials for Biological Structures, the disclosure of which is
`hereby incorporated by reference herein in its entirety and
`made part of the present U.S. utility patent application for all
`purposes.
`
`BACKGROUND OF THE INVENTION
`0002 Tissue engineering involves the synthesis of bio
`logically relevant tissue for a wide range of applications
`including wound healing and the replacement or Support of
`damaged organs. A common strategy is culturing target spe
`cific cells in vitro in a scaffold followed by implantation of the
`scaffold in a biological organism. As a logical cellular source
`for tissue engineering, stem cells have attracted a great deal of
`attention due to their relatively fast proliferation rate and
`diverse differentiation potential to various phenotypes. These
`include cells derived from several origins: induced pluripo
`tent stem cells from fibroblasts, mesenchymal stem cells from
`bone marrow and adult stem cells from adipose tissue. Stem
`cells distinctively self-renew and their terminal differentia
`tion depends on the influence of soluble molecules (e.g.,
`growth factors, cytokines) as well as physical and biochemi
`cal interactions with scaffolds.
`0003 Cellular behavior and subsequent tissue develop
`ment at the cell—scaffold interface therefore involve adhe
`Sion, motility, proliferation, differentiation and functional
`maturity. The physicochemical properties of a scaffold. Such
`as bulk chemistry, Surface chemistry, topography, three-di
`mensionality and mechanical properties, all influence cellular
`response. Bulk chemistry can control cytotoxicity, as most
`scaffolds are made of biodegradable materials and must even
`tually release the by-products of their degradation. The effect
`of surface chemistry is often mediated by instantly adsorbed
`proteins such as fibronectin, collagen, fibrinogen, vitronectin,
`and immunoglobulin that affect phenotype, viability, and
`morphology, as well as proliferation and differentiation.
`0004 Studies regarding the effect of surface topography
`and texture on cellular response have been conducted. Stem
`cells are known to recognize topographical features of the
`order of hundreds of nanometers to several micrometers, and
`exhibit distinctive genomic profiles in the absence of bio
`chemical differentiation cues and a commitment to terminal
`differentiation. Electrospun scaffolds are ideal matrices for
`two dimensional or three dimensional culture of the cells
`providing non-woven nano- to micro-sized fibrous micro
`structures typically having relative porosities of 70-90%.
`Natural biodegradable materials such as collagen, gelatin,
`elastin, chitosan, and hyaluronic acid, as well as Synthetic
`biodegradable polymers such as poly(e-caprolactone) (PCL),
`poly(glycolic) acid (PGA) and poly(lactic) acid (PLA), have
`been electrospun for chondral and Osseous applications.
`0005. In general, the broad utility of electrospun scaffolds
`for tissue engineering, wound healing, and organ replacement
`is clear (see Modulation of Embryonic Mesenchymal Pro
`genitor Cell Differentiation via Control Over Pure Mechani
`cal Modulus in Electrospun Nanofibers, Nama et al., Acta
`
`Biomaterialia 7, 1516-1524 (2011), which is incorporated by
`reference herein in its entirety, for all purposes) and the
`present invention provides polymer fiber constructs for these
`and other applications. Alignment of fibers produced during
`electrospinning has previously been achieved by various
`methods including, for example, high Velocity collection of
`fibers (e.g., on the Surface of a high Velocity rotating mandrel)
`and alternating collection of fibers from one grounded elec
`trode to another on an immobile surface or in the air. Current
`methods of electrospinning aligned fibers are not known to
`achieve the ideal alignment of fibers observed in the human
`body, such as, for example, in brain tissue. Therefore,
`improvements in alignment must be made in order to obtain
`the high degree of alignment necessary for an in vitro model
`of human tissue.
`
`SUMMARY OF THE INVENTION
`0006. The following provides a summary of certain exem
`plary embodiments of the present invention. This Summary is
`not an extensive overview and is not intended to identify key
`or critical aspects or elements of the present invention or to
`delineate its scope.
`0007. In accordance with one aspect of the present inven
`tion, a synthetic construct Suitable for transplantation into a
`biological organism is provided. This construct includes a
`synthetic construct Suitable for transplantation into a biologi
`cal organism, comprising: a two-dimensional or three-dimen
`sional polymer scaffold, wherein the shape and dimensions of
`the polymer scaffold are based on a native biological struc
`ture, wherein the polymer scaffold further includes at least
`one layer of polymer fibers that have been deposited by elec
`trospining, and wherein the orientation of the fibers in the
`scaffold relative to one another is substantially parallel; and
`wherein, optionally, the polymer scaffold has been preseeded
`with at least one type of biological cell prior to implantation
`into a biological organism, and wherein the at least one type
`of biological cell is operative to facilitate integration of the
`polymer scaffold into the organism so that the polymer scaf
`fold may function in a manner significantly similar to or the
`same as the native structure.
`0008. In accordance with another aspect of the present
`invention, a system for manufacturing an artificial construct
`Suitable for transplantation into a biological organism is pro
`vided. This system includes: a two or three three-dimensional
`preform that is based on the actual two or three-dimensional
`structure of a native mammalian tissue; and an electrospin
`ning apparatus, wherein the electrospinning apparatus is
`operative to depositat least one layer of polymer fibers on the
`preform to form a polymer scaffold, and wherein the orien
`tation of the fibers in the scaffold relative to one another is
`Substantially aligned or parallel.
`0009. In yet another aspect of this invention, a system for
`manufacturing an artificial construct suitable for transplanta
`tion into a biological organism for wound healing purposes is
`provided. This system includes a two or three three-dimen
`sional preform that is based on the actual two or three-dimen
`sional structure of a native mammalian tissue; an electrospin
`ning apparatus, wherein the electrospinning apparatus is
`operative to depositat least one layer of polymer fibers on the
`preform to form a polymer scaffold, and wherein the orien
`tation of the fibers in the scaffold relative to one another is
`Substantially parallel; and a least one type of biological cell
`for preseeding onto the polymer Scaffold, and wherein the at
`least one type of biological cell further includes autologous
`
`NANOFIBER 1014
`Nanofiber v Acera
`IPR2021-01016
`
`

`

`US 2014/003O315 A1
`
`Jan. 30, 2014
`
`cells or allogeneic cells, and wherein the autologous cells or
`allogeneic cells further include cord blood cells, embryonic
`stem cells, induced pluripotent cells, mesenchymal cells, pla
`cental cells, bone marrow derived cells, hematopoietic cell,
`epithelial cells, endothelial cells, fibroblasts and chondro
`cytes.
`0010 Additional features and aspects of the present inven
`tion will become apparent to those of ordinary skill in the art
`upon reading and understanding the following detailed
`description of the exemplary embodiments. As will be appre
`ciated by the skilled artisan, further embodiments of the
`invention are possible without departing from the scope and
`spirit of the invention. Accordingly, the drawings and associ
`ated descriptions are to be regarded as illustrative and not
`restrictive in nature.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`0.011 The accompanying drawings, which are incorpo
`rated into and form a part of the specification, Schematically
`illustrate one or more exemplary embodiments of the inven
`tion and, together with the general description given above
`and detailed description given below, serve to explain the
`principles of the invention, and wherein:
`0012 FIG. 1 depicts electrospun PCL fibers spun without
`an anti-static bar for 10 minutes at 0.5 ml/h, +4 kV on the
`needle, and -4 kV on the mandrel and the plot shows the angle
`of the fibers from horizontal;
`0013 FIG. 2 depicts electrospun PCL fibers spun with an
`anti-static bar for 10 minutes at 0.5 ml/h, +4 kV on the needle,
`and -4 kV on the mandrel and the plot shows the angle of the
`fibers from horizontal.
`0014 FIG.3 depicts electrospun PCL fibers spun without
`an anti-static bar at 1.0 ml/h and the plot shows the angle of
`the fibers from horizontal;
`0015 FIG. 4 depicts electrospun PCL fibers spun with an
`anti-static bar at 1.0 ml/h and the plot shows the angle of the
`fibers from horizontal;
`0016 FIG. 5 depicts electrospun PCL fibers spun using a
`dual syringe setup with two anti-static bars, wherein each
`needle has a flow rate of 1.0 ml/h and the plot shows the angle
`of the fibers from horizontal;
`0017 FIG. 6 depicts a dual syringe electrospinning setup,
`wherein the triangles indicate the relative positions of the first
`and second anti-static bars, wherein the straight arrows indi
`cate relative locations of the Syringe pump with polymer
`Solution, and wherein the clockwise arrows indicate the rota
`tion direction of the fiber collection mandrel at the center of
`the image:
`0018 FIG. 7 depicts electrospun PCL fibers spun with an
`anti-static bar at 1.0 ml/h, wherein an alternating ground was
`not applied during electrospinning;
`0019 FIG. 8 depicts electrospun PCL fibers spun with an
`anti-static bar at 1.0 ml/h, wherein an alternating ground was
`applied during electrospinning.
`0020 FIG. 9 is a scanning electromicrograph of the poly
`mer nanofibers used in the present invention;
`0021
`FIGS. 10-11 are scanning electromicrographs of the
`polymer nanofibers used in the present invention with a coat
`ing of marrow Stromal cells;
`0022 FIG. 12 is a scanning electromicrograph of the poly
`mer nanofibers used in the prior art; and
`0023 FIG. 13 is a scanning electromicrograph of the poly
`mer nanofibers used in the prior art with a coating of marrow
`stromal cells.
`
`DETAILED DESCRIPTION OF THE INVENTION
`0024 Exemplary embodiments of the present invention
`are now described with reference to the Figures. Although the
`following detailed description contains many specifics for the
`purposes of illustration, a person of ordinary skill in the art
`will appreciate that many variations and alterations to the
`following details are within the scope of the invention.
`Accordingly, the following embodiments of the invention are
`set forth without any loss of generality to, and without impos
`ing limitations upon, the claimed invention.
`0025. With reference to the Figures, this invention relates
`generally to the construction of implantable artificial tissues
`for humans and/or animals, and more specifically to a process
`or method for manufacturing two-dimensional polymer
`microScale and nanoscale structures for use as Scaffolds in the
`growth of biological structures. The use of these scaffolds in
`creating or repairing numerous and multiple biological tis
`Sues and structures, is contemplated by and included in this
`invention. Exemplary versions of the manufacturing process
`of this invention include preparing a preform or Substrate that
`is based on an actual native tissue and/or organ; electrospin
`ning one or more layers of nanoscale (less than 1000 nanom
`eters) or microscale (less than 50 microns) polymer fibers on
`the preform to form a nanofiber-based scaffold. The fibers are
`typically formed by electrospinning by extruding a polymer
`Solution from a fiberization tip; creating an electronic field
`proximate to the fiberization tip; and positioning a ground or
`opposite polarity within the preform. The preform may be
`rotated to align the fibers on the preform or a second ground
`or polarity may be placed in the preform and rapidly Switch
`ing the electric field to align the fibers. The microscale and
`nanoscale polymer fibers may be randomly aligned or maybe
`substantially parallel or both. These nanofiber structures may
`be seeded with one or more types of biological cells prior to
`implantation in the body to increase the rate of tissue growth
`into the scaffold. The polymer scaffold may include autolo
`gous or allogeneic cells Such as cord blood cells, embryonic
`stem cells, induced pluripotent cells, mesenchymal cells, pla
`cental cells, bone marrow derived cells, hematopoietic cell,
`epithelial cells, endothelial cells, fibroblasts, chondrocytes or
`combinations thereof. These biological cells may be applied
`to the surface of the scaffold or distributed throughout the
`scaffold matrix utilizing perfusion within a bioreactor. The
`polymer fibers may also be coated or otherwise treated with at
`least one compound that is operative to promote cellular
`attachment to the scaffold or promote engraftment of the
`scaffold into the biological organism. The at least one com
`pound may be selected from the group consisting of proteins,
`peptides, cytokines, growth factors, antibiotic compounds,
`anti-inflammatory compounds, and combinations thereof.
`0026 Choosing a material that accurately mimics the
`mechanical properties of the native tissue or organ may pro
`mote proper stem cell differentiation and facilitate normal
`function of the replacement tissue or organ. Included materi
`als may be non-resorbable for permanent implantation or may
`be designed to slowly degrade while the host body rebuilds
`the native tissue. In the latter case, the implanted prosthesis
`will eventually be completely resorbed. Permanent (i.e., non
`resorbable) polymers may include polyurethane, polycarbon
`ate, polyester terephthalate and degradable materials may
`include polycaprolactone, polylactic acid, polyglycolic acid,
`gelatin, collagen, or fibronectin. The fibers may be electro
`spun onto a preform with the desired prosthesis shape. An
`exemplary mandrel (preform) may be coated with Teflon or
`
`NANOFIBER 1014
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`IPR2021-01016
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`US 2014/003O315 A1
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`Jan. 30, 2014
`
`similar material to facilitate removal of the scaffold after
`deposition or a slight taper (e.g., about 1°) can be manufac
`tured into the mandrel. Nearly any size or shape can be pro
`duced from the electrospun fibers by using a pre-shaped form
`and the fiber deposition methods of the present invention.
`0027 Closely mimicking the structural aspects of the tis
`Sue or organ is important with regard to replicating the func
`tion of the native tissue or organ. By controlling the orienta
`tion of the fibers and assembling a composite structure of
`different materials and/or different fiber orientations it is pos
`sible to control and direct cell orientation and differentiation.
`Fiber orientation can be altered in each layer of a composite or
`sandwich scaffold in addition to the material and porosity to
`most closely mimic the native tissue. A properly constructed
`scaffold will permit substantially complete cellular penetra
`tion and uniform seeding for proper function and prevention
`of necrotic areas developing. If the fiber packing is too dense,
`then cells may not be able to penetrate or migrate from the
`exposed surfaces into the inner portions of the scaffold. How
`ever, if the fiber packing is not close enough, then attached
`cells may not be able to properly fill the voids, communicate
`and signal each other and a complete tissue or organ may not
`be developed. Controlling fiber diameter can be used to
`change scaffold porosity as the porosity Scales with fiber
`diameter. Alternatively, blends of different polymers may be
`electrospun together and one polymer preferentially dis
`solved to increase scaffold porosity. The properties of the
`fibers can be controlled to optimize the fiber diameter, the
`fiber spacing orporosity, the morphology of each fiber such as
`the porosity of the fibers or the aspect ratio, varying the shape
`from round to ribbon-like. The precursor solution described
`below may be controlled to optimize the modulus or other
`mechanical properties of each fiber, the fiber composition,
`and/or the degradation rate (from rapidly biosoluable to bio
`persitent). The fibers may also be formed as drug eluting
`fibers, anti-bacterial fibers or the fibers may be conductive
`fibers, radio opaque fibers to aid in positioning or locating the
`fibers in an X-ray, CT or other scan.
`0028. In accordance with this invention, the process of
`electrospinning is driven by the application of a high Voltage,
`typically between 0 and 30 kV, to a droplet of a polymer
`solution or meltata flow rate between 0 and 50 ml/h to create
`a condition of charge separation between two electrodes and
`within the polymer Solution to produce a polymer jet. A
`typical polymer Solution would consist of a polymer Such as
`polycaprolactone, polystyrene, or polyetherSulfone and a sol
`vent such as 1,1,1,3,3,3-Hexafluoro-2-propanol, N,N-Dim
`ethylformamide, acetone, or tetrahydrofuran in a concentra
`tion range of 1-50 wt %. As the jet of polymer solution travels
`toward the electrode it is elongated into small diameter fibers
`typically in the range of 0.1-100 um.
`0029. In preparing an exemplary scaffold, a polymer
`nanofiber precursor solution is prepared by dissolving 2-30
`wt % polyethylene terephthalate (PET) (Indorama Ventures)
`in a mixture of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and
`trifluoroacetic acid and the solution is heated to 60° C. fol
`lowed by continuous stirring to dissolve the PET. The solu
`tion may be cooled to room temperature and the Solution
`placed in a syringe (e.g., 60 cc) with a blunt tip needle (e.g., 20
`gauge). The nanofibers are formed by electrospinning using a
`high Voltage DC power Supply (Glassman High Voltage, Inc.,
`High Bridge, N.J.) set to 1 kV-40kV (e.g., +15 kV) positive or
`negative polarity, a 5-30 cm (e.g., 15 cm) tip-to-substrate
`distance, and a 1 ul/hr to 100 mL/hr (e.g., 10 ml/hr) flow rate.
`
`It is possible to use a needle array including a large number of
`needles (e.g., >1000) to increase system output. Fiber diam
`eter may be controlled by the viscosity of the precursor solu
`tion and the solvent used and suitable exemplary fibers are in
`the range of 100 nanometer 30 microns. Approximately 0.2-3
`mm (e.g., 1 mm) thickness of randomly oriented and/or
`highly-aligned fibers may be deposited onto the form, and
`polymer rings added, followed by an additional approxi
`mately 0.2-3.0 mm (e.g., 2 mm) of fiber added while the form
`is rotated. The scaffold may be placed in a vacuum overnight
`to ensure removal of residual solvent (typically less than 10
`ppm) and treated using a radio frequency gas plasma for 1
`minute to make the fibers more hydrophilic and promote cell
`attachment. Samples may be storied in re-closeable polyeth
`ylene bags, or the like.
`0030. In accordance with this invention, an exemplary
`preparation of electrospinning solution typically includes
`polyethylene terephthalate (PET), polycaprolactone (PCL),
`polylactic acid (PLA), polyglycolic acid (PGA), polyether
`ketoneketone (PEKK), polyurethane (PU), polycarbonate
`(PC), polyamide (Nylon), natural polymers such as fibronec
`tin, collagen, gelatin, hyaluronic acid or combinations thereof
`that are mixed with a solvent and dissolved. Suitable solvents
`include acetone, dimethylformamide, trifluoroacetic acid,
`hexafluoroisopropanol, acetic acid, dimethylacetamide, chlo
`roform, dichloromethane, water, ionic compounds, or com
`binations thereof. A form is prepared for the deposition of
`nanofibers. Optionally, simulated cartilage or other Support
`ive tissue may be applied to the form and the fibers are then
`sprayed onto or transferred onto a form to build up the scaf
`fold.
`0031. During electrospinning, polymer fibers are driven
`toward a collector by charge separation caused by applied
`Voltage. The collector typically is a conductive Surface. Such
`as, for example, aluminum or copper, and in this disclosure
`the collector is covered by a thin layer of plastic, ranging, for
`example, between about 0.001-0.1 inches thick. The charge
`that drives electrospinning toward the collector is derived
`from mobile ions within the polymer solution or melt. The jet
`of polymer that is produced has a net positive or negative
`charge, depending upon the polarity of the Voltage applied to
`the electrode(s). When the jet solidifies on the collector Sur
`face, a charge builds up as Subsequent fiber layers are col
`lected. As the charge builds up on the surface, fiber with
`similar charge is repelled leading to irregularly arranged
`fibers and thus a lower degree of alignment. To reduce the
`effects of surface charge, an anti-static device (e.g., bar) may
`be incorporated into the process to improve fiberalignment.
`The anti-static bar bombards the surface of a sample with
`positively and negatively charged ions in the form of for
`example, a plasma or corona discharge to neutralize the
`charge on the Substrate. Therefore, as fiber builds up. Succes
`sive layers of fibers will deposit more uniformly side-by-side
`(parallel relationship) to increase the alignment. The position
`of the anti-static bar is generally parallel to the surface of the
`collection mandrel, wheel, device, plate, etc. and is for
`example, about 0.5-3 inches away from the surface.
`0032 Experimental results demonstrate that fiber align
`ment was improved significantly with the addition of an anti
`static bar (or a device having similar functional properties),
`when compared to samples spun under the same conditions
`without the anti-static bar. FIG. 1 shows the alignment of a
`sample to be 83%, when electrospun without a static bar;
`while FIG. 2 shows a 12% increase in fiberalignment to 95%,
`
`NANOFIBER 1014
`Nanofiber v Acera
`IPR2021-01016
`
`

`

`US 2014/003O315 A1
`
`Jan. 30, 2014
`
`when a static bar treats the Surface during spinning. An addi
`tional benefit of anti-static bars is the ability to electrospin
`using multiple needles. FIG.3 shows a sample spun at 1 ml/h
`without a static bar; while FIG. 4 shows a sample spun at 1
`ml/h with an anti-static bar, and FIG. 5 shows a sample spun
`using a dual syringe configuration and two anti-static bars
`treating a wheel surface where the fibers are being deposited.
`When fibers are spun without an anti-static bar (see FIG. 3),
`the fiberalignment is low, with only 74% of fibers collected in
`a low angle orientation. With an anti-static bar, under the
`same spinning conditions, the alignment becomes 92% (see
`FIG. 4). Additionally, alignment is maintained at 92% (see
`FIG. 5) when multiple needles are used for electrospinning,
`and when two anti-static bars treat the wheel surface as illus
`trated in FIG. 6. This demonstrates the ability to scale-up the
`production rate by incorporating additional needles and anti
`static devices. Antistatic bars (or, alternately, one or more
`ionizing guns) may also be used to create continuous (i.e.,
`very long) fibers that are continuously aligned.
`0033. As previously indicated, the alternating collection
`offibers from one ground to the next will create aligned fiber.
`High velocity collection of fibers, such as on the surface of a
`rotating mandrel, will achieve similar results. When the two
`methods are combined, alternating grounds on a rotating
`mandrel, the fiberalignment is enhanced beyond what either
`method typically achieves. The combined method of fiber
`alignment is highly effective when the surface of the mandrel
`is coated in a thin insulating layer, such as, for example,
`polystyrene. An alternating ground is established by securing
`a continuous plastic sheet and wrapping conductive tape
`around the surface of the wheel, or a similar method that
`creates alternating layers of conductive and non-conductive
`Surface material. The conductive tape generally is made of
`for example, carbon or copper and ranges between about
`0.1-2 inches wide and is spaced uniformly around the wheel
`circumference. This tape should be connected to the charged/
`grounded wheel for the alternating ground effect to be
`obtained. FIGS. 7-8 illustrate the alternating ground effect.
`FIG. 7 shows a sample that was electrospun without the
`alternating ground and with an anti-static bar. FIG. 8 shows a
`sample that was electrospun with both the alternating ground
`and anti-static bar. These images demonstrate the dramatic
`improvement in alignment when using the alternating ground
`and anti-static bar on a high-speed mandrel.
`0034. With reference to FIGS. 9-13, the polymer fiber
`scaffolds of the present invention may be used to manufacture
`two-dimensional biocompatible patches of varying thickness
`for use in humans or animals (e.g., primates, cats, dogs,
`horses and cattle) as an aid in wound healing involving
`muscles, internal organs, bones, cartilage, and/or external
`tissues. Biocompatible materials, which are suitable for use in
`medical applications within the body or on external Surfaces,
`typically elicit little or no immune response in human or
`Veterinary applications. In one or more exemplary embodi
`ments, these patches include Substantially parallel electro
`spun nanoscale and microscale polymer fibers. These patches
`may be seeded with biological cells prior to use to increase the
`rate of tissue growth into the patch. Such biological cells may
`include autologous or allogenic cells such as cord blood cells,
`embryonic stem cells, induced pluripotent cells, mesenchy
`mal cells, placental cells, bone marrow derived cells, hemato
`poietic cells, epithelial cells, endothelial cells, fibroblasts and
`chondrocytes. Examples of internal uses include tissue, ocu
`lar tissue (lens, cornea, optic nerve or retina), intestinal tissue,
`
`internal organs such as the liver, kidney, spleen, pancreas,
`esophagus, trachea, uterus, stomach, bladder, muscles, ten
`dons, ligaments, nerves, dura matter and other brain struc
`tures, dental structures, blood vessels and other bodily struc
`tures. Examples of external uses may include wound
`dressings, burn and abrasion coverings, and recovery aides to
`inhibit the formation of scar tissue. External structures are
`typically the skin but may include the cornea or surface of the
`eye, the ear canal, the mouth and nasal passages or the nail
`bed.
`0035 An exemplary method for making the biocompat
`ible patches of this invention includes depositing a layer of
`Substantially parallel electrospun polymer fibers on a preform
`(i.e., a wheel or similar structure) to form a fiber patch or gap
`filling material; and applying donor cells to the patch. Pref
`erably, the fibers are formed byelectrospinning by extruding a
`polymer Solution from a fiberization tip; creating an elec
`tronic field proximate to the fiberization tip; and positioning a
`ground within the deposition surface. The preform may be
`rotated to align the fibers on the Surface or a second ground or
`opposite polarity may be placed in the preform and rapidly
`Switching the ground. To speed the growth of human tissue
`into the fiber preform, the fibers are aligned by rapidly spin
`ning the preform so that alignment of the structure produced
`by standard electrospinning while the fibers are drawn into a
`Substantially parallel ordering by the tension created by spin
`ning the form. See generally, Preparation and anisotropic
`mechanical behavior of highly-Oriented electrospun poly(bu
`tylene terephthalate) fibers, Journal of Applied Polymer Sci
`ence Volume 101, Issue 3, pages 2017-2021 (August 2006),
`which is incorporated by reference herein, in its entirety. A
`split ground technique, in which fiber deposition rapidly
`alternates between two separate grounding plates within the
`preform or by alternating the electric field is also an effective
`method of forming parallel fibers on the preform. See, gen
`erally, Electrospinning Nanofibers as Uniaxially Aligned
`Arrays and Layer-by-Layer Stacked Films, Advanced Mate
`rials Volume 16, Issue 4, pages 361-366 (February 2004),
`which is incorporated by reference herein, in its entirety.
`Fiber alignment can be further enhanced by controlling cer
`tain variables Such as humidity, Solvents, flow rates, and rota
`tional speeds. Lower relative humidity (e.g., 20%) results in
`better overall alignment than higher relative humidity (e.g.,
`50%). However, deposition efficiency is increased in higher
`humidity (e.g., deposition on plastic). The use of certain
`Solvents such as acetone typically reduce fiber alignment,
`while others such as 1,1,1,3,3,3-Hexafluoro-2-propanol typi
`cally increase alignment. Decreasing the flow rate at which
`the polymer Solution is being pumped typically increases
`fiberalignment, while increasing the flow rate decreases fiber
`alignment. Finally, increasing the rotational speed of the
`wheel upon which the fiber is being deposited typically
`increases fiberalignment.
`0036. The thickness of the patch may be from a few
`microns for application to Surfaces to speed cellular growth
`and inhibitscarring to several centimeters for use as a plug for
`insertion into a wound or to speed the growth of structures in
`a specific direction. High thickness patches are useful in
`repairing infracted cardiac tissue, esophageal or tracheal tis
`Sue or Supporting the growth of nerve in a predetermined
`direction. Depending on the material used in preparing the
`fibers the patch may dissolve within the body after a prede
`termined time or may be relatively permanent for longer term
`applications. It is also possible to fabricate a multipart struc
`
`NANOFIBER 1014
`Nanofiber v Acera
`IPR2021-01016
`
`

`

`US 2014/003O315 A1
`
`Jan. 30, 2014
`
`ture which includes one or more layers