WO2025073993A1

WO2025073993A1 - A layered implant and system to repair a perforation in a tympanic membrane

Info

Publication number: WO2025073993A1
Application number: PCT/EP2024/078121
Authority: WO
Inventors: Fergal O'brien; John Gleeson; Eamon SHEEHY; Cian O'CONNOR; Cian O'LEARY; Andrew Cameron; Ronaldo DO AMARAL; Elizabeth SAINSBURY; Alec BLANEY; Irina PASCU
Original assignee: Royal College of Surgeons in Ireland
Current assignee: Royal College of Surgeons in Ireland
Priority date: 2023-10-05
Filing date: 2024-10-07
Publication date: 2025-04-10
Anticipated expiration: 2026-04-05

Abstract

A layered implant to repair a perforation in a tympanic membrane comprises a dehydrated thin film layer comprising a biopolymer, and a porous body comprising a freeze-dried matrix comprising a biopolymer. The dehydrated thin film layer is mounted on and integrated with the porous body, and the porous body is resorbable in-vivo and resiliently compressible. The thin film layer functions as a regenerative scaffold for the tympanic membrane, and the porous body allows the implant to be resiliently compressed in a delivery device to allow it be delivered into a perforation in a tympanic membrane, and once delivered spring-back to its original shape where it anchors the implant and positions the thin film layer adjacent the perforation allowing the thin film layer to support the regeneration of the thin film membrane to close the perforation. A delivery device for the implant is also described.

Description

TITLE

A layered implant and system to repair a perforation in a tympanic membrane

Field of the Invention

The present invention relates to a layered implant and system to repair a perforation in a tympanic membrane.

Background to the Invention

The tympanic membrane, also known as the eardrum, is a thin tissue responsible for transmitting sound vibrations from the outer parts of the ear to the inner parts of the ear, which will eventually stimulate sensory nerve cells enabling us to hear. Ruptures in the eardrum (tympanic membrane perforation) can eventually result in hearing loss. In general, those ruptures heal by themselves without the need of specific treatment. However, in some case they remain open, resulting in what is known as chronic tympanic membrane perforations. In such situations, a surgery known as tympanoplasty is the only treatment option. It requires a specialist surgeon to (1 ) harvest a donor tissue known as temporalis fascia graft from the patient, (2) leave it aside to dry, (3) flap open the eardrum, (4) insert the graft inside the middle ear (ME), (5) insert packing material to support the graft from beneath and (6) suture the eardrum flap in place. It is a surgery that carries a wide risk of complications including, nerve damage and general anaesthesia side effects that restricts treatment in paediatric patients. Approximately 500,000/year tympanoplasties are performed in Europe, US, Japan, Canada and Australia. In developing countries, a shortage of trained professionals adds to the restriction of access to treatment.

It is an objective of the invention to overcome at least one of the above-referenced problems. Summary of the Invention

The objective is met by the provision of a bioresorbable implant having a dehydrated thin layer comprising a biopolymer such as collagen and optionally a second biopolymer and a porous body attached to the first layer that comprises a freeze-dried matrix comprising one or more biopolymers and that is resiliently compressible. The thin film layer functions as a regenerative scaffold for the tympanic membrane, and the porous body allows the implant to be resiliently compressed in a delivery device to allow it be delivered into a perforation in a tympanic membrane, and once delivered spring-back to its original shape where it anchors the implant and positions the thin film layer adjacent the perforation allowing the thin film layer to support the regeneration of the thin film membrane to close the perforation. The thin film layer generally comprises collagen and is generally formed by dehydration so that it is substantially non-porous. The porous body generally comprises collagen and is generally formed by freeze-drying such that it is highly porous and resiliently compressible, providing the spring-back functionality required to allow the implant to be compressed for delivery through a perforation and then self-expand to a deployed configuration where it anchors the implant with the thin film adjacent to the tympanic membrane.

LAYERED IMPLANT

In a first aspect, the disclosure provides a layered implant to repair a perforation in a tympanic membrane comprising:

(a) a thin film layer comprising a biopolymer; and

(b) a porous body comprising a freeze-dried matrix comprising a biopolymer,

In any embodiment, the thin film layer is of low porosity.

In any embodiment, the thin film layer is dehydrated. In any embodiment, the thin film layer is mounted on and integrated with the porous body.

In any embodiment, the layered implant is resorbable in-vivo.

In any embodiment, the porous body is resiliently compressible.

In any embodiment, the layered implant is configured for resilient deformation from a relaxed uncompressed state in which it is too large to fit within the perforation to be repaired to a compressed resiliently deformed state in which it is dimensioned to fit within the perforation to be repaired.

In any embodiment, the biopolymer of the porous body and dehydrated thin film layer are each, independently, selected from collagen, hyaluronic acid, and gelatin.

In any embodiment, the biopolymer of the freeze-dried matrix of the porous body and dehydrated thin film layer comprises collagen.

In any embodiment, the biopolymer of the freeze-dried matrix of the porous body comprises collagen, elastin, hyaluronic acid and a gelling agent.

In any embodiment, the biopolymer of the dehydrated thin film layer comprises collagen, elastin and hyaluronic acid.

In any embodiment, the dehydrated thin film layer comprises or is coated with one or more extracellular matrix proteins. Examples include collagen Type IV, Elastin and vitronectin.

In any embodiment, the porous body comprises a distal end, a proximal and, and a sidewall, and in which the dehydrated thin film layer is mounted on and integrated with the proximal end of the porous body. The porous body may be cylindrical and may taper inwardly towards a distal end or a proximal end. The cylinder may have a cross-sectional profile that is round, oval, rectangular, square, hexagonal,

In any embodiment, the dehydrated thin film layer comprises an annular flange portion that extends radially outwardly of the proximal end of the porous body. This allows the porous body extend through a perforation in an ear drum while the flange section abuts the ear drum around a periphery of the perforation.

In any embodiment, the layered implant has a height of 4-8mm and a width of 4- 10mm.

In any embodiment, the dehydrated thin film layer has a thickness of less than 1000, 500, 400, 300, 200, 100, 75 or 50 microns.

In any embodiment, the dehydrated thin film layer is a laminate having a plurality of thin film layers.

In any embodiment, the laminate comprises biopolymer fibres disposed between at least two thin film layers of the laminate.

In any embodiment, the biopolymer fibres are collagen fibres.

In any embodiment, the biopolymer fibres are directionally aligned in the laminate.

In any embodiment, the porous body is cylindrical. The cylindrical porous body may taper towards a distal or proximal end, or may have a waisted section.

In any embodiment, the porous body has a thickness of at least 5, 7, 9, 10, 12, 15, 17, 20, 30, 40, 50, 80 or 100 times the thickness of the thin non-porous regenerative film layer.

In any embodiment, porous body has a cross-sectional profile that is round or oval. In any embodiment, the thin film layer is attached to a proximal end of the porous body and comprises a flange portion that extends radially outwardly of the proximal end of the porous body. Thus, a diameter of the thin film may be 5 to 100% greater than a diameter of the proximal end of the porous body.

In any embodiment, the flange portion of the thin film is annular.

In any embodiment, the porous body comprises at least 20% or 25% collagen (w/w)

In any embodiment, the porous body comprises at least 40% or 50% gelling agent (e.g., gelatin) (w/w).

In any embodiment, the porous body comprises 1 to 5% hyaluronic acid (w/w).

In any embodiment, the porous body comprises 20-40% collagen, 50-80% gelling agent (e.g., gelatin) and 1 -5% hyaluronic acid (w/w).

In any embodiment, the porous body comprises about 27-37% collagen, about 60- 70% gelling agent (e.g., gelatin) and about 1 -5% hyaluronic acid (w/w).

In any embodiment, the dehydrated thin film layer comprises at least 80%, 85% or 90% collagen (w/w).

In any embodiment, the dehydrated thin film layer comprises 1 to 20%, 5 to 15%, or about 10% hyaluronic acid (w/w).

In any embodiment, the dehydrated thin film layer comprises 85-99% collagen and 1-15% hyaluronic acid (w/w).

In any embodiment, the dehydrated thin film layer comprises about 90-95% collagen and 5-10% hyaluronic acid (w/w). In any embodiment, the thin non-porous regenerative film layer comprises a laminate of at least two thin film layers.

In any embodiment, the implant has an uncompressed diameter of 4 to 20 mm and is typically resiliently compressible to a delivery diameter of 1 to 3 mm.

In any embodiment, the implant has an axial length of 2 to 20 mm.

In any embodiment, the implant has a width of 2 to 20 mm.

In any embodiment, the implant is bi-layered.

In any embodiment, the implant comprises three or more layers.

In any embodiment, the implant is not crosslinked or cured.

In any embodiment, the collagen employed in the present invention is Type 1 collagen.

In any embodiment, the collagen employed in the present invention is bovine or porcine collagen.

In any embodiment, the collagen employed in the present invention is microfibrillar collagen, preferably microfibrillar bovine tendon collagen.

The disclosure also provides a layered implant according to the invention, in a rehydrated form.

METHOD OF FORMING LAYERED IMPLANT The disclosure also provides a method of forming a layered implant (for example, a layered implant according to the disclosure), the method comprising the steps of: dehydrating a first slurry comprising a biopolymer to provide a dehydrated thin film layer; re-hydrating the dehydrated thin film layer; pouring a second slurry comprising a biopolymer on top of the re-hydrated regenerative film layer; and freeze-drying the re-hydrated regenerative film layer and second slurry together to form the layered implant.

In any embodiment, the rehydrated thin film layer is frozen prior to the freeze-drying step.

In any embodiment, the thin film layer is rehydrated with an acid, for example an organic acid. In any embodiment, the acid is acetic acid. In any embodiment, the acid has a molarity of 0.1 M to 1 .0M, or 0.25M to 0.75M, or about 0.5M.

In any embodiment, the thin film layer is laminated prior to the step of pouring the second slurry.

In any embodiment, the thin film layer is laminated prior to or after re-hydration (or after re-hydration and freezing) of the thin film layer.

In any embodiment, the thin film layer is laminated in a method comprising the steps of: optionally, rehydrating the dehydrated thin film layer; preparing a third slurry comprising a biopolymer; pouring the third slurry on top of the dehydrated or rehydrated thin film layer; and dehydrating the third slurry and the dehydrated or rehydrated thin film layer to form the laminated dehydrated thin film layer.

In any embodiment, the biopolymer is selected from collagen, elastin, hyaluronic acid and gelatin.

In any embodiment, biopolymer fibres are embedded between the layers of the laminated dehydrated thin film layer. The fibres may be collagen fibres. The fibres may be placed on the dehydrated or rehydrated thin film layer prior to the step of pouring the third slurry. The fibres may be directionally aligned.

The lamination step may be repeated one or more times.

In any embodiment, the first biopolymer comprises collagen and hyaluronic acid.

In any embodiment, the first slurry comprises 0.1% to 1 .0% of biopolymer (e.g., collagen) (w/v).

In any embodiment, the first slurry comprises 0.3% to 0.7%, 0.4% to 0.6%, or about 0.5% collagen (w/v).

In any embodiment, the first slurry comprises 0.01% to 0.1 % hyaluronic acid (w/v).

In any embodiment, the first slurry comprises 0.03% to 0.07%, 0.04% to 0.05%, or about 0.044% hyaluronic acid (w/v).

In any embodiment, the second slurry comprises hyaluronic acid. In any embodiment, the second slurry comprises collagen, hyaluronic acid and optionally a gelling agent.

In any embodiment, the gelling agent is gelatin.

In any embodiment, the first slurry and/or second slurry comprises a collagen to hyaluronic acid weight ratio of 5:1 to 20:1 or 8:1 to 13:1 or about 11 :1.

In any embodiment, the second slurry comprises collagen to gelatin weight ratio of 1 :1 to 1 :3 or about 1 :2 (w/w).

In any embodiment, the second slurry comprises a gelatin to hyaluronic acid weight ratio of 20:1 to 25:1 or about 23:1 (w/w).

In any embodiment, the second slurry comprises 0.1% to 1 .0% collagen (w/v).

In any embodiment, the second slurry comprises 0.3% to 0.7%, 0.4% to 0.6%, or about 0.5% collagen (w/v).

In any embodiment, the second slurry comprises 0.01% to 0.1% hyaluronic acid (w/v).

In any embodiment, the second slurry comprises 0.03% to 0.07%, 0.04% to 0.05%, or about 0.044% hyaluronic acid (w/v).

In any embodiment, the second slurry comprises 0.5% to 1 .5%, 0.8% to 1 .2%, or about 1 % gelling agent (w/v).

In any embodiment, the second slurry comprises:

0.3% to 0.7%, 0.4% to 0.6%, or about 0.5% collagen (w/v);

0.03% to 0.07%, 0.04% to 0.05%, or about 0.044% hyaluronic acid (w/v); and 0.5% to 1 .5%, 0.8% to 1 .2%, or about 1% gelling agent (w/v).

In any embodiment, the third slurry comprises a collagen, hyaluronic acid, or a gelling agent.

In any embodiment, the third slurry comprises a collagen, optionally hyaluronic acid, and a gelling agent.

In any embodiment, the third slurry comprises a collagen, elastin, hyaluronic acid, and a gelling agent.

In any embodiment, the gelling agent in the third slurry comprises gelatin.

In any embodiment, the method comprises: placing the rehydrated (non-porous) film layer in a base of a mould; pouring the second slurry on top of the re-hydrated (non-porous) film layer; and freeze-drying the re-hydrated (non-porous) film layer and second slurry together to form the implant.

In any embodiment, the mould comprises a porous body shaped recess.

In any embodiment, the method employs a mould comprising a base part having a top surface and a separate top part configured to abut the base part and comprising at least one porous body shaped through-hole.

In any embodiment, the method comprises: placing the rehydrated thin film layer on the top surface of the base part; placing the second part of the mould on top of the base part such that the rehydrated thin film layer is sandwiched between the top part and bottom part of the base and the at least one porous body shaped through-hole is disposed over a part of the rehydrated thin film layer; pouring the second slurry into the at least one porous body shaped through- hole. and freeze-drying the re-hydrated thin film layer and second slurry together to form the layered implant comprising the dehydrated thin film layer adhered to the porous body.

In any embodiment, the method comprises removing the implant from the mould, and cutting excess dehydrated thin film layer away.

In any embodiment, the cutting step comprises cutting excess dehydrated thin film layer away to leave a flange of dehydrated thin film layer around a base of the porous structural support scaffold.

In any embodiment, the top part of the mould comprises a plurality of porous body shaped through-holes.

MOULD TO FORM LAYERED IMPLANT

The disclosure also provides a mould to form a layered implant of the invention comprising a base part having a top surface and a top part configured to abut the base part and comprising at least one porous body shaped through-hole.

In any embodiment, the top part of the mould comprises a plurality of porous body shaped through-holes. In any embodiment, the or each porous body shaped through-hole is cylindrical.

In any embodiment, the porous body shaped through-holes have a depth of 2-20 (or 2-8) mm and a width of 2-20 (or 2-10)0 mm.

In any embodiment, the porous body shaped through-holes have a depth of 4-6 mm and a width of 4-8 mm.

In any embodiment, the porous body shaped through-holes are inwardly tapered towards a base of the top plate.

In any embodiment, the top part of the mould is configured to be secured to the bottom part of the mould.

SYSTEM TO REPAIR PERFORATION IN BODY MEMBRANE

The disclosure also provides a system to repair a perforation in a body membrane, for example a tympanic membrane, comprising: a layered implant according to the disclosure; and a delivery device for the layered implant configured to hold the layered implant and deliver the layered implant in a radially compressed form into a perforation in a tympanic membrane via the ear canal.

In any embodiment, the delivery device comprises: a handle; an elongated tube having a proximal end coupled to the handle and a distal end comprising a lumen; an ejection element disposed within the elongated tube and configured for axial movement along the elongated tube to eject the layered implant distally from the distal end of the lumen in a resiliently deformed (e.g., radially compressed) form; and optionally, an optic fibre camera to remotely view the distal end of the elongated tube.

In any embodiment, all or part of the lumen is inwardly tapered towards a distal end thereof.

In any embodiment, the delivery device comprises: a handle; an elongated stem having a proximal end coupled to the handle and a distal end; a cartridge configured to receive a layered implant and configured for detachable coupling to the distal end of the elongated stem; an ejection rod disposed within the elongated stem and configured for axial movement along the elongated stem to eject the layered implant distally from the cartridge in a resiliently deformed form; and optionally, a viewing module (e.g., an optic fibre camera) to remotely view the membrane to be repaired.

In any embodiment, the cartridge comprises a cylindrical body with open ends, a sidewall and an internal lumen.

In any embodiment, the sidewall comprises one or more apertures. In any embodiment, the layered implant in a relaxed un-tensioned configuration cannot fit within the cartridge without being resiliently deformed.

In any embodiment, the elongated stem is angled intermediate its ends at an angle 0 of 20° to 40° to a longitudinal axis of the elongated stem.

In any embodiment, the elongated stem is flexible and comprises an elongated stiffening mandrel.

In any embodiment, elongated stem comprises a proximal stem part detachably attachable to a distal stem part, wherein the distal stem part comprises a funnel shaped section, distal tip with distal opening, and a proximal opening dimensioned to receive the implant in an uncompressed configuration.

In any embodiment, the proximal stem part and distal stem part are configured to interlock when attached together.

In any embodiment, the method comprises a step of cutting around all or part of a periphery of the perforation in the membrane.

In any embodiment, the delivery device for the layered implant comprises a cutting blade configured to cut around all or part of a periphery of the perforation in the membrane.

In any embodiment, the cartridge comprises the cutting blade.

In any embodiment, the cutting blade is a curved blade.

In any embodiment, the system comprises a eustachian canal plug.

In any embodiment, the eustachian canal plug comprises gelatin. In any embodiment, the eustachian canal plug has a conical or frustoconical shape.

METHOD OF PREPARING LAYERED IMPLANT FOR IN-VIVO DELIVERY

The disclosure also provides a method comprising: providing a layered implant according to the disclosure; providing a cartridge for the layered implant comprising a cylindrical body with open ends, a sidewall and an internal lumen; inserting the layered implant into the internal lumen of the cylindrical body; coupling the cartridge to a delivery device for the layered implant.

In any embodiment, the method comprises the steps of immersing the cartridge containing the layered implant into an implant rehydration fluid for a suitable period of time to rehydrate the layered implant prior to coupling the cartridge to the delivery device.

In any embodiment, the cylindrical body comprises one or more apertures in the sidewall.

In any embodiment, the layered implant in a relaxed un-compressed configuration is too large to fit within the internal lumen of the cylindrical body without being resiliently deformed, wherein the method comprises resiliently deforming the layered insert and inserting the layered insert into the hollow cylindrical body in a resiliently deformed form. In any embodiment, the internal lumen of the hollow cylindrical body is dimensioned to receive the layered implant in an un-compressed relaxed configuration, wherein the internal lumen is inwardly tapered towards a distal end of the device such that when the layered implant is advanced distally along the internal lumen it is resiliently deformed.

METHOD OF TREATMENT

The disclosure also provides a method of repairing a perforation in a body membrane comprising the step of delivering a layered implant of the invention to the perforation such that the thin film layer is disposed adjacent to or abutting a periphery of the perforation.

In any embodiment, the method comprises resiliently compressing the layered implant, advancing the layered implant in a resiliently compressed form at least partly through the perforation, and then allowing layered implant to self-expand at a target location in which the thin film layer is disposed adjacent to or abutting a periphery of the perforation. The target location may be a position in which the layered implant spans the perforation (where the self-expended porous body anchors the implant in the perforation). Alternatively, the target location may be a position located at a distal side of the perforation.

In any embodiment, the implant is delivered by a delivery device configured to receive the layered implant and eject the layered implant at a desired location.

In any embodiment, the delivery device is configured to resiliently compress the implant during ejection of the implant. For example, the distal end of the delivery device may comprise a funnel configured to radially compress the implant during ejection.

In another embodiment, the delivery device is configured to receive and hold the layered implant in a resiliently compressed form. In any embodiment, the method comprises an initial step of hydrating the layered implant.

In any embodiment, the delivery device comprises an elongated stem and a cartridge having a lumen to receive the layered implant and configured for detachable coupling to a distal end of the elongated stem, wherein the method comprises inserting a layered implant into the lumen of the cartridge, optionally immersing the cartridge containing the layered implant into a rehydration liquid, and then coupling the cartridge containing the optionally-hydrated layered implant to the distal end of the elongated stem.

In any embodiment, the method comprises viewing using an imaging module the delivery of the layered implant, the imaging module may be an optic fibre camera forming part of the delivery device.

In any embodiment, the method comprises delivering a resiliently deformable eustachian canal plug into the eustachian canal to temporarily block the eustachian canal.

In any embodiment, the delivery device for the layered implant comprises a cutting blade.

In any embodiment, the cartridge comprises a cutting blade.

Hyaluronic acid has a number of benefits in this application, (i) improved mechanical stability and integrity of the membrane i.e., marginal stiffness augmentation but good control of biodegradation timing which is important for healing. Also serves as a better anchor during the crosslinking stiffening process i.e. works better than just collagen on its own, (ii) it can help with the small amount of fluid absorption at the wound site to encourage infiltration of local healing cells and (iii) it actively encourage cell infiltration to the centre of the scaffolds and membranes, a critical thing to help discouraging avascular necrosis of the healed tissue, i.e. improves patency of healing tissue and stops early unwanted cell death.

The thin film may be laminated (as described above). Lamination gives control of mechanical properties and resorption characteristics of the thin film.

The porous body may comprise a gelling agent such as gelatin. An important function of the porous body is to be as elastic as possible i.e., allow it to be compressed to a high degree but then that it can expand rapidly and firmly back to its original shape, to act as an anchor for the implant. The gelling agent gives a much better and firmer springback.

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

FIG. 1 A illustrates a thin regenerative film layer forming part of the implant of the invention.

FIG. 1 B illustrates a resiliently compressible porous supporting scaffold forming part of the implant of the invention.

FIG.1C illustrates a bilayered implant according to the invention.

FIG.2 is a photo of a bilayered implant according to the invention. FIG. 3 is an illustration of one embodiment of a layered implant according to the invention in which the regenerative film layer comprises a flange portion that extends before the base of the porous supporting scaffold.

FIG. 4A to 4C illustrate the use of one embodiment of a system of the invention to treat a perforation in a tympanic membrane of the human ear showing a hydrated implant according to Figure 3 in an elongated tube of a delivery device in a radially inwardly compressed form (FIG. 4A), a plunger pushing the implant distally out of the distal end of the elongated tube and through the perforation in the tympanic membrane where a distal end of the implant expands after it has passed through the tympanic membrane (FIG. 4B), and the implant fully released with the expended distal end of the implant anchoring the implant in the perforation and the regenerative film at this distal end flush with tympanic membrane around the perforation. (FIG. 4C).

FIG. 5A to 5C illustrate the use of another embodiment of a system of the invention to treat a perforation in a tympanic membrane of the human ear showing a hydrated implant in an elongated tube of a delivery device (FIG. 3A), a plunger pushing the implant distally toward a distal tip of the elongated tube with consequent radial compression of the implant in the inwardly tapering funnel section of the tube (FIG. 3B), and the hydrated implant after being ejected from the tip of the tube through the perforation where the implant springs-back to a deployed radially expanded configuration where the regenerative thin film layer abuts and spans the perforation causing the membrane to self-regenerate (FIG. 3C).

FIG. 6 illustrates the anatomy of a human ear showing the external auditory canal separated from the internal auditory canal by a perforated tympanic membrane, and an otoscope positioned to view the tympanic membrane.

FIG. 7 illustrates a system comprising a delivery device according to one embodiment of the invention in use delivering an implant through a perforation in the tympanic membrane. FIG. 8 illustrates a system comprising a delivery device according to one embodiment of the invention in use delivering an implant through a perforation in the tympanic membrane.

FIG. 9 illustrates a system comprising a delivery device according to one embodiment of the invention in use delivering an implant through a perforation in the tympanic membrane.

FIG. 10 illustrates a system comprising a delivery device according to one embodiment of the invention in use delivering an implant through a perforation in the tympanic membrane.

FIG. 11 illustrates a delivery device of the invention comprising a detachable cartridge having a lumen for holding a layered implant in a compressed form and a plurality of windows to allow hydration of the layered implant while it is inside the cartridge.

FIG. 12 illustrates a delivery device according to one embodiment of the invention having a plurality of different plungers.

FIG. 13 illustrates two different types of plungers.

FIG. 14 illustrates a number of plunger types.

FIG. 15A illustrates a shuttle for the implant having an implant receiving lumen and configured for radial contraction as it is advanced distally along the elongated tube.

FIG. 15B illustrates the shuttle in the elongated tube of the delivery device.

FIG. 16 illustrates a delivery device with a detachable cartridge incorporating a funnel shaped section that functions as an implant hydration part. In use, an implant is inserted into a proximal end of the cartridge, which is then placed in a hydrating liquid, and once the implant is fully hydrated the cartridge is attached to a distal end of the elongated stem for delivery of the implant.

FIG. 17 and 18 are images of the delivery device of FIG. 16 with the detachable distal part detached from the elongated tune (FIG. 17) and attached to the elongated tube (FIG. 18).

FIG. 19 is an image of the detachable distal part of the delivery device of FIG. 16 containing an implant, FIG. 20 shows the detachable distal part immersed in a hydration solution, and FIG. 21 shows the detachable distal part about to be attached to a distal end of the elongated tube.

FIG. 22 shows the detachable distal part being attached to a distal end of the elongated tube, FIG. 23 shows the plunger advancing the hydrated implant distally along the funnel shaped section of the detachable distal tube, and FIG. 24 shows the implant deployed proud of the distal tip of the detachable distal tube where it has expanded radially to a deployed configuration.

FIG. 25 illustrates a delivery device according to another embodiment of the invention showing the handle and elongated stem (FIG. 25A) and the cartridge and ejection module (FIG. 25B).

FIG. 26 illustrates the delivery device of FIG. 25 in more detail showing the cartridge and layered implant just prior to insertion into the lumen of the cartridge (FIG. 26B) and the delivery device and layered implant in an exploded view (FIG.

26 A).

FIG. 27 illustrates a method of forming a layered implant according to the invention comprising the steps of: pouring a first slurry into a first mould (FIG. 27A); dehydrated thin film layer (FIG. 27B); rehydrating (or freezing) the thin film layer (FIG. 27C); placing the rehydrated (or frozen) thin film layer in a base of a second mould (FIG.

27D); pouring a second slurry into the second mould (FIG. 27E); freeze-drying the thin film and second slurry to form the layered implant (FIG. 27F); and the layered implant after removal from the second mould (FIG. 27G).

FIG. 28 illustrates another method and apparatus for forming a layered implant according to the invention showing: the dehydrated thin film layer after dehydration (FIG. 28A); the thin film placed on top of a base part of the mould (FIG. 28B); the top part of the mould having 49 porous body forming through-holes (FIG. 28C); the top part of the mould placed on the base part of the mould sandwiching the dehydrated thin film between the mould parts (FIG. 28D); the mould after the porous body forming slurry has been poured into the through- holes (FIG. 28E); a sheet of layered implants after removal from the mould (FIG. 28F); and a single layered implant cut from the sheep of layered implants (FIG. 28G).

FIG. 29 illustrates a first method of preparing a layered implant for delivery.

FIG. 30 illustrates a second method of preparing a layered implant for delivery.

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full. Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open- ended and does not exclude additional, unrecited integers or method/process steps.

As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, age, poisoning or nutritional deficiencies.

As used herein, the term "treatment" or "treating" refers to an intervention (e.g., the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s). In this case, the term is used synonymously with the term “therapy”. Additionally, the terms "treatment" or "treating" refers to an intervention (e.g., the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis”.

In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human. As used herein, the term “equine” refers to mammals of the family Equidae, which includes horses, donkeys, asses, kiang and zebra.

As used herein, the term “biopolymer” refers to a polymer produced by the body or a derivative thereof, for example a collagen, a glycosaminoglycan such as hyaluronic acid, or a collagen by-product such as gelatin.

As used herein, the term “non-porous” as applied to the film layer means that the scaffold is substantially free of pores. The film layer is usually obtained by preparing a slurry of a biomaterial such as collagen and then allowing the slurry to dry (without sublimation) to provide a dehydrated non-porous film layer. As used herein the term “dehydrated” as applied to the film layer means that the film is dried without sublimation, for example drying at room temperature or above and at ambient pressure or below (e.g., vacuum drying). This means that ice crystals do not form in the film layer during drying and the film layer is therefore substantially non-porous. In the methods of the invention, the film layer is usually formed by dehydration without sublimation, and then re-hydrated prior to placing in a mould. A slurry is then poured into the mould on top of the film layer, and the contents of the mould is then freeze-dried which enables the film layer adhere to the porous matrix layer. During freeze-drying, ice crystals do not form in the film layer as it is too thin (thus, no sublimation) and the resultant construct comprises a film layer that is dehydrated and substantially non-porous. In one embodiment, the rehydrated film layer is frozen before slurry is poured on top.

The thin-film layer is typically regenerative. As used herein, the term “regenerative” as applied to the film layer means that it comprises a material that is capable of supporting host cell migration and proliferation. An example is a collagen, gelatin, or a mixture of collagen or gelatin, and hyaluronic acid. Other materials suitable for use in the regenerative film layer include chondroitin sulfate and chitosan.

As used herein, the term “thin” as applied to the film layer generally should be understood to mean a thickness of less than 1 mm, 0.75 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.075 mm or 0.005 mm. In any embodiment, the thin film layer has a thickness of about 20 microns to about 200 microns, about 20 microns to about 100 microns, about 20 microns to about 80 microns, or about 40 microns to 60 microns.

As used herein, the term “porous” as applied to the resiliently compressible porous body means that the body has a porosity that is characteristic of being formed by freeze-drying where ice crystals formed in the body are removed by sublimation leaving a 3-D body that is resiliently deformable/compressible. The implant, including the porous body, may be hydrated prior to use which increases the resilient deformability of the implant for delivery.

As used herein, the term “implant advancement module” should be understood to mean a system for advancing the implant along the elongated tube of the delivery device. It is generally a plunger, but may also be provided by a hydraulic or pneumatic force generator configured to push the implant along the tube.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Referring to the drawings and initially to Figures 1 and 2, an implant according to the invention, and indicated generally by the reference numeral 1 , is illustrated. The implant is a bilayered implant comprising a regenerative thin-film layer 2 (FIG. 1 A) and a cylindrical porous body 3 (FIG. 1 B). In this embodiment, the implant (FIG.

10) has a height H of about 4 mm and a diameter D of about 6 mm. The thin layer has a thickness of about 0.5 mm and the porous scaffold has a thickness of about 3.5 mm. The regenerative thin-film layer 2 comprises collagen and hyaluronic acid and is formed from a collagen-hyaluronic acid aqueous slurry that is dehydrated in a mould as described below. The cylindrical porous body 3 is formed from a slurry comprising collagen, hyaluronic acid and gelatin which is freeze-dried as described below, making the porous body spongy and resiliently compressible when rehydrated.

Figure 3 illustrates another embodiment of an implant according to the invention in which parts described with reference to the previous embodiment are assigned the same reference numerals. In this embodiment, the implant, indicated by the reference numeral 10, has a “top hat” structure due to the regenerative thin-film layer 2 having a peripheral flange section 11 that extends radially outwardly of a periphery of a distal end 12 of the cylindrical porous body 3. In addition, the cylindrical porous body 3 has a proximal end 13 having a greater diameter that the distal end 12, a distal frustoconical section 14 and a proximal cylindrical section 15. Figures 4A to 4C illustrate the use of a first system of the invention comprising an implant 10 of Figure 3 and a delivery device 20 for the implant to treat a perforation in a tympanic membrane of the human ear. The delivery device 20 comprises an elongated tube 21 having a distal end 22 comprising a lumen and the implant 10 (of Figure 3) disposed in the lumen in a radially inwardly compressed form. Figure 4A shows the distal end 22 of the elongated tube 21 approaching a tympanic membrane 23 of a human ear having a perforation 24. The implant 10 has already been re-hydrated (the details of re-hydration are provided below). FIG. 4B shows the delivery device advanced towards the proximal side of the perforation in the tympanic membrane and a plunger 25 forming part of the delivery device pushing the implant 10 distally out of the distal end 22 of the elongated tube and through the perforation where distal section 14 of the implant expands after it has passed through the tympanic membrane. Figure 4G shows the implant fully released from the delivery device and the expended distal section 14 of the implant anchoring the implant in the perforation and the flange section 11 regenerative film 2 abutting the tympanic membrane around the perforation (FIG. 4C) where it acts to promote migration of cells into the thin film layer aiding with regeneration of the tympanic membrane to close the perforation.

Figures 5A to 5C illustrate the use of a second system of the invention to treat a perforation in a tympanic membrane of the human ear. In this embodiment, the system comprises the implant 1 of Figures 1 and 2 (e.g., without the flange section) and a delivery device 30. The delivery device comprises an elongated tube 31 with a distal funnel shaped section 32 having a distal end with a distal aperture 34. Figure 5A shows the distal end 7 approaching a tympanic membrane 23 of a human ear having a perforation 24. An implant 1 (which has been hydrated |) is shown in the elongated tube 31 proximal of the funnel shaped section 32 in an uncompressed configuration. FIG. 5B shows the delivery device advanced towards the tympanic membrane and a plunger 25 pushing the implant distally toward a distal tip of the elongated tube with consequent radial compression of the implant in the inwardly tapering funnel section 32. FIG. 5C shows the implant 1 after being ejected from the tip of the tube through the perforation where the implant springs- back to a deployed radially expanded configuration where the regenerative thin film layer 2 abuts and spans the perforation 24 causing the membrane to selfregenerate by the formation of new tissue (not shown).

FIG. 6 illustrates the anatomy of a human ear showing the external auditory canal 37 separated from the internal auditory canal 38 by a tympanic membrane 23 with a perforation 24, and an otoscope 39 positioned to view the tympanic membrane.

FIG. 7 illustrates a system comprising a delivery device according to one embodiment of the invention in use delivering an implant through a perforation in the tympanic membrane, in which parts described with reference to the previous embodiment are assigned the same reference numerals. In this embodiment, the delivery device comprises the elongated tube 31 with a funnel section 32 that extend the full length of the tube from the proximal end with proximal opening to the distal end with distal opening, and an elongated plunger 25. The implant 1 is shown in a compressed configuration adjacent the distal end of the tube 31 .

FIG. 8 illustrates a system comprising a delivery device according to another embodiment of the invention in use delivering an implant through a perforation in the tympanic membrane, in which parts described with reference to the previous embodiment are assigned the same reference numerals. In this embodiment, the elongated tube 31 is a rigid plastic tubing with a distal part with a narrow internal diameter (2mm) and a funnel shaped proximal part 32, a handle part 40 with an external sliding button 41 , and a plunger 25 with a proximal end 42 coupled to the sliding button 41 for axial movement therewith. In use, a user can hold the handle in their hand and actuate the movement of the plunger axially along the lumen of the tube 31 with their thumb.

FIG. 9 illustrates a system comprising a delivery device according to another embodiment of the invention in use delivering an implant through a perforation in the tympanic membrane, in which parts described with reference to the previous embodiment are assigned the same reference numerals. In this embodiment, the elongated tube 31 is a rigid plastic tubing with a distal part 50 with a narrow internal diameter (2mm), a proximal part 51 cranked an at angle 0 of about 70° to the distal part, and a funnel shaped proximal end 52. The plunger 25 is made from flexible tubing.

The embodiment illustrated in FIG. 10 is similar to the embodiment of FIG. 9 with the exception of the elongated tube 31 being a flexible tube and having a steel mandrel 45 dimensioned to be inserted into and run the length pf the elongated tube. In use, the steel mandrel can be shaped into a desired configuration that suits the anatomy of the patient’s ear, and then inserted into the elongated tube which will then adapt the elongated shape of the mandrel.

FIG. 11 illustrates a system comprising a delivery device according to another embodiment of the invention, in which parts described with reference to the previous embodiment are assigned the same reference numerals. In this embodiment, the delivery device 60 comprises an elongated tube 61 with a distal end 62 and an implant receiving cartridge 63 coupled to the distal end 62 of the elongated tube 61 . The cartridge 63 has a hollow cylindrical body with a proximal opening 64, a distal opening 65, and a sidewall 66 comprising a plurality of windows 67. An implant 1 is shown loaded into the cartridge 63. Before the cartridge is coupled to the elongated tube 61 of the delivery device, the implant 1 is radially compressed and inserted one end of the cartridge where it is held within the cartridge in a radially compressed form. The cartridge containing the implant may then be immersed in a hydration liquid where the windows 67 facilitate access of the hydration liquid to the implant. Once the implant has been hydrated, the cartridge is coupled to the distal end of the elongated tube.

FIG. 12 illustrates a system comprising a delivery device according to another embodiment of the invention in which parts described with reference to the previous embodiment are assigned the same reference numerals. In this embodiment, the elongated tube 71 has a distal part 72 and a funnel shaped proximal part 73 configured to couple together. A plurality of plungers 25 having different diameters are provided to assist advancing the implant through the inwardly tapering funnel shaped proximal part 73.

FIG. 13 illustrates different designs of plungers, one with a convex end (FIG. 13A) and one with base and sidewalls (FIG. 13B).

FIG. 14 illustrates more designs of plungers of different length and bore size, and including different design of plunger heads including a spherical head, a flat head, and a convex head.

FIG. 15A illustrates a shuttle 80 comprising a split tube 81 with an internal lumen 82 dimensioned to receive an implant 1 in a non-radially compressed configuration and configured for radial contraction as it is advanced distally along a funnel shaped section of the delivery device. FIG. 15B illustrates an elongated tube 84 of a delivery device with the shuttle 80 containing an implant 1 inserted into a proximal end of the tube. As the shuttle is advanced along the funnel shaped tube, it compressed radially compressing the implant contained in the shuttle. A distal end of the tube 84 defines a radially inward step which in practice acts as a stop for the shuttle allowing the radially compressed implant 1 to be advanced out of the distal end of the tube.

FIGS. 16 to 18 illustrate a system comprising a delivery device according to another embodiment of the invention in which parts described with reference to the previous embodiment are assigned the same reference numerals. In this embodiment, the elongated tube comprises a proximal tube part 80 with a non-tapering internal lumen and a distal tube part 81 configured for detachable attachment to a distal end 82 of the proximal tube part 80. The distal tube part 81 can function as an implant hydration tube (cartridge) and has a proximal opening 83 dimensioned to receive an uncompressed implant, a funnel shaped section 84 and a distal end 85 with distal opening 86. The distal and proximal tube parts are configured to couple together in a twist-lock manner. The implant 1 is shown disposed in distal tube part 81 just proximal of the funnel shaped section 84. In use, the implant 1 is loaded into the distal tube part 81 in an uncompressed configuration, the distal tube part 81 containing the implant 1 is then immersed in a hydration liquid, before being coupled to the proximal tube part 80 as shown in FIG. 16. The plunger 25 can then be advanced to push the implant 1 along the funnel shaped section 84 to radially compress the implant before it is ejected through distal opening 86. In FIG. 16, the proximal tube part is angled and in FIGS. 17 and 18 the proximal tube part is straight.

FIG. 19 shows the detachable distal tube part being attached to a distal end of the proximal tube part, FIG. 20 shows the plunger advancing the hydrated implant distally along the funnel shaped section of the detachable distal tube part, and FIG. 21 shows the implant deployed proud of the distal tip of the detachable distal tube part where it has expanded radially to a deployed configuration.

FIG. 22 shows the distal tube part containing an implant and detached from the proximal tube part. FIG. 23 shows the distal tube part containing the implant immersed in a hydration liquid. FIG. 24 shows the distal part of the elongated tube being attached to the proximal end of the elongated tube after the implant has been hydrated.

FIGS. 25 and 26 illustrate a delivery device and system according to one embodiment of the invention. The delivery device 90 comprises a handle 91 , first elongated tube 92 having a proximal end 93A coupled to the handle 91 and a distal end 93B, second elongated tube 94 having a proximal end 95 configured for coupling to the distal end 93B of the first elongated tube 92, and a cylindrical cartridge 96 having a lumen 97, a proximal end 98 configured for coupling to a distal end of the second elongated tube 92 and a funnel shaped distal end 99. The second elongated tube 92 has an internal lumen housing a plunger 99 that is movable axially in response to actuation of a button 100 on the handle, and also comprises a plurality of windows 101 spaced around a circumference of the funnel shaped distal end 99. FIG. 26A illustrates the system of the invention comprising the delivery device 90 of FIG. 25 and a bi-layered implant 10 of FIG. 3, in an exploded form. FIG. 26B is a detailed view of the implant 10 and cartridge 96.

In use, a layered implant (not shown) is loaded into a proximal end of the cartridge 96 in an un-compressed form and the cartridge housing the implant is immersed in a hydration liquid to hydrate the implant. The delivery device is than assembled by coupling the first and second elongated tubes together and then coupling the proximal end of the cartridge to the distal end of the second elongated tube. The second elongated tube may then be inserted into the ear canal of a patient under guidance and advanced until the distal end of the cartridge is disposed adjacent a perforation in the patient’s tympanic membrane. The button 100 on the handle is then pressed to actuate the plunger and advance the implant along the funnel- shaped section of the cartridge to eject the implant in a radially compressed form into the perforation. Once released from the cartridge, the implant self-expends to anchor the implant in the perforation with the thin film layer disposed almost flush with the periphery of the tympanic membrane.

FIG. 27 illustrates a method of forming a layered implant according to the invention comprising the steps of pouring a first slurry 110 into a first mould 1 11 to a depth of about 0.5mm (FIG. 27A), dehydrated the first slurry in the mould to form a dehydrated thin film layer 2 (FIG. 27B), placing the thin film layer 2 into a rehydration bath 112 containing a hydration liquid 113 to rehydrate the thin film layer (FIG. 27C). Once fully re-hydrated, the thin film layer 2 is placed onto a base 115 of a second mould 116 (FIG. 27D), a second slurry 117 is poured into the second mould 116 (FIG. 27E), before the mould is placed on a shelf of a freeze- dryer 118 to freeze-dry the thin film and second slurry (FIG. 27F), before the layered implant 10 is removed from the second mould (FIG. 27G).

FIG. 28 illustrates another method and apparatus for forming a layered implant according to the invention that employs a mould apparatus have a bottom plate 120 having a top surface 121 and a top plate 122 configured to be coupled together with the bottom plate in a face-to-face relationship. The top plate 122 comprises a plurality of porous body shaped through-holes 123. FIG. 28A shows a dehydrated thin film layer 2 after dehydration. FIG. 28B shows the thin film 2 placed on the top surface 121 of the bottom plate 120. FIG. 28C shows the top plate 122 having forty- nine porous body forming through-holes 124. FIG. 28D shows the top plate 122 placed on top of the bottom plate 120 sandwiching the dehydrated thin film 2 between the plates. A second slurry is then poured into the apertures 124 in the top plate 122 and the mould apparatus is placed in a freeze-dryer 126 and a freeze- drying procedure performed. FIG. 28F shows a sheet of layered implants 128 after removal from the mould and FIG. 28G shows a single layered implant 129 cut out of the sheet of layered implants 128.

FIG. 29 illustrates a method of preparing a layered implant for delivery to a target locus in the body. In this embodiment, the implant 1 is radially compressed by applying a force in the direction of the arrows marked A as illustrated in FIG. 29A from a diameter of about 8mm to a diameter of about 4mm (FIG. 29B). The implant is then inserted into a cartridge 130 in a radially compressed form (FIG. 29C), and the cartridge 130 is then immersed into a hydration fluid 131 in a hydration bath 132 (FIG. 29D). In one embodiment, this hydration step is not required. The cartridge 130 is then attached to a distal end of an elongated tube 133 of a delivery device (FIGS. 29E and 29F).

FIG. 30 illustrates a further method of preparing a layered implant for delivery to a target locus in the body. In this embodiment, the cartridge 140 has a lumen comprising a proximal lumen section 141 that is dimensioned to receive the implant 10 in an uncompressed form and a distal funnel-shaped lumen section 142 configured to radially compress the implant as it is advanced through the distal funnel-shaped lumen section 142. In use, the implant 10 (FIG. 30A) is inserted into the proximal lumen section 141 of the cartridge 140 (FIG. 30B). The cartridge 140 is then attached to a distal end of an elongated tube 133 of a delivery device and an ejection mechanism is actuated to push the implant 10 distally along the lumen of the cartridge to radially compress the implant prior to ejection. This allows the implant to be ejected into the perforation and self-expend to anchor the implant in the perforation (FIG. 30C).

Example 1

Preparation of collagen-hyaluronic acid (CHyA) slurry (0.5% Collagen 0.044%, HyA)

Supplies:

1 . 2g dry weight Type 1 Collagen

2. 400ml 0.5M acetic acid

3. 0.176g sodium salt hyaluronic acid (Sigma-Aldrich)

Procedure:

1 . Soak 2g of collagen in 340ml 0.5M acetic acid and place in the fridge at 4°C overnight

2. Soak 0.176g HyA in the remaining 0.5M acetic acid (60ml) and place in the fridge at 4°C overnight

3. Next morning, fill an ice box with ice and water, using a retort stand and clamp, hold the beaker of collagen in the ice box

4. Blend 2g of collagen with 340ml of 0.5M acetic acid with the blender (Ultra Turrax T25 overhead blender, IKA works Inc., Wilmington, NC) at 12,000rpm for 90 minutes, keeping an eye on the temperature of the collagen slurry

5. Blend the HyA in 60ml 0.5M acetic acid for 10-15 minutes on its own to ensure HyA is dispersed throughout

6. Using a 5ml pipette and pipette gun, slowly add the HyA solution into the collagen slurry, 1 ml at a time

7. Once all the HyA has been added, blend the slurry for an additional 90 minutes at 12,000rpm at 4°C

8. Degas the slurry and store at 4°C

Film collagen-HyA film fabrication

1 . Clean Telfon molds and metal frames with 0.5M acetic acid

2. Clamp the Telfon molds and metal frames using the metal clips to ensure no slurry is lost during drying time

3. For a dry film of collagen 0.5%- HyA 0.044% w/v with an~thickness of 50 micron, pour 18.4ml of slurry into 6cm x 6cm metal tray

4. Allow the suspension 72h at room temperature to dry in a flow hood

5. Remove the metal clamps and slowly lift the metal frame from the Telfon molds, using a forceps or scalpel gently remove the edges of the film from the metal frame

6. Clean the molds and metal frames using 0.5M acetic acid

7. Films can be stores at room temperature in an aluminum foil pocket

Example 3

Direct freeze-drying of a bilayer non-crosslinked collage/hyaluronic acid/ gelatin scaffold

1 . Rehydrate the collagen-hyaluronic film formed according to Example 2 in 0.5M acetic acid for 1 h.

2. Clean the metal mold and frame 6cm x 6cm with 0.5 M acetic acid.

3. Dissolve gelatin in dH2O by heating on a hot plate between 45-50°C and stirring constantly with a stir bar, to make 1% gelatin in 12ml

4. Carefully slide the hydrated film onto the metal mold to avoid trapping air bubbles.

5. Pour 12ml of degassed 0.5% CHyA slurry into a beaker, followed by the 12ml of slightly cooled 1 % gelatin solution to give 24ml of 0.5% CHyA slurry and 1% gelatin slurry. Mix this solution gently to avoid creating air bubbles with a spatula

6. Pour 20ml of the mixed slurry on top of the hydrated film in the 6cm x 6cm mold without trapping bubbles of air.

7. Place the mold in the freeze dryer and use the -40°C programme 8. Once the cycle has finished, remove the scaffolds from the mold.

9. Clean the mold using 0.5M acetic acid.

10. Store the bi-layered scaffolds in custom-made pockets of aluminium foil.

Example 4

Direct freeze-drying of a bilayered material with cylindrical scaffold on top of rectangular film

1 . Cut film to dimensions of 11 cm x 1 1 cm (outer dimensions of 05mm cylindrical mould)

2. Rehydrate the collagen-hyaluronic film in 0.5M acetic acid for 1 h.

3. Clean the 05mm cylindrical mould with 0.5 M acetic acid.

4. Dissolve gelatin in dH2O by heating on a hot plate between 45-50°C and stirring constantly with a stir bar, to make 1% gelatin in 12ml

5. Carefully slide the hydrated film onto the base metal mould to avoid trapping air bubbles.

6. Place the top part of the 05mm cylindrical mould onto the base part (do not screw on)

7. Pour 12ml of degassed 0.5% CHyA slurry into a beaker, followed by the 12ml of slightly cooled 1 % gelatin solution to give 24ml of 0.5% CHyA slurry and 1% gelatin slurry. Mix this solution gently to avoid creating air bubbles with a spatula

8. Pipette 100 pl of the mixed slurry into each 05mm inset.

9. Place the mold in the freeze dryer and use the -40°C programme

10. Once the cycle has finished, remove the scaffolds from the mold.

11 . Clean the mold using 0.5M acetic acid.

12. Store the bi-layered scaffolds in custom-made pockets of aluminium foil.

Lamination of collagen-HyA film 1 . Cut film according to Example 2 to dimensions of 10cm x 10cm (outer dimensions medium rectangular mould)

2. Rehydrate the collagen-hyaluronic film in 0.5M acetic acid for 1 h.

3. Carefully slide the hydrated film onto the Telfon mold base.

4. Place the top part of the medium rectangular mould onto the base part trapping the first film layer and clamp

5. For a 2nd dry film layer of collagen 0.5%- HyA 0.044% w/v with an~thickness of 50 micron, pour 18.4ml of slurry into medium rectangular mould

6. Allow the suspension 72h at room temperature to dry in a flow hood.

7. Store the bi-layered films in custom-made pockets of aluminium foil.

Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1 . A layered implant to repair a perforation in a tympanic membrane comprising:

(a) a dehydrated thin film layer having a thickness of less than 0.5 mm and comprising a biopolymer; and

(b) a porous body comprising a freeze-dried matrix comprising a biopolymer, wherein the dehydrated thin film layer is mounted on and integrated with the porous body, wherein the porous body is resorbable in-vivo and resiliently compressible, and wherein the biopolymer of the porous body and the dehydrated thin film layer are each, independently, selected from collagen, hyaluronic acid, and gelatin.

2. A layered implant according to Claim 1 , in which the porous body is cylindrical and comprises a distal end, a proximal and, and a sidewall, and in which the dehydrated thin film layer is mounted on and integrated with the proximal end of the porous body.

3. A layered implant according to Claim 2, in which the cylindrical porous body tapers inwardly towards its proximal end.

4. A layered implant according to any preceding Claim, in which the dehydrated thin film layer comprises an annular flange portion that extends radially outwardly of the proximal end of the porous body.

5. A layered implant according to any preceding Claim, in which the porous body has a thickness that is at least 10 times greater than a thickness of the dehydrated thin film layer.

6. A layered implant according to any preceding Claim, in which the biopolymer of the freeze-dried matrix of the porous body and dehydrated thin film layer comprises collagen.

7. A layered implant according to any preceding Claim, in which the biopolymer of the freeze-dried matrix of the porous body comprises collagen, hyaluronic acid and a gelling agent.

8. A layered implant according to any preceding Claim, in which the biopolymer of the dehydrated thin film layer comprises collagen and hyaluronic acid.

9. A layered implant according to any preceding Claim, in which the porous body comprises at least 20% collagen (w/w) and at least 40% gelling agent (w/w).

10. A layered implant according to any preceding Claim, in which the porous body comprises 1 to 5% hyaluronic acid (w/w).

11 . A layered implant according to any preceding Claim, in which the porous body comprises 20-40% collagen, 50-80% gelling agent and 1 -5% hyaluronic acid (w/w).

12. A layered implant according to any preceding Claim, in which the dehydrated thin film layer comprises at least 80% collagen (w/w).

13. A layered implant according to any preceding Claim, in which the dehydrated thin film layer comprises 5 to 15% hyaluronic acid (w/w).

14. A layered implant according to any preceding Claim, in which the dehydrated thin film layer is a laminated thin film having a plurality of layers.

15. A layered implant according to Claim 14, in which the laminated thin film comprises biopolymer fibres disposed between at least two layers of the laminate.

16. A layered implant according to Claim 15, in which the biopolymer fibres are collagen fibres.

17. A layered implant according to Claim 15 or 16, in which the biopolymer fibres are directionally aligned in the laminate.

18. A layered implant according to any preceding Claim, in which the dehydrated thin film layer is coated with one or more extracellular matrix proteins.

19. A method of forming a layered implant according to any of Claims 1 to 18, the method comprising the steps of: dehydrating a first slurry comprising a biopolymer to provide a dehydrated thin film layer; re-hydrating the dehydrated thin film layer; placing the re-hydrated thin film layer on to a base of mould pouring a second slurry comprising a biopolymer into the mould on top of the re-hydrated thin film layer; and freeze-drying the re-hydrated thin film layer and second slurry together to form the layered implant.

20. A method according to Claim 19, in which the re-hydrated thin film layer is frozen prior to being placed on to the base of the mould.

21 . A system to repair a perforation in a tympanic membrane comprising: a layered implant according to any of Claims 1 to 18; and a delivery device for the layered implant configured to hold the layered implant and deliver the layered implant in a radially compressed form into a perforation in a tympanic membrane via the ear canal.