WO2022146730A1

WO2022146730A1 - Implantable pouch device and methods of making thereof

Info

Publication number: WO2022146730A1
Application number: PCT/US2021/064227
Authority: WO
Inventors: Nathaniel REMLINGER; Thomas W. Gilbert; Andrea S. MARKS; D. Adam Young; Luai HULEIHEL; Nichol REISHER; Jiayu TANG
Original assignee: Acell, Inc.
Priority date: 2020-12-29
Filing date: 2021-12-17
Publication date: 2022-07-07

Abstract

Implantable pouch devices for encasing a cardiac implantable electronic device (CIED) or other medical device are formed from one or more bioresorbable sheets of material comprising an extracellular matrix (ECM) for maintaining and supporting a healing environment. Each bioresorbable sheet may be a medical graft assembly which comprises at least first and second sheets of ECM material layered together such that the epithelial basement membrane sides face outward.

Description

IMPLANTABLE POUCH DEVICE AND METHODS OF MAKING THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit under 35 U.S.C. 119(e) to U.S. Provisional Application No. 63/131,583, filed December 29, 2020 and U.S. Provisional Application No. 63/131,578, filed December 29, 2020, the entire contents of which are each incorporated by reference herein.

FIELD

[0002] The present disclosure relates to implantable pouch devices comprising a bioresorbable, extracellular matrix (ECM) for encasing a medical device, such as a cardiac implantable electronic device.

BACKGROUND

[0003] Cardiac implantable electronic devices (CIEDs), such as pacemakers, defibrillators, generators, and neuromodulators, deliver a stimulus to the heart when the heart’s own natural pacemaker and/or conduction system fails to provide synchronized atrial and ventricular contractions at desirable rates and intervals. A surgeon generally implants CIEDs subcutaneously or sub-muscularly in or near a patient’s pectoral or abdominal region. Prior to implantation, some surgeons encase the CIED within a biocompatible pouch for securing the device in position, facilitating removal of the device, inhibiting or reducing bacterial growth, preventing corrosion, and/or inhibiting scarring or fibrosis on or around the CIED.

[0004] Some biocompatible pouches formed from natural tissues ensure that the patient’s body does not reject the CIED or that the device does not trigger an adverse response, such as inflammation, infection or blood clot formation.

[0005] Medical graft assemblies formed from dried sheets of bioabsorbable tissue comprising an extracellular matrix (ECM) can serve as tissue grafts such as in wound healing applications and can be used to form other medical devices. Some examples of such tissue derived from mammals can be processed to contain an epithelial basement membrane layer on one side and a lamina propria layer on an opposite side of the tissue sheet. The lamina propria is a thin layer of connective tissue that forms part of the mucous membranes or mucosa that line various organ systems in the body, such as the respiratory tract, the gastrointestinal tract, and the urogenital tract. The lamina propria lies beneath the epithelium and, together with the epithelium and epithelium basement membrane, constitutes the mucosa. Some prior studies indicated an enhanced capacity of ECM material to support cellular infiltration on the lamina propria side of the material and cellular spreading along the epithelial basement membrane side of the material. [0006] A need continues for implantable pouches designed to accommodate various CIEDs having different shapes, sizes and weights, or having sharp edges, connecting conduits, wires, or tubes. Preferably, manufacture of such pouches would not require sealant, stitches, staples or the like. Furthermore, the material forming the implantable pouches would ideally be bioresorbable and have the ability to maintain and support a healing environment by helping to remodel functional tissue post-surgery and/or to minimize tissue attachment.

SUMMARY

[0007] The present disclosure describes various examples of implantable pouch devices for encasing a CIED or other medical device formed from one or more bioresorbable sheets of material comprising an extracellular matrix (ECM) for maintaining and supporting a healing environment. Pouch devices may also be referred to herein as pouches or pouch structures. The ECM may be synthetic or naturally-occurring. Naturally-occurring bioresorbable sheets may have an epithelial basement membrane side and an opposing luminal side preferably surrounding the pouch area. The bioresorbable sheets may further have regions of varying density. Manufacture of the pouch devices advantageously does not require sealant, stitches or staples. [0008] In examples, an implantable pouch device of this disclosure includes at least a first and a second bioresorbable sheet of material made of an extracellular matrix (ECM). Each of the at least first and second bioresorbable sheets has corresponding top, bottom, and side edges. The corresponding bottom and side edges of the at least first and second bioresorbable sheets are laminated together, forming a cavity between the at least first and second bioresorbable sheets for receipt of a medical device. In examples, the implantable pouch device further includes a plurality of through holes defined by the at least first and second bioresorbable sheets for allowing passage of bodily fluids and/or wires. In examples, at least one of the corresponding top, bottom and side edges of the at least first and second bioresorbable sheets have a denser matrix structure than a remainder of the at least first and second bioresorbable sheets. In examples, the ECM is synthetic or naturally-occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least first and second sheets.

[0009] In examples, a method of making an implantable pouch device of this disclosure includes providing at least a first and a second hydrated bioresorbable sheet of material made of an extracellular matrix (ECM). Each of the at least first and second bioresorbable sheets has corresponding top, bottom, and side edges. A non-stick insert is placed between the at least first and second bioresorbable sheets. The corresponding bottom and side edges of the pair of bioresorbable sheets are laminated together, forming a cavity about the non-stick insert. The at least first and second bioresorbable sheets are dried and the non-stick insert is removed from the cavity. In examples, laminating together the corresponding bottom and side edges of the at least first and second bioresorbable sheets is performed by lyophilization and/or vacuum pressing. In examples, through holes are formed in the at least first and second sheets for allowing passage of bodily fluids and/or wires. In examples, at least one of the corresponding bottom and side edges of the at least first and second bioresorbable sheets have a denser matrix structure than a center of the at least first and second bioresorbable sheets. In examples, the ECM is synthetic or naturally-occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least first and second sheets. In examples, drying the at least first and second bioresorbable sheets is air drying.

[0010] In other examples, an implantable pouch device of this disclosure includes at least one bioresorbable sheet of material made of an extracellular matrix (ECM). The at least one bioresorbable sheet includes a body flap portion having a lower region and an upper region, a right arm flap portion, and a left arm flap portion. The body flap portion, the right arm flap portion, and the left arm flap portion are folded into a three-dimensional structure, forming a cavity for receipt of a medical device. In examples, the at least one bioresorbable sheet defines a plurality of alignment holes for aligning folds of the three-dimensional structure. In examples, the ECM is synthetic or naturally-occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least one bioresorbable sheet. [0011] In other examples, a method of making an implantable pouch device of this disclosure includes providing at least one bioresorbable sheet of material made of an extracellular matrix (ECM). The at least one bioresorbable sheet includes a body flap portion having a lower region and an upper region, a right arm flap portion, and a left arm flap portion. The lower region of the body flap portion is folded over the upper region of the body flap portion. One of the right arm flap portion or the left arm flap portion is folded over the folded lower region of the body flap portion. The other of the right arm flap portion or the left arm flap portion is folded over the folded lower region of the body flap portion such that a cavity is formed between the lower and upper regions of the body flap portion, the right arm flap portion, and the left arm flap portion for receipt of a medical device. In examples, folding the lower region of the body flap portion over the upper region of the body flap portion includes aligning right and left alignment holes defined through the lower region with corresponding right and left alignment holes defined through the upper region. In examples, folding the right arm flap portion over the folded lower region of the body flap portion includes aligning an alignment hole defined through the right arm flap portion with the left alignment holes defined through the lower and upper regions of the body flap portion. In examples, folding the left arm flap portion over the folded lower region of the body flap portion includes aligning an alignment hole defined through the left arm flap portion with the right alignment holes defined through the lower and upper regions of the body flap portion. In examples, the ECM is synthetic or naturally-occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least first and second sheets.

[0012] In yet further examples, an implantable pouch device of this disclosure includes at least one bioresorbable sheet of material made of an extracellular matrix (ECM). The pouch device has a tubular shape with at least one open end. The tubular shape forms a cavity for receipt of a medical device. In examples, the ECM is synthetic or naturally-occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least one bioresorbable sheet.

[0013] In yet further examples, a method of making an implantable pouch device of this disclosure includes providing at least a first hydrated, bioresorbable sheet of material made of an extracellular matrix (ECM). The at least first bioresorbable sheet is wrapped around a cylindrical member at least one time. The at least first bioresorbable sheet is dried and the cylindrical member is removed to form a cavity for receipt of a medical device. In examples, prior to drying the at least first bioresorbable sheet, at least a second hydrated bioresorbable sheet is wrapped around an end of the cylindrical member and overlapping the at least first bioresorbable sheet to form a closed end. In examples, the ECM is synthetic or naturally- occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least first bioresorbable sheet.

[0014] In additional examples, an implantable pouch device of this disclosure includes at least one bioresorbable sheet of material made of an extracellular matrix (ECM). A periphery of the at least one bioresorbable sheet defines a plurality of thread holes for lacing of a flexible member about the periphery, such that tensioning the flexible member cinches the periphery of the at least one bioresorbable sheet to form a cavity between sides of the at least one bioresorbable sheet for receipt of a medical device. In examples, the implantable pouch device further includes a plurality of through holes defined by the at least one bioresorbable sheet for allowing passage of bodily fluids and/or wires. In examples, the ECM is synthetic or naturally-occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least one bioresorbable sheet. In examples, a shape of the at least one bioresorbable sheet is selected from: round, triangular, polygonal, or star-shaped.

[0015] In additional examples, a method of making an implantable pouch device of this disclosure includes providing at least one bioresorbable sheet of material made of an extracellular matrix (ECM). A plurality of thread holes is created about a periphery of the at least one bioresorbable sheet for passage of a flexible member such that tensioning of the flexible member cinches the periphery of the at least one bioresorbable sheet to form a cavity between sides of the at least one bioresorbable sheet for receipt of a medical device. In examples, the method further includes forming a plurality of through holes in the at least one bioresorbable sheet for allowing passage of bodily fluids and/or wires. In examples, the ECM is synthetic or naturally-occurring. In examples, the ECM has a luminal side and an opposing epithelial basement membrane side, and the cavity is formed from the luminal side of the at least one bioresorbable sheet. [0016] The present disclosure also describes medical graft assemblies, also referred to as medical graft devices, comprising at least two sheets of ECM material having an epithelial basement membrane (EBM) side and an opposing lamina propria (LP) side. The sheets are layered one on top of the other such that either the two EBM sides, or the two LP sides, face outwards on both sides of the device. Such orientation advantageously helps to control tissue attachment and/or cellular/vascular ingrowth when used in a surgical repair. Such oriented or directional ECM assemblies can also each be used as a bioresorbable sheet for forming implantable pouch devices of the present disclosure.

[0017] In examples, medical graft assemblies of this disclosure include at least a first and second sheet of extracellular matrix (ECM) material having an epithelial membrane side and an opposing lamina propria side. The at least first and second sheets of ECM material are layered such that either the epithelial basement membrane sides face outward and the lamina propria sides face inward, or the lamina propria sides face outward and the epithelial basement membrane sides face inward. In examples, the epithelial basement membrane sides of the at least first and second sheets face outward and the lamina propria sides face inward. In other examples, the lamina propria sides of the at least first and second sheets face outward and the epithelial basement membrane sides face inward. In examples, the ECM is selected from the group consisting of a urinary bladder matrix (UBM), submucosa of the intestine (SIS), liver or dermis. In examples, the medical graft device is a surgical graft or a wound dressing device. In examples, the at least first sheet has a density and a resorption rate selected to differ from a density and a resorption rate of the at least second sheet. In examples, the epithelial basement membrane sides of the at least first and second sheets comprise mammalian epithelial basement membrane free of epithelial cells and cellular elements of the epithelial cells.

[0018] Examples of a method of making a medical graft assembly of this disclosure include providing at least a first hydrated sheet of ECM material having an epithelial basement membrane side and an opposing lamina propria side. At least a second hydrated sheet of ECM material is then provided having an epithelial basement membrane side and an opposing lamina propria side. The at least first sheet and second sheets of ECM material are layered such that either the epithelial basement membrane sides face outward and the lamina propria sides face inward, or the lamina propria sides face outward and the epithelial basement membrane sides face inward. Regions of the at least first and second sheets are then compressed together. The at least first and second sheets of the ECM material are then dried to form the device. In examples, the epithelial basement membrane sides of the at least first and second sheets face outward and the lamina propria sides face inward. In other examples, the lamina propria sides of the at least first and second sheets face outward and the epithelial basement membrane sides face inward. In examples, the ECM is selected from the group consisting of a urinary bladder matrix (UBM), submucosa of the intestine (SIS), dermis and liver. In examples, compressing the regions of the at least first and second sheets together and drying the at least first and second sheets occurs simultaneously. In examples, compressing the at least first and second sheets comprises one or more of vacuum pressing, lyophilizing, clamping, crushing or stitching. In examples, drying the at least first and second sheets comprises one or more of vacuum pressing, lyophilizing or air drying. In examples, the at least first sheet has a density and a resorption rate selected to differ from a density and a resorption rate of the at least second sheet. In examples, the epithelial basement membrane sides of the at least first and second sheets comprise mammalian epithelial basement membrane free of epithelial cells and cellular elements of the epithelial cells.

[0019] Examples of a method for inducing repair of diseased, defective or damaged tissue in a mammal of this disclosure include implanting on a defect site a medical graft assembly. The medical graft assembly includes at least a first and second sheet of ECM material having an epithelial membrane side and an opposing lamina propria side. The at least first and second sheets of ECM material are layered such that either the epithelial basement membrane sides face outward and the lamina propria sides face inward, or the lamina propria sides sheets face outward and the epithelial basement membrane sides face inward. In examples, implanting the medical graft assembly on a defect site includes implanting the medical graft assembly on a tissue surface such that the epithelial basement membrane side of either the at least first or second sheet faces the tissue surface. In examples, a percentage of tissue attachment of the epithelial basement membrane side to the tissue surface is about 0% after 14 days of implantation and between about 0% and 33% after 90 days of implantation. In examples, implanting the medical graft assembly on a defect site includes implanting the medical graft assembly on a tissue surface such that the lamina propria side of either the at least first or second sheet faces the tissue surface.

[0020] Further examples of implantable pouch devices are those wherein at least one of the at least a first and a second bioresorbable sheet of material is a medical graft assembly described above. An embodiment includes two medical graft assemblies laminated together along at least a portion of the edges. The lamination may be performed by vacuum pressing, and the implantable pouch device does not comprise any suture or other fixation means. Further, the epithelial basement membrane sides of each medical graft assembly faces outward and the lamina propria sides face inward. Regarding the implantable pouch device, the epithelial basement membrane sides of the ECM (for example, UBM) face both outward from the implantable pouch device and inward toward the cavity.

[0021] The present disclosure also describes examples of single channel or multi-channel nerve guides made of at least one bioresorbable sheet of material comprising an extracellular matrix (ECM). Each bioresorbable sheet of material may be a medical graft assembly described herein. [0022] The present disclosure further describes methods for making nerve guides of two or more channels. The method may comprise providing a first ECM sheet, for example with a flat surface with the epithelial basement membrane side facing down and the luminal side facing up, or with an opposite orientation; placing a first mandrel on the first ECM sheet; rolling for at least one turn to form a first channel; placing a second mandrel on a second ECM sheet adjacent to the first mandrel; rolling for at least one turn to form a second channel; optionally repeating the previous steps until a desired number of channels is formed; wrapping a last ECM sheet around the mandrels to form a conduit; optionally drying the conduit; and removing the mandrels. Depending on which side, the epithelial basement membrane (EBM) side or lamina propria (LP) side, of each ECM sheet faces down in the rolling process, the nerve guide may have an external surface of EBM or LP, as desired. Each channel may have a channel wall with EMB or LP facing the channel cavity. In examples, the external surface of the nerve guide, as well as the wall of each channel facing the channel cavity comprises an EBM surface. Alternatively, the nerve guide outer surface and one or more channels may have an LP surface.

[0023] A reading of the following detailed description and a review of the associated drawings will make apparent the advantages of these and other features. Both the foregoing general description and the following detailed description serve to explain the disclosure only and do not restrict aspects of the disclosure as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS [0024] Reference to the detailed description, in conjunction with the following figures, will make the disclosure more fully understood, wherein:

[0025] FIG. 1 A illustrates an example of the implantable pouch device of this disclosure in a front view;

[0026] FIG. IB illustrates a method of making the implantable pouch device of FIG. 1 A;

[0027] FIGS. 1C-E illustrate examples of differing shapes and manufacturing methods of the implantable pouch device.

[0028] FIGS. 1F-H illustrate regions of varying density of the implantable pouch device of FIG.

1A;

[0029] FIG. 2A illustrates a second example of the implantable pouch device of this disclosure in an isometric view;

[0030] FIGS. 2B-D show multiple examples of tissue sources that can be used to create the device of FIG. 2A

[0031] FIGS. 2E-F illustrate the features and dimensions of the tissue material for arriving at the geometry of the implantable pouch of FIG. 2 A;

[0032] FIGS. 2G-K illustrate a method of forming the implantable pouch device of FIG. 2 A;

[0033] FIG. 3 A illustrates a third example of the implantable pouch device of this disclosure in a perspective view;

[0034] FIGS. 3B-D illustrate a method of forming the implantable pouch device of FIG. 3A;

[0035] FIGS. 4A and 4B illustrates a fourth example of the implantable pouch device of this disclosure in a deployed view (FIG. 4A) and a pre-deployed view (FIG. 4B);

[0036] FIGS. 4C and 4D illustrate a method of forming the implantable pouch device of FIG.

4A; and

[0037] FIGS. 4E-4H illustrate alternative examples of the implantable pouch device of FIG. 4 A.

[0038] FIG. 5A illustrates an example of a single-channel nerve guide of this disclosure;

[0039] FIG. 5B shows an example of a multi-channel nerve guide, more specifically a three- channel nerve guide, of this disclosure;

[0040] FIG. 5C is a side view of the 3 three-channel nerve guide of FIG. 5B;

[0041] FIG. 5D shows an example of a multi-channel nerve guide, more specifically a five- channel nerve guide, of this disclosure;

[0042] FIG. 5E shows another example of a five-channel nerve guide of this disclosure; [0043] FIG. 6 illustrates an example of the medical graft assembly of this disclosure in which the epithelial basement membrane sides of the device face outwards;

[0044] FIG. 7 illustrates two medical graft assemblies of FIG. 6 arranged to form at least a portion of an implantable pouch or other device such that the epithelial basement membrane sides face both outward and inward toward a cavity of the device; and

[0045] FIG. 8 illustrates an example of the medical graft assembly of this disclosure in which the lamina propria sides of the device face outwards.

DETAILED DESCRIPTION

[0046] In the description that follows, like components have the same reference numerals, regardless of whether they present in different examples. To illustrate examples in a clear and concise manner, the drawings may not necessarily illustrate scale and may show certain features in somewhat schematic form. Features described and/or illustrated with respect to one example may exist in the same way or in a similar way in one or more other examples and/or in combination with or instead of the features of the other examples.

[0047] As used in the specification and claims, for the purposes of describing and defining the invention, the terms “about” and “substantially” represent the inherent degree of uncertainty attributed to any quantitative comparison, value, measurement, or other representation. The terms “about” and “substantially” also represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. “Comprise,” “include,” “have” and variations of each word include the listed parts and can include additional parts not listed. “And/or” includes one or more of the listed parts and combinations of the listed parts. The terms “upper,” “lower,” “left,” “right,” and the like serve to clearly describe the disclosure but do not limit the structure, positioning and/or operation of the disclosure in any manner.

[0048] As used in the specification and claims, the term “laminate” describes both the process and the end result of two or more independent pieces of tissue bonding together. The term “lamination” refers to the adherence of a region of between two or more tissues. Lamination can occur during any process that employs a mechanical force during a drying process, or that includes compression of tissues and the subsequent drying of the tissues in the compressed state. In tissues comprising an ECM, lamination occurs by crushing the matrix structure of the ECM. This compression produces van der Waals forces between the tissue layers, causing the layers to stick together when dry. Lamination produces a directed area of connection between the tissues that would not occur unless intentionally created.

[0049] As used in the specification and claims, “vacuum pressing” consists of compressing hydrated, remoldable material while subjecting the material to a vacuum. Tissue compressed by vacuum pressing generally has a higher tensile strength and lower strain value compared to tissue compressed by other methods because of its more compressed matrix structure.

[0050] As used in the specification and claims, “lyophilization” consists of drying tissue by sublimation, a process of changing ice crystals from a solid directly to a gas without passing through an intermediate liquid phase. During lyophilization, a vacuum applied to frozen tissue at low temperatures causes the ice crystals to sublimate from the frozen tissue, leaving behind small pockets of open space formerly occupied by the ice. Hence, the resultant dried tissue retains its native pore structure, and has a more open matrix structure compared to tissue dried by other methods due to pores created in the frozen tissue after extraction of the ice crystals and lack of compressive forces. Surgeons often use lyophilized devices for wound management since their open matrix structure results in a faster resorption time and their open spaces allow for increased integration and interaction with local cells and tissue.

[0051] FIG. 1A shows an example of an implantable pouch device 100 of this disclosure made from a hydrated first sheet 102 and a hydrated second sheet 104 of bioresorbable material. The bioresorbable material comprises an extracellular matrix (ECM). Examples of the ECM material may be synthetic (for example, synthetic collagen) or naturally-occurring (for example, submucosa of the intestine (SIS), dermis or liver) having a luminal side and an opposite epithelial basement membrane side. Some non-limiting examples of naturally-occurring ECM may include the urinary bladder matrix (UBM) described in U.S. Patent No. 6,576,265, incorporated herein by reference. Non-limiting examples of preparing solid sheets and strips of ECM material may include the methods described in U.S. Patent No. 5,711,969, incorporated herein by reference. Each of the first sheet 102 and the second sheet 104 may contain one or more layers of the ECM. The first sheet 102 and the second sheet 104 may define fenestrations or through holes 114 for allowing passage of bodily fluids, advantageously reducing the risk fluid build-up. The through holes 114 may also allow for the passage of connecting conduits, wires, tubes, or the like. Each of the first sheet 102 and the second sheet 104 have a corresponding top edge 106, bottom edge 108, left side edge 110 and right side edge 112, with a cavity 116 (shown more clearly in FIG. 1G) formed between the first sheet 102 and the second sheet 104. The cavity 116 is sized and shaped for receipt of a CIED or other medical device (not shown). In alternative examples, the pouch device 100 comprises only a first sheet 102 folded into equal halves such that the folded edge forms the bottom edge 108.

[0052] FIG. IB illustrates a method of forming the implantable pouch device 100. The first sheet 102 is placed over the second sheet 104, preferably with the luminal sides facing inward towards the cavity 116 and the epithelial basement membrane sides facing outwards to minimize adherence to surrounding tissue. A biocompatible, non-stick insert 118 placed between the first sheet 102 and the second sheet 104. Examples of the non-stick insert 118 can be made from polytetrafluoro-ethylene (PTFE), non-stick foil, parchment paper, or any suitable material that allows liquid and gas to pass through the insert 118. The corresponding bottom edges 108 and side edges 110, 112 of the first and second sheets 102, 104 are pressed together (for example, by vacuum pressing or by compression followed by lyophilization) until dry to laminate the edges 108, 110, 112 together without the need for sealant, staples, stitches or the like. Removing the non-stick insert 118 creates the cavity 116. The pouch device 100 is trimmed to its final shape and the through holes 114 are created (for example, with a biopsy punch) to complete the final steps.

[0053] As shown in FIGS. 1C-1E, examples of the pouch device 100 can include different pouch shapes, sizes and through hole patterns.

[0054] As shown in FIGS. 1F-1H, the first sheet 102 and the second sheet 104 of the implantable pouch device 100 may have regions of varying density. For example, the top, bottom and side edges 106, 108, 110, 112 may have a more compressed matrix structure than the center 120 of the implantable pouch device 100, which may have a more open matrix structure. Further, the bottom and side edges 108, 110, 112 may have a more compressed matrix structure than the top edge 106. Advantageously, the regions of varying density allow for a more robust matrix structure at the edges 106, 108, 110, 112 of the pouch device 100, helping to prevent damage and wear around its periphery.

[0055] FIG. 2A shows another example of an implantable pouch device 200 of this disclosure made from at least one hydrated sheet 202 of bioresorbable material. In examples, the sheet 202 comprises an ECM, and has a luminal side and an opposite epithelial basement membrane side. As shown in FIG. 2B, the sheet 202 may have regions of varying density. In the example of FIG. 2A, the sheet 202 is folded into a three-dimensional structure, forming a cavity 216 between the folded sides of the sheet 202 for receipt of a CIED or other medical device 500. Preferably, the luminal side of the sheet 202 faces inward toward the cavity 216 and the epithelial basement membrane side of the sheet 202 faces outwards. Drying of the sheet 202 may occur by freeze drying (lyophilization) (FIG. 2C), vacuum pressing (FIG. 2D), or combinations of the two (FIG. 2B), which may occur simultaneously. Sheets 202 prepared by a combination of freeze drying and vacuum pressing have a more open matrix structure throughout the majority of the sheet 202 but more compressed edges. The sheet 202 can be cut using a die or guide that produces the desired flat “teddy bear” configuration, as further described below.

[0056] Non-compressed regions have a preserved matrix structure, which is a more open matrix structure and has a lower density, than the matrix structure of compressed regions. Compressed regions have a more collapsed matrix structure, and also a higher density, than the matrix structure of the non-compressed regions. Compressed regions also have a more collapsed matrix structure than the native matrix structure of the tissue before it is processed. The tissue matrix structure is generally maintained or preserved after the tissue is lyophilized. The process of lyophilization maintains the native matrix structure. In other words, the original porosity and thickness of the tissue is preserved in areas that are uncompressed. The preservation or compression of the tissue matrix can be assessed by scanned electron microscope (SEM) imaging.

[0057] Advantageously, adjustments to the size of the cut sheet 202 can produce a pouch device 200 to accommodate CIEDs 500 of any size. The geometry of the cut sheet 202 derives from calculations based on the actual measurements of the CIED 500. This equation-driven design allows the pouch device 200 to work for many different-sized CIEDs 500 to produce a geometrically proportional configuration based on a variety of inputs. FIG. 2E, as well as Table 1, illustrate a non-limiting example of dimensions, inputs and equations based on a pace maker having a length of 1.75 cm, a width of 1.75 cm, and a thickness of 0.3 cm. Table 1: Description of Input Variables for Flat Device Configuration Geometry

[0058] FIGS. 2F-K illustrate a method of forming the implantable pouch device 200 from the cut sheet 202. As shown in FIG. 2F, the cut sheet 202 includes a body flap portion 222 having a lower region 222a and an upper region 222b, a left arm flap 224, and a right arm flap 226. A bottom edge of the lower region 222a further defines a left alignment hole 228 and a right alignment hole 230, while a top edge of the upper region 222b defines a left alignment hole 232 and a right alignment hole 234. A top edge of the left arm flap 224 defines a single alignment hole 236, and a top edge of the right arm flap 226 defines a single alignment hole 238. In examples of the sheet 202, such as the sheet 202 of FIG. 2B, a center of the lower region 222a, the upper region 222b, the left arm flap 224 and the right arm flap 226 away from the edges may form a slight pocket or depression in the sheet 202.

[0059] Turning now to FIG. 2G, the pouch device 200 is formed by placing the CIED (not shown) on the upper region 222b of the body flap portion 222. Alternatively, the CIED is inserted into the pouch device 200 after completely folding the sheet 202. Fold lines 240 can be marked around the upper region 222b, as shown, or the sheet 202 can be folded without the use of fold lines 240. As shown in FIG. 2H, the lower region 222a is then folded up and over the upper region 222b so that the left alignment hole 228 aligns with the left alignment hole 232, and the right alignment hole 230 aligns with the right alignment hole 234. Notably, the folds will leave some clearance in a clearance region 242 to allow for the passage of bodily fluids, wires, etc. Next, as shown in FIG. 21, the right arm flap 226 is folded over the folded lower region 222a, aligning the alignment hole 238 with the left alignment holes 228, 232. As shown in FIG. 2 J, the left arm flap 224 is then folded over the folded lower region 222a, aligning the alignment hole 236 with the right alignment holes 230, 234. Alternatively, the right arm flap 226 and the left arm flap 224 can be folded over lower region 222a in the reverse order. As shown in FIG. 2K, completion of the folds creates the cavity 216 for receipt of the CIED. A flexible member, such as a suture (not shown), can then be threaded through the alignment holes 228, 230, 232, 234, 236, 238 in the pouch device 200 to close the cavity 216 and/or to attach the pouch device 200 to the heart tissue during surgery.

[0060] FIG. 3A shows another example of an implantable pouch device 300 of this disclosure made from at least a first hydrated sheet 302. Optionally, the implantable pouch device 300 can be made with additionally a second hydrated sheet 304 of bioresorbable material, and optionally one or more additional sheets of bioresorbable material. In examples, each of the first sheet 302 and the second sheet 304 comprises an ECM, and has a luminal side and an opposite epithelial basement membrane side. The sheets 302, 304 may have regions of varying density. In the example of FIG. 3 A, the pouch device 300 has a tubular shape, preferably with an open first end 300a and a closed second end 300b, forming a cavity 316 for receipt of a CIED or other medical device (not shown). However, either of the first end 300a or the second end 300b may be open or closed. Advantages of the pouch device 300 include ease of production of various sizes in large quantities, reduced labor time, and a shape unspecific to any one medical device, allowing for flexibility in use.

[0061] FIGS. 3B-D illustrate an example of a method of forming the implantable pouch device 300. Initially, the first sheet 302 is cut to the desired size for wrapping. The first sheet 302 is then laid on a flat surface, preferably with the epithelial basement membrane side facing down and the luminal side facing up. As shown in FIG. 3B, the luminal side of the first sheet 302 is then wrapped around a cylindrical mandrel 344 comprised of any rigid material (for example, a steel rod) covered by a flexible outer tube (for example, silicone), which prevents the first sheet 302 from sticking to the mandrel 344. A diameter of the mandrel 344 is selected to be slightly larger than a desired diameter of the cavity 316 (i.e., slightly larger than the CIED). A diameter of the outer tube (not shown) is selected to be slightly larger than the diameter of the mandrel 344 so that the outer tube easily inserts over the mandrel 344. The mandrel 344 with the outer tube is laid at the edge of the first sheet 302, and the edge of the first sheet 302 is placed on top of the mandrel 344. The mandrel 344, with the first sheet 302 attached, is rolled at a minimum of one complete turn until a desired thickness is achieved, so that the first sheet 302 connects with the starting end to form the tubular shape (FIG. 3C). [0062] As shown in FIG. 3D, in examples, the second sheet 304 wraps around an end of the mandrel 344 to create the closed second end 300b of the pouch device 300. Preferably, the pouch device 300 is then dried (for example, air dried) to laminate the overlapping edges of the first and second sheets 302, 304. Once the pouch device 300 is dried, the outer tube is removed, creating a clearance region between the pouch device 300 and the mandrel 344. The mandrel 344 is then removed and the pouch device 300 can be cut to a desired length. The surgeon can then place the CIED inside the pouch device 300 during or prior to implantation. The surgeon can also create one or more thread holes (not shown) in the desired areas of the pouch device 300 for passage of a suture prior to suturing the pouch device 300 into place.

[0063] In addition to encasing a CIED, examples of the pouch device 300 could also house an orthopedic device (such as a joint replacement) or serve as a single or multi-channel nerve guide - i.e., an open-ended tubular structure having a single or multiple inner tubes. The multi-channel nerve guide offers increased surface area (compared to the single channel conduit) upon which nerve cells and/or nerve “support” cells can attach to regenerate. FIGS. 4A and 4B show another example of an implantable pouch device 400 of this disclosure made from at least one hydrated sheet 402 of bioresorbable material. In examples, the sheet 402 comprises an ECM, and has a luminal side and an opposite epithelial basement membrane side. The sheet 402 may have regions of varying density. In examples, the sheet 402 can be cut using a die or guide that produces the desired shape.

[0064] In the examples of FIGS. 4A and 4B, the sheet 402 has a substantially round shape to accommodate a generally round or oval -shaped CIED 500. A periphery of the sheet 402 defines a plurality of thread holes 446 for lacing of a flexible member 448 about the periphery. Examples of the flexible member 448 may include a suture, an elongated piece of UBM, or any biocompatible string-like structure. Tensioning of the flexible member 448 causes the flexible member 448 to cinch the periphery of the sheet 402 to form a cavity 416 between sides of the sheet 402 for receipt of the CIED or other medical device 500. One or more knots 450 tied in the flexible member 448 secure the pouch device 400 closed. The sheet 402 may define additional through holes 414 (FIG. 4B) for allowing passage of bodily fluids or connecting conduits, wires, tubes and the like.

[0065] FIGS. 4C and 4D illustrate a method of forming the implantable pouch device 400 from a cut sheet 402 with the flexible member 448 pre-laced through the plurality of thread holes 446 about the periphery of the sheet 402. Alternatively, the sheet 402 may be provided to the surgeon without the pre-laced flexible member 448 so that the surgeon may use a flexible member of choice. As shown in FIG. 4C, the surgeon preferably places the CIED or other medical device 500 on the luminal side of the sheet 402. As shown in FIG. 4D, the surgeon then applies tension to the flexible member 448 to cinch the sheet 402 into the pouch device 400. Advantageously, adjustments to the size of the cut sheet 402 can produce a pouch device 400 to accommodate CIEDs 500 of any size.

[0066] Further examples of the pouch device 400’ are shown in FIGS. 4E-4G. As shown in FIG. 4E, the sheet 402’ has a substantially square shape with rounded corners. A periphery of the sheet 402’ defines a plurality of thread holes 446’ for lacing of a flexible member 448’ about the periphery. Tensioning of the flexible member 448’ causes the flexible member 448’ to cinch the periphery of the sheet 402’ to form a cavity 416’ between sides of the sheet 402’ for receipt of the CIED 500 (FIG. 4F). One or more knots 450’ tied in the flexible member 448’ secure the pouch device 400’ closed (FIG. 4G). FIG. 4H shows another example of a sheet 402” having a substantially star shape. This configuration allows for less bunching or overlap of the sheet 402” following cinching of the flexible member 448”. The disclosure also contemplates other shapes of the sheet 402, not shown, including triangular or polygonal.

[0067] The first sheet 102 of bioresorbable material and second sheet 104 of bioresorbable material may each be a medical graft assembly as described below. In general, medical graft assemblies may comprise an ECM and having surfaces of similar properties facing outwards on both sides of the device.

[0068] As shown in FIG. 5A, to construct a single channel nerve guide 352, the first sheet 302 is laid on a flat surface with the epithelial basement membrane side facing down and the luminal side facing up. The mandrel (not shown), covered with the outer tube, is placed at the end of the sheet 302 and rolled for the desired number of turns to form the single channel nerve guide 352. [0069] FIGS. 5B and 5C show cross-sections of an example of a three-channel nerve guide and FIGS. 5D and 5E show cross-sections of examples of five-channel nerve guides. Each channel of the 5-channel nerve guide may be approximately 1 mm in diameter, and the overall device may have a diameter of about 2.5 mm. The final conduit is rigid when dry, and preserves structural integrity after potential hydration. [0070] To form the multi-channel nerve guide 354, another mandrel (not shown) is placed on another sheet adjacent to the first mandrel, and again rolled for the desired number of turns. This process is repeated with additional sheets and additional mandrels until the desired number of channels 356 is formed (for example, three as shown). Once the desired number of channels 356 is formed, another sheet 358 is wrapped around all of the mandrels to form a conduit, which is then dried. After drying, the mandrels and the outer tubes are removed to form the multi-channel nerve guide 354.

[0071] A nerve guide means a channeled conduit comprising ECM tissue intended to aid in nerve regeneration. Nerve guides of the present disclosure comprises peeled and thin ECM sheets that are rolled around minimally one mandrel that may be made of a Teflon, similar material or other suitable material to reduce tissue adherence. The final product may have single or mutli-channels, such as up to 5-channel forms. Multi-channel nerve guides may have 2, 3, 4, 5, 6 or more channels. Multi-channel ECM nerve conduit is intended to aid in peripheral nerve regeneration by acting as a scaffold for nerve growth.

[0072] Degeneration of the peripheral nervous system such as neuropathy, disease, and trauma can result in loss of motor control and/or sensation. The current gold standard for peripheral nerve regeneration is accomplished through the autograft procedure and it does have a high success rate. However, nerve autografting procedures require the damage of an alternative nerve in order to repair the damage nerve. The lack of options that would not require autografting nerves has led to the development of a nerve conduit made of an extracellular matrix, specifically comprised of Urinary Bladder Matrix (UBM). By eliminating autografting, nerve conduits made of a biodegradable material may eliminate donor site morbidity and increased operative time associated with nerve autografting. Additionally, the donor does not have the availability of expendable donor nerves. By having another option from autografting made of a biocompatible material that degrades over time, this would solve a lot of the issues with nerve autografting. The UBM serves as a scaffold to augment sensory nerve growth. It is hypothesized that by providing a larger surface area as a scaffold for nerve growth the results could be enhanced.

[0073] Methods of making multi-channel nerve guides are described in more detail as examples. Non-sterile sheets of urinary bladder matrix (UBM) are cut into 5 cm x 10 cm rectangle sheets. Sheets are laid on a flat surface with the luminal side down and basement membrane side facing up. A mandrel is placed on the bottom edge of the sheet, about 2-3mm from the edge. The mandrel could be a steel rod (1/64” diameter) covered with silicone tubing (0.02” inner diameter, 0.037” outer diameter) or ETFE tubing (1/16” outer diameter). The mandrel should be comprised of a rigid material that will allow the UBM to form into a cylindrical shape but should also be covered by or have a coating that the UBM will not permanently stick to. The bottom edge of the sheet is placed over the mandrel about halfway around with forceps and then that mandrel is rolled with UBM for 2.5 turns. Then, a second mandrel is placed on the UBM next to the rolled mandrel and again rolled with the UBM for 2.5 turns. This is repeated until the desired amount of channels is formed (as many as desired, more than 2). After the last mandrel, the UBM is wrapped around all mandrels to form the complete conduit and to ensure that the cylindrical conduit will all stay together. The conduit is left to air dry overnight. After, the mandrels are pulled from the conduit, which results in the channels created inside the UBM conduit. The conduit should be cut to the desired length (i.e. the size of the gap of a degenerative nerve).

[0074] FIG. 6 shows an example of a medical graft assembly 10 of this disclosure made from at least a first sheet 12 and a second sheet 14 of a bioresorbable ECM (for example, submucosa of the intestine (SIS), dermis, or liver), each sheet having an EBM side 16 and an opposite LP side 18. Some non-limiting examples of the ECM may include the urinary bladder matrix (UBM) described in U.S. Patent No. 6,576,265, incorporated herein by reference. Non-limiting examples of preparing solid sheets and strips of ECM material may include the methods described in U.S. Patent No. 5,711,969, incorporated herein by reference. In examples, the EBM side 16 of the first and second sheets 12, 14 comprises a mammalian epithelial basement membrane free of epithelial cells and cellular elements of the epithelial cells. In examples, the first sheet 12 may have a density and resorption rate that differs from the density and resorption rate of the second sheet 14. In examples, the medical graft assembly 10 is in the form of a surgical graft or a wound dressing device.

[0075] As shown in FIG. 6, examples of the first and second sheets 12, 14 of the medical graft assembly 10 are layered one on top of the other such that the EBM sides 16 of the device face outwards and the LP sides 18 face inward. Advantageously, a surgeon may use this “EBM-out” medical graft assembly 10 for reinforcement of soft tissue between tissue layers to separate tissue from a foreign body or nearby organs where attachments may have a negative impact. Alternatively, a surgeon may use the medical graft assembly 10 in applications where tissue ingrowth needs to be delayed, such as in traumatic muscular injuries at the interface between distinct muscular groups, articulating surfaces, and/or surrounding device implants. The medical graft assembly 10 may also have added utility in current indications such as the intraperitoneal onlay match (IPOM) device placement and anastomotic wraps, where a surgeon desires mitigation of attachment to surrounding tissue. In addition, the medical graft assembly 10 may have benefit for new indications, such as a repair intending to maintain separation of skeletal muscle tissue layers.

[0076] As shown in the EXAMPLE below, in cases where the ECM is selected from UBM, the percentage of tissue attachment of the EBM side 16 to the surrounding tissue may be about 0% after 14 days of implantation and between about 0% and 33% after 90 days of implantation. [0077] In examples of making the medical graft assembly 10, hydrated first and second sheets 12, 14 layer one on top of the other such that the EBM sides 16 of the medical graft assembly 10 face outwards and the LP sides 18 face inward. Subsequent compression and drying of the medical graft assembly 10 causes the first and second sheets 12, 14 to laminate together. For example, a mechanical force in the form of vacuum pressing, clamping, stitching, crushing or other suitable means applies consistent pressure across the surfaces of the first and second sheets 12, 14 to compress the first and sheets 12, 14 together. Drying may occur by placing the hydrated medical graft assembly 10 in a sealed bag (such as a vacuum bag) and vacuum pressing on a vacuum pressing system. In other examples, drying may occur by air drying, or by lyophilizing the medical graft assembly 10. Drying causes the compressed regions of the sheets to laminate together. In some examples, compression and freeze drying of the various regions of the medical graft assembly 10 may occur simultaneously.

[0078] As shown in FIG. 7, two medical graft assemblies can be arranged to define a cavity 30 of an implantable pouch device therebetween for insertion of a medical device, such as a pacemaker (not shown). In this case, multiple first and second sheets 12, 14 layer such that the EBM sides 16 face both outward and inward toward the cavity 30. This configuration advantageously minimizes attachment to both of the medical device and the surrounding tissue. [0079] FIG. 8 shows an example of a medical graft assembly 20 of this disclosure made from at least a first sheet 22 and a second sheet 24 of a bioresorbable ECM having an EBM side 26 and an opposite LP side 28. In examples, the EBM side 26 of at least one of the first and second sheets 22, 24 comprises a mammalian epithelial basement membrane free of epithelial cells and cellular elements of the epithelial cells. In examples, the medical graft assembly 20 may be in the form of a tissue graft or a wound dressing device.

[0080] As shown in FIG. 8, the first and second sheets 22, 24 of the device are layered one on top of the other such that the LP sides 28 of the medical graft assembly 20 face outwards and the EBM sides 26 face inward. Subsequent compression and drying of the medical graft assembly 20, as described above with regard to the medical graft assembly 10, causes the first and second sheets 22, 24 to laminate together. Advantageously, a surgeon may use this “LP-ouf ’ medical graft assembly 20 for reinforcement of soft tissues or treatment of wounds where the surgeon desires to promote tissue attachment or ingrowth between tissue layers, such as between a wound bed and a skin graft or within a skeletal muscle defect. A surgeon may also use the medical graft assembly 20 for reinforcement of soft tissue in the retrorectus space (abdominal hernia repair) or where seroma might form, which can be mitigated by the medical graft assembly 20 creating tissue attachments or natural “tacking.”

[0081] EXAMPLE

[0082] The purpose of this GLP cecal abrasion study was to evaluate the performance of EBM Surgical Matrix devices in a rabbit model of intraperitoneal tissue attachments. This rabbit model utilized excision of the peritoneum around a midline incision, as well as abrasion of the cecum, to induce the formation of intraabdominal tissue attachments between the bowel and the peritoneum, as described in Kim et al. Efficacy and safety of hyaluronate membrane in the rabbit cecum-abdominal wall adhesion model. 2013. 85:51-57. Test articles for GLP the study are listed in Table 2 below. These devices were off-the-shelf finished product offered by ACell, Inc.

Table 2: Test Articles for GLP Cecal Abrasion Study

[0083] GENTRIX® Surgical Matrix Thin, GENTRIX® Surgical Matrix, and GENTRIX® Surgical Matrix Plus were placed against the peritoneum to reinforce a midline defect repair. The device was positioned such that the basement membrane side of the device was facing the viscera. At time points of 14 and 90 days, the materials were assessed for the extent of tissue attachments between the device and the surrounding viscera. As shown in Table 3 below, the study indicated that ECM material oriented with the epithelial basement membrane side facing the tissue of interest (viscera in this case) minimized tissue attachment when placed into the peritoneum (body cavity) of a rabbit model.

Table 3. Percentage of Animals with Tissue Attachments

[0084] One skilled in the art will realize the disclosure may embody other specific forms without departing from the spirit or essential characteristics thereof. The foregoing examples in all respects illustrate rather than limit the disclosure described herein. The appended claims, rather than the foregoing description, thus indicate the scope of the disclosure, and embrace all changes that come within the meaning and range of equivalency of the claims.

Claims

1. An implantable pouch for encasing a medical device, the pouch device comprising: at least a first and a second bioresorbable sheet of material comprising an extracellular matrix (ECM), each of the at least first and second bioresorbable sheets having corresponding top, bottom, and side edges; wherein the corresponding bottom and side edges of the at least first and second bioresorbable sheets are laminated together, forming a cavity between the at least first and second bioresorbable sheets for receipt of a medical device.

2. The implantable pouch device of claim 1, further comprising a plurality of through holes defined by the at least first and second bioresorbable sheets for allowing passage of bodily fluids and/or wires.

3. The implantable pouch device of claim 1, wherein at least one of the corresponding top, bottom and side edges of the at least first and second bioresorbable sheets comprise a denser matrix structure than a remainder of the at least first and second bioresorbable sheets.

4. The implantable pouch device of claim 1, wherein at least one of the at least a first and a second bioresorbable sheet of material is a medical graft assembly comprising: at least a first and second sheet of extracellular matrix (ECM) material having an epithelial membrane side and an opposing lamina propria side; wherein the at least first and second sheets of ECM material are layered such that the epithelial basement membrane sides face outward and the lamina propria sides face inward.

5. The implantable pouch device of claim 1, wherein the ECM is selected from the group consisting of: a urinary bladder matrix (UBM), submucosa of the intestine (SIS), liver or dermis.

6. The implantable pouch device of claim 4, wherein the epithelial basement membrane sides of the at least first and second sheets comprise mammalian epithelial basement membrane free of epithelial cells and cellular elements of the epithelial cells.

23

7. The implantable pouch device of claim 4, comprising two medical graft assemblies laminated together along at least a portion of the edges.

8. The implantable pouch device of claim 7, wherein the lamination is performed by vacuum pressing; and wherein the implantable pouch device does not comprise or require any suture or other fixation means.

9. The implantable pouch device of claim 7, wherein at least one of the medical graft assemblies comprises two UBM sheets; and wherein epithelial basement membrane sides of the UBM sheets face both outward from the implantable pouch device and inward toward the cavity.

10. A method of making an implantable pouch device for encasing a medical device, the method comprising: providing at least a first and a second hydrated bioresorbable sheet of material comprising an extracellular matrix (ECM), each of the at least first and second bioresorbable sheets having corresponding top, bottom, and side edges; placing a non-stick insert between the at least first and second bioresorbable sheets; laminating together the corresponding bottom and side edges of the pair of bioresorbable sheets, forming a cavity about the non-stick insert; drying the at least first and second bioresorbable sheets; and removing the non-stick insert from the cavity.

11. The method of claim 7, wherein each of the at least a first and a second bioresorbable sheet of material is a medical graft assembly comprising at least a first and second sheet of extracellular matrix (ECM) material having an epithelial membrane side and an opposing lamina propria side, and wherein the at least first and second sheets of ECM material are layered such that the epithelial basement membrane sides face outward and the lamina propria sides face inward; the method further comprising the steps of making the medical graft assembly: providing at least a first hydrated sheet of ECM material having an epithelial basement membrane side and an opposing lamina propria side; providing at least a second hydrated sheet of ECM material having an epithelial basement membrane side and an opposing lamina propria side; layering the at least first sheet and second sheets of ECM material such that either the epithelial basement membrane sides face outward and the lamina propria sides face inward, or the lamina propria sides face outward and the epithelial basement membrane sides face inward; compressing regions of the at least first and second sheets together; and drying the at least first and second sheets of the ECM material to form the device.

12. The method of claim 7, wherein the ECM is selected from the group consisting of: a urinary bladder matrix (UBM), submucosa of the intestine (SIS), dermis and liver.

13. The method of claim 8, wherein compressing the regions of the at least first and second sheets together and drying the at least first and second sheets occurs simultaneously.

14. The method of claim 8, wherein compressing the at least first and second sheets comprises one or more of vacuum pressing, lyophilizing, clamping, crushing or stitching.

15. The method of claim 7, wherein drying the at least first and second sheets comprises one or more of vacuum pressing, lyophilizing or air drying.