WO2015120405A1 - Patterned tissue membranes and methods and system for their preparation - Google Patents

Abstract

The present disclosure provides tissue membranes that have a gridded pattern on their surface and methods for their preparation and use. The patterned tissue membranes are useful for aiding repair of living tissue, including for use as allograft tissue for aid in tissue repair. The patterned tissue membranes include human amnion tissue membrane. A related drying system for preparation of the tissue membranes is also provided.

Description

PATTERNED TISSUE MEMBRANES AND METHODS AND SYSTEM FOR THEIR

PREPARATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States provisional patent application no.

61/936,900 titled Methods for Preparing Tissue Membranes filed on February 7, 2014, and to United States provisional patent application no. 62/062,676 titled Gaseous Drying Assembly for Biological Samples filed on October 10, 2014, the contents of both applications are incorporated by reference herein.

TECHNICAL FIELD

[0002] The present disclosure relates to patterned tissue membranes for aiding repair of soft living tissue. Methods and a system for preparing the patterned tissue membranes are described.

BACKGROUND

[0003] Tissue membranes such as collagen membranes are used for many purposes, including medical and health related purposes. In some instances, drying the collagen membranes is desirable to achieve stable preservation of the membrane at ambient temperature prior to its use in clinical or research applications.

[0004] Current methods for removing moisture from collagen tissues, including allograft tissue, range from use of heat convection and use of lyophilization. However, there is a need for improved methods that allow for better maintenance of the integrity of the collagen membrane, such as preservation of extracellular matrix proteins and retained bioavailability of growth factors. Thus, improved methods and systems for drying collagen membranes are desirable. The present disclosure provides such an improved system and methods.

SUMMARY

[0005] This summary is provided to introduce in a simplified form concepts that are further described in the following detailed descriptions. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it to be construed as limiting the scope of the claimed subject matter.

[0006] According to at least one embodiment, a patterned tissue membrane is provided, the patterned tissue membrane including: a top surface and a bottom surface; a gridded pattern on each of the top and bottom surfaces; and a residual moisture content based on gram weight of about 6% or below.

[0007] According to at least one embodiment, a patterned tissue membrane is provided, the patterned tissue membrane produced by a process including: supporting a tissue membrane on a sample holding member defining a gridded top surface, wherein the sample holding member is elevated from a bottom of an enclosure in the presence of a forced airflow generated by an inert gas, for a length of time sufficient to reduce the moisture content of the tissue membrane below a predetermined threshold and in which a pattern of the gridded top surface is generated on the tissue membrane.

[0008] According to at least one embodiment, a method for preparing patterned tissue membrane is provided. The method includes supporting a tissue membrane on a sample holding member defining a gridded top surface, wherein the sample holding member is elevated from a bottom of an enclosure in the presence of a forced airflow generated by an inert gas, for a length of time sufficient to reduce the moisture content of the tissue membrane below a predetermined threshold and in which a pattern of the gridded top surface is generated on the tissue membrane.

[0009] According to at least one embodiment, a method is provided for aiding repair of a soft tissue in a living body, the method including: hydrating a patterned tissue membrane having a top surface and a bottom surface, a gridded pattern on each of the top and bottom surfaces, and a residual moisture content based on gram weight of about 6% or below, with a solution suitable for repair of the soft tissue; and wrapping, placing on, embedding into, or filling the soft tissue of the living body in need of repair with the patterned tissue membrane, to aid in repair of the tissue.

[00010] According to at least one embodiment, a drying system is provided. The system includes a chamber defining a sealed enclosure, a spray member configured for translation about the sealed enclosure and spraying treatment fluids therein, and a sample holding member elevated from a bottom of the enclosure and defining a gridded top surface to which a sample rests on.

[00011] According to one or more embodiments, the system includes a fluid source in communication with the spray member.

[00012] According to one or more embodiments, the fluid source includes an inert gas.

[00013] According to one or more embodiments, the inert gas includes nitrogen gas.

[00014] According to one or more embodiments, the system includes a translation assembly that translates the spray member about the sealed enclosure. The translation assembly includes a threaded axle extending across the chamber and a support carried by the threaded rod.

[00015] According to one or more embodiments, the support is engaged with the spray member.

[00016] According to one or more embodiments, the support is further carried by a second axle extending across the enclosure.

[00017] According to one or more embodiments, a method of using the system described herein is provided. The method includes spraying fluids through the spray member and monitoring the moisture content of the sample.

[00018] According to one or more embodiments, the method includes applying a vacuum with a vacuum source to the chamber.

[00019] According to one or more embodiments, spraying fluids is carried out continuously.

[00020] According to one or more embodiments, spraying fluids is carried out in an on- off-on-off pattern.

[00021] According to one or more embodiments, the method includes monitoring the temperature of at least one of the enclosure or the sample.

[00022] According to one or more embodiments, spraying is carried out until the moisture content of the sample is under 6%.

BRIEF DESCRIPTION OF THE DRAWINGS

[00023] The previous summary and the following detailed descriptions are to be read in view of the drawings, which illustrate particular exemplary embodiments and features as briefly described below. The summary and detailed descriptions, however, are not limited to only those embodiments and features explicitly illustrated.

[00024] FIG. 1 is a front view of a drying system according to one or more embodiments disclosed herein. The image is shown with a front portion that encloses and seals the system removed for illustration purposes;

[00025] FIG. 2 is a system diagram of a drying system according to one or more embodiments disclosed herein;

[00026] FIG. 3 is a flowchart depicting one or more methods according to one or more embodiments disclosed herein;

[00027] FIG. 4 A and 4B are sequential views showing a fluid spraying member translating from one side of drying system enclosure to another side according to one or more embodiments disclosed herein;

[00028] FIG. 5 illustrates an end view of the translation assembly disclosed herein. The fluid spraying member is shown extending laterally across the image;

[00029] FIG. 6 illustrates an end view of the translation assembly, including a driver motor that provides translation to the threaded rod;

[00030] FIG. 7 illustrates an enlarged view of the screened top surface;

[00031] FIG. 8 is a view of the support, threaded rod, bushing, and second rod according to one or more embodiments disclosed herein;

[00032] FIG. 9 is a bottom view of the apertures on the spray member that allow flow through of fluids;

[00033] FIG. 10 is an illustration of an alternate embodiment of the drying system provided herein;

[00034] FIG. 11 is an illustration of an alternate screen assembly according to one or more embodiments disclosed herein; and

[00035] FIG. 12 is an illustration of a membrane made according to one or more methods disclosed herein.

DETAILED DESCRIPTIONS

[00036] These descriptions are presented with sufficient details to provide an

understanding of one or more particular embodiments of broader inventive subject matters. These descriptions expound upon and exemplify particular features of those particular embodiments without limiting the inventive subject matters to the explicitly described embodiments and features. Considerations in view of these descriptions will likely give rise to additional and similar embodiments and features without departing from the scope of the inventive subject matters. Although the term "step" may be expressly used or implied relating to features of processes or methods, no implication is made of any particular order or sequence among such expressed or implied steps unless an order or sequence is explicitly stated.

[00037] Any dimensions expressed or implied in the drawings and these descriptions are provided for exemplary purposes. Thus, not all embodiments within the scope of the drawings and these descriptions are made according to such exemplary dimensions. The drawings are not made necessarily to scale. Thus, not all embodiments within the scope of the drawings and these descriptions are made according to the apparent scale of the drawings with regard to relative dimensions in the drawings. However, for each drawing, at least one embodiment is made according to the apparent relative scale of the drawing.

[00038] The presently disclosed subject matter provides tissue membranes having a screen-like, screened, or gridded pattern on their surface, and a system and methods for their preparation and use. The terms "screen-like", "screened", and "gridded" are herein used interchangeably for the purposes of the specification and claims. The patterned tissue membranes can be useful for aiding the repair of living soft tissue, and can be useful as allograft tissue for aiding the repair of living soft tissue. The patterned tissue membranes can demonstrate enhanced durability and handling characteristics. The patterned tissue membranes can include human amnion tissue membrane.

[00039] In one embodiment, a method is provided for preparing patterned tissue membranes. The method includes supporting a tissue membrane on a sample holding member defining a gridded top surface, wherein the sample holding member is elevated from a bottom of an enclosure in the presence of a forced airflow generated by an inert gas, for a length of time sufficient to reduce the moisture content of the tissue membrane below a predetermined threshold and in which a pattern of the gridded top surface is generated on the tissue membrane.

[00040] In one embodiment, the presently disclosed subject matter provides a patterned tissue membrane, the patterned tissue membrane produced by a process that includes: supporting a tissue membrane on a sample holding member defining a gridded top surface, wherein the sample holding member is elevated from a bottom of an enclosure in the presence of a forced airflow generated by an inert gas, for a length of time sufficient to reduce the moisture content of the tissue membrane below a predetermined threshold and in which a pattern of the gridded top surface is generated on the tissue membrane.

[00041] The method for preparing the patterned tissue membranes can be carried out free of an application of heat. The method can be carried out at a room temperature. The method can be carried out at a temperature range of about 1 to about 10 degrees Celsius.

[00042] In the method for making patterned tissue membranes, the inert gas can include nitrogen gas.

[00043] In the method for making patterned tissue membranes, the tissue membrane can be a mammalian tissue membrane. The mammalian tissue membrane can be an amnion tissue membrane. The mammalian tissue membrane can be a human tissue membrane. The mammalian tissue membrane can be a human amnion tissue membrane.

[00044] In the method for making patterned tissue membranes, the pattern of the gridded top surface can include a honeycomb pattern or a circular pattern. In the method, the gridded top surface can define at least one portion that defines an area of increased thickness of a frame of the gridded top surface to impart a corresponding portion on the tissue membrane.

[00045] In the method for making patterned tissue membranes, the predetermined threshold for moisture reduction can be equal to or less than 6% residual moisture based on gram weight.

[00046] In one embodiment, a patterned tissue membrane is provided. The patterned tissue membrane includes: a top surface and a bottom surface; a gridded pattern on each of the top and bottom surfaces; and a residual moisture content based on gram weight of about 6% or below. The patterned tissue membrane can be a mammalian tissue membrane. The patterned tissue membrane can be an amnion tissue membrane. The patterned tissue membrane can be a human tissue membrane. The patterned tissue membrane can be a human amnion tissue membrane. The gridded pattern can include a honeycomb pattern or a circular pattern. The gridded pattern of the patterned tissue membrane can define at least one portion that defines an area of increased thickness of the gridded pattern to impart a corresponding portion on the tissue membrane.

[00047] In one embodiment, the presently disclosed subject matter provides a method for aiding repair of a soft tissue in a living body. The method includes: hydrating a patterned tissue membrane having a top surface and a bottom surface, a gridded pattern on each of the top and bottom surfaces, and a residual moisture content based on gram weight of about 6% or below with a solution suitable for repair of the soft tissue; and one of wrapping, placing on, embedding into, and filling the soft tissue of the living body in need of repair with the patterned tissue membrane to aid the repair of the tissue.

[00048] In the method for aiding repair of a soft tissue in a living body, the tissue membrane can be a mammalian tissue membrane. The mammalian tissue membrane can be an amnion tissue membrane. The mammalian tissue membrane can be a human tissue membrane. The mammalian tissue membrane can be a human amnion tissue membrane. The pattern on each of the top and bottom surfaces of the tissue membrane can include a honeycomb pattern or a circular pattern. The gridded pattern of the patterned tissue membrane can define at least one portion that defines an area of increased thickness of the gridded pattern to impart a

corresponding portion on the tissue membrane.

[00049] In the method for aiding repair of a tissue in a living body, the tissue of the living body can include epidermis, cornea, dura mater, tendon, ligament, fascia, nerve, vascular, organs, or bone. In the method for aiding repair of a tissue in a living body, the tissue of the living body can be a structural tissue.

[00050] FIG. 1 is a drying system 10. The system 10 includes a chamber 12 that defines a sealed enclosure 14. The chamber 12 may be constructed of any appropriately configured material and in any appropriately configured way. A spray member 16 is configured for translation about the sealed enclosure and spraying treatment fluids therein. As used herein, treatment fluids or fluids may include gaseous materials. The spray member 16 is in

communication with a fluid source 26. Fluid source 26 may include any appropriately configured fluid, including gaseous fluids such as nitrogen gas. [00051] A translation assembly 15 translates the spray member 16 about the sealed enclosure 14. As illustrated, this translation is generally linear in nature and may include side-to- side linear translation, but may take on any other appropriately configured translation. In this manner, spraying of gases or fluids can be done from a first position and a second position relative to the sample.

[00052] The translation assembly 15 includes a threaded axle 28 extending across the chamber and a support 18 carried by the threaded axle 28. The support 18 is engaged with the spray member 15. The support 18 is further carried by a second axle 24 extending across the enclosure 14. A motor 31 may provide rotational translation to the threaded rod 28.

[00053] A sample holding member 20 is elevated from a bottom of the enclosure 14 and defines a screened top surface 22 to which a sample rests on. By elevating the sample with the sample holding member 20 and providing the screened top surface 22, spray fluids may circulate beneath the sample and provide consistent drying across the entire surface sample.

[00054] Outflow valves 29A, 29B may be provided for cycling fluids within the chamber

12.

[00055] A system diagram is shown in FIG. 2 and generally designated 100. As illustrated, the enclosure 14 contains the sample. A control module 102 is in communication with a fluid source 104, vacuum 106, and translation device 108 that is in communication with the translation assembly 15. The vacuum 106 may be in communication with the outflow valve 29 A, 29B.

[00056] The control module 102 may be any appropriately configured computer and may be in communication with the components illustrated in FIG. 2. The control module 102 may be in wireless or wired communication with the drying system 10. As used herein, control module may be a computing system or may be an electronic or mechanical control system controlled by an operator.

[00057] A scale 110 may be provided inside of the enclosure 14 or outside of the enclosure

14 and is provided for measuring one of the sample or the enclosure containing the sample. A moisture detector 112 may be provided in fluid communication with the enclosure 14. The moisture detector 112 may be separate and may measure the moisture of the sample and not the fluid. While each of control module 102, fluid supply 104, vacuum 106, translator 108, scale 110, and moisture detector 112 are shown exterior of the enclosure 14, at least one of or all may be placed within the enclosure 14.

[00058] In operation, a sample is placed within the enclosure 14. Before being placed in the enclosure 14, the sample may be treated with one or more treatment steps such as cutting the sample to size, weighing the sample using scale 110, applying one or more chemicals or materials, or other pretreatments. In one or more embodiments, the sample may be any appropriately selected biological material such as allograft tissue.

[00059] One or more methods 300 are illustrated in the flow chart of FIG. 3. As illustrated, the method includes weighing the sample 302. This may be carried out with scale 110 and may be a human directed procedure or may be carried out and information recorded with the control module 102. Once the sample is placed within the enclosure 14, the control module 102 may then direct spraying of fluid 304. Simultaneously, the spray member 16 may be translated within the enclosure 14 in a linear fashion. This is illustrated sequentially in FIGS. IB, 1C, and ID. When the spray member 16 reaches an end of the enclosure 14, the translation mechanism 15 is configured to change the direction of rotation of axle 22 in order to impart translation of spray member 16 in the opposite direction. [00060] During the step of spraying fluids 304, the moisture content of the sample is measured. This may be carried out by the moisture detector 112 or a scale 110 may be used to determine the weight of the sample at any given time in order to determine the moisture content thereof.

[00061] The method 300 may optionally further include applying a vacuum with a vacuum source to the enclosure 306. The vacuum may be applied with vacuum 106 and may be provided for removing moisture from the enclosure 14, and additionally for removing cooled fluids from the enclosure 14. During the step of spraying fluid 104, the temperature inside of the enclosure 14 will drop and, in order to moderate temperature drops, the vacuum may remove cooled air. The step of applying vacuum 106 may be provided in a step relationship to spraying fluid or may be continuous or may be in response to a measurement by moisture detector 112. Applying vacuum 306 may be a human directed action or may be controlled by control module 102.

[00062] Fluid is sprayed 304 until the moisture content is below a predetermined threshold

308. In the flowchart of method 300 in FIG. 3, step 308 has a predetermined moisture content of less than 6%, though this ratio is indicative of only one possible predetermined content. When the moisture content is below the predetermined threshold 308, the process is complete 310. If the moisture content is not below the predetermined threshold 308, then the method 300 restarts at any of steps 302, 304, and 306. Determining whether the method 300 restarts at any step may be based on one or more monitored characteristics. For example, if it is determined that the sample temperature is lower than desired, the control module 102 may direct spraying fluid 304 as opposed to vacuuming 306 that will likely cause further drop in temperature. Additionally, particularly after going through a first complete spraying, vacuuming, and measuring step, only spraying fluid 304 may be carried out without vacuum. In these embodiments, fluid is sprayed and moisture is continuously monitored.

[00063] Spraying fluids 304 may be carried out continuously or in an on-off-on-off pattern. Additionally, the pressure and volume of flow during spraying fluids 304 may be altered according to one or more determined characteristics. For example, the control module 102 may detect that the drying rate of the sample is too fast for optimal performance and may reduce either the pressure or volume of spraying fluids. Alternatively, if the control module 102 detects that the drying rate of the sample is too slow for optimal performance, the control module 102 may increase the pressure or volume of spraying fluids or stop spray all-together for a period of time.

[00064] The temperature of the sample may be monitored within the enclosure 14.

Alternatively, the temperature of the enclosure 14 may be monitored. During spraying of fluids 304, the temperature within the enclosure 14 will tend to drop due to evaporation of moisture in the sample. In order to maintain the temperature of the sample at some amount to where the integrity of the sample is not compromised, the sample or the enclosure may have heating applied to it.

[00065] The control module 102 may be configured for monitoring and directing the various components necessary to carry out the one or more steps and methods shown in the flowchart of FIG. 3. For example, the control module 102 may be configured to direct the spray member 16 as already described. The control module 102 may be configured to vacuum 306, detect moisture 308, and all of the remaining steps shown in the flowchart of FIG. 3.

[00066] In one or more embodiments, a desiccant may be provided within chamber 12.

[00067] The system is shown where the spray member 16 is translating from one side of the enclosure 14 (FIG. 4A) to the other side of the enclosure 14 (FIG. 4B). The translation assembly 15 is configured such that when the spray member 16 reaches an end of the enclosure 14, the translation assembly 15 then reverses direction until the spray member 16 reaches the other end of the enclosure 14 such that the spray member 16 translates in a zig-zag side-to-side motion within the enclosure 14.

[00068] FIG. 5 illustrates an end view of the translation assembly 15 disclosed herein.

The fluid spraying member 16 is shown extending laterally across the image.

[00069] FIG. 6 illustrates an end view of the translation assembly 15, including a driver motor 31 that provides translation to the threaded rod 28. The driver motor 31 may provide on- off same speed rotation or may vary the speed of translation depending on one or more monitored characteristics such as weight, temperature, humidity, and the like.

[00070] FIG. 7 illustrates an end view of the translation assembly 15 and a larger view of the chamber 12.

[00071] FIG. 8 is an overhead view of the fluid spray member 16 and FIG. 9 is a bottom, upward facing view. The spray member 16 includes one or more apertures 42 that are in fluid communication with the fluid source 24 to apply fluids into the sealed enclosure 14. In one or more embodiments, the apertures are defined on a top-facing surface of the spray member 16 in order to spray fluids upwardly away from the sample. In one or more embodiments, the apertures are defined on a bottom- facing surface of the spray member 16 in order to spray fluids downwardly towards the sample. In one or more embodiments, the apertures may be defined on a top and bottom surface of spray member 16, or extending circumferentially around the surface of the spray member 16.

[00072] FIG. 10 illustrates an alternate embodiment of system 10 where a second spraying apparatus 116 is carried by a support 118. A spray member 116 is configured for translation about the sealed enclosure and spraying treatment fluids therein. In this manner, fluids and gases can be sprayed from both an upper and lower position relative to the sample.

[00073] FIG. 11 illustrates an alternate sample holder 210. The sample holder 210 includes a lower portion 216 hinged to an upper portion 220 by hinge 222. A grid pattern 212 is defined on the sample holder 210. The grid pattern 212 further defines areas of increased thickness or other structural property or the like that are provided for making scoring or cutting lines on the sample as illustrated in FIG. 12. As used herein, structural properties may be wiring or grid portions that have increased thickness, rigidity, depth, and the like. As illustrated, the sample may include grid 240 with intersecting scoring or cutting lines 242. In this manner, this allows a user to cut along the scoring or cutting lines 242 to select a desired size of a membrane. For example, if a user desires a 3 centimeter by 4 centimeter section, a scoring line 242 may allow for separation of the sample in such an arrangement. Alternatively, a template may be overlaid a dried sample for determining a manner to cut or otherwise separate.

EXAMPLES

[00074] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. Example 1

Preparation of Dry Human Amnion Membrane

[00075] The following experiments describe the generation of an improved method for drying tissue for use as an allograph.

[00076] Preparation for Drying: The membrane used in this experiment was isolated from fresh human chorion tissue. Prior to the drying process, the membrane was cleansed of residual debris through a cycle of isotonic buffered rinses and manual massaging in which the fibroblast layer was retained. The appearance of the membrane after the rinses and massaging was very translucent.

[00077] Desiccator. To facilitate the membrane drying process, a desiccator was purchased from BEL- ART SCIENCEWARE made from polycarbonate, model number 424002021 (Lab Companion Round Style Vacuum Desiccator).

[00078] Desiccation using Vacuum Suction; Trial 1. Initially, the amnion membrane was placed into a petri dish inside of the desiccator and a vacuum suction pump (GOMCO 789 Suction Unit) with pressure set at 10 PSI was used to create negative pressure inside the desiccator. With this procedure, the drying did not appear to be very effective. For this experiment, the chamber pressure observed was -0.04Mpa and the pressure was applied at 10- minute increments. The weights of the membranes initially and after the drying procedure were measured on a TREE PRECISION Balance Model: HRB903 200g capacity, 0.00 lg divisions, and are shown in Table I below. It was noticeable in this experiment that the underside of the membrane did not dry due to sticking of the membrane to the petri dish which prevented airflow underneath the membrane. Table I.

[00079] Desiccation using Nitrogen; Trial 2. Based on the results of Trial 1, a new experiment was performed in which an industrial grade Nitrogen gas (AIRGAS, INC.) was connected using 5 ' of clear tubing from a beveled valve on the Nitrogen tank regulator to a flow meter (CLEATECH, LLC) that had 2 beveled valves (in/out) to measure the flow of nitrogen. An additional 5 ' of tubing ran from the flow meter to the desiccator. A sample piece of the membrane was placed on the drying chamber bottom shelf of the desiccator in order to allow airflow to get underneath the membrane (there were approximately ½" holes in the bottom shelf of the desiccators). The membrane in this case was placed directly on the bottom shelf rather than inside a petri dish as in Trial 1.

[00080] The nitrogen gas flow was controlled by the flow meter and the 3 -way valve was set to allow for a small amount of gas in-flow and out- flow into and out of the desiccation device. After the first run, a 3/8" hole was drilled into the polycarbonate side of the desiccator and an additional 3 -way valve was added. The gauge on the top of the flow meter indicated a positive pressure although the particular gauge used did not indicate the degree of positive pressure. While the results of this Trial were an improvement over Trial 1, it was believed that optimal drying had not been achieved after testing several batches (see the results and observations provided in Table II below). Table II.

[00081] While the membrane was dried effectively in this trial, the membrane had a frail handling characteristic.

[00082] Trial 3. After observing the varied outcomes of the drying and the failure to achieve a membrane which was both durable and dry on both sides, several conclusions were made. One conclusion was that the chamber likely created a homeostatic environment with laminar flow conditions. No matter if there was a positive air pressure or a negative air pressure in the desiccator, the airflow was not disruptive enough to effectively dry the membrane. In addition, there was insufficient exposure of the underside of the membrane to air. Thus, it was decided that a device that could provide a more disruptive and turbulent airflow as well as a screen in which to support the membrane might provide a solution.

[00083] In Trial 3, the membrane was transferred from isotonic phosphate-buffered saline onto a circular screen. The screen material used in this experiment was an aluminum wire screen purchased from LOWES hardware. The screen material was cut into a circular shape designed to fit inside the desiccator at varying angles ranging from flat on the desiccator floor shelf to about 45-degrees.

[00084] The membrane was spread out onto the screen such that the membrane was completely flat with few to no folds at the edges. Multiple experiments (Runs) were then performed as described in Trial 2 using the device with the screen inside the desiccator and the results are provided in Table III below. A visual and touch observation of the membrane was made at various time intervals.

Table III.

[00085] During Step 3 of Run 2, the top was removed from the desiccator and the hose with nitrogen was evenly distributed across the membrane by running a tube from the nitrogen regulator and manually moving the tube back and forth over the membrane. The even distribution of gas onto the membrane resulted in an effective and fast drying time.

[00086] After the first 2 Runs, the flow meter was removed from the device configuration to allow for a faster airflow and thus a more turbulent airflow. In order to allow for the faster airflow, the 3-way valve was removed leaving open the 3/8" hole that had been drilled into the side of the desiccator. The 3/8" aperture allowed for nitrogen gas flow out of the desiccator in Run 3 and Run 4. In Run 3 and Run 4, due to the removal of the flow meter, and a direct hose connection to the drying chamber, it was estimated that approximately 25-30 PSI nitrogen gas was being run into the desiccator. An exact number for the PSI was not determined because the regulator being used did not allow for small incremental changes in the gas flow.

[00087] After Run 3, a pattern was consistently observed on the dried membrane that was the same as the pattern of the screen. The screened pattern was observed on the membranes that were dried using the faster and more forceful airflow conditions. FIG. 12 illustrates a

representative image of the final dried membrane showing the screen-like pattern which was achieved consecutively over multiple runs. Unexpectedly, it was observed that the membranes having the screened pattern were more durable and thus easier to handle than the membranes produced in the previous processing methods.

[00088] In order to measure residual moisture levels present in the dried membrane tissue, an OHAUS MB45 Moisture Analyzer was used to analyze the vacuum sealed preserved samples. The analyzer was transferred into a Class 1000 cleanroom and introduced under a non-laminar forced air biologic safety cabinet. This was done to protect the integrity of the sample from air movement and particulate matter on the weighing surface. All samples were opened from their vacuum-seal packs under the biologic safety cabinet and individually removed from their packets using stainless steel pick-ups. Approximate transfer times from the time the pack was opened until the graft was introduced onto the moisture balance and sealed for analysis was

approximately 10 seconds. The analysis readout time took approximately 30 seconds. Prior to the analysis of each sample, the MB45 was recalibrated to 0.0 g to ensure each sample had an accurate reading. The moisture levels are presented in Tables II and III above. After the analysis was completed, the graft was transferred back into the same packet and sealed under heat-seal only, no vacuum. After completing the heat seal, the next graft was opened for analysis which ensured no transfer of particulate or moisture from one graft to the other.

[00089] According to the 13^th Edition American Association of Tissue Banks (AATB) Standards reference, any and all human tissue intended for transplant must meet a standard equal to or less than 6% residual moisture based on gram weight. If these samples had been produced for therapeutic use, they would have all met the AATB Standards. Therefore, the method of drying described herein can be used in compliance with the latest edition of AATB Standards.

[00090] In summary, use of the turbulent airflow while having the membrane supported on the screen resulted in a surprisingly effective and efficient method for producing dry membranes with good handling characteristics.

[00091] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. These patents and publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[00092] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present Examples along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

[00093] Particular embodiments and features have been described with reference to the drawings. It is to be understood that these descriptions are not limited to any single embodiment or any particular set of features, and that similar embodiments and features may arise or modifications and additions may be made without departing from the scope of these descriptions and the spirit of the appended claims.

Claims

CLAIMS What is claimed is:

1. A method for making patterned tissue membranes, the method comprising supporting a tissue membrane on a sample holding member defining a gridded top surface, wherein the sample holding member is elevated from a bottom of an enclosure in the presence of a forced airflow generated by an inert gas, for a length of time sufficient to reduce the moisture content of the tissue membrane below a predetermined threshold and in which a pattern of the gridded top surface is generated on the tissue membrane.

2. The method of claim 1, wherein the method is carried out free of an application of heat.

3. The method of claim 1, wherein the method is carried out at a room temperature.

4. The method of claim 1, wherein the method is carried out at a temperature of about 1 to about 10 degrees Celsius.

5. The method of claim 1, wherein the inert gas comprises nitrogen gas.

6. The method of claim 1, wherein the tissue membrane is a mammalian tissue membrane.

7. The method of claim 6, wherein the mammalian tissue membrane is an amnion tissue membrane.

8. The method of claim 6, wherein the mammalian tissue membrane is a human tissue membrane.

9. The method of claim 6, wherein the mammalian tissue membrane is a human amnion tissue membrane.

10. The method of claim 1, wherein the pattern of the gridded top surface comprises a screened or gridded pattern, honeycomb pattern, or a circular pattern.

11. The method of claim 1 , wherein the pattern of the gridded top surface defines at least one portion that defines an area of increased thickness of a frame of the gridded top surface to impart a corresponding portion on the tissue membrane.

12. The method of claim 1, wherein the predetermined threshold is equal to or less than 6% residual moisture based on gram weight.

13. A patterned tissue membrane, the patterned tissue membrane produced by a process comprising: supporting a tissue membrane on a sample holding member defining a gridded top surface, wherein the sample holding member is elevated from a bottom of an enclosure in the presence of a forced airflow generated by an inert gas, for a length of time sufficient to reduce the moisture content of the tissue membrane below a predetermined threshold and in which a pattern of the gridded top surface is generated on the tissue membrane.

14. The tissue membrane of claim 13, wherein the process is carried out free of an application of heat.

15. The tissue membrane of claim 13, wherein the process is carried out at a room

temperature.

16. The tissue membrane of claim 13, wherein the process is carried out at a temperature range of about 1 to about 10 degrees Celsius.

17. The tissue membrane of claim 13, wherein the inert gas comprises nitrogen gas.

18. The tissue membrane of claim 13, wherein the tissue membrane is a mammalian tissue membrane.

19. The tissue membrane of claim 18, wherein the mammalian tissue membrane is an amnion tissue membrane.

20. The tissue membrane of claim 18, wherein the mammalian tissue membrane is a human tissue membrane.

21. The tissue membrane of claim 18, wherein the mammalian tissue membrane is a human amnion tissue membrane.

22. The tissue membrane of claim 13, wherein the pattern of the gridded top surface comprises a honeycomb pattern or a circular pattern.

23. The tissue membrane of claim 13, wherein the pattern of the gridded top surface defines at least one portion that defines an area of increased thickness of a frame of the gridded top surface to impart a corresponding portion on the tissue membrane.

24. The tissue membrane of claim 13, wherein the predetermined threshold is equal to or less than 6% residual moisture based on gram weight.

25. A method for repair of a tissue in a living body, the method comprising:

hydrating a patterned tissue membrane according to claim 12 or claim 32 with a solution suitable for repair of the tissue; and

one of wrapping, placing on, embedding into, suturing in place, and filling the tissue of the living body in need of repair with the patterned tissue membrane to aid the repair.

26. The method of claim 25, wherein the tissue membrane is a mammalian tissue membrane.

27. The method of claim 26, wherein the mammalian tissue membrane is an amnion tissue membrane.

28. The method of claim 26, wherein the mammalian tissue membrane is a human tissue membrane.

29. The method of claim 26, wherein the mammalian tissue membrane is a human amnion tissue membrane.

30. The method of claim 25, wherein the pattern comprises a honeycomb pattern or a circular pattern.

31. The method of claim 25, wherein the tissue of the living body comprises epidermis, cornea, dura mater, tendon, ligament, fascia, nerve, vascular, organs, or bone.

32. A patterned tissue membrane, the patterned tissue membrane comprising:

a top surface and a bottom surface;

a gridded pattern on each of the top and bottom surfaces; and

a residual moisture content based on gram weight of about 6% or below.

33. The patterned tissue membrane of claim 32, wherein the tissue membrane is a mammalian tissue membrane.

34. The patterned tissue membrane of claim 33, wherein the mammalian tissue membrane is an amnion tissue membrane.

35. The patterned tissue membrane of claim 33, wherein the mammalian tissue membrane is a human tissue membrane.

36. The patterned tissue membrane of claim 33, wherein the mammalian tissue membrane is a human amnion tissue membrane.

37. The patterned tissue membrane of claim 32, wherein the gridded pattern comprises a honeycomb pattern or a circular pattern.

38. The patterned tissue membrane of claim 32, wherein the gridded pattern defines at least one portion that defines an area of increased thickness of the gridded pattern to impart a corresponding portion on the tissue membrane.

39. A drying system comprising :

a chamber defining a sealed enclosure;

a spray member configured for translation about the sealed enclosure and spraying treatment fluids therein; and

a sample holding member elevated from a bottom of the enclosure and defining a gridded top surface to which a sample rests on.

40. The system of claim 39, further including a fluid source in communication with the spray member.

41. The system of claim 39, wherein the fluid source includes an inert gas.

42. The system of claim 41, wherein the inert gas includes nitrogen gas.

43. The system of claim 39, further including a translation assembly that translates the spray member about the sealed enclosure, wherein the translation assembly includes a threaded axle extending across the chamber and a support carried by the threaded rod.

44. The system of claim 43, wherein the support is engaged with the spray member.

45. The system of claim 44, wherein the support is further carried by a second axle extending across the enclosure.

46. The system of claim 39, comprising an additional spray member, wherein the spray member is positioned on one side of the sample holding member and the additional spray member is positioned on another side of the sample holding member.

47. The system of claim 39, wherein the sample holding member comprises a honeycomb grid pattern or a circular grid pattern.

48. The system of claim 39, wherein the gridded top surface defines at least one portion that defines an area of increased thickness of a frame of the gridded top surface to impart a corresponding portion on the sample.

49. The system of claim 46 wherein the sample holding member comprises a first portion and a second portion for receiving the sample therebetween.

50. A method comprising :

spraying treatment gases onto a sample from at least a first position and a second position;

measuring one of a weight and moisture content of the sample;

wherein the sample is positioned on a sample holding member that defines a gridded pattern, wherein the sample defines a corresponding gridded pattern after being sprayed by the treatment gases.

51. The method of claim 50, further including applying a vacuum with a vacuum source to the chamber.

52. The method of claim 50, wherein spraying treatment gases is carried out continuously.

53. The method of claim 50, wherein spraying treatment gases is carried out in an on-off-on- off pattern.

54. The method of claim 50, further including monitoring the temperature of at least one of an enclosure that carries the sample or the sample.

55. The method of claim 50, wherein spraying treatment gases is carried out until the moisture content of the sample is under 6%.