AU2002354915B8 - Porous extracellular matrix scaffold and method - Google Patents

Description

DEP747/71152 "i'j ILI I AL 11:3:: '13:Si ii"11.1 rE::I r 11 :11 i -1- POROUS EXTRACELLULAR MATRIX SCAFFOLD AND METHOD CROSS REFERENCE TO RELATED APPLICATIONS Cross reference is made to copending U.S. patent applications Serial No. 10/195,794 entitled "Meniscus Regeneration Device and Method" (Attorney Docket No. 265280-71141, DEP-745); Serial No. 10/195,719 entitled "Devices from Naturally Occurring Biologically Derived Materials" (Attorney Docket No. 265280- 71142, DEP-748); Serial No. 10/195,347 entitled "Cartilage Repair Apparatus and Method" (Attorney Docket No. 265280-71143, DEP-749); Serial No. 10/195,344 entitled "Unitary Surgical Device and Method" (Attorney Docket No. DEP-750); Serial No. 10/195,341 entitled "Hybrid Biologic/Synthetic Porous Extracellular Matrix Scaffolds" (Attorney Docket No. 265280-71144, DEP-751); Serial No.

10/195,606 entitled "Cartilage Repair and Regeneration Device and Method" (Attorney Docket No. 265280-71145, DEP-752); Serial No. 10/195,334 entitled "Cartilage Repair and Regeneration Scaffolds and Method" (Attorney Docket No.

265280-71180, DEP-763); and Serial No. 10/195,633 entitled "Porous Delivery Scaffold and Method" (Attorney Docket No. 265280-71207, DEP-762), each of which is assigned to the same assignee as the present application, each of which is filed concurrently herewith, and each of which is hereby incorporated by reference.

Cross reference is also made to U.S. Patent Application Serial No. 10/172,347 entitled "Hybrid Biologic-Synthetic Bioabsorbable Scaffolds" which was filed on June 14, 2002, which is assigned to the same assignee as the present application, and which is hereby incorporated by reference.

FIELD OF THE DISCLOSURE The present disclosure relates generally to an extracellular matrix scaffold, and more particularly to a porous extracellular matrix scaffold for repairing or regenerating body tissue and a method for making such a scaffold.

BACKGROUND OF THE INVENTION Naturally occurring extracellular matrices (ECMs) are used for tissue repair and regeneration. One such extracellular matrix is small intestine submucosa (SIS). SIS has been used to repair, support, and stabilize a wide variety of anatomical AMENDVD

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DEP747/71152 -2defects and traumatic injuries. Commercially available SIS material is derived from porcine small intestinal submucosa that remodels to the qualities of its host when implanted in human soft tissues. Further, it is taught that the SIS material provides a natural matrix with a three-dimensional microstructure and biochemical composition that facilitates host cell proliferation and supports tissue remodeling. Indeed, SIS has been shown to contain biological molecules, such as growth factors and glycosaminoglycans that aid in the repair of soft tissue of the human body. The SIS material currently being used in the orthopaedic field is provided in a dried and layered configuration in the form of a patch to repair or regenerate soft tissue such as tendons, ligaments and rotator cuffs.

While small intestine submucosa is readily available, other sources of ECM are known to be effective for tissue remodeling. These sources include, but are not limited to, stomach, bladder, alimentary, respiratory, or genital submucosa, or liver basement membrane. See, U.S. Patents Nos. 6,379,710, 6,171,344; 6,099,567; and 5,554,389, each of which is hereby incorporated by reference.

Further, while SIS is most often porcine derived, it is known that various submucosa materials may also be derived from non-porcine sources, including bovine and ovine sources. Additionally, the ECM material may also include partial layers of laminar muscularis mucosa, muscularis mucosa, lamina propria, stratum compactum and/or other such tissue materials depending upon factors D such as the source from which the ECM material was derived and the delamination procedure.

As used herein, it is within the definition of a naturally occurring extracellular matrix to clean, delaminate, and/or comminute the extracellular matrix, or to cross-link the collagen or other components within the extracellular matrix. It is also within the definition of naturally occurring extracellular matrix to fully or partially remove one or more components or subcomponents of the naturally occurring matrix. However, it is not within the definition of a naturally occurring extracellular matrix to separate and purify the natural components or subcomponents and reform a matrix material from purified natural components or subcomponents.

Thus, while reference is made to SIS, it is understood that other naturally occurring extracellular matrices stomach, bladder, alimentary, respiratory, and genital submucosa, and liver basement membrane), whatever the source bovine, ^W^DFDn SIE^T WO 03/007789 PCT/US02/22393 -3porcine, ovine) are within the scope of this disclosure. Thus, in this application, the terms "naturally occurring extracellular matrix" or "naturally occurring ECM" are intended to refer to extracellular matrix material that has been cleaned, processed, sterilized, and optionally crosslinked. The following U.S. patents, hereby incorporated by reference, disclose the use of ECMs for the regeneration and repair of various tissues: 6,379,710; 6,187,039; 6,176,880; 6,126,686; 6,099,567; 6,096,347; 5,997,575; 5,993,844; 5,968,096; 5,955,110; 5,922,028; 5,885,619; 5,788,625; 5,762,966; 5,755,791; 5,753,267; 5,733,337; 5,711,969; 5,645,860; 5,641,518; 5,554,389; 5,516,533; 5,460,962; 5,445,833; 5,372,821; 5,352,463; 5,281,422; and 5,275,826.

The manipulation of scaffold pore size, porosity, and interconnectivity is an important science contributing to the field of tissue engineering (Ma and Zhang, 2001, J Biomed Mater Res, 56(4):469-477; Ma and Choi, 2001 Tissue Eng, 7(1):23- 33) because it is believed that the consideration of scaffold pore size and density/porosity influences the behavior of cells and the quality of tissue regenerated.

In fact, several researchers have shown that different pore sizes influence the behavior of cells in porous three-dimensional matrices. For example, it has been demonstrated in the art that for adequate bone regeneration to occur scaffold pore size needs to be at least 100 microns (Klawitter et al., 1976, J Biomed Mater Res, 10(2):311-323). For pore sizes and interconnectivity less than that, poor quality bone is regenerated and if pore size is between 10-40 microns bone cells are able to form only soft fibrovascular tissue (White and Shors, 1991, Dent Clin North Am, 30:49-67). The consensus of research for bone regeneration indicates that the requisite pore size for bone regeneration is 100 600 microns (Shors, 1999, Orthop Clin North Am, 30(4):599-613; Wang, 1990, Nippon Seikeigeka Gakki Zasshi, 64(9):847-859). It is generally known in the art that optimal bone regeneration occurs for pore sizes between 300 600 microns.

Similarly, for the regeneration of soft orthopaedic tissues, such as ligament, tendon, cartilage, and fibro-cartilage, scaffold pore size is believed to have a substantial effect. For example, basic research has shown that cartilage cells (chondrocytes) exhibit appropriate protein expression (type II collagen) in scaffolds with pore sizes of the order of 20 microns and tend to dedifferentiate to produce type I collagen in scaffolds with nominal porosity of about 80 microns (Nehrer et al., 1997, 00 CI Biomaterials, 18(11):769-776). More recently, it has been shown that cells that form ligaments, tendons, and blood vessels (fibroblasts and endothelial cells) exhibit significantly different activity when cultured on scaffolds with differing pore sizes C, ranging from 5 to 90 microns (Salem et al., 2002, J Biomed Mater Res, 61(2):212- 217).

SUMMARY OF THE INVENTION 0 According to the invention, there is provided a method of making an C 10 implantable scaffold for repairing or regenerating body tissue, the method comprising the steps of: providing an extracellular matrix material selected from the group consisting of: small intestine submucosa, stomach submucosa, bladder submucosa, respiratory submucosa, alimentary submucosa, genital submucosa, and liver basement membrane; physically comminuting the extracellular matrix in the presence of a ligand to form a slurry comprising intertwined, ribbon-like, cohesive pieces of extracellular matrix; and lyophilizing the cohesive pieces of extracellular matrix to form an open cell foam of extracellular matrix.

The invention also provides a product made from the aforementioned method.

27/0208,at 3850.specipgs,2 BRIEF DESCRIPTION OF T1HE DRAWINGS The detailed description particularly refers to the accompanying figures in which: 09/08/07,l13850p4.5, WO 03/007789 PCT/US02/22393 -6- FIG. 1 is an image from a scanning electron microscope which shows the surface of a porous reticulated SIS open cell foam scaffold having a relatively large pore size and a relatively low material density; FIG. 2 is an image from a scanning electron microscope which shows the surface of a porous reticulated SIS open cell foam scaffold having a relatively moderate pore size and a relatively moderate material density; FIG. 3 is an image from a scanning electron microscope which shows the surface of a porous reticulated SIS open cell foam scaffold having a relatively small pore size and a relatively high material density; FIG. 4 is an image from a scanning electron microscope which shows a cross-section of a porous reticulated SIS open cell foam scaffold, with an example of a pore indicated by the arrow, the image being at a greater magnification than the images of FIGS. 1-3; FIG. 5 is an image from a scanning electron microscope which shows a cross-section of a porous reticulated SIS open cell foam scaffold, with examples of pores indicated by the arrows, the image being at a greater magnification than the images of FIGS. 1-3; FIG. 6 is an image from a scanning electron microscope which shows a cross-section of a porous reticulated SIS open cell foam scaffold, with examples of pores indicated by the arrows, the image being at a greater magnification than the images of FIGS. 1-3; FIG. 7 is an image from a scanning electron microscope which shows a cross-section of a porous reticulated SIS open cell foam scaffold, the image being at a greater magnification than the images of FIGS. 1-3; FIG. 8 is an image from a scanning electron microscope which shows a surface of a porous reticulated SIS open cell foam scaffold, the image being at a greater magnification than the images of FIGS. 1-3; and FIGS. 9 and 10 are images from a scanning electron microscope which show a mass of cohesive SIS pieces.

WO 03/007789 PCT/US02/22393 -7- DETAILED DESCRIPTION OF THE DRAWINGS The present disclosure relates to a porous scaffold for implanting into the body of a patient to repair or regenerate damaged or diseased tissue. The porous scaffold is constructed from a naturally occurring extracellular material. For example, the scaffold may be constructed from SIS. As will be discussed herein in greater detail, both the material density and the pore size of the porous scaffold may be varied to fit the needs of a given scaffold design.

Such porous scaffolds may be fabricated by suspending pieces of an extracellular matrix material in a liquid. As used herein, the term "piece" is intended to mean any fiber, strip, ribbon, sliver, filament, shred, bit, fragment, part, flake, slice, cut, chunk, or other portion of solid or solid-like material. Also, as used herein, the term "suspending" is intended to include any placement of a solid pieces of ECM) in a liquid whether or not an actual suspension is created. As such, the term "suspending" is intended to include any mixing of a solid in a liquid or any other placement of a solid in a liquid. As a result, the term "suspension" is likewise not intended to be limited to suspensions, but rather is intended to mean any mass having a solid present in a liquid.

In any event, the suspension of the pieces of extracellular matrix material and the liquid forms a mass in the form of, for example, a "slurry". The liquid may then be subsequently driven off of the mass so as to form interstices therein. The liquid may be driven off in a number of different manners. For example, as will herein be described in greater detail, the liquid may be driven off via sublimation in a freeze drying process. Alternatively, the liquid may also be driven off by subjecting the suspension to either an unheated vacuum process or a vacuum under a controlled heating process. The liquid may also be driven off from the suspension ultrasonically. Microwave energy, RF energy, UV energy, or any other type of energy (or combination thereof) may also be utilized to drive the liquid off of the suspension. Liquid may also be driven off of the suspension by forcing or drawing air through the suspension. The suspension may be centrifuged to drive off the liquid. Moreover, the liquid may include a water-soluble filler which is driven off, for example, by use of an alcohol. In short, the present disclosure contemplates the driving off of the liquid from the suspension by any liquid removal process.

WO 03/007789 PCT/US02/22393 -8- As alluded to above, while any of the aforementioned processes for driving off the liquid from the suspension may be utilized, along with any other process known by one skilled in the art, the processes of the present disclosure will herein be exemplary described in regard to a lyophilization process freeze drying). However, it should be understood that such a description is merely exemplary in nature and that any one or more of the aforedescribed processes for driving off the liquid from the suspension may be utilized to fit the needs of a given scaffold design or process design.

As alluded to above, one useful process for fabricating the porous scaffolds of the present disclosure is by lyophilization. In this case, pieces of an extracellular matrix material are suspended in a liquid. The suspension is then frozen and subsequently lyophilized. Freezing the suspension causes the liquid to be turned to ice crystals. These ice crystals are then sublimed under vacuum during the lyophilization process thereby leaving interstices in the material in the spaces previously occupied by the ice crystals. The material density and pore size of the resultant scaffold may be varied by controlling, amongst other things, the rate of freezing of the suspension and/or the amount of water in which the extracellular matrix material is suspended in at the on-set of the freezing process.

As a specific example of this process, fabrication of a porous SIS scaffold by lyophilization will be described in detail. However, it should be appreciated that although the example is herein described in regard to an SIS scaffold, fabrication of a scaffold constructed from other extracellular matrix materials may also be performed in a similar manner.

The first step in fabricating a porous scaffold with a desired pore size and density is the procurement of comminuted SIS. Illustratively, scissor-cut SIS runners long) are positioned in a 1700 series ComitrolTM machine, commercially available from Urschel Laboratories (Valparaiso, Indiana). The SIS material is processed in the presence of a liquid and thereafter collected in a receptacle at the output of the machine. The material is then processed through the machine a second time under similar conditions. In one exemplary process, a liquid water) is introduced into the input of the machine contemporaneously with the SIS material.

The resultant material is a "slurry" of SIS material (thin, long SIS fibers -200 microns thick x 1-5 mm long) suspended in a substantially uniform manner in water.

WO 03/007789 PCT/US02/22393 -9- Although the suspension is herein described as being formed as a byproduct of the comminuting process, it should be appreciated that the pieces of SIS may be suspended in the liquid water) in other manners known to those skilled in the art.

Furthermore, while other methods are known for comminuting SIS, it is understood that for the purposes of the present disclosure, comminuted SIS comprises, ribbonlike or string-like fibers wherein at least some of the individual pieces of ECM and SIS material have lengths greater than their widths and thicknesses. Such fibers may be interlaced to provide a felt-like material, if desired.

Process parameters can be varied using the above-identified 1700 series ComitrolTM machine, including the choice of blade used, whether water is used, the amount of water used, the speed at which the blades turn, and the number of times the material is passed through the machine. As an example, cutting head 140084-10 and a Vericut, sealed impeller from Ursehel Laboratories may be used, with a flow of water of about two gallons per minute, with the blade running at a constant speed of about 9300 rpm. A first pass through the machine at these parameters will produce fibrous SIS material of varying sizes, and a second pass will produce SIS fibers of a more uniform size. By way of example, the comminuted material may be tested to determine if it has the consistency of that which is desired for use in regard to the illustrative embodiments described herein by the following process: the comminuted SIS suspension or slurry is centrifuged, excess water is poured off and the remaining slurry is poured into a dish. By hand, a small amount of the comminuted SIS material in the dish is pinched between the thumb and index finger and gently lifted from the dish. Illustratively, at least a small amount of additional SIS, beyond the portion pinched between the thumb and index finger, will lift along with the material that has been pinched ("pinch test"). This additional comminuted SIS material lifts with the material that is between the thumb and index finger because the individual pieces of comminuted SIS material are commingled or intertwined.

The terms "cohesive ECM", "cohesive SIS", "cohesive ECM pieces" and "cohesive SIS pieces" are used herein to respectively denote ECM or SIS material that has been comminuted or otherwise physically processed to produce ECM or SIS pieces that are capable of comingling or intertwining (in the wet or dry state) to form a mass of discrete pieces of ECM or SIS that remain massed together under some conditions (such as under gravity), regardless of the shape or shapes of the individual WO 03/007789 PCT/US02/22393 ECM or SIS pieces. One method of demonstrating that the ECM or SIS material comprises cohesive pieces is the "pinch test" described in the preceding paragraph.

Examination of the final ECM or SIS product produced may also provide evidence that the base material comprised cohesive ECM or SIS pieces. Illustratively, the ECM or SIS pieces are sufficiently cohesive to each other (or to other pieces in the mix or slurry) that they remain unified throughout the process used to produce the foam structure. Examples of cohesive SIS pieces are shown in the scanning electron microscopic images of FIGS. 9 and Thereafter, the comminuted SIS suspension is frozen and lyophilized freeze dried). In particular, the SIS suspension is frozen at a controlled rate of temperature drop to control the size of the formed ice crystals. Once frozen, and without allowing the material to thaw, the lyophilization process sublimes the ice crystals directly to a vapor under vacuum and low temperatures. This leaves voids or interstices in the spaces previously occupied by the ice crystals.

Any commercially available frcczcr for freezing the suspension to a desired temperature may be used. Likewise, any commercially available lyophilizer may be used for the lyophilization process. One exemplary machine for performing the lyophilization process is a Virtis Genesis TM Series lyophilizer which is commercially available from SP Industries, Inc. of Gardiner, New York.

The process parameters of the aforedescribed fabrication process may be varied to produce scaffolds of varying pore sizes and material densities. For example, the rate at which the suspension is frozen, the amount of water present in the suspension, or the compactness of the extracellular matrix material may be varied to produce scaffolds of varying pore sizes and material densities.

For instance, to produce scaffolds having a relatively large pore size and a relatively low material density, the extracellular matrix suspension may be frozen at a slow, controlled rate -l°C/min or less) to a temperature of about followed by lyophilization of the resultant mass. To produce scaffolds having a relatively small pore size and a relatively high material density, the extracellular matrix material may be tightly compacted by centrifuging the material to remove a portion of the liquid water) in a substantially uniform manner prior to freezing.

Thereafter, the resultant mass of extracellular matrix material is flash-frozen using liquid nitrogen followed by lyophilization of the mass. To produce scaffolds having a WO 03/007789 PCT/US02/22393 -11moderate pore size and a moderate material density, the extracellular matrix material is first tightly compacted by centrifuging the material to remove a portion of the liquid water) in a substantially uniform manner prior to freezing. Thereafter, the resultant mass of extracellular matrix material is frozen at a relatively fast rate -l°C/min) to a temperature of about -80 0 C followed by lyophilization of the mass.

EXAMPLE 1 Example 1 demonstrates the fabrication of a porous SIS scaffold having a relatively large pore size and a relatively low material density. Such scaffolds are obtained by freezing a comminuted SIS suspension at a slow, controlled rate (-1°C/min or less) to -20 0 C, followed by lyophilization. The procedure is as follows. First, comminuted SIS is fabricated as described above. Specifically, scissor-cut SIS runners long) are placed in a suitable comminuting machine such as the Urschel Comitrol machine described above. The comminuted SIS is collected in a receptacle at the output of the machine. The collected material is then processed through the machine a second time, under the same conditions as before. The resultant mass is a "slurry" of SIS material (thin, long SIS fibers -200 microns thick x mm long) suspended relatively uniformly in water.

Next, a slow-freeze ethanol bath is prepared as follows. Pour enough ethanol to obtain about a 1 centimeter head in a flat-bottomed plastic container large enough to hold four 24-well culture plates. Place the container in a -20°C freezer.

The mass of each of four empty twenty-four well plates is then recorded. Under a sterile hood using sterile conditions, an approximately 3ml aliquot of the comminuted SIS material is placed in each well of the tissue culture plates. The mass of each full plate of material is then recorded. The four culture plates are then placed into the ethanol freeze bath and allowed to freeze overnight.

The frozen plates are then removed from the ethanol bath and placed in a suitable lyophilization machine such as the Virtis Genesis Series lyophilizer described above. Without allowing the frozen SIS material to thaw, the process of lyophilization sublimes ice crystals directly to vapor under vacuum and low temperatures. This leaves voids or interstices in the spaces previously occupied by the ice crystals. In this case, the parameters used in the lyophilization process include DIP747/71152 r,'2///i1y i!EiI iiiit i5 1 f 3 iii 3ii ',iiii: m ICiii i iii? itE I 10li 3,tl: -12a first period at a primary drying temperature of-13°C for 8 hours, followed by a second period at a secondary drying temperature of 35°C for 4 hours.

After the lyophilization cycle is complete, the plates are removed from the lyophilization machine and the mass of each plate is determined and recorded.

The results from this process are summarized in the following table: Average Volume Average Mass Average Density 1.249 ml 0.007396 g 0.006 (g/cc) A scanning electron image of the surface of the samples was taken to visualize the relative pore sizes. These pore sizes are about 600 microns to about 700 microns. An image indicative of the samples prepared in accordance with Example 1 is shown in FIG. 1.

Pore sizes can be determined from scanning electron microscope images of the exterior surface of the foam, as in FIGS. 1-3 and 8, and of crosssections of the foam, as in FIGS. 4-7. These images may be used in conjunction with standard commercially available image analysis software to determine the ranges of pore sizes. FIGS. 4-6 illustrate the results of using suitable commercially available software to measure or estimate the pore sizes in the foam. This technique was used to determine that the Example 1 foam had pores in the range of 600-700 microns.

The sample may also include smaller pores.

D EXAMPLE 2 Example 2 demonstrates the fabrication of a porous SIS scaffold having a relatively moderate pore size and a relatively moderate material density.

Such scaffolds are obtained by compacting the comminuted SIS material by centrifugation, freezing at a faster rate (relative to Example 1) and to a lower temperature to followed by lyophilization of the resultant mass. The procedure is as follows. First, comminuted SIS is fabricated as described above in regard to Example 1. Specifically, scissor-cut SIS runners long) is comminuted by two passes through a suitable comminuting machine to produce a "slurry" of SIS material (thin, long SIS fibers -200 microns thick x 1-5 mm long) suspended relatively uniformly in water.

AMENDED SHEET DIP747/71152

I

"il' it R '11 11-:L 1 1[3 iii iiii :i i! ii:1:11. C.11t:3 %j -13- Next, the mass of each of four empty twenty-four well plates is recorded. Under a sterile hood using sterile conditions, an approximately 3ml aliquot of the comminuted SIS material is placed in each well of the tissue culture plates.

The mass of each plate full of material is then recorded.

The plates are then balanced for centrifuging by use of the following technique. The two plates are placed on the balance, and RO water is added to the area in between the wells of the lighter plate until the two plates are balanced. The two plates are then placed across from one another in the centrifuge, and centrifuged at 3000rpm for seven minutes. Once done, the plates are removed from the centrifuge, and the water is emptied therefrom. The mass of each of the plates is then recorded. The centrifuging and mass measurement process is then repeated for the remaining plates.

The plates are then placed in a -80°C freezer until the specimen is fully frozen. Depending upon the bulk of the material, the time for full freezing can vary from about 1 to about 30 minutes, for example. The frozen plates are then removed from the freezer and placed in a suitable lyophilization machine and lyophilized under similar parameters to as described above in regard to Example 1 for a first period at a primary drying temperature of-13°C for 8 hours, followed by a second period at a secondary drying temperature of 35 0 C for 4 hours).

After the lyophilization cycle is complete, the plates are removed from the lyophilization machine and the mass of each plate is determined and recorded.

The results from this process are summarized in the following table: Average Volume Average Mass Average Density 0.285 ml 0.010104 g 0.035 g/cc As with the samples of Example 1, a scanning electron image of the surface of each of the samples prepared in accordance with Example 2 was taken to visualize the relative pore sizes. An image indicative of the samples prepared in accordance with Example 2 is shown in FIG. 2. Using the technique described above for determining pore size, this sample was found to have pores in the range of about 100-150 microns.

AMENDED SHEF WO 03/007789 PCT/US02/22393 -14- EXAMPLE 3 Example 3 demonstrates the fabrication of a porous SIS scaffold having a relatively small pore size and a relatively high material density. Such scaffolds are obtained by compacting the comminuted SIS material to an even higher density than in Example 2, flash-freezing the samples using liquid nitrogen, followed by lyophilization. The procedure is as follows. First, comminuted SIS is fabricated as described above in regard to Examples 1 and 2. Specifically, scissor-cut SIS runners long) is comminuted by two passes through a suitable comminuting machine to produce a "slurry" of SIS material (thin, long SIS fibers -200 microns thick x 1-5 mm long) suspended relatively uniformly in water. Once done, the resultant mass is centrifuged under a dead-weight. The dead weights are prepared as follows. Forty-eight strips of Coban are cut into pieces that measure 50mm in length and 5mm in width (unstretched). Thereafter, the pieces are stretched and wrapped around the outer edges of a polyethylene disk measuring 1cm in diameter and 2 mm in thickness. Each strip is trimmed, if need be, so that the Coban strips wrap around the disk two times.

Next, the mass of each of four empty twenty-four well plates is recorded. Under a sterile hood using sterile conditions, an approximately 3ml aliquot of the comminuted SIS material is placed in each well of the tissue culture plates.

The mass of each full plate of material is then recorded. The plates are then balanced and centrifuged as described above in regard to Example 2. Thereafter, the water is drained from the plates, and the mass of each of the centrifuged plates is recorded.

Once this is completed, the wells of the plates are combined at a ratio of 2:1 thereby reducing the number of plates from four to two. An attempt is made to combine low material wells with high material wells in order to have a somewhat consistent amount of SIS material in each well. The mass of each full plate is then recorded. The Coban-wrapped polyethylene disks are then placed into each well.

The two plates are then balanced using the technique described above in regard to Example 2. The plates are then centrifuged at 3000rpm for five minutes. Thereafter, the plates are removed from the centrifuge, the water is emptied therefrom, and the polyethylene disks are also removed. The mass of each plate is again recorded. The two plates are balanced once again (in a manner similar to as described above in regard to Example and the plates are again centrifuged at 3000rpm for seven L~II LI ~e DEP747/71152 rtitiT:,; il::l :11":ijl minutes. Once done, the water is emptied again from each of the plates, and the mass of each plate is again recorded.

Contemporaneously with the centrifuging process, a liquid nitrogen bath is prepared by pouring liquid nitrogen into a wide-mouthed liquid nitrogen container. The plates are kept in the centrifuge until the bath is ready. Thereafter, each plate is dipped into the bath and held in the liquid for approximately 15 seconds.

Upon removal from the nitrogen bath, the plates are immediately placed in a freezer to prevent thawing. The frozen plates are then removed from the freezer and placed in a suitable lyophilization machine and lyophilized under similar parameters to as described above in regard to Examples 1 and 2 for a first period at a primary drying temperature of -13°C for 8 hours, followed by a second period at a secondary drying temperature of 35 0 C for 4 hours).

After the lyophilization cycle is complete, the plates are removed from the lyophilization machine and the mass of each plate is determined and recorded.

The results from this process are summarized in the following table: Average Volume Average Mass Average Density 0.786 ml 0.071563 g 0.091 g/cc As with the samples of Examples 1 and 2, a scanning electron image of the surface of each of the samples prepared in accordance with Example 3 was taken to visualize the relative pore sizes. An image indicative of the samples prepared in accordance with Example 3 is shown in FIG. 3. Using the technique described above for determining pore size, this sample was found to have pores in the range of about 40-60 microns. Such pore sizes are illustrated in FIGS. 4-6, indicated by the arrows.

As shown in the scanning electron microscope images of FIGS. 1-8, each of the illustrated ECM foams comprises a three-dimensional network of reticulated naturally occurring ECM defining a plurality of interconnected pores. The foam has these pores throughout its height, width, and thickness. The pores are open and interconnected to define a plurality of irregularly shaped interconnected passageways leading from the exterior surface of the foam (see FIGS. 1-3 and 8) into the interior of the foam (see cross-sections FIGS. These interconnected passageways are three-dimensional. As discussed above, the sizes of the pores, and WO 03/007789 PCT/US02/22393 -16therefore the maximum size for the interconnected passageways, can be controlled by controlling the manufacturing process as described above.

These interconnected passageways facilitate cell migration through the implant and enable efficient nutrient exchange in vivo. These interconnected passageways also provide a means of transmitting bioactive agents, biologically derived substances stimulants), cells and/or biological lubricants, biocompatible inorganic materials, synthetic polymers and biopolymers collagen) throughout the length, width and thickness of the ECM prior to implantation. The interconnected passageways defined by the pores also serve as passageways for materials used during the manufacturing process, such as compounds used for chemically cross-linking the foam. These interconnected passageways as well as the outer surfaces of the foam may also serve as sites on which the above materials are carried.

As shown in the above examples, the process parameters can be varied to produce an ECM foam that has the desired porosity for the particular application.

For example, it may be desirable to produce a foam with lower density (and higher porosity) for applications involving osteocytes and to produce a foam with higher density (and lower porosity) for applications involving chondrocytes.

Moreover, the ECM foams described herein may be crosslinked.

Specifically, the ECM foams described herein may be either chemically or physically crosslinked.

As can be seen in the scanning electron microscopic images of FIGS.

1-8, each of the illustrated ECM foams comprises interconnected pieces of naturally occurring extracellular matrix. As shown in the scanning electron microscope images of cross-sections of the ECM foams of FIGS. 4-7, these interconnected pieces of naturally occurring extracellular matrix provide the foam with an interior surface having an three-dimensional topography of irregular shape. As shown in the scanning electron microscope images of the surfaces of the ECM foam, these interconnected pieces of naturally occurring extracellular matrix provide the foam with exterior surfaces having three-dimensional topographies of irregular shapes. With these irregular three-dimensional topographies and the interconnected passageways, the ECM foams of the present disclosure provide a relatively large surface area of naturally occurring ECM. Such a large surface area of naturally occurring ECM can be advantageous in providing a relatively large surface area to which biological II- I I I -s DIP747/71152 11 H D 'I I El;:F iiti;'' i~ iiii i3'ii ll I~:iii~ iii C~i C:lt:i -17agents, biologically derived agents, cells, biocompatible polymers and biocompatible inorganic materials can be affixed pre-implantation. In addition, the illustrated ECM foams provide a relatively large surface of area of naturally occurring ECM to which cells may attach in vivo.

ECM foam products can be made with substantially lower densities than those of other ECM products. For comparison, the density of the commercially available RESTORE@ product, an ECM laminate, is 0.466 0.074 g/cc. An ECM product consisting of comminuted and hardened SIS as described in U.S. Patent Application Serial No. 10/195,719 entitled "Devices from Naturally Occurring Biologically Derived Materials" (Attorney Docket No. 265280-71142, DEP-748), has been made with a density of can be 0.747 0.059 g/cc. And, an ECM product consisting of toughened SIS laminate as described in U.S. Patent Application Serial No. 10/195,794 entitled "Meniscus Regeneration Device and Method" (Attorney Docket No. 265280-71141, DEP-745) has been made with a density of 0.933 0.061 g/cc.

As discussed above, the ECM foams of the present disclosure may be combined with bioactive agents, biologically derived substances, cells and/or stimulants, biocompatible inorganic materials and/or biocompatible polymers biocompatible synthetic polymers and biopolymers) and combinations of two or more of these materials at the time of manufacture. Illustratively, cells can be seeded throughout the three-dimensional volume of the ECM foam; the biological materials can be dried on the ECM foam at manufacture; the biological materials and the ECM foam can be co-lyophilized; and the biological materials can be covalently linked to the ECM foam. It is contemplated to bond, cross-link, or otherwise incorporate one or more of these materials to the raw ECM material prior to formation of the ECM foam. Alternatively, the materials could be bonded, cross-linked, or otherwise incorporated to the final ECM foam after lyophilization. Finally, combinations of the above methods may be used. For example, an implant of covalently linked ECM foam and biological lubricant can be implanted and additional intra-articular injections of the same or different biological lubricants can be made at surgery, postoperatively, or both at surgery and post-operatively.

"Bioactive agents" include one or more of the following: chemotactic agents; therapeutic agents antibiotics, steroidal and non-steroidal analgesics and ADIENDD S 1

CE'

WO 03/007789 PCT/US02/22393 -18anti-inflammatories, anti-rejection agents such as immunosuppressants and anticancer drugs); various proteins short chain peptides, bone morphogenic proteins, glycoprotein and lipoprotein); cell attachment mediators; biologically active ligands; integrin binding sequence; ligands; various growth and/or differentiation agents epidermal growth factor, IGF-I, IGF-II, TGF-3 I-III, growth and differentiation factors, vascular endothelial growth factors, fibroblast growth factors, platelet derived growth factors, insulin derived growth factor and transforming growth factors, parathyroid hormone, parathyroid hormone related peptide, bFGF; TGFo superfamily factors; BMP-2; BMP-4; BMP-6; BMP-12; sonic hedgehog; GDF5; GDF6; GDF8; PDGF); small molecules that affect the upregulation of specific growth factors; tenascin-C; hyaluronic acid; chondroitin sulfate; fibronectin; decorin; thromboelastin; thrombin-derived peptides; heparin-binding domains; heparin; heparan sulfate; DNA fragments and DNA plasmids If other such substances have therapeutic value in the orthopaedic field, it is anticipated that at least some of these substances will have use in concepts of the present disclosure, and such substances should be included in the meaning of "bioactive agent" and "bioactive agents" unless expressly limited otherwise.

"Biologically derived agents" include one or more of the following: bone (autograft, allograft, and xenograft) and derivates of bone; cartilage (autograft, allograft and xenograft), including, for example, meniscal tissue, and derivatives; ligament (autograft, allograft and xenograft) and derivatives; derivatives of intestinal tissue (autograft, allograft and xenograft), including for example submucosa; derivatives of stomach tissue (autograft, allograft and xenograft), including for example submucosa; derivatives of bladder tissue (autograft, allograft and xenograft), including for example submucosa; derivatives of alimentary tissue (autograft, allograft and xenograft), including for example submucosa; derivatives of respiratory tissue (autograft, allograft and xenograft), including for example submucosa; derivatives of genital tissue (autograft, allograft and xenograft), including for example submucosa; derivatives of liver tissue (autograft, allograft and xenograft), including for example liver basement membrane; derivatives of skin tissue; platelet rich plasma (PRP), platelet poor plasma, bone marrow aspirate, demineralized bone matrix, insulin derived growth factor, whole blood, fibrin and blood clot. Purified ECM and other collagen sources are also intended to be included within "biologically derived DEP747/71152 r I JL E ii1:111 11"'It T3 V ur-, R.P; tiff"",E) :3 2.4 Q -19agents." If other such substances have therapeutic value in the orthopaedic field, it is anticipated that at least some of these substances will have use in the concepts of the present disclosure, and such substances should be included in the meaning of "biologically derived agent" and "biologically derived agents" unless expressly limited otherwise.

"Biologically derived agents" also include bioremodelable collagenous tissue matrices. The expressions "bioremodelable collagenous tissue matrix" and "naturally occurring bioremodelable collagenous tissue matrix" include matrices derived from native tissue selected from the group consisting of skin, artery, vein, pericardium, heart valve, dura mater, ligament, bone, cartilage, bladder, liver, stomach, fascia and intestine, tendon, whatever the source. Although "naturally occurring bioremodelable collagenous tissue matrix" is intended to refer to matrix material that has been cleaned, processed, sterilized, and optionally crosslinked, it is not within the definition of a naturally occurring bioremodelable collagenous tissue matrix to purify the natural fibers and reform a matrix material from purified natural fibers. The term "bioremodelable collagenous tissue matrices" includes "extracellular matrices" within its definition.

"Cells" include one or more of the following: chondrocytes; fibrochondrocytes; osteocytes; osteoblasts; osteoclasts; synoviocytes; bone marrow cells; mesenchymal cells; stromal cells; stem cells; embryonic stem cells; precursor cells derived from adipose tissue; peripheral blood progenitor cells; stem cells isolated from adult tissue; genetically transformed cells; a combination of chondrocytes and other cells; a combination of osteocytes and other cells; a combination of synoviocytes and other cells; a combination of bone marrow cells and other cells; a combination of mesenchymal cells and other cells; a combination of stromal cells and other cells; a combination of stem cells and other cells; a combination of embryonic stem cells and other cells; a combination of precursor cells isolated from adult tissue and other cells; a combination of peripheral blood progenitor cells and other cells; a combination of stem cells isolated from adult tissue and other cells; and a combination of genetically transformed cells and other cells. If other cells are found to have therapeutic value in the orthopaedic field, it is anticipated that at least some of these cells will have use in the concepts of the present disclosure, and such cells AMENDED SHEET DEP747/71152 C' 11 41'2 i.,11, E41 ii"R, 1911 ER1 11,11 1E.

should be included within the meaning of "cell" and "cells" unless expressly limited otherwise.

"Biological lubricants" include: hyaluronic acid and its salts, such as sodium hyaluronate; glycosaminoglycans such as dermatan sulfate, heparan sulfate, chondroiton sulfate and keratan sulfate; synovial fluid and components of synovial fluid, including as mucinous glycoproteins lubricin), vitronectin, tribonectins, articular cartilage superficial zone proteins, surface-active phospholipids, lubricating glycoproteins I, II; and rooster comb hyaluronate. "Biological lubricant" is also intended to include commercial products such as ARTHREASE T high molecular weight sodium hyaluronate, available in Europe from DePuy International, Ltd. of Leeds, England, and manufactured by Bio-Technology General (Israel) Ltd., of Rehovot, Israel; SYNVISC® Hylan G-F 20, manufactured by Biomatrix, Inc., of Ridgefield, New Jersey and distributed by Wyeth-Ayerst Pharmaceuticals of Philadelphia, Pennsylvania; HYLAGAN® sodium hyaluronate, available from Sanofi-Synthelabo, Inc., ofNew York, New York, manufactured by FIDIA of Padua, Italy; and HEALON® sodium hyaluronate, available from Pharmacia Corporation of Peapack, New Jersey in concentrations of 1.4% and 2.3% (for opthalmologic uses). If other such substances have therapeutic value in the orthopaedic field, it is anticipated that at least some of these substances will have use in the concepts of the present disclosure, and such substances should be included in the meaning of "biological lubricant" and "biological lubricants" unless expressly limited otherwise.

"Biocompatible polymers" is intended to include both synthetic polymers and biopolymers collagen). Examples ofbiocompatible polymers include: polyesters of [alpha]-hydroxycarboxylic acids, such as poly(L-lactide) (PLLA) and polyglycolide (PGA); poly-p-dioxanone (PDO); polycaprolactone (PCL); polyvinyl alcohol (PVA); polyethylene oxide (PEO); polymers disclosed in U. S.

Pats. Nos. 6,333,029 and 6,355,699; and any other bioresorbable and biocompatible polymer, co-polymer or mixture of polymers or co-polymers that are utilized in the construction of prosthetic implants. In addition, as new biocompatible, bioresorbable materials are developed, it is expected that at least some of them will be useful materials from which orthopaedic devices may be made. It should be understood that AMENDED

SHEET

DEP747/71152 IP' itl 1, F 4_ 11 11, :11 ER l i~ti~~i' iji~ i'R 13 ru";!il 10 1ER~1 ijitt 1: r::l i! -21the above materials are identified by way of example only, and the present invention is not limited to any particular material unless expressly called for in the claims.

"Biocompatible inorganic materials" include materials such as hydroxyapatite, all calcium phosphates, alpha-tricalcium phosphate, beta-tricalcium phosphate, calcium carbonate, barium carbonate, calcium sulfate, barium sulfate, polymorphs of calcium phosphate, ceramic particles, and combinations of such materials. If other such substances have therapeutic value in the orthopaedic field, it is anticipated that at least some of these substances will have use in the concepts of the present disclosure, and such substances should be included in the meaning of "biocompatible inorganic material" and "biocompatible inorganic materials" unless expressly limited otherwise.

It is expected that various combinations of bioactive agents, biologically derived agents, cells, biological lubricants, biocompatible inorganic materials, biocompatible polymers can be used with the scaffolds of the present disclosure.

It is expected that standard sterilization techniques may be used with the products of the present disclosure.

Illustratively, in one example of embodiments that are to be seeded with living cells such as chondrocytes, a sterilized implant may be subsequently seeded with living cells and packaged in an appropriate medium for the cell type used.

For example, a cell culture medium comprising Dulbecco's Modified Eagles Medium (DMEM) can be used with standard additives such as non-essential aminoacids, glucose, ascorbic acid, sodium pyrovate, fungicides, antibiotics, etc., in concentrations deemed appropriate for cell type, shipping conditions, etc.

It is anticipated that the ECM foams of the present disclosure may be combined with the concepts disclosed in the following applications for U.S. patent, filed concurrently herewith, which are incorporated by reference herein in their entireties: Serial No. 10/195,794 entitled "Meniscus Regeneration Device and Method" (Attorney Docket No. 265280-71141, DEP-745); Serial No. 10/195,719 entitled "Devices from Naturally Occurring Biologically Derived Materials" (Attorney Docket No. 265280-71142, DEP-748); Serial No. 10/195,347 entitled "Cartilage Repair Apparatus and Method" (Attorney Docket No. 265280-71143, DEP-749); Serial No. 10/195,344 entitled "Unitary Surgical Device and Method" AMENDED

SHEET

L

DEP747/71152 -22- (Attorney Docket No. DEP-750); Serial No. 10/195,341 entitled "Hybrid Biologic/Synthetic Porous Extracellular Matrix Scaffolds" (Attorney Docket No.

265280-71144, DEP-751); Serial No. 10/195,606 entitled "Cartilage Repair and Regeneration Device and Method" (Attorney Docket No. 265280-71145, DEP-752); Serial No. 10/195,334 entitled "Cartilage Repair and Regeneration Scaffolds and Method" (Attorney Docket No. 265280-71180, DEP-763); and Serial No. 10/195,633 entitled "Porous Delivery Scaffold and Method" (Attorney Docket No. 265280- 71207, DEP-762), along with U.S. Patent Application Serial No. 10/172,347 entitled "Hybrid Biologic-Synthetic Bioabsorbable Scaffolds" which was filed on June 14, 2002. For example, for orthopaedic uses, it may be desirable to accompany or follow implantation with a treatment regime involving administering hyaluronic acid to the implantation site.

As can be seen from the forgoing description, the concepts of the present disclosure provide numerous advantages. For example, the concepts of the present disclosure provide for the fabrication of a porous implantable scaffold which may have varying mechanical properties to fit the needs of a given scaffold design.

For instance, the pore size and the material density may be varied to produce a scaffold having a desired mechanical configuration. In particular, such variation of the pore size and the material density of the scaffold is particularly useful when designing a scaffold which provides for a desired amount of cellular migration therethrough, while also providing a desired amount of structural rigidity. In addition, according to the concepts of the present disclosure, implantable devices can be produced that not only have the appropriate physical microstructure to enable desired cellular activity upon implantation, but also has the biochemistry (collagens, growth factors, glycosaminoglycans, etc.) naturally found in such ECMs.

Although it is believed that naturally occurring extracellular matrix provides advantages over purified extracellular matrix, it is contemplated that the teachings of the present disclosure can be applied to purified extracellular matrix as well. Thus, it is expected that the naturally occurring extracellular matrix could be purified prior to physically comminuting the extracellular matrix. This purification could comprise treating the naturally occurring extracellular matrix to remove substantially all materials other than collagen prior to physically comminuting the extracellular matrix. The purification could be carried out to substantially remove WEDED SHEET -23 glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins and nucleic acids (DNA and RNA).

While the disclosure is susceptible to various modifications and alternative forms, specific exemplary embodiments thereof have been shown by way of example in the drawings and has herein be described in detail. It should be understood, however, that there is no intent to limit the disclosure to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure.

There are a plurality of advantages of the present disclosure arising from the various features of the apparatus and methods described herein. It will be noted that alternative embodiments of the apparatus and methods of the present disclosure may not include all of the features described yet still benefit from at least some of the advantages of such features. Those of ordinary skill in the art may readily devise their own implementations of an apparatus and method that incorporate one or more of the features of the present disclosure and fall within the spirit and scope of the present disclosure.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form or suggestion that the prior art forms part of the common general knowledge in Australia.

09108/07,n'I 3850.pg2 3 2 3

Claims (14)

1. A method of making an implantable scaffold for repairing or regenerating body tissue, the method comprising the steps of: providing an extracellular matrix material selected from the group consisting of: small intestine submucosa, stomach submucosa, bladder submucosa, Srespiratory submucosa, alimentary submucosa, genital submucosa, and liver basement membrane; physically comminuting the extracellular matrix in the presence of a ligand to form a slurry comprising intertwined, ribbon-like, cohesive pieces of extracellular matrix; and lyophilizing the cohesive pieces of extracellular matrix to form an open cell foam of extracellular matrix.

2. The method of claim 1, further comprising purifying the extracellular matrix prior to physically comminuting the extracellular matrix.

3. The method of claim 1, further comprising treating the extracellular matrix to substantially remove glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins and nucleic acids.

4. The method of claim 1, further comprising treating the extracellular matrix to remove substantially all materials other than collagen prior to physically comminuting the extracellular matrix.

The method of claim 1, wherein physically comminuting the extracellular matrix in the presence of a liquid comprises suspending the extracellular matrix in the liquid.

6. The method of claim 1, wherein the lyophilizing step comprises lyophilizing the intertwined, ribbon-like, cohesive pieces of extracellular matrix and the liquid to form the open cell foam of extracellular matrix.

7. The method of claim 1, wherein the physically comminuting step comprises physically comminuting the extracellular matrix to form intertwined, ribbon-like, cohesive pieces of extracellular matrix having a length which is greater than each of the width and the thickness of the pieces.

8. A product made by the method of claim 1. 27/0208,at 13850.spccipgs,24 00 O cN

9. The product of claim 8, further comprising at least one of the Sfollowing: a bioactive agent; a biologically derived agent; cells; a biological lubricant; a biocompatible inorganic material; and a biocompatible polymer. C

10. The product of claim 8, wherein the open cell foam of extracellular matrix defines pores having a nominal pore size of 100-700 microns.

11. The product of claim 8, wherein the open cell foam of extracellular matrix defines pores having a nominal pore size of 300-700 microns. C

12. The product of claim 8, wherein the open cell foam of extracellular 0 matrix defines pores having a nominal pore size of less than 100 microns. N 10

13. A method of making an implantable scaffold for repairing or regenerating body tissue, substantially as hereinbefore described with reference to any one of the specific examples.

14. A product made by the method of claim 13. DATED this 2 7 th day of February, 2008 DEPUY PRODUCTS, INC. By their Patent Attorneys: CALLINANS 27/0208,at 13850.specipgs,25