WO2005005609A2

WO2005005609A2 - Multi-staged absorbable nonwoven structures for culturing pancreatic cells

Info

Publication number: WO2005005609A2
Application number: PCT/US2004/021044
Authority: WO
Inventors: Alireza Rezania; Mark Zimmerman
Original assignee: Liefscan, Inc.
Priority date: 2003-06-30
Filing date: 2004-06-29
Publication date: 2005-01-20
Also published as: WO2005005609A3; EP1644478A4; EP1644478A2

Abstract

The present invention provides an implantable biodegradable device containing a fibrous matrix, which matrix is constructed from fibers A and fibers B, wherein fibers A biodegrade faster than fibers B, and fibers A and fibers B are present in relative amounts and are organized such that the fibrous matrix possesses properties desirable for seeding and subsequent transplantation of cells bearing at least one characteristic of a pancreatic cell. The present invention also provides an implantable biodegradable device containing a fibrous matrix, which matrix has been seeded with cells bearing at least one characteristic of a pancreatic cell. Furthermore, the present invention provides a method for treating diabetes by implanting a device with seeded cells in a diabetic patient.

Description

Multi-Staged Absorbable Non oven Structures For Culturing Pancreatic Cells

FIELD OF THE INVENTION The present invention relates to biodegradable, implantable devices, e.g. tissue scaffolds which facilitate seeding and subsequent transplantation of cells bearing at least one characteristic of a pancreatic cell, including islets and pancreatic ductal cells, for treatment of diabetes. The present invention also relates to seeding cells bearing at least one characteristic of a pancreatic cell into the bioabsorbable, implantable medical device.

Furthermore, the present invention relates to a method for treating diabetes by implanting such device in a diabetic patient .

BACKGROUND OF THE INVENTION Diabetes mellitus (DM) results from destruction of beta cells (Type I) in the pancreas or from insensitivity of muscle or adipose tissues to the hormone insulin (Type II) . Current methods of treatment include diet and exercise, oral hypoglycemic agents, insulin injections, insulin pump therapy, and whole pancreas or islet transplantation. The most common treatment of DM involves daily injections of endogenous source such as porcine, bovine, or human insulin. This treatment prevents severe hyperglycemia and ketoacidosis, but does not completely normalize blood glucose levels. In addition, this treatment fails to prevent the complications of the disease process, including premature vascular deterioration. A second approach^' of treating diabetes is by transplantation of whole pancreas organ. However, transplanting a whole adult pancreas is a major, technically complex operation which requires aggressive immunosuppressive drugs. In addition, the applicability of this approach is restricted by the limited availability of cadaver pancreas. A third treatment method involves transplanting islets of Langerhans cells into a diabetic patient. There are many advantages of cellular over whole pancreas transplantation, including lower tissue mass, less invasive therapy, access to immunomanipulation, and engineering of graft composition. However, until recently, islet grafting has been generally unsuccessful due to aggressive immune rejection of islets. Recent reports (N. Eng. J. Mecl. 343:230-238, 2000; Diabetes, 50:710-719, 2001) indicate that a glucocorticoid- free immunosuppressive regimen can significantly benefit the patients with brittle type I diabetes. However, this regimen requires a large number of islets (~9000 islet equivalents/kg) to induce normoglycemia, and the patients are prone to renal complications and mouth ulcers. Thus, there has been an intense effort to devise strategies for islet cell transplantation that avoid the large doses of immunosuppressive drugs and use a commercially viable islet cell source. This has led to the concept of immunoisolation (de Vos et al . , Diabetologia, 45:159-173, 2002), which involves shielding of the islets with a selectively permeable membrane. The membrane allows passage of small molecules, such as nutrients, oxygen, glucose, and insulin, while restricting the passage of larger humoral immune molecules and immune cells. In theory, one could use an immunoisolation device with an abundant animal islet cell source, such as porcine islet cells, to treat DM. However, in practice this approach has had little success in large animal models or in clinic due to fibrosis of the device, limited oxygen supply within the pouch, and passage of small humoral immune molecules which ultimately lead to the loss of islet cells or islets. An alternative approach to immunoisolation is the creation of an immunologically privileged site by transplanting Sertoli cells into a nontesticular site in a mammal. See, e.g., US 5,849,285, US 6,149,907, and US 5,958,404. Insulin-producing islets can be subsequently introduced to such immunologically privileged site. The immunologically privileged site would allow transplantation of either human or animal derived islets. One of the drawbacks of this approach is that the transplanted Sertoli and islet cells are not physically restricted to site of transplantation. This can lead to migration of these cells to unwanted tissue sites and ultimately to the loss of islets. Furthermore, the immunologically privileged environment created by Sertoli cells is most effective only when the islets are in the close vicinity of the Sertoli

CC-2 - -1_ • The recent emergence of tissue engineering may offer alternative approaches to treat diabetes. Tissue engineering strategies typically utilize biomaterials in combination with cells and/or growth factors to develop biological substitutes that can ultimately restore or improve tissue function. Scaffold materials have been extensively studied as tissue templates, conduits, barriers, and reservoirs useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth (see, e.g., US5770417, US6022743, US5567612, and US5759830) . In preparing a scaffold, it is important to match the rate of degradation of the scaffold to the rate of proliferation and matrix formation of seeded cells in vivo . It is also essential that the degradation products of the scaffold do not negatively affect cell function. Previous attempts at using nonwoven fibrous structures have been primarily focused on a polyglycolic acid rich matrix which starts degrading a few weeks following transplantation. There remains a need for a three-dimensional construct that can be seeded with a large number of insulin-producing cells, retains the majority of the cells following implantation, and provides a matrix containing fibers with various degradation profiles. The biodegradable construct of the present invention provides such a three-dimensional porous matrix.

SUMMMIY OF THE INVENTION In one aspect, the present invention provides a biodegradable, bioabsorbable, implantable medical device containing a fibrous matrix suitable for seeding cells bearing at least one characteristic of a pancreatic cell. The device of the present invention contains a fibrous matrix made from fibers A and fibers B, wherein fibers A biodegrade faster than fibers B, and wherein fibers A and fibers B are present in such relative amounts and combined in such fashion that the resulting matrix possesses properties desired for seeding cells bearing at least one characteristic of a pancreatic cell to be used in the treatment of diabetes . In a preferred embodiment of the invention, fibers A are formed from a copolymer of PGA and PLA, where PGA constitutes from about of 50 to about 95 weight percent and PLA constitutes from about 5 to about 50 weight percent; and fibers B are formed from a copolymer of PGA and PLA, where PGA constitutes from about 2 to about 50 weight percent and PLA constitutes from about 50 to about 98 weight percent. Preferably, the weight ratio of fibers A versus fibers B ranges from about 19:1 to about 1:19, more preferably from about 9:1 to about 1:9. In another aspect of the present invention, the bioabsorbable, implantable medical device has been seeded with cells bearing at least one characteristic of a pancreatic cell. In still another aspect, the present invention provides a method for treating diabetes by implanting in a diabetic patient a bioabsorbable, implantable medical device, seeded with cells bearing at least one characteristic of a pancreatic cell. BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts mice islets seeded within a 100% 90/10 PGA/PLA nonwoven mat. The cells were viable as evidenced by positive fluorescent staining for Calcein. Figure 2 depicts rat ductal cells seeded within a 100% 90/10 PGA/PLA nonwoven mat. The cells were viable as evidenced by positive fluorescent staining for Calcein.

Detailed Description of the Invention The present invention provides a biodegradable bioabsorbable, implantable medical device containing a fibrous matrix suitable for incorporating or seeding cells bearing at least one characteristic of a pancreatic cell. The present invention also provides a bioabsorbable, implantable medical device containing a fibrous matrix incorporated with cells bearing at least one characteristic of a pancreatic cell, as well as methods for making such device incorporated with the desired cells. Furthermore, the present invention provides a method for treating diabetes by implanting such device in a diabetic patient. In one aspect, the present invention provides a bioabsorbable, implantable medical device containing a fibrous matrix suitable for seeding cells bearing at least one characteristic of a pancreatic cell. The terms "matrix", "scaffold" and "construct" are used herein interchangeably and refer to a three-dimensional porous material that is suitable for seeding cells bearing at least one characteristic of a pancreatic beta cell and is biocompatible, biodegradable and resorbable by the body. By "biocompatible" is meant that the matrix of the present invention does not substantially adversely affect any desired characteristics of the cells to be seeded within the matrix, or the cells or tissues in the area of a living subject where the device is to be implanted, or any other areas of the living subject. By "biodegradable" or "absorbable" is meant that the matrix will be gradually degraded or absorbed after the device made of the matrix is delivered to a site of interest inside the body of a living subject. By "a living subject" is meant to include any mammalian subject, including a primate, porcine, canine or urine subject, and particularly a human subject. The matrix of the present device permits tissue ingrowth in order for the growing tissue to replace the resorbing matrix. The matrix of the present device is also capable of providing and maintaining structural support required for a particular device for so long as is required to affect the repair and/or regeneration of the tissue, including that time in which the matrix is being resorbed by the body. In other words, the device of the present invention has a desirable rate of resorption which approximates the rate of replacement of the fibrous matrix by tissue. Thus, devices of the present invention advantageously balance the properties of biodegradability, resorption, structural integrity over time and the ability to facilitate tissue in-growth. According to the present invention, the matrix is constructed from at least two different fibrous materials, e.g. fibers, one of which iodegrades faster than the other. The fibers are of such composition and structure and are combined in such a way, with respect to both relative fiber amounts and matrix structure, to enhance retention and function of seeded cells bearing at least one characteristic of a pancreatic cell, especially a pancreatic beta cell, and to facilitate infiltration and ingrowth of tissue. Biodegradable materials suitable for use in the preparation of fibers and fibrous matrices include various biodegradable polymers such as aliphatic polyesters, poly (amino acids), copoly (ether-esters) , polyalkylene oxalates, polyamides, poly (iminocarbonates) , polyorthoesters, polyoxaesters, polyamidoesters, poly (anhydrides) , polyphosphazenes, or copolymers or blends thereof. Certain polyoxaester copolymers can further include amine groups. According to the present invention, for seeding insulin-producing cells, fibers and fibrous matrices are preferably made of biodegradable polymers selected from polylacetic acid (PLA) , polyglycolic acid (PGA) , ε- polycaprolactone (PCL) , polydioxanone (PDO) , polyoxaesters, or copolymers or blends thereof. The scaffold, matrix or construct of the present device is characterized by having interconnecting pores or voids, which facilitate the transport of nutrients and/or invasion of cells into the scaffold. The interconnected voids range in size from about 20 to 500 microns, preferably 50 to 400 microns, and constitute about 70 to 95 percent of the total volume of the construct. The range of the void size in the construct can be manipulated by modifying process steps during the preparation of the construct. According to the present invention, the fibrous matrix of the present device is characterized as an organized network in the form of threads, yarns, nets, laces, felts and nonwovens, or a combination of these various forms. The fibrous matrix of the present device is constructed by combining at least two different bioabsorbable fibrous materials, e.g. fibers or filaments, one of which biodegrades faster than the other. The terms "fibers A" and "fibers B" used herein refer to the two different types of fibers used to make the matrix of the present device. The fibers may be solid, or hollow, or may be of a sheath/core construction. Methods for making individual filaments are well known to those skilled in the art. For example, filaments may be co-extruded to produce a sheath/core construct, where each filament contains a sheath of biodegradable polymer that surrounds one or more cores made of another biodegradable polymer. Filaments with a fast-absorbing sheath surrounding a slow-absorbing core may be desirable in instances where extended support is necessary for tissue ingrowth. Alternatively, filaments can be formed by coating biodegradable fibers, e.g., biodegradable glass fibers, with a biodegradable polymer. In one embodiment of the invention, a continuous multifilament yarn (Yarn A) is formed from a copolymer of PGA and PLA, where PGA constitutes from about of 50 to about 95 weight percent and PLA constitutes from about 5 to about 50 weight percent. Another continuous multifilament yarn (Yarn B) is formed from a copolymer of PGA and PLA, where PGA constitutes from about 2 to about 50 weight percent and PLA constitutes from about 50 to about 98 weight percent. As a result of the differences in the copolymer composition, Yarn A degrades faster than Yarn B. Both types of filaments are of a diameter from about 2 to about 200 microns, preferably from about 5 to about 100 microns. Yarn A and Yarn B are both cut into uniform lengths between 1/4" and 2". Fiber in this form is known as "staple fiber". Predetermined amounts of staple fiber produced from Yarn A and Yarn B are combined to form the matrix. The predetermined amounts of fibers from Yarn A and Yarn B, respectively, may vary depending upon, for example, the composition of the respective fibers, the construction of the respective fibers, and the particular organization of the respective fibers, which determines the structure of the fibrous matrix produced from the organized fibers. The relative amounts of the two types of fibers are selected in order to produce a fibrous matrix with the desired properties for seeding cells bearing at least one characteristic of a pancreatic cell. For example, the selection is such that the matrix produced not only possesses the structural integrity required for tissue repair and/or regeneration, but also enhances tissue growth and infiltration into the matrix. In addition, the selection is such that the rate of resorption of the matrix approximates the rate of replacement of the matrix by tissue, thus preserving the structural integrity of the implant throughout the treatment period. Preferably, the weight ratio of fibers A (e.g., staple fiber from Yarn A) versus fibers B (e.g., staple fiber from Yarn B) ranges from about 19:1 to about 1:19, more preferably from about 9:1 to about 1:9. The two different types of fibers can be combined by any means convenient and suitable, e.g., by a wet lay process or a dry lay process. The wet lay method has been described in "Nonwoven Textiles," by Radko Krcma, Textile Trade Press, Manchester, England, 1967 ppl75-176, the content of which is incorporated herein by reference. When a wet lay process is used, predetermined amounts of fibers A and fibers B are dispersed into water. Additional processing aids, such as viscosity modifiers, surfactants and defoaming agents, may be added to the water. The purpose of such processing aids is to allow a uniform dispersion of the filaments within the water without causing foaming, which in turn may cause defects in the final product. A bioabsorbable thermoplastic polymer or copolymer, such as Polycaprolactone (PCL) in powder form, also may be added to the water. This powder possesses a low melting temperature and acts as a binding agent later in the process to increase the tensile strength and shear strength of the nonwoven structure, or fibrous matrix. The preferred particulate powder size of PCL is in the range of 10-500 μm in diameter, and more preferably 10-150 μm in diameter. Additional binding agents include a biodegradable polymeric binders selected from the group consisting of polylactic acid, polydioxanone and polyglycolic acid. Once the fibers are uniformly dispersed within the water, the mixture is drained through a screen. The screen allows water to pass through, but traps the fiber. If PCL powder is included in the mixture, some of the powder is trapped as well. After the water has drained through the screen, the mat of fibers is removed. The mat containing PCL powder fibers is then subjected to heat in order to melt the PCL. The melt temperature range is between about 60°C and about 100°C, preferably between 60-80°C. It is crucial to perform this step at a temperature that is above the melting point of PCL powder or similar binding agent, and below the softening point of the fibers. This is necessary to avoid damaging the staple fibers. The powder melts, flows around the filaments and cools to a solid state. When the molten powder returns to a solid state, some of the points where the filaments intersect are encapsulated in solid polymer and locked in place. The powder thus acts as a binding agent, increasing the strength of the matrix. The matrix is rinsed overnight in water, followed by another overnight incubation in ethanol to remove any residual chemicals or processing aids used during the manufacturing process. The matrix may then be sterilized by a number of standard techniques, such as exposure to ethylene oxide or gamma radiation. When a dry lay process is used to form the matix, predetermined amounts of the two different types of fibers are opened and carded on standard nonwoven machinery, resulting in webbed staple fibers. The webbed staple fibers are needle punched to form a dry lay needle-punched, fibrous nonwoven mat or matrix. The nonwoven fibrous matrices, made by either a wet lay process or a dry lay process, may be formed into different shapes, or configurations, such as disks, rectangles, squares, stars and tubes, by thermal or non-thermal punching of the nonwoven sheets with dies of appropriate shape and dimension. In yet another embodiment, the fibrous matrix has a gradient structure. For example, a fibrous implant may have a gradual or rapid, but continuous, transition from rapidly degrading fibers at the periphery of the implant, to slowly degrading fibers at the center, relatively speaking. Alternatively, the transition may occur between the top of the matrix to the bottom of the matrix. One profile for transition from rapidly degrading fibers to slowly degrading fibers may be, for instance, from about 100% rapidly degrading fibers, to about 75% rapidly degrading fibers/25% slowly degrading fibers, to about 50% rapidly degrading fibers/50% slowly degrading fibers, to about 25% rapidly degrading fibers/75% slowly degrading fibers, to about 100% slowly degrading fibers, proceeding from the periphery of the implant to the center. In yet another embodiment, the three-dimensional matrices of the present invention may be coated with a biodegradable, fibrous and porous polymer coating, e.g. a sheet, preferably produced by an electrostatic spinning process. The electrostatically spun polymer coating can provide the nonwoven matrices with enhanced mechanical properties and the ability to hold sutures. Exemplary biodegradable polymeric coats may be prepared from polymers selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone and copolymers thereof. It should be recognized that the device of the present invention may include a homogenous mixture of filaments in the form of a sheet, or nonwoven matrix. However, the mixture need not be homogenous and the final form need not be a sheet. Devices containing a non-homogenous mixture of filaments may be desirable in applications where total absorption time and/or loss of strength over time varies throughout the material. Therefore, in yet another embodiment, a multi-layered device composed of a first layer in which the majority of filaments are prepared from a (90/10) PGA/PLA copolymer, and a second layer in which the majority of filaments are prepared from a (95/5) PLA/PGA copolymer. Such device, when implanted, has a first, e.g. top, layer that is absorbed more quickly than the second, e.g. bottom, layer. Similar structures may be produced in any shape. In other embodiments, cylinders or prisms with fast (or slow) absorbing cores may be produced during a nonwoven process by segregating the different filaments during the forming process . In yet another embodiment of the invention, the porous nonwoven matrix can be chemically crosslinked or combined with hydrogels, such as alginates, hyaluronic acid, collagen gels, and poly (N-isopropylacryalmide) . In another embodiment of the invention, the matrix may be modified, either through physical or chemical means, to contain biological or synthetic factors that promote attachment, proliferation, differentiation, and/or matrix synthesis of targeted cell types. The bioactive factors can be included in the matrix for controlled release of the factor to elicit a desired biological function. Growth factors, extracellular matrix proteins, and biologically relevant peptide fragments that can be used with the matrices of the current invention include, but are not limited to, members of TGF-β family, including TGF-βl, 2, and 3, bone morphogenic proteins (BMP-2, -4, 6, -12, -13 and -14), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -8, -10), angiogen, erythropoiethin, placenta growth factor, angiogenic factors such as vascular endothelial cell-derived growth factor (VEGF) , glucacgon- like peptide I, exendin-4, pleiotrophin, endothelin, parathyroid hormone, stem cell factor, colony stimulating factor, tenascin-C, tropoelastin, thrombin-derived peptides, anti-rejection agents; analgesics, anti-inflammatory agents such as acetoaminophen, anti-apoptotic agents, statins, cytostatic agents such as Rapamycin and biological peptides containing cell- and heparin-binding domains of adhesive extracellular matrix proteins such as fibronectin and vitronectin. The biological factors can be obtained either through a commercial source, isolated and purified from a tissue or chemically synthesized. In another aspect of the present invention, the three- dimensional device of the present invention is incorporated or seeded with cells bearing at least one marker characteristic of a pancreatic cell. The term "cells", as used herein, refers to isolated cells, cells lines (including cells engineered in vi tro) , any preparation of living tissue, including primary tissue explants and preparations thereof. By "pancreatic cell" is meant to include cells of both endocrine and exocrine pancreatic tissues. The endocrine pancreas is composed of hormone-producing cells arranged in clusters or islets of Langerhans . Of the four main types of cells that form the islets ("islet cells") , the alpha cells produce glucagons, the beta cells produce insulin, the delta cells produce somatostatin, and the PP cells produce pancreatic polypeptide (PP). The endocrine pancreas includes the pancreatic acini and the pancreatic duct. Pancreatic acinar cells synthesize a range of digestive enzymes. Ductal cells secret bicarbonate ions and water in response to the hormone secreted from the gastrointestinal tract. Thus, "pancreatic cells" as used herein include alpha cells, beta cells, delta cells, PP cells, acinar cells, ductal cells or other cells in a mammalian pancreas. Markers characteristic of a pancreatic cell include the expression of cell surface proteins or the encoding genes, the expression of intracellular proteins or the encoding genes, cell morphological characteristics, and the production of secretory products such as glucagons, insulin and somatostatin. Those skilled in the art will recognize that known immunofluorescent, immunochemical, polymerase chain reaction, in si tu hybridization, Northern blot analysis, chemical or radiochemical methods can readily ascertain the presence of absence of a islet cell specific characteristic. In a preferred embodiment, the device of the present invention is incorporated with pancreatic islets. The term "islets" as used herein includes both islets isolated from a mammalian pancreas as masses formed by alpha cells, beta cells, delta cells and PP cells, and islets formed in vitro from isolated or engineered islet cells or cells that bear at least one marker, preferably two or more markers, characteristic of an islet cell. In another preferred embodiment, the present device is seeded with cells engineered in vi tro having at least one marker characteristic of a pancreatic islet cell, preferably, a pancreatic beta cell. Markers characteristic of pancreatic beta cells have been described, and include the expression of the Pdxl , Ngn3 _f Hlxb9 , Nkx6 , Isll , Pax 6 , Neurod, Hnfla _r Hnf6 genes and the encoded proteins, and the secretion of insulin, among others. Such cells can be produced in vi tro by, e.g., differentiating adipose stromal cells . In still another preferred embodiment, the device of the present invention is incorporated with pancreatic ductal cells . Other cells, such as Sertoli cells, can be co-seeded with the cells having at least one marker characteristic of a pancreatic cell. To introduce the cells into a scaffold, the scaffold is contacted and incubated with a suspension containing the cells, or clusters of cells (e.g., islets) or the tissue preparation to be seeded. The incubation can be performed for a short period of time (< 1 day) just prior to implantation, or for longer (> 1 clay) period to allow for enhanced cell attachment and matrix synthesis within the nonwoven scaffold prior to implantation. In a further aspect, the present invention provides a method for treating a human diabetic patient by implanting at a site in the patient, a device of the present invention which is seeded with cells bearing at least one characteristic of a pancreatic cell. The cells seeded in the device for treating the patient can be of an autologous, allogenic or xenogenic origin. The site where the device can be implanted can be any clinically relevant site, such as the liver, the natural pancreas, the renal subcapsular space, the mesentery, the omentum, a subcutaneous pocket, or the peritoneum. The site can be an immunologically privileged site, either naturally existing or created using, e.g., Sertoli cells. The following examples are merely illustrative of the principles and practices of the present invention and are not intended to limit the scope of the invention. Example 1 Preparation Of Three-Dimensional Nonwoven Fibrous Matrices Or Mats A needle-punched nonwoven mat (2 mm in thickness) composed of 90/10 PGA/PLA fiber and 95/5 PLA/PGA was made as described below. A copolymer of PGA/PLA (90/10) was melt- extruded into a continuous multifilament yarn by conventional methods of making yarn and subsequently oriented in order to 'increase strength, elongation and energy required to rupture. The same method was used to prepare yarns of a 95/5 PLA/PGA copolymer. The yarns comprised filaments of approximately 20 microns in diameter. These yarns were then cut and crimped into uniform 2-inch lengths to form a 2-inch staple fiber. A dry lay needle-punched nonwoven mat was then prepared utilizing the 90/10 PGA/PLA copolymer staple fiber and the 95/5 PLA/PGA fiber. The staple fibers were opened and carded on standard nonwoven machinery. The resulting mat was in the form of webbed staple fibers. The webbed staple fibers were needle punched to form the dry lay needle- punched, fibrous nonwoven mat. A number of dry lay nonwoven matrices were then prepared utilizing fiber selection as follows: (a) 100% of fiber prepared from the (90/10) PGA/PLA copolymer; (b) 100% of fiber prepared from the (95/5) PLA/PGA copolymer; (c) a fiber mixture of 50% by weight of fibers prepared from the (95/5) PLA/PGA copolymer and 50% by weight of fibers prepared from the (90/10) PGA/PLA copolymer; d) 100% of the fiber prepared from PDO polymer. Example 2 Seeding Of Murine Islets Within Nonwoven Scaffolds Islets were isolated from Balb/c mice by collagenase digestion of the pancreas and Ficoll density gradient centrifugation followed by hand picking of islets. Nonwoven scaffolds comprised of 100% 90/10 PGA/PLA, 100% PDO, or 50:50 mix of 100% 90/10 PGA/PLA: 95/5 PLA/PGA, were prepared as described in Example 1 and seeded with 500 fresh islets and cultured for 1 day in Ham' s-FlO medium (Gibco Life Technologies, Rockville, MD) supplemented with bovine serum albumin (0.5%), nicotinamide (10 mM) , D-glucose (10 mM) , L-glutamine (2 mM) , IBMX (3-Isobutyl-l- methylxanthine, 50 mM) , and penicillin/streptomycin. The islets residing in the scaffolds were stained with calcein and ethidium homodimer (Molecular Probes, Oregon) to determine the viability of the seeded cells. Majority of the islets stained positive for calcein, indicating viable cells within the lumen of the pouch. Figure 1 depicts viable islets seeded within a 100% 90/10 PGA/PLA nonwoven scaffold.

Example 3 Seeding Of Murine Islets Within Nonwoven Scaffolds And Subsequent Implantation Male Balb/c mice recipients were (8-10 wk old) rendered diabetic by intraperitoneal injection of Streptozotocin (Sigma, St. Louis, MO) at a dose of 250mg/Kg body weight. Diabetes was confirmed by two consecutive days of nonfasting blood glucose levels exceeding 300 mg/dl. Pancreatic islets were isolated from Balb/c mice (8-12 wk old) and seeded into nonwoven scaffolds comprised of 100% 90/10 PGA/PLA fibers. The cells were seeded for ~2 hrs on the scaffolds prior to transplantation. One scaffold was transplanted into the epididymal fat pad of each of the five recipient mice (N=5) . In a control group (N=5), the recipients were transplanted with 500 syngeneic islets suspended in a MATRIGEL gel and transplanted into the epididymal fat pad. Recipient mice were anesthetized with an intraperitoneal injection of a Ketamine/Xylazine cocktail. The cell seeded scaffolds (8 mm in diameter) or MATRIGEL gel were wrapped with the thin layer of the epididymal fat pad and sutured to the surrounding fat tissue. The incision was sutured and the skin closed with surgical clips. Tail vein blood was collected every 2 days to measure non-fasting blood glucose levels. Grafts were removed at various times to confirm a return to hyperglycemia and also for histological analysis. Following 60 days of transplantation, 5 out of 5 animals transplanted with the nonwoven scaffold were normoglycemic; whereas all of the mice transplanted with islets seeded in the MATRIGEL gel reverted to hyperglycemia after 6 days of transplantation. Upon removal of the graft from one of the mice transplanted with a cell-seeded scaffold, the blood glucose levels reverted to hyperglycemic levels (>500 mg/dl) .

Example 4 Seeding Of Murine Pancreatic Ducts Within A Nonwoven Scaffold Pancreatic ducts were isolated from Sprague Dawley rats (SD) by collagenase digestion of the pancreas and Ficoll density gradient centrifugation followed by hand picking of pancreatic ducts. About 100 ducts were seeded onto a nonwoven scaffold comprised of 100% PGA/PLA fibers. The ducts were cultured on the scaffold (5 mm in diameter) for 4 wks in Ham' s-FlO medium (Gibco Life Technologies, Rockville, MD) , supplemented with bovine serum albumin (0.5%), nicotinamide (10 mM) , D-glucose (10 mM) , L-glutamine (2 mM) , IBMX (3-Isobutyl-l-methylxanthine, 50 mM) , and penicillin/streptomycin. The cells residing in the scaffolds were stained with calcein and ethidium homodimer (Molecular Probes, Oregon) to determine the viability of the seeded cells. Majority of the cells stained positive for calcein, indicating viable cells within the lumen of the pouch. Figure 2 depicts viable ductal cells seeded within a 100% 90/10 PGA/PLA nonwoven scaffold.

Claims

We claim :

1. An implantable, biodegradable device comprising a fibrous matrix, said fibrous matrix comprising fibers A and fibers B, wherein fibers A biodegrade faster than fibers B, and wherein fibers A and B are present in relative amounts and are organized such that the fibrous matrix is provided with properties useful for seeding and subsequent transplantation of cells bearing at least one characteristic of a pancreatic cell.

2. The device of claim 1 wherein the weight ratio of fibers A to fibers B is the range of from about 9:1 to about 1:9.

3. The device of claim 1 wherein the porosity of the fibrous matrix is effective to facilitate uniform tissue growth therein.

4. The device of claim 3 wherein the pores of said matrix are interconnected, range in size from about 20 microns to about 500 microns, and constitute from about 70 percent to about 95 percent of the fibrous matrix.

5. The device of claim 1 wherein fibers A and fibers B comprise a biodegradable polymer.

6. The device of claim 5 wherein the biodegradable polymer is selected from the group consisting of aliphatic polyesters, poly(amino acids), copoly (ether-esters) , polyalkylene oxalates, polyamides, poly (iminocarbonates) , polyorthoesters, polyoxaesters, polyamidoesters, poly (anhydrides) , polyphosphazenes and biopolymers .

7. The device of claim 5 wherein the fibrous matrix comprises from about 50 to about 99 weight percent of fibers A made from a polyglycolic acid/polylactic acid (PGA/PLA) copolymer, and from about 50 to about 1 weight percent of fibers B made from a polylactic acid/polyglycolic acid (PLA/PGA) copolymer.

8. The device of claim 7 wherein the PGA/PLA copolymer comprises about 90 percent PGA and about 10 percent PLA, and the PLA/PGA copolymer comprises about 95 percent PLA and about 5 percent PGA.

9. The device of claim 1 wherein the fibrous matrix comprises an organized network selected from the group consisting of threads, yarns, nets, laces, felts and nonwovens .

10. The device of claim 1 wherein the fibrous matrix comprises a configuration selected from the group consisting of a disk, a rectangle, a square, a tube and a star.

11. The device of claim 1 wherein the diameters of fibers A and fibers B range from about 5 microns to about 100 microns .

12. The device of claim 1 wherein fibers A and fibers B are bonded together by a biodegradable polymeric binder.

13. The device of claim 12 wherein the biodegradable polymeric binder is selected from the group consisting of polycaprolactone, polylactic acid, polydioxanone and polyglycolic acid.

14. The device of claim 1 wherein the fibrous matrix comprises a gradient structure.

15. The device of claim 1 wherein said fibrous matrix comprises a continuous transition from fibers A at the periphery of the device to fibers B at the center of the device.

16. The device of claim 1 wherein said fibrous matrix comprises a continuous transition from fibers A at the top of the device to fibers B at the bottom.

17. The device of claim 1 wherein the fibrous matrix further comprises a biodegradable, fibrous polymeric coating.

18. The device of claim 17 wherein the biodegradable polymeric coating is selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone and copolymers thereof.

19. The device of claim 1 wherein the fibrous matrix is chemically crosslinked or combined with hydrogels.

20. The device of claim 1 wherein the fibrous matrix is coated with an adhesive biological factor selected from the group consisting of fibronectin, vitronectin, hyaluronic acids, glycosaminoglycans, collagens, peptide fragments, pleiotrophin, laminin, endothelin and tenascin-C.

21. The device of claim 1 wherein the fibrous matrix is coated with a growth factor selected from the group consisting of members of TGF-β family, bone morphogenic proteins, fibroblast growth factors-1 and -2, platelet- derived growth factor-AA, and -BB, platelet rich plasma and vascular endothelial cell-derived growth factor (VEGF) .

22. The device of claim 1 wherein said fibrous matrix comprises a first layer of filaments, the majority of which are prepared from a 90/10 PGA/PLA copolymer, and a second layer of filaments, the majority of which are prepared from a 95/5 PLA/PGA copolymer.

23. The device of claim 1 wherein fibers A and fibers B comprise a sheath/core construction, where each filament comprises a sheath of a first biodegradable polymer surrounding one or more cores comprising a second biodegradable polymer.

24. The implantable, biodegradable device according to any of claims 1-23, wherein said fibrous matrix is seeded with cells bearing at least one marker characteristic of a pancreatic cell.

25. The device of claim 24, wherein said pancreatic cell is selected from an alpha cell, a beta cell, a delta cell, a PP cell, an ancinar cell or a ductal cell.

26. The device of claim 24, wherein said cells are pancreatic islets or pancreatic ductal cells, isolated from a mammalian pancreas.

27. The device of claim 26, wherein said mammalian pancreas is a primate, canine, porcine or murine pancreas.

28. The device of claim 27, wherein said primate is human.

29. A method of treating a human diabetic patient by implanting at a site in the patient, the device of claim 24.

30. The method of claim 29, wherein the cells seeded in said device have at least one marker characteristic of a pancreatic cell and said pancreatic cell is selected from an alpha cell, a beta cell, a delta cell, a PP cell, an ancinar cell or a ductal cell.

31. The method of claim 30, wherein said cells seeded in said device are islets isolated from a mammalian pancreas.

32. The method of claim 31, wherein said mammalian pancreas is a primate, canine, porcine or murine pancreas.

33. The method of claim 31, wherein the cells seeded in the device are of an autologous, allogenic or xenogenic origin.

34. The method of claim 29, wherein said site is selected from the liver, the natural pancreas, the renal subcapsular space, the mesentery, the omentum, a subcutaneous pocket, or the peritoneum.