Title:  Porous biodegradable polymeric materials for cell transplantation

United States Patent:  6,689,608

Issued:  February 10, 2004

Inventors:  Mikos; Antonios G. (Houston, TX); Langer; Robert S. (Newton, MA); Vacanti; Joseph P. (Winchester, MA); Griffith; Linda G. (Cambridge, MA); Sarakinos; Georgios (Maastricht, NL)

Assignee:  Massachusetts Institute of Technology (Cambridge, MA)

Appl. No.:  669760

Filed:  September 26, 2000

Abstract

Polymeric materials are used,to make a pliable, non-toxic, injectable porous template for vascular ingrowth. The pore size, usually between approximately 100 and 300 microns, allows vascular and connective tissue ingrowth throughout approximately 10 to 90% of the matrix following implantation, and the injection of cells uniformly throughout the implanted matrix without damage to the cells or patient. The introduced cells attach to the connective tissue within the matrix and are fed by the blood vessels. The preferred material for forming the matrix or support structure is a biocompatible synthetic polymer which degrades in a controlled manner by hydrolysis into harmless metabolites, for example, polyglycolic acid, polylactic acid, polyorthoester, polyanhydride, or copolymers thereof. The rate of tissue ingrowth increases as the porosity and/or the pore size of the implanted devices increases. The time required for the tissue to fill the device depends on the polymer crystallinity and is less for amorphous polymers versus semicrystalline polymers. The vascularity of the advancing tissue is consistent with time and independent of the biomaterial composition and morphology.

SUMMARY OF THE INVENTION

Polymeric materials are used to make a pliable, non-toxic, implantable porous template for vascular ingrowth and into which cells can be injected. The pore size, usually between approximately 100 and 300 microns, allows vascular and connective tissue ingrowth throughout approximately 10 to 90% of the matrix following implantation, and the injection of cells uniformly throughout the implanted matrix without damage to the cells or patient. The introduced cells attach to the connective tissue within the matrix and are fed by the blood vessels. The preferred material for forming the matrix or support structure is a biocompatible synthetic polymer which degrades in a controlled manner by hydrolysis into harmless metabolites, for example, polyglycolic acid, polylactic acid, polyorthoester, polyanhydride, or copolymers thereof. The elements of these materials can be overlaid with a second material to enhance cell attachment. The polymer matrix is configured to provide access to ingrowing tissues to form adequate sites for attachment of the required number of cells for viability and function and to allow vascularization and diffusion of nutrients to maintain the cells initially implanted.

As described in the examples, highly-porous biocompatible and biodegradable polymers forms were prepared and implanted in the mesentery of rats for a period of 35 days to study the dynamics of tissue ingrowth and the extent of tissue vascularity, and to explore their potential use as substrates for cell transplantation. The advancing fibrovascular tissue was characterized from histological sections of harvested devices by image analysis techniques. The rate of tissue ingrowth increased as the porosity and/or the pore size of the implanted devices increased. The time required for the tissue to fill the device depended on the polymer crystallinity and was less for amorphous polymers versus semicrystalline polymers. The vascularity of the advancing tissue was consistent with time and independent of the biomaterial composition and morphology.

DETAILED DESCRIPTION OF THE INVENTION

As described in more detail below, the present invention is the preparation and use of synthetic, biocompatible, biodegradable polymeric matrices for implantation into a patient, followed by seeding of cells. In the preferred method, the matrix is implanted, vascularized by ingrowth of capillaries and connective tissue from the recipient, then the cells are seeded.

Methods described herein relate to the manufacture of polymeric membranes having a thickness that is preferably between 500 and 2000 microns, formed of biocompatible, preferably biodegradable, synthetic polymers, which have interconnected interstices with a pore size range between greater than 0 and 500 microns, most preferably for seeking of cells, in excess of 100 to 150 microns.

Poly(L-actic acid) (PLLA) porous membranes were prepared with sodium chloride, sodium tartrate, and sodium citrate particles, without any heat treatment to determine the effect of the initial salt weight faction and particle size on the porosity, median pore diameter, and surface/volume ratio. The membrane porosity increased monotonically with the initial salt weight fraction, and was independent of the salt particle size, as shown by FIG. 6. By varying the initial sodium chloride weight fraction from 0.5 to 0.9, as shown by FIG. 6A, the porosity of the PLLA membranes increased from 0.45 to 0.93 when salt particles between 0 and 53 microns were used, from 0.48 to 0.92 when salt particles between 53 and 106 microns were used, and from 0.48 to 0.91 when salt particles between 106 and 150 microns were used. A similar dependence was also observed for PLLA membranes prepared with sodium tartrate or sodium citrate particles.

As demonstrated by the examples, the preferred matrix is an amorphous or semicrystalline polymer such as poly(lactic acid-glycolic acid) having a porosity (defined herein as the fraction of void volume) in the range of 50 to 95% and median pore diameter of 100 to 300 microns, more preferably a median pore size between approximately 150 and 250 microns and a porosity between 75 and 95%, which allows vascular ingrowth and the introduction of cells into the matrix without damage to the cells or patient. As used herein, an amorphous polymer is not crystallized; a semi-crystallized polymer is where the degree of crystallinity (fraction of mass of crystallites) is less than 100%. In general, the greater the porosity, the faster the rate of ingrowth of capillaries and connective tissue. The rate of ingrowth is also increased by pre-wetting of the matrix with a surfactant or alcohol followed by saline wash. At this time the most preferred embodiment is an amorphous polylactic acid having 90% porosity and 200 micron median pore diameter.

Polymers

Biodegradable, biocompatible polymers that degrade by hydrolysis can provide temporary scaffolding to transplanted cells and by so doing allow the cells to secrete extracellular matrix enabling a completely natural tissue replacement to occur. Their macromolecular structure is selected so that they are completely degraded and eliminated as the need for an artificial support diminishes. Polymer templates for use in cell transplantation must be highly porous with large surface/volume ratios to accommodate a large number of cells. In addition to being biocompatible, they must promote cell adhesion and allow retention of differentiated function of attached cells. The formation of a vascularized bed within the matrix for cell attachment results in an adequate supply of nutrients to transplanted cells which is essential to their maintenance. They must also be resistant to compression and yet semi-flexible to provide adequate support without discomfort within the recipient.

Studies have been performed with poly(vinyl alcohol), although this material demonstrates some of the drawbacks of using non-degradable materials which may cause formation of a fibrous scar or tissue infection.

Examples of useful polymers include poly(lactic acid), poly(glycolic acid), copolymers thereof, polyanhydrides, polyorthoesters, and polyphosphazines. These are all available commercially or can be manufactured by standard techniques.

In some embodiments, attachment of the cells to the polymer is enhanced by coating the polymers with compounds such as basement membrane components, agar, agarose, gelatin, gum arabic, collagens types I, II, III, IV, and V, fibronectin, laminin, glycosaminoglycans, mixtures thereof, and other materials known to those skilled in the art of cell culture.

All polymers for use in the matrix must meet the mechanical and biochemical parameters necessary to provide adequate support for the cells with subsequent growth and proliferation. The polymers can be characterized with respect to mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy, with respect to toxicology by initial screening tests involving Ames assays and in vitro teratogenicity assays, and implantation studies in animals for immunogenicity, inflammation, release and degradation studies.

In a preferred embodiment, the matrix contains catheters for injection of the cells into the interior of the matrix after implantation and ingrowth of vascular and connective tissue. Catheters formed of medical grade silastic tubing of different diameters and of differing exit ports to allow even distribution of cells throughout the matrix, as described in the following examples, are particularly useful. Other methods can also be used, such as molding into the matrix distribution channels from the exterior into various parts of the interior of the matrix, or direct injection of cells through needles into interconnected pores within the matrix.

Shaping of the Matrix

The matrix is formed by methods such as casting a polymer solution containing salt crystals into a mold, then leaching out the salt crystals after the polymer is hardened, to yield a relatively rigid, non-compressible structure. This method is described in more detail in co-pending application U.S. Ser. No. 08/012,270, filed Feb. 1, 1993, the teachings of which are incorporated herein.

The polymer matrix can be heated after removal of the solvent to decrease or increase the crystallinity of the matrix. Membranes having high crystallinity, i.e., greater than 20%, will be stronger and will therefore degrade slower than matrixes having reduced crystallinity. To obtain matrices with a lower crystallinity and faster rate of degradation after implantation, the salt and polymer mixture is heated at a temperature that will melt the polymer without affecting the particles. Preferably, the mixture is heated at a temperature between 15^oC. and 20^oC. higher than the melting temperature T_m of the polymer. A temperature approximately 15^oC. higher than the polymer melting temperature is most preferred. The mixture is heated for a sufficient amount of time to uniformly melt the polymer. One hour is normally sufficient.

The melted polymer is cooled to room temperature at a predetermined constant rate. The rate of cooling will also affect crystallinity and the rate of biodegradation after implantation. Preferably, the mixture is cooled at a rate of between 5^o and 20^oC. per minute. The preferred cooling rate for the formation of a membrane for use in liver or cartilage cells transplants is large enough to yield amorphous PLLA matrices. The mixture cooled at the predetermined rate will have the desired degree of crystallinity for the intended use.

As described in more detail below, since many of the useful polymers are hydrophobic, it may be useful in some embodiments to pre-wet the matrix prior to seeding of cells within the matrix. Suitable surfactants include any of the FDA approved surfactants, including polyols, alcohols, and, in some cases, saline.

Sources of Cells

In a preferred embodiment, cells are obtained either from the recipient for autologous transplantation or from a related donor. Cell transplantation can provide an alternative treatment to whole organ transplantation for failing or malfunctioning organs such as liver and pancreas. Because many isolated cell populations can be expanded in vitro using cell culture techniques, only a very small number of donor cells may be needed to prepare an implant. Consequently, the living donor need not sacrifice an entire organ.

Cells can also be obtained from established cell lines which exhibit normal physiological and feedback mechanisms so that they replicate or proliferate only to a desired point.

For gene therapy, gene transfer vectors can be introduced into different cell types, such as endothelial cells and myoblasts, which are transplanted back to the host for the production and local release of proteins and other therapeutic drugs. Methods for gene transfer are well known to those skilled in the art and have been approved by Food and Drug Administration.

Cells types that are suitable for implantation include most epithelial and endothelial cell types, for example, parenchymal cells such as hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary axis, heart muscle cells, kidney epithelial cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, and smooth and skeletal muscle.

In one variation of the method using a single matrix for attachment of one or more cell lines, the matrix is configured such that initial cell attachment and growth occur separately within the matrix for each population. Alternatively, a unitary scaffolding may be formed of different materials to optimize attachment of various types of cells at specific locations. Attachment is a function of both the type of cell and matrix composition. Cell attachment and viability can be assessed using scanning electron microscopy, histology, and quantitative assessment with radioisotopes.

Although the presently preferred embodiment is to utilize a single matrix implanted into a host, there are situations where it may be desirable to use more than one matrix, each implanted at the most optimum time for growth of the attached cells to form a functioning three-dimensional organ structure from the different matrices.

The function of the implanted cells, both in vitro as well as in vivo, must be determined. In vivo liver function studies can be performed by placing a cannula into the recipient's common bile duct. Bile can then be collected in increments. Bile pigments can be analyzed by high pressure liquid chromatography looking for underivatized tetrapyrroles or by thin layer chromatography after being converted to azodipyrroles by reaction with diazotized azodipyrroles ethylanthranilate either with or without treatment with P-glucuronidase. Diconjugated and monoconjugated bilirubin can also be determined by thin layer chromatography after alkalinemethanolysis of conjugated bile pigments. In general, as the number of functioning transplanted hepatocytes increases, the levels of conjugated bilirubin will increase. Simple liver function tests can also be done on blood samples, such as albumin production. Analogous organ function studies can be conducted using techniques known to those skilled in the art, as required to determine the extent of cell function after implantation. Studies using labelled glucose as well as studies using protein assays can be performed to quantitate cell mass on the polymer scaffolds. These studies of cell mass can then be correlated with cell functional studies to determine what the appropriate cell mass is. In most cases it is not necessary to completely replace the function of the organ from which the cells are derived, but only to provide supplemental or partial replacement therapy.

Methods of Implantation

The technique described herein can be used for delivery of many different cell types to achieve different tissue structures. For example, islet cells of the pancreas may be delivered in a similar fashion to that specifically used to implant hepatocytes, to achieve glucose regulation by appropriate secretion of insulin to cure diabetes. Other endocrine tissues can also be implanted. The matrix may be implanted in many different areas of the body to suit a particular application. Sites other than the mesentery for hepatocyte injection in implantation include subcutaneous tissue, retroperitoneum, properitoneal space, and intramuscular space.

Implantation into these sites may also be accompanied by portacaval shunting and hepatectomy, using standard surgical procedures. The need for these additional procedures depends on the particular clinical situation in which hepatocyte delivery is necessary. For example, if signals to activate regeneration of hepatocytes are occurring in the patient from his underlying liver disease, no hepatectomy would be necessary. Similarly, if there is significant portosystemic shunting through collateral channels as part of liver disease, no portacaval shunt would be necessary to stimulate regeneration of the graft. In most other applications, there would be no need for portacaval shunting or hepatectomy.

Claim 1 of 7 Claims

We claim:

1. A polymeric matrix formed of a biodegradable, biocompatible, synthetic, amorphous polymer or semi-crystalline polymer with a degree of crystallinity in the range of from 0 to 24.5% having a porosity of between 45 to 93% and a pore size range of greater than zero to 500 microns that is suitable for attachment and proliferation of dissociated cells.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.