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Polymeric membrane materials for artificial organs

References Publications referenced by this paper. Competitive plasma protein adsorption onto fluorinated polyimide surfaces. Development of a fluorinated polyimide hollow fiber for medical devices Hiroyoshi Kawakami , Toshiyuki Kanamori , Sunao Kubota.

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Biocompatibility of fluorinated polyimide. Dissociation between complement activation, integrin expression and neutropenia during hemodialysis. Yannick Y. Kazatchkine , Nicole Haeffner-Cavaillon. Influence of synthetic polymers on neutrophil migration in three-dimensional collagen gels. Jian Cheng Tan , W. Autotransplantation of the adrenal tissue or injection of fetal cell suspensions into the brain appears to be of benefit. Loss, deformation or obstruction of blood vessels is another frequent cause of disease, such as high blood pressure or aneurysm. In the past, surgeons have primarily dealt with this problem by grafting blood vessels from another portion of the body to the affected area or by implanting cloth substitutes as permanent replacements.

Disadvantages include the requirement of multiple operations as well as the associated pain to the patient. Even though these techniques do not have many of the problems associated with transplantation of organs such as the liver or intestine, the results are still often imperfect. Although different from organs such as the liver and intesting in a number of ways, skin is also an organ subject to damage by disease or injury which performs the vital role of protecting the body from fluid loss and disease.

Although skin grafts have been prepared from animal skin or the patient's skin, more recently "artificial skin" formed by culturing epidermal cells has been utilized. One method for forming artiiicial skin is by seeding a fibrous lattice with epidermal cells. For example, U.

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Patent No. A disadvantage to the first two methods is that the matrix is formed of a "permanent" synthetic polymer. The ' patent has a feature that neither of the two prior patents has, a biodegradable matrix which can be formed of any shape, using the appropriate cells to produce an organ such as the skin. Unfortunately, there is a lack of control over the composition and configuration of the latter matrices since they are primarily based on collagen.

Further, since collagen is degraded by enzymatic action as well as over time by hydrolysis, the degradation is quite variable. Moreover, the matrix is completely infiltrated with cells and functional in the absence of the moisture controlling polymer overlay only when it is grafted onto the patient and capillaries have formed a vascular network through the entire thickness of the matrix.

The limitation of these matrices as a function of diffusion is discussed in the article by Yannas and Burke in J. Although the authors recognized that the pore size and thickness of the matrix were controlling factors in determining viability and successful engraftment, their only ways of dealing with the lack of sufficient nutrient supply to the interior portions of the matrix at the time of engraftment were either to ignore the problem and hope the graft was thin enough and porous enough to allow sufficient capillary growth along with migration of the epithelial cells into the matrix, or to seed the graft with additional epithelial cells after sufficient capillary growth into the matrix had occurred.

Although skin is considered to be an "organ" of the body, these methods for making artificial skin have not been used to make other types of organs such as a liver or pancreas, despite the all encompassing statements in the patents that the disclosed or similar techniques could be utilized to do so. It is postulated that, when these methods are used to construct organs having a larger overall three dimensional structure, such as a liver or pancreas, the cells within the center of the organs tend to die after a period of time and that the initial growth rate is not maintained, in a manner analogous to the situation with very large tumors which are internally necrotic due to a decrease in diffusion of nutrientsinto the growing three-dimensional structure as the cell density and thickness increase.

Indeed, in view of the Yannas and Burke article, it appears that growth within a matrix, even one as thin as a skin graft, presented problems until vascularization had occurred, even at relatively low cell densities. It is therefore an object of the present invention to disclose a method and means for creating a variety of organs, including skin, liver, kidneys, blood vessels, nerves, and muscles, which functionally resemble the naturally occurring organ. It is a further object of the present invention to provide a method and means for designing, constructing and utilizing artificial matrices as temporary scaffolding for cellular growth and implantation.

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It is a still further object of the invention to provide biodegradable, non-toxic matrices which can be utilized for cell growth, both in vitro and iri vivo, as support structures in transplant organs immediately following implantation. It is another object of the present invention to provide a method for configuring and constructing biodegradable artificial matrices such that they not only provide a support for cell growth but allow and enhance vascularization and differentiation of the growing cell mass following implantation.

It is yet another object of the invention to provide matrices in different configurations so that cell behavior and interaction with other cells, cell substrates, and molecular signals can be studied in vitro. The present invention is a method and means whereby cells having a desired function are grown on polymer scaffolding using cell culture techniques, followed by transfer of the polymer-cell scaffold into a patient at a site appropriate for attachment, growth and function, after attachment and equilibration, to produce a functional organ equivalent.

Success depends on the ability of the implanted cells to attach to the surrounding environment and to stimulate angiogenesis. Nutrients and growth factors are supplied during cell culture allowing for attachment, survival or growth as needed. After the structure is implanted and growth and vascularization take place, the resulting organoid is a chimera formed of parenchymal elements of the donated tissue and vascular and matrix elements of the host.

The polymer scaffolding used for the initial cell culture is constructed of a material which degrades over time and is therefore not present in the chimeric organ. Vascular ingrowth following implantation allows for normal feedback mechanisms controlling the soluble products of the implanted cells.

The preferred material for forming the matrix or support structure is a biodegradable artificial polymer, for example, polyglycolic acid, polyorthoester, or polyanhydride, which is degraded by hydrolysis at a controlled rate and reabsorbed. These materials provide the maximum control of degradability, manageability, size and configuration. In some embodiments these materials are overlaid with a second material such as gelatin or aparose to enhance cell attachment. The polymer matrix must be configured to provide both adequate sites for attachment and adequate diffusion of nutrients from the cell culture to maintain cell viability and growth until the matrix is implanted and vascularization has occurred.

The presently preferred structure for organ construction is a branched fibrous tree-like structure formed of polymer fibers having a high surface area. The preferred structure results in a relatively shallow concentration gradient of nutrients, wastes, and gases, so as to produce uniform cell growth and proliferation. Theoretical calculations of tne maximum cell attachment suggest that fibers 30 microns in diameter and one centimeter in length can support ,, cells and still provide access of nutrients to all of the cells.

Another advantage of the biodegradable material is that compounds may be incorporated into the matrix for slow release during degradation of the matrix. For example, nutrients, growth factors, inducers of differentiation or dedifferentiation, products of secretion, immunomodulators, inhibitors of inflammation, regression factors, biologically active compounds which enhance or allow ingrowth of the lymphatic network or nerve fibers, and drugs can be incorporated into the matrix or provided in conjunction with the matrix, in solution or incorporated into a second biodegradable polymer matrix.

Cells of one or more types can be selected and grown on the matrix. The matrix structure and the length of time and conditions under which the cells are cultured in vitro are determined on an individual basis for each type of cell by measuring cell attachment only viable cells remain attached to the polymers , extent of proliferation, and percent successful engraftment.

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Examples of cells which are suitable for implantation include hepatocytes and bile duct cells, islet cells of the pancreas, parathyroid cells, thyroid cells, cells of the adrenal- hypothalmic-pituitary axis including hormone-producing gonadal cells, epithelial cells, nerve cells, heart muscle cells, blood vessel cells, lymphatic vessel cells, kidney cells, and intestinal cells, cells forming bone and cartilage, smooth and skeletal muscle.

Initially growing the cells in culture allows manipulation of the cells which may be beneficial following implantation of the matrix cell structure. Presently available technology allows the introduction of genes into the cells to make proteins which would otherwise be absent, such as those resulting from liver protein deficiencies and metabolic defects such as cystic fibrosis. Repression of gene expression may also be used to modify antigen expression on the cell surface, and thereby the immune response, so that cells are not recognized as foreign.

The present invention also provides techniques and matrices for in. Although current methods of cell culture have provided valuable insight into fundamental aspects of cell organization and function, studies of cell behavior, communication, control, and morphogenesis have been difficult for lack of a system controllable in three dimensions. Artificial matrices which have been coated with attached cells can be embedded in extracellular matrices such as collagen, basement membrane complexes such as Matrigel Tm , or other materials.

Various combinations of cell types, biochemical signals for growth, differentiation, migration, and extracellular matrix components can then be examined in vitro in a three-dimensional system. Brief Description of the Drawings. Figure 1 is a schematic of the process of the present invention to produce a chimeric organ, in this diagram, a liver, pancreas or intestine: 1 the appropriate parenchymal cells are harvested, dispersed, and seeded onto the polymer matrix in cell culture, where attachment and growth occur and 2 a partial hepatectomy is performed to stimulate growth of the transplant and the polymer-cell scaffold is then implanted into the recipient animal where neovascularization, cell growth, and reabsorption of the polymer matrix occurs.

Figure 2 are the chemical structures of polymers which have been used for biodegradable cellular matrices: a polygalactin; b polyorthoester; and c polyanhydride. Figure 3 is a diagram demonstrating the slow release of biologically active factors from the polymer matrix. Figure 4 -is a diagram of a technique to study in vitro morphogenesis using biodegradable polymers, cells, and matrix. Figure 5 is a photograph x of hepatocytes attached to fibers of polyglactin after 4 days in culture. Cells are stained with Hematoxylin and Eosin.

Figure 6 is a photograph of bile duct epithelial cells cultured on polymer fibers for one month. Figure 7 is a photograph X of an implant of hepatocytes from an adult rat donor into omenturn. The polymer-cell implant has been in place for 7 days before sacrifice.

Artificial organ

Hepatocytes are healthy and several mitotic figures can be seen. Blood vessels are present in the mass. To the left, an inflammatory infiltrate in the area of the polymer is observed. Figure 8 is a scanning electron micrograph X of hepatocytes attached to polymer fibers for one week. Figure 9 is a higher magnification X of the hepatocytes on polymer fibers of Figure 8. Figure 10 is a photomicrograph 10X of an intestinal cell implant into omentum ten days after implantation. It shows a 6 mm cystic structure that has formed in the omentum with blood vessels streaming into it.

Polymer fibers can be seen in the wall of the cyst.

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Figure 11 is a photograph X of a cross- section of the cyst of Figure 10 demonstrating a luminal structure lined by intestinal epithelial cells. These cells show polarity. The lumen contains cellular debris and mucous. The white oval areas to the left of the lumen represent polymer fibers. They are surrounded by an inflammatory infiltrate and new blood vessels. A layer of smooth muscle can be seen to the right of the lumen, suggesting that this cyst may have arisen from a small intestinal fragment.

Hematoxylin and Eosin.

Self-healing polymers - AccessScience from McGraw-Hill Education

Figure 12 is a photograph of Islets of the pancreas attached to polymer fibers after four weeks in culture, showing some secretion of insulin in response to glucose, Figure 13 is a photograph of polymer fibers seeded with bovine aortic endothelial cells in a biomatrix. The cells can be seen migrating off the polymer into the matrix in a branch-like orientation.

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Figure 14 is a photograph of bovine aortic endothelial cells attached to polymer fibers after one month in culture. Figure 15 is a phase contrast photomicrogarph showing polymer fibers coated with mouse fetal fibroblasts.