| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | March 25, 2003 |
| PATENT TITLE |
Tissue-engineered tubular construct having circumferentially oriented smooth muscle cells |
| PATENT ABSTRACT | Improved methods for the production of tissue-engineered constructs, including muscular tissue constructs such as vascular constructs, are disclosed. The methods include the use of improved substrates for cell growth, improved cell culture media for cell growth, and the use of distensible bodies to impart pulsatile stretching force to lumens of constructs during growth. Also disclosed are improved products and methods for making those products, including substrates and cell culture media, for tissue engineering and tissue culture generally. Improved muscular tissue constructs, including vascular constructs, are also disclosed, which may be used in medicine for the repair or replacement of damaged natural structures. In an embodiment, a muscular, tubular tissue-engineered construct is prepared having a wall of mammalian smooth muscle cells oriented circumferentially about a lumen of the construct at a cell density of at least 10.sup.7 cells/cc |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 2, 1998 |
| PATENT REFERENCES CITED |
Barera et al, J. Am. Chem. Soc., 115:11010-11011 (1993). Bell, J. Cellular Biochem., 56:147-149 (1994). Cao et al., Transplantation Proc., 26(6):3390-3391 (1994). Chen et al., Circulation, 89:1922-1928 (1994). Cima et al., Biotechnol. and Bioeng., 38:145-158 (1991). Cima et al., Chem. Eng. Prog., 46-53 (1993). Connolly et al., Trans. ASAIO, 34:1043-1046 (1988). D'Amore et al., Growth Factors, 8:61-75 (1993). Edelman et al., Biomaterials, 12:619-626 (1991). Freed et al., J. Cell. Biochem., 51:257-264 (1993). Freed et al., J. Biomed. Mat. Res., 28:891-899 (1994). Freed et al., Bio/Technology, 12:689-693 (1994). Gilbert et al.. Transplantation, 56(2):423-427 (1993). Gilding et al., Polymer, 20:1459-1464 (1979). Greisler et al., Circulation, 78(Suppl. I):16-112 (1988). Jarrell et al., ASAIO Trans., 113:120-122 (1991). Langer et al., Science, 260:920-926 (1993). Massia et al., Ann. N.Y. Acad. Sci., 589:261-270 (1990). Mazzucotelli et al., Artificial Organs, 17(9):787-790 (1993). Mooney et al., Mat. Res. Soc. Symp. Proc., 252:345-352 (1992). Mooney et al., Cell Transplantation, 3(2):203-210 (1994). Mooney et al., Transplantation Proc., 26(6):3425-3426 (1994). Niklason et al., Transplant Immunology, 5:303-306 (1997). Ott et al., Surgery, 117:334-339 (1995). Rogelj et al., J. Cell Biol., 109:823-831 (1989). Shayani et al., J. Surg. Res., 57:495-504 (1994). Takeda et al., Transplantation Proc., 27(1):635-636 (1995). Vacanti et al., Transplantation Proc., 26(6):3434-3435 (1994). Weinberg et al., Science, 231:397-400 (1986). Wintermantel et al., ASAIO Trans., 37:M334-M336 (1991). Greisler et al., Biomaterials, 17:329-336 (1996). Hirai et al., Cell Transplantation, 4(6):597-608 (1995). L'Heureux et al., The FASEB Journal, 12:47-56 (1998). L'Heureux et al., Jour. of Vascular Surgery, 17:499-509 (1993). Ziegler et al., Annals of Biomedical Engineering, 23:216-225 (1995). Mooney et al., Biomaterials, 17(2):115-124 (1996). Kanda et al., Cell Transplantation, 2(6):475-484 (1993). Kanda K and Matsuda T. Behavior of arterial wall cells cultured on periodically stretched substrates. Cell Transplantation 1993; 2: No. 6: 475-484. Mooney DJ, et al. Design and fabrication of biodegradable polymer devices to engineer tubular tissues. Cell Transplantation 1994; 3: No. 2: 203-210. Mooney DJ, et al. Stabilized polyglycolic acid fibre-based tubes for tissue engineering. Biomaterials 1996; 17: No. 2: 115-214. Niklason L and Langer RS. Advances in tissue engineering of blood vessels and other tissues. Transplant Immunology 1997; 5: 303-306. |
| PATENT GOVERNMENT INTERESTS |
GOVERNMENT SUPPORT This invention was made with government support under Grant Number BES-9525913 awarded by the National Science Foundation and Grant Number HL03492-02 awarded by the National Institute of Health. The government has certain rights in the invention. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A muscular, tubular tissue-engineered construct comprising: a substantially tubular construct of living mammalian tissue having a first end and a second end, an inner surface and an outer surface; wherein the first end, the second end, and the inner surface of the construct define a lumen passing through the construct; and wherein tissue between said inner surface and said outer surface defines a wall of mammalian smooth muscle cells; wherein said wall comprises said mammalian smooth muscle cells oriented circumferentially about said lumen; and wherein said mammalian smooth muscle cells in said wall have a cell density of at least 10.sup.7 cells/cc. 2. A muscular, tubular tissue-engineered construct as in claim 1 wherein said tubular construct is capable of withstanding an internal pressure of at least 100 mm Hg for a sustained period without rupturing. 3. A muscular, tubular tissue-engineered construct as in claim 1 wherein said tubular construct is capable of withstanding an internal shear force of at least 5 dynes/cm.sup.2 for a sustained period without rupturing. 4. A muscular, tubular tissue-engineered construct as in claim 1 wherein said wall further comprises a biocompatible synthetic polymeric material. 5. A muscular, tubular tissue-engineered construct as in claim 1 wherein said outer surface is substantially free of an adventitia. 6. A muscular, tubular tissue-engineered construct as in claim 1 wherein said wall is substantially free of an intermediate layer of an intima. 7. A muscular, tubular tissue-engineered construct as in claim 1 wherein said wall is substantially free of an internal elastic lamina of an intima. 8. A muscular, tubular tissue-engineered construct as in claim 1 wherein said wall is substantially free of fibroblasts in an intimal layer. 9. A muscular, tubular tissue-engineered construct as in claim 1 wherein said wall is substantially free of fibroblasts in a medial layer. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention is directed generally to the art of tissue engineering, or the production of organized mammalian tissues in vitro. BACKGROUND OF THE INVENTION Tissue engineering is emerging as a new field in the biomedical sciences. Langer and others have demonstrated the feasibility of seeding and culturing various cell types on biocompatible, biodegradable polymer films and three-dimensional scaffolds or substrates (Takeda et al. (1995); Vacanti et al. (1994); Mooney et al. (1994); Cao et al. (1994); Bell (1994); Gilbert et al. (1993); Freed et al. (1994a); Mooney et al. (1994); Cima et al. (1991); Cima and Langer (1993); Wintermantel et al. (1991); Mooney et al. (1992); Freed et al. (1994b); Freed et al. (1993)). Cell attachment, spreading and replication have been demonstrated to occur on these polymers, and the formation of solid tissue masses of up to one millimeter in thickness has been demonstrated for tissues such as cartilage (Freed et al. (1994a); Freed et al. (1994b); Freed et al. (1993)). Many cell types have been implanted successfully in vivo, including hepatocytes, chondrocytes, fibroblasts, enterocytes, smooth muscle cells and endothelial cells (Takeda et al. (1995); Mooney et al. (1994); Gilbert et al. (1993); Mooney et al. (1994)). Tissue-engineered constructs may be used for a variety of purposes both in vivo and in vitro. For example, such constructs may serve as prosthetic devices for the repair or replacement of damaged organs or tissues, such as in coronary bypasses or liver grafts. In addition, tissue-engineered constructs can serve as in vivo delivery systems for proteins or other molecules secreted by the cells of the construct. Alternatively, tissue-engineered constructs can serve as in vitro models of tissue function or as models for testing the effects of various treatments or pharmaceuticals. Of particular interest are vascular tissue-engineered constructs. There are 1.4 million surgical procedures performed annually in this country that require arterial prostheses (Langer and Vacanti (1993)). Small arteries with diameters less than five to six mm cannot be replaced with artificial materials due to high rates of thrombosis (Connolly et al. (1988); Greisler et al. (1988)). Thus, autologous vein or artery grafts are generally used to replace small arteries in the coronary or peripheral circulations. Vein grafts have thin walls that are sometimes damaged when transplanted into the arterial system, and suitable veins are not available in all patients due to amputation or previous vein harvest. Internal mammary arteries, which comprise the majority of arterial grafts, are useful only in the coronary circulation. Thus, there remains a need for developing methods for culturing autologous arterial grafts from a small biopsy of the patient's own tissue, or heterologous arterial grafts from histocompatible cells derived from a donor or cell line. SUMMARY OF THE INVENTION The present invention is directed to improved methods for the production of tissue-engineered constructs, including muscular tissue constructs such as vascular constructs. The methods include the use of improved substrates for cell growth, improved cell culture media for cell growth, and the use of distensible bodies to impart pulsatile stretching force to the lumens of constructs during growth. Also provided are improved products, including substrates and cell culture media, for tissue engineering and tissue culture generally. Improved muscular tissue constructs, including vascular constructs, are also provided, which may be used in medicine for the repair or replacement of damaged natural structures. Thus, in one aspect, the invention provides a method for producing a muscular tissue-engineered construct in which a porous substrate, comprising a biocompatible material, and having an inner surface and an outer surface, is first provided. The inner surface of the porous substrate defines a lumen. Within the lumen, a distensible body is provided which is capable of distending within the lumen so as to contact the inner surface of the substrate. The porous substrate, either before or after inserting the distensible body, is contacted with a suspension comprising muscle cells which adhere to and infiltrate the porous substrate, thereby forming a primary cell-seeded construct. The primary cell-seeded construct is then maintained for a first growth period in an environment suitable for growth of the muscle cells to form a primary tissue-engineered construct. During the first growth period, cyclical increases in pressure within the distensible body are provided, thereby causing the distensible body to distend within the lumen of the construct and to apply pulsatile stretch to the construct. This pulsatile stretch mimics natural pulsatile stretching forces encountered in the body, and aids the growing construct in developing strength and/or an appropriate phenotype. In another aspect, the invention provides a method for producing a muscular tissue-engineered construct in which a porous substrate comprising a biocompatible material, and having an inner surface and an outer surface, is first provided. The inner surface of the porous substrate defines a lumen. The porous substrate is contacted with a suspension comprising muscle cells which adhere to and infiltrate the porous substrate, thereby forming a primary cell-seeded construct. Rather than a distensible body within the lumen of the construct, a sleeve is provided, either before or after cell-seeding, around a portion of the exterior of the porous substrate. The sleeve is capable of resisting distension of the substrate in response to pressure within the lumen. The primary cell-seeded construct is then maintained for a first growth period in an environment suitable for growth of the smooth muscle cells to form a primary tissue-engineered construct. During the first growth period, intralumenal flow is provided within the lumen, thereby causing the substrate to distend within the sleeve, and to contact the sleeve. The sleeve, by resisting the distension, provides mechanical support to the growing construct. Optionally, during the first growth period, cyclical increases in pressure are also provided within the lumen, thereby causing the substrate to cyclically distend within the sleeve, and thereby applying pulsatile stretch to the construct. This intralumenal flow, and optional pulsatile stretch, mimic natural flow and pulsatile stretching forces encountered in the body, and aids the growing construct in developing strength and/or an appropriate phenotype. In another aspect, the invention provides a method for producing a muscular tissue-engineered construct in which a porous substrate comprising a biocompatible material, and having an inner surface and an outer surface, is first provided. The inner surface of the porous substrate defines a lumen. Rather than a distensible body or sleeve, an inner surface of the lumen (or a medial layer of the substrate) is provided which is substantially less porous than the outer surface, and this inner surface (or medial layer) is also capable of resisting distension of the substrate in response to pressure within the lumen. The porous substrate is contacted with a suspension comprising smooth muscle cells which adhere to and infiltrate the porous substrate, thereby forming a primary cell-seeded construct. The primary cell-seeded construct is then maintained for a first growth period in an environment suitable for growth of the smooth muscle cells to form a primary tissue-engineered construct. During the first growth period, intralumenal flow within the lumen is provided, thereby causing the substrate to distend. The inner surface (or medial layer), by resisting the distension, provides mechanical support to the growing construct. Optionally, during the first growth period, cyclical increases in pressure are also provided within the lumen, thereby causing the substrate to cyclically distend, and thereby applying pulsatile stretch to the construct. This intralumenal flow, and optional pulsatile stretch, mimic natural flow and pulsatile stretching forces encountered in the body, and aids the growing construct in developing strength and/or an appropriate phenotype. Preferably, in each of the above described embodiments, the porous substrate comprises a synthetic polymeric material having a hydrophilic surface, as described below. In addition, optionally in each of the above-described embodiments, the methods include the additional steps of contacting the resulting primary cell-seeded construct or primary tissue-engineered construct with a suspension comprising a second type of mammalian cells capable of adhering to and/or infiltrating the substrate, thereby forming a secondary cell-seeded construct, and maintaining the secondary cell-seeded construct for a second growth period in an environment suitable for growth of the second type of cells to form a secondary tissue-engineered construct. In preferred embodiments, the above-described muscular tissue-engineered constructs are vascular tissue constructs. Therefore, in these preferred embodiments, the porous substrate is a substantially tubular substrate, the first type of mammalian cells are smooth muscle cells, and the second type of mammalian cells are endothelial cells which are contacted with the inner surface of the lumen. In each of the embodiments applying pulsatile stretch to the growing tissue construct, it is preferred that the pulsatile stretch causes an increase in an inner diameter of the construct of between approximately 1-10%, more preferably between approximately 2-6%. The present invention also provides improved methods for producing a tissue-engineered construct, whether muscular or non-muscular, employing substrates which comprise biocompatible synthetic polymers having hydrophilic surfaces. Thus, in another aspect, the invention provides a method for producing a tissue-engineered construct in which a substrate, porous or non-porous, is provided which comprises a biocompatible synthetic polymer having a hydrophilic surface. The substrate is contacted with a suspension comprising a first type of mammalian cells which are capable of adhering to and/or infiltrating the substrate to form a primary cell-seeded construct. The primary cell-seeded construct is maintained for a first growth period in an environment suitable for growth of the mammalian cells to form a primary tissue-engineered construct. In these methods, it is found that the biocompatible synthetic polymers with hydrophilic surfaces result in much improved cell seeding densities and/or much improved cell density in the final tissue-engineered construct. Optionally, the resulting primary cell-seeded construct or said primary tissue-engineered construct is contacted with a suspension comprising a second type of mammalian cells which are capable of adhering to or infiltrating the construct to form a secondary cell-seeded construct, and this secondary cell-seeded construct is maintained for a second growth period in an environment suitable for growth of the second type of cells to form a secondary tissue-engineered construct. In each of the foregoing embodiments, a variety of cells may be seeded onto the substrates. These include smooth muscle cells, epithelial cells, endothelial cells, fibroblasts, myoblasts, hepatocytes, bile duct cells, pancreatic islet cells, thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian, testicular, or salivary cells, cardiac muscle cells, renal cells, chondrocytes, nerve cells, and progenitor cells. In each embodiment described above, it is preferred that the polymeric substrate material comprises a polymer selected from polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxy carboxylic acids and dicarboxylic acids. In particularly preferred embodiments, the polymeric material is selected from the polymers or copolymers of glycolic acid, lactic acid, and sebacic acid. In those embodiments employing a porous substrate, it is preferred that the substrate comprises a porous mesh of fibers having diameters of between approximately 5-20 .mu.m, preferably between approximately 10-15 .mu.m, and most preferably about 13 .mu.m. It is also preferred that the substrate comprises a porous mesh of fibers in which substantially parallel fibers in the mesh are separated by approximately 20-200 .mu.m, preferably approximately 50-100 .mu.m. It is also preferred that the porous substrate is characterized by a void volume of greater than 90%, preferably greater than 95%. It is also preferred that the substrate has an average pore size of less than 200 .mu.m, preferably less than 175 .mu.m, and more preferably less than 150 .mu.m. In those embodiments employing a substrate of polymeric material having a hydrophilic surface, it is preferred that the surface comprises a multiplicity of hydrophilic chemical groups selected from carboxyl, hydroxyl, thiol, amine, sulfonyl, guanidine, and amide groups. In preferred embodiments, these hydrophilic groups have a density of at least 5 pmol/cm.sup.2, preferably at least 10 pmol/cm.sup.2, and generally between 5 and 20 pmol/cm.sup.2. It is also preferred that the hydrophilic surface has a contact angle of less than 20.degree., preferably less than 15.degree., more preferably less than 10.degree., and most preferably less than 5.degree.. In another aspect, the present invention provides for improved growth media for producing muscular tissue-engineered constructs. Therefore, in those embodiments described above in which smooth muscle cells are cultured, a standard cell culture medium is employed which is supplemented with about 0.01-0.1 g/L, preferably about 0.02-0.06 g/L, of at least one amino acid selected from proline, glycine, and alanine. In addition, a standard cell culture medium is employed which is supplemented with about 0.01-0.1 g/L, preferably about 0.02-0.06 g/L, of vitamin C. Further, a standard cell culture medium is employed which is supplemented with about 0.5-5.0 .mu.g/L, preferably about 1.0-3.0 .mu.g/L, of a copper salt. In another aspect, the present invention provides substrates for use in tissue culture, which comprise three-dimensional scaffolds of a biocompatible synthetic polymer having a hydrophilic surface. As described above, these substrates preferably comprise a polymer selected from the polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, and copolymers of hydroxy carboxylic acids and dicarboxylic acids. Most preferably, the polymeric material is selected from the polymers or copolymers of glycolic acid, lactic acid, and sebacic acid. In those embodiments in which the substrate is a porous substrate, it is preferred that the substrate comprises a porous mesh of fibers having diameters of between approximately 5-20 .mu.m, preferably between approximately 10-15 .mu.m, and most preferably about 13 .mu.m. It also preferred that the substrate comprises a porous mesh of fibers in which substantially parallel fibers in the mesh are separated by approximately 20-200 .mu.m, preferably approximately 50-100 .mu.m. It is also preferred that the porous substrate is characterized by a void volume of greater than 90%, preferably greater than 95%. It is also preferred that the substrate has an average pore size of less than 200 .mu.m, preferably less than 175 .mu.m, and more preferably less than 150 .mu.m. In particularly preferred embodiments, a substrate is provided comprising a biocompatible polymeric material with a hydrophilic surface hydrophilic, in which the surface comprises a multiplicity of hydrophilic chemical groups selected from the carboxyl, hydroxyl, thiol, amine, sulfonyl, guanidine, and amide groups. It preferred that these hydrophilic groups have a density of at least 5 pmol/cm.sup.2, preferably at least 10 pmol/cm.sup.2, and generally between 5 and 20 pmol/cm.sup.2. It is also preferred that the hydrophilic surface has a contact angle of less than 20.degree., preferably less than 15.degree., more preferably less than 10.degree., and most preferably less than 5.degree.. In another aspect, the present invention provides substrates for cell culture and tissue-engineering, and methods for making such substrates, in which the substrate comprises a multiplicity of polyester or polyanhydride bonds, and the hydrophilic surface is formed by at least partial hydrolysis of the bonds at the surface. In another aspect, the present invention provides a muscular, tubular tissue-engineered construct comprising a substantially tubular construct of living mammalian tissue having a first end and a second end, an inner surface and an outer surface. In these constructs, the first end, the second end, and the inner surface of the construct define a lumen passing through the construct, and the tissue between the inner surface and outer surface defines a wall of the construct. The wall comprises mammalian smooth muscle cells oriented circumferentially about the lumen. In preferred embodiments, a muscular tissue-engineered construct is provided in which the smooth muscle cells in the wall have a cell density of at least 10.sup.7 cells/cc, preferably at least 10.sup.8 cells/cc. It is also preferred that the tubular construct is capable of withstanding, for a sustained period without rupturing (e.g., at least one hour), an internal pressure of at least 100 mm Hg, preferably at least 110 mm Hg, more preferably at least 120 mm Hg, and most preferably at least 130 mm Hg. It is also preferred that the tubular construct is capable of withstanding, for a sustained period without rupturing, an internal shear force of at least 5 dynes/cm.sup.2, preferably at least 10 dynes/cm.sup.2, more preferably at least 20 dynes/cm.sup.2, and most preferably at least 30 dynes/cm.sup.2. In other aspects, the present invention provides such constructs in which the wall further comprises a synthetic polymeric material, in which the outer surface is substantially free of an adventitia, in which the wall is substantially free of an intermediate layer of an intima, in which the wall is substantially free of an internal elastic lamina of an intima, in which the wall is substantially free of fibroblasts in an intimal layer, and/or in which the wall is substantially free of fibroblasts in a medial layer. These and other aspects of the present invention will be apparent to one of ordinary skill in the art from the following detailed description of the invention and certain preferred embodiments. |
| PATENT PHOTOCOPY | Available on request |
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