Source: https://patents.google.com/patent/US6309670?oq=6181294
Timestamp: 2018-03-25 02:27:59
Document Index: 99426217

Matched Legal Cases: ['art) 5', 'arts 5', 'arts 1', 'art, 5', 'art, 5', 'art) 5']

US6309670B1 - Collagen-polysaccharide matrix for treatment of bone tumors - Google Patents
Collagen-polysaccharide matrix for treatment of bone tumors Download PDF
US6309670B1
US6309670B1 US09324792 US32479299A US6309670B1 US 6309670 B1 US6309670 B1 US 6309670B1 US 09324792 US09324792 US 09324792 US 32479299 A US32479299 A US 32479299A US 6309670 B1 US6309670 B1 US 6309670B1
US09324792
As used in this discussion, “treatment of bone tumor” refers to minimizing or eliminating the presence of tumor cells at the site of administration. Bone tumor includes osteosarcoma and neuroectodermal tumors. Administration encompasses injection of a gel-like matrix as well as implantation of a sponge like matrix.
As used in this discussion, “repair” is defined as growth of new tissue. The new tissue may or may not be phenotypically or genotypically identical to the original lost tissue. As used herein, “regeneration of tissue” means that the new tissue grown is identical to the lost tissue. Tissue repair can also be the result of replacing lost tissue with non-identical tissues. The basic cellular properties involved in repair include adhesion, proliferation, migration and differentiation.
By “conduction”, it is meant that the host tissue, e.g.,bone, grows by extension of existing tissue onto or into the crosslinked collagen-polysaccharide matrix. In conduction, repair cells move onto and into the matrix to synthesize and remodel new tissue identical to the surrounding host tissue. By induction, it is meant that the growth and differentiation of progenitor repair cells is stimulated. These progenitor cells go on to synthesize and remodel new tissue to be continuous with the surrounding host tissue.
The usefulness of the matrices according to the present invention can be shown by both in vitro and in vivo tests. For in vitro evaluation, candidate matrices about 45 mm3 in size are injected with 2×105 bone tumor cells, such as the SK-ES-1 cell line. The cell-seeded matrices are placed in 24 well TransWell plates (Costar) and cultured in DMEM containing 10% FBS and the specific differentiation factor at concentrations of 10 and 100 ng/ml. After 21-28 days, the growth properties of the tumor cells are monitored using a mitogenic or MTS assay. The expression of differentiation markers is examined at the protein level using an alkaline phosphatase activity assay and immunoassays for osteocalcin. The effect of matrices on the expression of genetic markers for osteoblastic differentiation is performed based on semi-quantitative RT-PCR analysis using specific primers to alkaline phosphatase, type I collagen, and osteocalcin.
Semed F collagen (0.9 part) Hyaluronate/polyaldehyde solution (1 part,
Semed S collagen (0.1 part) 5% of the repeat units oxidized) Solids
Semed F collagen (0.9 part) Hyaluronate/polyaldehyde solution (2
Semed S collagen (0.1 part) parts 5% of the repeat units oxidized)
Semed F collagen (0.9 part) Hyaluronate/polyaldehyde solution (4
Semed F collagen (0.9 part) Hyaluronate/polyaidehyde solution (4
Semed S collagen (0.1 part) parts 1% of the repeat units oxidized)
Collagen Type II (9 parts) Hyaluronate/polyaldehyde solution (1 part,
Collagen Type II (1 part) Hyaluronate/polyaldehyde solution (1 part,
Semed F collagen (7 parts) Hyaluronate/polyaldehyde solution (1 part,
Collagen Type II (2 parts) 5% of the repeat units oxidized) Solids
Semed F collagen (8.1 parts) Dextran/polyaldehyde solution (1 part, 5%
Semed S collagen (0.9 part) of the repeat units oxidized) Solids
Semed F collagen (8.1 parts) Dextran sulfate/polyaldehyde (1 part, 5%
Semed F collagen (8.1 parts) Chondroitin sulfate/polyaldehyde (1 part,
Semed S collagen (0.9 part) 5% of the repeat units oxidized) Solids
The activity of crude BMPs and GDF-5 is monitored based on their ability to enhance bone formation in situ. The cell lines, Human osteosarcoma SK-ES-1 and MNNG/HOS are utilized to determine the inhibitory effects of candidate matrix /differentiation factor combinations. The SK-ES-1 cell line undergoes a dramatic change in cellular morphology in response to antiproliferative growth factors. This change in cellular morphology is marked by a reduction in the number of transformed foci which correlates directly to the loss of anchorage-independent growth and a decrease in tumorogenicity (Freedman, V. H., and Shin, S.: Cell 3:355-359, 1974.). Fetal Rat Calvarial (FRC) cells are utilized as normal osteoblasts. The cells are maintained in DMEM with non-essential amino acids, supplemented with 10% FBS, 2 mM glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin, and 5 ug/ml gentamicin. The cells are incubated at 37° C in a humidified atmosphere of 95% air, 5% CO2 and subcultured weekly using 0.05% trypsin and 0.53 mM EDTA.
Tissue culture plates are coated for 3 hours at 37° C with a solution of PBS or 0.1 M sodium bicarbonate containing various protein substrates (?et al. J.Cell Physiol. 153:256-365, 1992). Nonadsorbant proteins are removed by rinsing twice with PBS. The tumor cells are seeded in 12-well plates at an initial density of 20-25000 cells/cm2 in DMEM supplemented with 10% FBS. After 12 hours, the media is replaced with DMEM, 1% FBS supplemented with various growth factors including TGF-β GDF-5 and crude BMPs. The ability of other mitogens including Platelet Derived Growth Factor (PDGF), to change the cellular morphology of osteosarcoma cell lines is assessed in parallel as a control.
For analysis of the effect of different growth factor/ECM combinations on proliferation of tumor cell lines in vitro, SK-E-ES-1 cells (1×105) are suspended in 0.4% agarose (SeaPlaque) in DME containing 10% calf serum along with various concentrations of ECM proteins. Cells are fed with DME containing 10% calf serum in the presence or absence of indicated growth factors once a week. Colonies are stained with p-iodonitrotetrazolium violet and scored after 2 weeks of culture (Yu et al., 1994, J. Cell. Biol. 127: 479-487).
DNA synthesis is measured in 12-well cell culture plates pre-coated with various ECM proteins prior to cell seeding in the presence of indicated growth factors as described above. After indicated time periods, the cultures are placed in serum free growth medium containing an equal mixture of MCBD 401, LeibovitzÕs L-15 and Ham's F-12 supplemented with 10 nM selenium (Gibco) and 10 ug/ml transferrin (Collaborative Research) and [3HMethyl-thymidine] (2 uCi/ml; Amersham) for 2-4 hours at 37° C. Cells are harvested by washing twice in PBS followed by three washes in cold 5% trichloroacetic acid. Trichloroacetic acid-precipitable radioactivity is solubilized by adding 200 ul of 0.25 M sodium hydroxide followed by addition of 10 ml Biofluor (NEN). Samples are counted in Beckman scintillation counter (Fleming et al. Oncogene 7:1355-1359). In parallel, the viability of cells is measured using MTS assays as described previously (1992 Scudiero, D. A. Cancer Research 48:4827-4829, 1988). Cell differentiation: The analysis of differentiation markers is performed at the protein and transcriptional level. The specific markers to be analyzed include: 1) type I, II, and X collagen 2) alkaline phosphatase, 3) osteonectin and 4) osteocalcin.
SK-ES-1 osteosarcoma cell line (obtained from the ATCC, Rockville, Md.) was expanded in Dulbeco Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) using a T75 flask for 5 days. The confluent cells were then washed with PBS/EDTA solution briefly, followed by trypsinization using a mixture of Trypsin/EDTA for 2 minutes at room is temperature. The trypsination experiment was terminated by addition of excess growth media. The cells were then washed and resuspended in the appropriate final volume of growth media. For viability assays, 12 well plates were coated with or without 0.1 mg/ml soluble type I collagen (Collagen Corp., Palo Alto, Calif.) for 2 hours at 37 C. The excess collagen was removed and the harvested cells were either plated at density of 1×105 cells per well or loaded onto the Collagen/HA matrix (24 mm3). SK-ES-1 cells were then cultured for 10 days in the growth media supplemented with or without 100 ng/ml of TGF-β (R&D Systems Minneapolis, Minn.). For quantitation of cell viability, an aliquot of culture media was used for MTS assay.
Twelve well plates were coated overnight with 0.01% (w/v) collagen type I in PBS. After removal of nonadsorbant protein, SK-ES-l cells were plated at a density of 2×105 cell/well in DMDM supplemented with 10% FBS. Cells were then treated with vehicle or TGF-β at 100 ng/ml. Cell cultures were maintained for 21 days in the presence of factor. Plates were then stained with Giesma and photographed. The results indicate that TGF-β acts in synergy with the type I collagen to reduce the number of transformed foci. Under these experimental conditions, type I collagen alone partially inhibits focal transformation and the TGF-β alone fails to reduce focal transformation. Since there is a direct correlation between formation of foci in vitro and tumor formation in vivo, these findings suggest that as matrix composed of type I collagen and TGF-β suppress the growth of Ewing sarcoma in vivo.
1. A method for the treatment of a bone tumor comprising the steps of administering at a site of desired treatment a matrix comprising:
a) collagen covalently crosslinked to an exogenous polysaccharide, wherein said polysaccharide is crosslinked to said collagen through oxidized sugar rings on said polysaccharide which form covalent linkages to said collagen; and
b) a differentiation factor.
2. The method of claim 1, wherein said bone tumor comprises sarcomas and carcinomas.
US09324792 1997-01-15 1999-06-03 Collagen-polysaccharide matrix for treatment of bone tumors Expired - Lifetime US6309670B1 (en)
US6309670B1 true US6309670B1 (en) 2001-10-30
ID=26677320
US09324792 Expired - Lifetime US6309670B1 (en) 1997-01-15 1999-06-03 Collagen-polysaccharide matrix for treatment of bone tumors
US (1) US6309670B1 (en)
Dietz, U., et al., 1993, "Alterations of Collagen mRNA Expression During Retinoic Acid Induced Chondrocyte Modulation: Absence of Untranslated alpha1(I) mRNA in Hyaline Chondrocytes," J. Cell Biol. 52(1):57-68.
Lee, M.K., et al., 1991, "Analysis of affinity and structural selectivity in the binding of proteins to glycosaminoglycans: Development of a sensitive electrophoretic approach " Proc. Natl. Acad. Sci USA 88 (7):2768-72.
Sandell, L.J., et al., 1991, "Alternatively Spliced Type II Procollagen mRNAs Define Distinct Populations of Cells during Vertebral Development: Differential Expression of the AMino-Propeptide," J. Cell Biol. 114 (4):1307-19.
Spiro, R.C., et al., 1991, "Uncoupling of Chondroitin Sulfate Glycosaminoglycan Synthesis by Brefeldin A," J. Cell. Biol., 115 :(5)1463073
Thyberg and Moskalewski, 1979, "Bone Formation in Cartilage Produced by Transplanted Epiphyseal Chodrocytes,"Cell Tissue Res. 204(1): 77-94.
Wong and Cohn, 1975, "Target cells in bond for parathormore and calcitonin are different: Enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces," Proc. Natl. Acad. Sci. USA 72:3167-71.
DeLong et al. 2005 Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HEIDARAN, MOHAMMAD;SPIRO, ROBERT C.;REEL/FRAME:010268/0087;SIGNING DATES FROM 19990827 TO 19990901