Method for enhancing the implantation and differentiation of marrow-derived mesenchymal cells

Claim 1

A method for inducing human marrow-derived mesenchymal stem cells to differentiate into bone-forming cells, comprising: a) providing human marrow-derived mesenchymal stem cells that have been isolated, purified and culturally expanded from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the isolated, purified and culturally expanded human marrow-derived mesenchymal stem cells to a porous carrier; and, c) implanting the porous carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into an environment containing factors necessary for differentiating the human mesenchymal stem cells into bone cells.

Claim 2

A method for inducing human marrow-derived mesenchymal stem cells to differentiate into bone-forming cells, comprising: a) providing human marrow-derived mesenchymal stem cells that have been isolated, purified and culturally expanded from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the isolated, purified and culturally expanded human marrow-derived mesenchymal stem cells to a porous carrier; and, c) implanting the porous carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into an environment containing factors necessary for differentiating the human mesenchymal stem cells into bone cells.

Claim 5

A method for repairing skeletal defects comprising: a) providing marrow-derived human mesenchymal stem cells that have been culturally expanded from isolated and purified human marrow-derived mesenchymal stem cells which have been isolated from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the culturally expanded marrow-derived human mesenchymal stem cells to a porous carrier; and, c) implanting the porous carrier containing the culturally expanded purified human marrow-derived mesenchymal stem cells into the defective skeletal tissue.

Claim 6

A method for repairing skeletal defects comprising: a) providing marrow-derived human mesenchymal stem cells that have been culturally expanded from isolated and purified human marrow-derived mesenchymal stem cells which have been isolated from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the culturally expanded marrow-derived human mesenchymal stem cells to a porous carrier; and, c) implanting the porous carrier containing the culturally expanded purified human marrow-derived mesenchymal stem cells into the defective skeletal tissue.

Claim 8

A method for repairing skeletal defects comprising: a) providing a bone marrow specimen containing human marrow-derived mesenchymal stem cells and bone pieces; b) adding the bone marrow specimen to a medium thereby producing a bone marrow specimen-medium mixture, wherein said medium contains factors which stimulate human marrow-derived mesenchymal stem cell growth without differentiation and allows, when cultured, for selective adherence of only the human marrow-derived mesenchymal stem cells to a substrate surface; c) separating the bone pieces from the bone marrow medium mixture; d) dissociating marrow cells in the bone marrow specimen-medium mixture into single cells; e) culturing the dissociated marrow cells in the bone marrow specimen-medium mixture thereby selectively adhering only the human mesenchymal stem cells to the substrate surface; f) separating non-adherent matter from the substrate surface, thereby producing isolated culturally expanded human mesenchymal stem cells; g) removing remaining adherent isolated and culturally expanded human mesenchymal stem cells from the substrate surface with a releasing agent; h) applying the isolated and culturally expanded human marrow-derived mesenchymal stem cells to a porous carrier comprised of about 60% hydroxyapatite and about 40% tricalcium phosphate; and, i) implanting the porous carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into the defective skeletal tissue.

Claim 9

A method for repairing skeletal defects comprising: a) providing a bone marrow specimen containing human marrow-derived mesenchymal stem cells and bone pieces; b) adding the bone marrow specimen to a medium thereby producing a bone marrow specimen-medium mixture, wherein said medium contains factors which stimulate human marrow-derived mesenchymal stem cell growth without differentiation and allows, when cultured, for selective adherence of only the human marrow-derived mesenchymal stem cells to a substrate surface; c) separating the bone pieces from the bone marrow medium mixture; d) dissociating marrow cells in the bone marrow specimen-medium mixture into single cells; e) culturing the dissociated marrow cells in the bone marrow specimen-medium mixture thereby selectively adhering only the human mesenchymal stem cells to the substrate surface; f) separating non-adherent matter from the substrate surface, thereby producing isolated culturally expanded human mesenchymal stem cells; g) removing remaining adherent isolated and culturally expanded human mesenchymal stem cells from the substrate surface with a releasing agent; h) applying the isolated and culturally expanded human marrow-derived mesenchymal stem cells to a porous carrier comprised of about 60% hydroxyapatite and about 40% tricalcium phosphate; and, i) implanting the porous carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into the defective skeletal tissue.

Claim 13

A method for inducing marrow-derived human mesenchymal stem cells to differentiate into cartilage-forming cells, comprising: a) providing human marrow-derived mesenchymal stem cells that have been culturally expanded from isolated and purified human marrow-derived mesenchymal stem cells which have been isolated from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the culturally expanded human marrow-derived mesenchymal stem cells to a carrier formatted to promote round cell morphology; c) implanting the carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into an environment containing factors necessary for differentiating the human mesenchymal stem cells into cartilage-forming cells.

Claim 14

A method for inducing marrow-derived human mesenchymal stem cells to differentiate into cartilage-forming cells, comprising: a) providing human marrow-derived mesenchymal stem cells that have been culturally expanded from isolated and purified human marrow-derived mesenchymal stem cells which have been isolated from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the culturally expanded human marrow-derived mesenchymal stem cells to a carrier formatted to promote round cell morphology; c) implanting the carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into an environment containing factors necessary for differentiating the human mesenchymal stem cells into cartilage-forming cells.

Claim 15

A method for repairing damaged articular cartilage comprising: a) providing human marrow-derived mesenchymal stem cells that have been culturally expanded from isolated and purified human marrow-derived mesenchymal stem cells which have been isolated from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the culturally expanded human marrow-derived mesenchymal stem cells to a carrier formatted to promote round cell morphology; and, c) implanting the carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into the damaged articular cartilage.

Claim 16

A method for repairing damaged articular cartilage comprising: a) providing human marrow-derived mesenchymal stem cells that have been culturally expanded from isolated and purified human marrow-derived mesenchymal stem cells which have been isolated from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; b) applying the culturally expanded human marrow-derived mesenchymal stem cells to a carrier formatted to promote round cell morphology; and, c) implanting the carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into the damaged articular cartilage.

Claim 17

A method for repairing damaged articular cartilage comprising: a) providing a bone marrow specimen containing human mesenchymal stem cells and bone pieces; b) adding the bone marrow specimen to a medium thereby producing a bone marrow specimen-medium mixture, wherein said medium contains factors which stimulate human marrow-derived mesenchymal stem cell growth without differentiation and allows, when cultured, for selective adherence of only the human marrow-derived mesenchymal stem cells to a substrate surface; c) separating the bone pieces from the bone marrow specimen-medium mixture; d) dissociating marrow cells in the bone marrow specimen-medium mixture into single cells; e) culturing the dissociated marrow cells in the bone marrow medium specimen-mixture thereby selectively adhering only the human mesenchymal stem cells to the substrate surface; f) separating non-adherent matter from the substrate surface, thereby producing isolated culturally expanded human marrow-derived mesenchymal stem cells; g) removing remaining adherent isolated culturally expanded human mesenchymal stem cells from the substrate surface with a releasing agent; h) applying the isolated culturally expanded human marrow-derived mesenchymal cells to a carrier formatted to promote round cell morphology; and, i) implanting the carrier containing the culturally expanded human marrow-derived mesenchymal cells into the damaged articular cartilage.

Claim 18

A method for repairing damaged articular cartilage comprising: a) providing a bone marrow specimen containing human mesenchymal stem cells and bone pieces; b) adding the bone marrow specimen to a medium thereby producing a bone marrow specimen-medium mixture, wherein said medium contains factors which stimulate human marrow-derived mesenchymal stem cell growth without differentiation and allows, when cultured, for selective adherence of only the human marrow-derived mesenchymal stem cells to a substrate surface; c) separating the bone pieces from the bone marrow specimen-medium mixture; d) dissociating marrow cells in the bone marrow specimen-medium mixture into single cells; e) culturing the dissociated marrow cells in the bone marrow medium specimen-mixture thereby selectively adhering only the human mesenchymal stem cells to the substrate surface; f) separating non-adherent matter from the substrate surface, thereby producing isolated culturally expanded human marrow-derived mesenchymal stem cells; g) removing remaining adherent isolated culturally expanded human mesenchymal stem cells from the substrate surface with a releasing agent; h) applying the isolated culturally expanded human marrow-derived mesenchymal cells to a carrier formatted to promote round cell morphology; and, i) implanting the carrier containing the culturally expanded human marrow-derived mesenchymal cells into the damaged articular cartilage.

Claim 21

A method for repairing skeletal defects comprising: a) providing a bone marrow specimen containing human mesenchymal stem cells; b) adding the bone marrow specimen to a medium thereby producing a bone marrow specimen-medium mixture, wherein said medium contains factors that stimulate mesenchymal stem cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; c) adding the bone marrow specimen-medium mixture to a density gradient which separates cells into low, medium and high density cell fractions based on differences in density; d) removing the low density cell fraction from the density gradient; e) adding the low density cell fraction to the medium used in step (b) to produce a low density cell fraction-medium mixture; f) culturing the low density cell fraction-medium mixture, thereby selectively adhering only the mesenchymal stem cells to the substrate surface; g) removing any non-adherent matter from the substrate surface; h) removing remaining adherent mesenchymal stem cells from the substrate surface with a releasing agent, thereby allowing for the isolated mesenchymal stem cells to be recovered; i) applying the recovered isolated mesenchymal stem cells to a porous carrier comprised of about 60% hydroxyapatite and about 40% tricalcium phosphate; and, j) implanting the porous carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into the skeletal defect.

Claim 22

A method for repairing skeletal defects comprising: a) providing a bone marrow specimen containing human mesenchymal stem cells; b) adding the bone marrow specimen to a medium thereby producing a bone marrow specimen-medium mixture, wherein said medium contains factors that stimulate mesenchymal stem cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; c) adding the bone marrow specimen-medium mixture to a density gradient which separates cells into low, medium and high density cell fractions based on differences in density; d) removing the low density cell fraction from the density gradient; e) adding the low density cell fraction to the medium used in step (b) to produce a low density cell fraction-medium mixture; f) culturing the low density cell fraction-medium mixture, thereby selectively adhering only the mesenchymal stem cells to the substrate surface; g) removing any non-adherent matter from the substrate surface; h) removing remaining adherent mesenchymal stem cells from the substrate surface with a releasing agent, thereby allowing for the isolated mesenchymal stem cells to be recovered; i) applying the recovered isolated mesenchymal stem cells to a porous carrier comprised of about 60% hydroxyapatite and about 40% tricalcium phosphate; and, j) implanting the porous carrier containing the culturally expanded human marrow-derived mesenchymal stem cells into the skeletal defect.