Production of tissue engineered digits and limbs

• US 20060257377A1
• Filed: 03/10/2006
• Published: 11/16/2006
• Est. Priority Date: 03/11/2005
• Status: Active Grant

• Current Assignee: Wake Forest University Health Sciences
• Original Assignee: Wake Forest University Health Sciences
• Inventors: Lee, Sang Jin, Atala, Anthony, Yoo, James J., Lim, Grace
• Granted Patent: US 8,728,463 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61F 2/28 Bones joints A61F2/30
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61K 38/18 Growth factors; Growth regulators
• CPC Class Codes: A61L 2430/02 for reconstruction of bones; weight-bearing implants
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3817 Cartilage-forming cells, e.g. pre-chondrocytes
• CPC Class Codes: A61L 27/3821 Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
• CPC Class Codes: A61L 27/3826 Muscle cells, e.g. smooth muscle cells
• CPC Class Codes: A61L 27/383 Nerve cells, e.g. dendritic cells, Schwann cells
• CPC Class Codes: A61L 27/3891 as distinct cell layers
• CPC Class Codes: A61L 27/48 with macromolecular fillers
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: A61L 27/58 Materials at least partially resorbable by the body

Abstract

The invention pertains to methods of producing artificial composite tissue constructs that permit coordinated motion. Biocompatable structural matrices having sufficient rigidity to provide structural support for cartilage-forming cells and bone-forming cells are used. Biocompatable flexible matrices seeded with muscle cells are joined to the structural matrices to produce artificial composite tissue constructs that are capable of coordinated motion.

Claims

1. A method of producing an artificial composite tissue construct permitting coordinated motion, comprising:

providing a first biocompatable structural matrix having sufficient rigidity to provide structural support;

seeding the first matrix with an isolated population of cells selected from the group consisting of cartilage-forming cells, bone-forming cells and combinations thereof;

providing a second biocompatable flexible matrix;

seeding the flexible matrix with an isolated population of muscle progenitor cells (MPCs); and

joining the structural matrix and the flexible matrix to produce an artificial composite tissue construct capable of coordinated motion.

2. The method of claim 1, further comprising joining a flexible matrix to two separate structural matrices to provide a flexible linkage therebetween.

3. The method of claim 1, further comprising joining a flexible matrix to a structural matrix and a natural bone structure to provide a flexible linkage therebetween.

4. The method of claim 1, wherein the structural matrix is selected from the group consisting of an electrospun substrate, a decellularized substrate, and a synthetic polymer substrate.

5. The method of claim 1, wherein the flexible matrix is selected from the group consisting of a decellularized bladder submucosa substrate and an electrospun substrate.

6. The method of claim 1, wherein the step of joining the structural matrix and the flexible matrix comprises using a joining technique selected from the group consisting of suturing, heating, and gluing with biological glue.

7. The method of claim 1, wherein the structural matrix and the flexible matrix further comprise a biological agent selected from the group consisting of nutrients, growth factors, cytokines, extracellular matrix components, inducers of differentiation, products of secretion, immunomodulators, proteins, antibodies, nucleic acids molecules, carbohydrates, and biologically-active compounds which enhance or allow growth of the cellular network or nerve fibers.

8. The method of claim 7, wherein the biological agent is a growth factor selected from the group consisting of transforming growth factor-alpha (TGF-α

), transforming growth factor-beta (TGF-β

), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain derived neurotrophic factor, cartilage derived factor, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), and skeletal growth factor.

9. The method of claim 1, wherein the rigid tissue layer is selected from the group consisting of a cartilage tissue layer and a bone tissue layer.

10. The method of claim 1, wherein the bone-forming cells are selected from the group consisting of osteogenic cells, osteoblasts, osteocytes, osteoclasts, and bone-lining cells.

11. The method of claim 1, wherein the cartilage-cells are selected from the group consisting of chondrocytes and chondroblasts.

12. The method of claim 1, wherein the first biocompatable structural matrix is a composite scaffold.

13. A method for treating a subject with a limb or digit disorder, comprising:

providing an artificial composite tissue construct having coordinated motion comprising a first biocompatable structural matrix having sufficient rigidity to provide structural support seeded with an isolated population of cells selected from the group consisting of cartilage-forming cells, bone-forming cells and combinations thereof, and a second biocompatable flexible matrix seeded with an isolated population of muscle progenitor cells (MPCs), wherein the structural matrix and the flexible matrix are joined;

joining the flexible matrix to the structural matrix and a natural bone structure to provide a flexible linkage therebetween; and

monitoring the subject for an improvement in the limb or digit disorder.

14. The method of claim 13, further comprising joining a flexible matrix to two separate structural matrices to provide a flexible linkage therebetween.

15. The method of claim 13, wherein the step of monitoring the improvement comprises monitoring parameters selected from the group consisting of the mobility of the digit or limb, the extensor and flexor function, the motor function, the sensory function, the contractile response, and the tensile response.

16. A biocompatible composite scaffolding system capable of providing structural support for engineered bone tissue comprising a biodegradable synthetic polymer and naturally derived collagen matrix.

17. The biocompatible composite scaffolding system of claim 16, wherein the synthetic polymer is selected from the group comprising poly(lactide-co-glycolide) (PLGA), poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates, degradable polyurethanes, hydroxyapatite (HA), tricalcium phosphate (TCP), and calcium sulfate.

18. The biocompatible composite scaffolding system of claim 16, wherein the naturally derived collagen matrix is submucosa.

19. The biocompatible composite scaffolding system of claim 18, wherein the submucosa is bladder submucosa (BSM).

20. The biocompatible composite scaffolding system of claim 16, wherein the composite scaffolding system is fabricated using a solvent casting/particulate leaching process.

21. The biocompatible composite scaffolding system of claim 16, wherein the biocompatible composite scaffolding system has substantially uniform porous structures having an average pore diameter ranging from about 50 to about 250 μ

M and a porosity greater than about 50%.

22. The biocompatible composite scaffolding system of claim 21, wherein the average pore diameter ranges from about 90 to about 150 μ

m.

23. The biocompatible composite scaffolding system of claim 21, wherein the porosity is greater than about 80%.

24. A method of fabricating a biocompatible composite scaffolding system capable of providing structural support for regenerated bone tissue comprising:

selecting a pore size,

obtaining porogens of said selected size;

adding the porogens to a solution containing a synthetic polymer and submucosa and mixing to distribute said porogens and form a composite scaffold;

drying said composite scaffold to remove residual solvent; and

removing said porogens from said composite scaffold.

25. The method of claim 24, wherein said porogens are selected from the group consisting of salts, sodium hydroxide, sugars, waxes, gelatins, naphthalene, natural or synthetic water soluble polymers, natural or synthetic non-water soluble polymers, degradable polymers, non-degradable polymers, partially degradable polymers, and mixtures thereof.

26. The method of claim 25, wherein the salts are selected from the group consisting of sodium chloride, potassium chloride, sodium fluoride, potassium fluoride, sodium iodide, sodium nitrate, sodium sulfate, sodium iodate, and mixtures thereof.

27. The method of claim 24, wherein the method further includes sieving porogens through a sieve to obtain said selected size porogens.

28. The method of claim 24, wherein said synthetic polymer is selected from the group comprising poly(lactide-co-glycolide) (PLGA), poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates, degradable polyurethanes, hydroxyapatite (HA), tricalcium phosphate (TCP), and calcium sulfate.

29. The method of claim 24, wherein the synthetic polymer is poly(D,L-lactide-co-glycolide) (PLGA) and the submucosa is bladder submucosa (BSM).

30. The method of claim 29, wherein the weight ratio of PLGA and BSM to NaCl is about 1:

10.

31. The method of claim 24, wherein the step of removing said porogens from said composite scaffold comprises immersion in water.