Ultrasonic modification of soft tissue matrices
The present invention provides a method of, and apparatus for, utilizing ultrasonic energy so as to mechanically disrupt the collagenous architecture of biological matrices in a controlled manner so that the tissue can be rapidly recellularized in continuous form. The present invention also provides for the production of tissue matrices with improved recellularization properties, cell stratification or patterning.
1. A method of preparing an acellular, biomechanically intact, collagenous tissue matrix having a scaffold ultrastructure for recellularization, the method comprising subjecting an intact, acellular collagenous tissue matrix having a scaffold ultrastructure to ultrasonic (US) energy ex vivo, wherein the ultrasonic energy is applied in an amount which is sufficient to form gaps between collagen bundles while leaving the collagenous matrix ultrastructure intact, wherein subjecting said acellular collagenous tissue matrix having a scaffold ultrastructure to the ultrasound energy improves the rccellularization and/or cellular infiltration of the tissue matrix.
2. A method according to claim 1 wherein the US power on the surface of the acellular tissue matrix is in the range of 1 to 1,000 Watts.
3. A method according to claim 2 wherein the US power range is in the range of 10 to 600 Watts.
4. A method according to claim 3 wherein the US power is about 300 Watts.
5. A method according to claim 1 wherein intensity of US energy on the surface of the acellular tissue matrix is in the region of 0.01 to 50 Watts/mm 2 .
6. A method according to claim 5 wherein the US energy is in the region of 0.01 to 50 Watts/mm 2 .
7. A method according to claim 6 wherein the US energy is about 3.0 Watts/mm 2 .
8. A method according to claim 1 wherein the US energy is supplied to the surface of the acellular tissue matrix for a period of time in a range from 1 second to 1 hour.
9. A method according to claim 8 wherein the US energy is supplied for a period of time in a range from 1 to 10 minutes.
10. A method according to claim 1 wherein the US energy is applied as pulsed or continuous energy.
11. A method according to claim 10 wherein pulsed US energy is applied in the range 0.5 on 1 off to 10 on 1 off.
12. A method according to claim 11 wherein the energy is applied as 3 on 1 off.
13. A method according to claim 1 wherein the acellular tissue matrix is bathed in a physiological medium.
14. A method according to claim 13 wherein the medium is physiological saline.
15. A method according to claim 1 further including the step of recellularizing the acellular tissue matrix either in vitro or, post-implantation, in vivo.
16. A method according to claim 15 wherein the acellular tissue matrix is recellularized by at least one cell type selected from the group consisting of: mesenchymal or stromal cells, fibroblasts, smooth muscle cells, tenocytes, ligament cells, skeletal or cardiac myocytes, reticulo-endothelial cells, chondrocytes, neuroectodermal cells, neurons, glial cells, astrocytes, endocrine cells, melanocytes, adrenal, pituitary, or islet cells and stem cells.
17. A method according to claim 1 further comprising one or more additional treatments of the acellular tissue matrix to facilitate recellularization or successful implantation.
18. A method according to claim 17 wherein the additional treatment is a growth factor and/or an adhesion molecule.
19. A method according to claim 1 wherein the acellular tissue matrix has a cell penetration depth after recellularization of between 5 to 100% of the total acellular tissue matrix thickness.
20. A method according to claim 1 , wherein the acellular tissue matrix is configured for implantation.
21. A method according to claim 1 wherein the gaps between collagen bundles are formed without damage to the collagen.
22. A method according to claim 1 wherein after subjecting the acellular, intact, tissue matrix to ultrasonic (US) energy, the biomechanical function of the acellular tissue matrix scaffold ultrastructure is retained.
23. A method according to claim 1 wherein collagen of the acellular tissue matrix is not damaged by ultrasonication.
24. A method according to claim 1 wherein the acellular tissue matrix is configured to direct a migration of cells and to predetermine a distribution of the cells to achieve uniformly dispersed, localized or stratified cell patterning within the acellular tissue matrix.
25. A method according to claim 1 further comprising applying the US energy using an ultrasound probe configured to contact or be held in close proximity to the acellular tissue matrix.
26. A method according to claim 20 further comprising applying the US energy using an ultrasound probe configured to contact or be held in close proximity to the acellular tissue matrix.