WO2012145442A1

WO2012145442A1 - Tissue engineering methods, systems and devices employing ultrasound

Info

Publication number: WO2012145442A1
Application number: PCT/US2012/034136
Authority: WO
Inventors: Clark T. Hung; Elisa E. Konofagou
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2011-04-18
Filing date: 2012-04-18
Publication date: 2012-10-26

Abstract

Embodiments of tissue engineering articles, methods, systems, and devices employ ultrasound to deliver biological agents to selected regions of a tissue scaffold, deliver mechanical stimulation to cells growing in a tissue scaffold, and enhance the perfusion of fluids through tissue scaffolds.

Description

Attorney Docket No. T4356-1 8312WO01 Tissue Engineering Methods, Systems, and Devices Employing Ultrasound

Cross Reference to Related Applications

This application claims the benefit of US Provisional Application 61/476,573 filed 18 April 201 1 , the entirety of which is hereby incorporated by reference herein.

Background

Acoustic radiation force has been used to induce motion in living tissue to allow the non-invasive characterization of tissue properties in a live host. In U.S. Patent Publication No. 2007/0276242 to Konofagou, which is attached as Appendix I to the present provisional and incorporated herein by reference as if set forth in its entirety herein, Konofagou describes systems, methods and apparatus which are used to focus ultrasound in a selected volume of tissue remote from an externally applied transducer to generate motion in the tissue. The techniques and devices of this reference may be employed in the new subject matter described in the disclosure below.

Tissue engineering methods devices and system that employ hydrogels incorporating microbubbles have been described in PCT Patent Publication No. WO 201 1 /028690 (PCT/US2010/047263) to Borden, et al, which is attached as Appendix II to the present provisional and incorporated herein by reference as if set forth in its entirety herein. In this application, Borden, et al., describe tissue scaffolds with microbubbles and seeded with cells. The bubbles may be gas-filled to alter the mechanical properties of the tissue scaffold, for example, by making it compressible. Also, the microbubbles can ameliorate the movement (as by diffusion) of fluids such Attorney Docket No. T4356-1 8312WO01 as perfusate through the tissue scaffold. Microbubbles of a suitable form are described in the incorporated (and attached) reference (Appendix II).

Tissue engineering requires the cultivation and development of cells in realistic environments. For example, some kinds of tissues may require mechanical stimulation or signaling in order to develop properly. Also, thick three-dimensional structures may make it difficult for chemical signaling and nutrient perfusion.

Brief Description of the Drawings

Embodiments will hereinafter be described in detail below with reference to the accompanying drawings, wherein like reference numerals represent like elements. The accompanying drawings have not necessarily been drawn to scale. Where applicable, some features may not be illustrated to assist in the description of underlying features.

Fig. 1 shows an agent depot which comprises an agent-containing material encased in an encapsulating shell that may be disrupted by ultrasound to permit the diffusion of the agent into a surrounding environment.

Fig. 2 shows the agent depot in a tissue scaffold, for example, a

polymerizable material such as a hydrogel such as alginate which may be cell- seeded.

Fig. 3A shows a cell-seeded tissue scaffold with embedded agent depots with an ultrasonic emitter configured to focus ultrasound energy on selected parts of the tissue scaffold to release an agent into the tissue scaffold and influence the development of cells. Attorney Docket No. T4356-1 8312WO01

Figs. 3B and 3C are close-ups of the configuration of Fig. 3A showing, respectively, intact agent depots with isolated contents and agent depots whose encapsulating shells have been opened.

Figs. 4A, 4B, and 4C illustrate a tissue scaffold and agent store separated by a barrier layer with microbubbles which can be disrupted at selected portions to allow the transport of an agent across the barrier layer to affect the activity of cells in the tissue scaffold.

Fig 5A shows a tissue scaffold seeded with cells and microbubbles with an ultrasonic emitter configured to focus ultrasound energy on selected parts of the tissue scaffold to disrupt selected microbubbles causing them to fill with fluid from the scaffold thereby enhancing diffusion or convection through channels or low diffusion resistance regions defined thereby.

Figs. 5B and 5C show close-ups of the configuration of Fig. 5A and, respectively, intact microbubbles and selectively disrupted microbubbles.

Figs. 6A, 6B, and 6C illustrate a method of forming a three dimensional tissue scaffold with agent depot and barrier layer features.

Figs. 7 and 8 illustrate a tissue scaffold supported in an acoustical coupling medium/nutrient bath in an arrangement which may mechanically stimulate the tissue scaffold and/or generate acoustic streaming of the coupling medium/nutrient bath fluid.

Figs. 9 and 10 illustrate the use of ultrasound create pulsed, shifting acoustic streaming, to create channels by disruption of microbubbles and/or use of a mechanical focusing alone or in conjunction with electronic focusing.

Figs. 9 and 10 illustrate mechanical stimulation of engineered tissue structures or tissue scaffolds. Attorney Docket No. T4356-1 8312WO01

Detailed Description of the Embodiments

Microbubbles may be employed in agent delivery devices, methods, and systems. They may allow on-demand ultrasound-triggered release of agents, enzymes, and other factors that are useful in tissue engineering. Methods and systems for controlling the release of drugs in a patient using encapsulated drug depots a described in International Patent Publication WO/201 1 /075557 to Kohane, et al., which is hereby incorporated herein by reference as if set forth in its entirety herein. This publication describes injectable or implantable drug delivery vehicles that permit the release of drugs by ultrasound in a patient. The described systems employ a drug depot and a drug-encapsulated in an encapsulating material. The present embodiments may employ similar depot structures and systems to release bioactive agents into tissue scaffolds.

Referring to Fig. 1 , presently disclosed are agent depots, one of which is shown at 1 It contains an agent 104 (depicted as a separate structure 1 04 but may be a single structure combining the encapsulating material and agent as well) that influences the growth or behavior of cells in an engineered tissue or precursor thereof. The agent 1 04 may include a hydrogel or other material, including polymers that form non-hydrogel matrices following crosslinking. The agent 104 may contain one or more agents, enzymes, nutrients, or other biologically active agents. An encapsulating shell 1 02 may be of similar or identical material as combined with the agent to form the core 104, but which further incorporates microbubbles 106 such as liposomes. The microbubbles may contain any biocompatible gas or mixture of gases. The microparticles enhance the release of agent from the core 104 when ultrasound is applied to the shell and the energy absorbed by the microbubbles Attorney Docket No. T4356-1 8312WO01 causing them to disrupt the shell or fill the microbubbles thereby permitting or enhancing diffusion or convection of the core materials into the surrounding material.

Referring to Fig. 2, agent depots 100 may be formed and incorporated in a tissue scaffold 1 12 such as by combining with a hydrogel precursor prior to forming of the tissue scaffold. The tissue scaffold may be seeded with cells. Referring to Fig. 3A, a tissue scaffold 130 contains agent depots 100 and cells 1 Referring also to Figs. 3B and 3C, an ultrasound transducer 1 28 directs focused sound energy into the tissue scaffold causing the encapsulated agent in agent depots 100 to be released into the tissue scaffold 130 at the parts of the tissue scaffold where the ultrasound energy is focused. A disrupted agent depot is indicated at 1 The ultrasound transducer 128 may employ electronic focusing or may use a reflector or other device to mechanically focus the ultrasound energy.

The depot may be a hydrogel. However, other materials, including polymers that form non-hydrogel matrices following crosslinking, may be used. The depot contains one or more agents or biologies to be delivered encapsulated in an encapsulating material. In a preferred embodiment the agent is encapsulated in liposomes. However, other encapsulating materials, such as nanoparticles, microparticles, or particles greater than 500 microns in size may be used. In a particularly preferred embodiment, the depot also contains microbubbles. The microbubbles may contain any biocompatible gas or mixture of gases. The microparticles enhance agent release from the encapsulating material in response to ultrasound by increasing the difference between baseline and peak release rates compared to the release from the same agent depot in the absence of the

microparticles. Attorney Docket No. T4356-1 8312WO01

Agents incorporated in the depots 100 may include hormones, nutrients, growth factors, angiogenesis factors, or any biological agent that may influence cell growth, movement, or other behaviors. Scaffolds may include any suitable hydrogels or other water soluble polymers or other cross-linking materials with water. Agent depots may be formed by any of the mechanisms and using any of the materials described in International Patent Publication WO/201 1 /0755 Formed agent depots may be combined with tissue scaffold precursor prior to formation of a tissue scaffold which may include molding three-dimensional structures. Agent depots can be substantially larger than the drug depot described in the foregoing publication. This may permit the use of further casting and assembly techniques. For example, depots may be a millimeter or more in size.

Referring to Figs. 4A and 4B, agent depots may be macro-sized substructures adjacent to, or included in, a tissue scaffold structure such as one based on a hydrogel. A tissue scaffold 204 with cells 202 is adjacent to, or incorporates, a barrier structure 206 filled with air filled microbubbles 207 which isolates from target cells 202 an agent containing structure 208 which may be of the material used for storing agents in the foregoing agent depot embodiments. The structures 204, 206, and 208 may be of any arbitrary form and variegated dimensionally along any and all of three orthogonal axes.to form arbitrary three dimensional shapes. At an outer boundary, an ultrasound transducer may be contacted to one of the structures 204, 206, and 208, through a coupling medium such as a liquid if desired, and ultrasound energy focused onto selected parts of the barrier structure 2 The dissipation of ultrasound energy in the microbubbles 207 may disrupt the material of the barrier structure 206 (e.g., same material as described above such as hydrogel) forming channels therein. The channels may permit the convection or diffusion of agent from Attorney Docket No. T4356-1 8312WO01 the agent-containing structure 208 as indicated by arrows 21 2 in Fig. 4B. Ultrasound energy may be directed to focus narrowly on the barrier structure to form one or more channels by disrupting the microbubbles and/or surrounding regions of the barrier structure. Note that barrier structures may be part of one or more macro- sized (e.g., multiple millimeter or centimeter sized) castings 226 embedded in tissue scaffold 222 as indicated in Fig. 4C. The barrier structure may include microbubbles.

Figs. 6A through 6C illustrate one method of forming the tissue scaffold, barrier structure, and agent-containing structures described above. A tissue scaffold 306 structure may be formed such as by casting in a mold 302, 3 A flowing precursor 308 of the barrier layer with incorporated gas-filled microbubbles may be added and molded using the formed tissue scaffold to shape an adjacent surface thereof as shown in Fig. 6B. After forming the barrier structure 309, the mold 302 can be removed. Then the agent-containing precursor 310 can be flowed into an adjacent volume and formed as illustrated in Fig. 6C.

Referring now to Figs. 5A and 5B, a cell (1 14)-containing tissue scaffold 132 includes microbubbles 1 A focusing ultrasound emitter 128 is in direct contact with the scaffold 132 or contacts the scaffold 132 through a coupling material such as a perfusate bath. The ultrasound emitter 1 28 (shown in part at 129 in the zoomed in illustration of Figs. 5B and 5C) direct energy to specific internal regions of the tissue scaffold 132 to disrupt the microbubbles as indicated in 139 causing them to fill with fluid from the surrounding scaffold. The diffusion or convection of fluid through the now-filled microbubbles is enhanced. Multiple microbubbles may be disrupted along a path to form channels 140 in the tissue scaffold 1 The same ultrasound emitter 1 28 or a different one may, upon forming the channels by so disrupting the microbubbles, may induce convective motion of fluid through the channels or more porous medium Attorney Docket No. T4356-1 8312WO01 using acoustic streaming by applying a constant or slowly changing acoustic radiation force to the scaffold where the channels are defined. Cell seeded hydrogel tissue scaffolds with microbubbles according to the present disclosure may be as formed and described in International Patent Publication WO201 1028690 to Mark Borden, et al, filed 21 August 20 This publication also describes using ultrasound to cause the microbubbles to be disrupted using ultrasound and to enhance diffusive transport.

In preferred embodiments, the agent depot contains microbubbles that encapsulate a gas. The microbubbles enhance the agent release when ultrasound is applied compared to the same system in the absence of microbubbles. In a preferred embodiment, the agent delivery system, contains an encapsulating material, preferably liposomes, an agent to be delivered, microbubbles, and at least two hydrogel- forming precursor components. In embodiments, the agent depot also contains microbubbles that encapsulate one or more gases. The microbubbles enhance the agent release when ultrasound is applied compared to the same system in the absence of microbubbles. As used herein, microbubbles refer to micron range-sized spherical gas-filled particles, which can be stabilized by an organic coating, such as a lipid shell, at the gas- liquid interface. Microbubbles having a diameter of 10 microns or less can be generated and used as contrast agents. Depending on controlled ultrasound parameters, the microbubbles may be destroyed by externally applied ultrasound of sufficient intensity so as to release shell material as well as any gas contained by the microbubble shell.

In further embodiments, in vivo, implanted, and ex vivo engineered tissues or tissue scaffolds are mechanically stimulated using ultrasound. Apparatus and methods that are suitable for this are described in United States Patent Application Attorney Docket No. T4356-1 8312WO01

20070276242 to Elisa Konofagou et al, filed 6 April 20 Therein acoustic radiation force is applied to tissue structure and modulated at high frequency for purposes of measuring tissue properties. In the present subject matter, engineered tissue constructs or scaffolds with growing cells are loaded mechanically using the same force but modulated at power levels and frequencies suitable for the mechanical stimulation of the growing cells. Referring to Fig. 7, a tissue scaffold 338 with cells is stimulated mechanically by an oscillating acoustic radiation force from an ultrasonic emitter 3 The acoustic energy may be coupled to the tissue scaffold 338 via a medium or applied directly. The coupling may be through a living host tissue 337 where the tissue scaffold or engineered tissue 338 is implanted in a living host. The ultrasonic emitter may be pressed against the skin 362 or to the wall of a body lumen. Fig. 1 1 shows a tissue construct 404 (scaffold or engineered tissue) to which acoustic radiation force is applied by an emitter having a transducer 400 and focusing reflector 4 As illustrated in Fig. 12, a tissue scaffold or engineered tissue is attached to or resting on a membrane 430 and held in a liquid medium 4 An ultrasound emitter applies regular patterns of acoustic radiation force to the tissue scaffold 424 to stimulate growth, migration, differentiation, signaling or any other biological activity. The acoustic radiation force may also be used to enhance movement of perfusate through the tissue construct, for example, the coupling medium 428 may be a perfusate bath. Focusing by the emitter 422 may be electronic or mechanical.

Fig. 8 shows a tissue scaffold or tissue construct 338 supported by a permeable support 348 in a bath of coupling material 340 which may also provide nutrients and or signaling to cells in the construct 3 The bath, in a container 336, may be replenished by flow through inlet and outlet ports 334 and 3 A focused Attorney Docket No. T4356-1 8312WO01 acoustic radiation force may provide acoustic streaming within the construct 338 or channels molded therein (not shown separately). This streaming may enhance the movement of fresh perfusate into the interior of the construct 3 Fig. 9 shows a similar arrangement, with the identical element labeled identically, wherein an ultrasound emitter, which may employ a reflector 356, scans the acoustic radiation force over a range of angles indicated figuratively by arrows 352, 353, and 3 A single or multiple lobes may be scanned electronically across a tissue construct 338 to induce a continuously varying strain in the construct. Multiple emitters or patterns may be combined to induce different strain spatial and temporal strain patterns in the tissue construct 3 The oscillatory force-inducing local oscillatory motion may be a single amplitude modulated ultrasound beam. The magnitude of the acoustic wave emitted by the source depends on the radiation force and the mechanical frequency response of the target construct. A system may be configured to include a transducer for inducing localized oscillatory motion of tissue through the application of the oscillatory radiation force. The mechanical stimulation can also be generated by varying the phase of two ultrasonic emitters to modulate the interference pattern. A variety of interference modes are possible to produce mechanical strain or forcing as would be apparent to those skilled in the art.

Fig. 1 0 a tissue scaffold or tissue construct 338, which may be supported by a support (not shown) in a bath of coupling material 340 which may also provide nutrients and or signaling to cells in the construct 3 The bath, in a container 336, may be replenished by flow through inlet and outlet ports 334 and 3 The tissue construct 338 incorporates microbubbles. An ultrasonic emitter generates a focused ultrasound beam 370 that may be controlled to create one or more flow channels such as 371 , 372, and 373 in the construct 3Then the same, or a different, emitter Attorney Docket No. T4356-1 8312WO01 produces acoustic streaming. The ultrasound emitter may include a reflector 356 and transducer 3

In further embodiments, hydrogels containing microbubbles and cells which have been homogeneously mixed in hydrogel scaffold are subjected to acoustic forces to cause dissolution of gas-filled microbubbles, creating fluid-filled pores that enhance nutrient diffusion by creating a microporosity in the hydrogel scaffold (which has a nanoporosity). Timing of microbubble dissolution can be controlled by microbubble design (size, lipid composition) that dictates microbubble stability in culture, or "on-demand" using applied hydrostatic pressure or ultrasound. Example: microbubbles (2-4 micron diameter); agarose hydrogel (-0.2 micron or 200 nm pores). The context and suitable mechanisms of the latter set of embodiments is set forth in PCT Patent Publication No. WO 201 1 /028690 (PCT/US2010/047263) to Borden, et al which is incorporated by reference as if fully set forth herein.

In further embodiments, ultrasound is used to cause "on-demand" oscillation of gas-filled microbubbles encapsulated in hydrogel scaffold. This can cause local tissue deformations that a) enhance convection of nutrients from the surrounding, bathing culture media into the engineered tissue construct; b) help to distribute cell synthesized products within the scaffold; c) provide a mechanobiological signal to cells encapsulated in the gel.

Ultrasound may be applied to whole regions of the engineered construct, or on small selected subvolumes using High Intensity Focused Ultrasound (HIFU) or Harmonic Motion Imaging Focused Ultrasound (HMIFU) as described in Appendix I, incorporated and attached. Here, ultrasound can be targeted at specific spatial regions and tissue depths only for localized effects. Using HMIFU. This technique Attorney Docket No. T4356-1 8312WO01 may also be extended to rasterize the targeted subvolume or move the targeted subvolume in a time sequence.

In a further embodiment, a steady acoustic radiation force is applied to a tissue scaffold to induce movement of perfusate therein. This may be combined with the other techniques, systems, and methods described herein.

The foregoing descriptions apply, in some cases, to examples generated in a laboratory, but these examples can be extended to production techniques. For example, where quantities and techniques apply to the laboratory examples, they should not be understood as limiting.

Features of the disclosed embodiments may be combined, rearranged, omitted, etc., within the scope of the invention to produce additional embodiments. Furthermore, certain features may sometimes be used to advantage without a corresponding use of other features. It is, thus, apparent that there is provided, in accordance with the present disclosure, mechanisms for use in tissue engineering. Many alternatives, modifications, and variations are enabled by the present disclosure. While specific embodiments have been shown and described in detail to illustrate the application of the principles of the invention, it will be understood that the invention may be embodied otherwise without departing from such principles. Accordingly, Applicants intend to embrace all such alternatives, modifications, equivalents, and variations that are within the spirit and scope of the present invention.

Claims

Attorney Docket No. T4356-1 8312WO01 Claims

1 . A method for engineering a tissue, comprising:

providing a cell-seeded tissue scaffold;

applying ultrasound to create an acoustic radiation force of such magnitude at a selected volume of the tissue scaffold that a mechanical motion below the ultrasonic frequency of the applied ultrasound is induced.

2. The method of claim 1 , wherein the applying includes modulating a magnitude of the applied ultrasound.

3. The method of any of the above claims, wherein the mechanical motion is an oscillatory motion in the range of 1 to 20000 Hz.

4. The method of any of the above claims, wherein the tissue scaffold includes a hydrogel.

5. The method of either of claims 1 or 4, wherein the ultrasound induces a 0 to 1000 Hz acoustic radiation pressure for a period of time which induces a flow of a perfusate.

6. The method of claim 1 , 4 or 5 further comprising creating channels in the tissue scaffold by disrupting the tissue scaffold using ultrasound.

7. The method of claim 6, wherein the disrupting includes focusing ultrasound in regions of the tissue scaffold containing air-filled microbubbles to cause the air-filled microbubbles to fill with fluid from the tissue scaffold.

8. The method of claim 7, wherein the perfusate includes nutrients.

9. The method of any of the above claims, wherein the tissue scaffold includes a hydrogel.

10. The method of claims 6 or 7, wherein the applying is effective to cause air filled microbubbles to fill with a fluid. Attorney Docket No. T4356-1 8312WO01

1 1 . The method of claims 6 through 8, wherein the tissue scaffold includes liposomes and the applying is also effective to release a material from the liposomes that chemically affects the behavior of the cells.

12. A method for engineering a tissue, comprising:

providing a cell-seeded tissue scaffold, the scaffold incorporating

microbubbles;

growing the cells ex vivo;

applying ultrasound so as to disrupt the microbubbles in a selected volume of the tissue scaffold during the growing.

13. A method for engineering a tissue, comprising:

providing a cell-seeded tissue scaffold, the scaffold incorporating liposomes with a biologically active agent;

growing the cells ex vivo;

applying ultrasound so as to disrupt the liposomes in a selected volume of the tissue scaffold during the growing thereby to release the agent.

14. The method of any of the above claims, wherein the tissue scaffold includes a hydrogel.

15. The method of claims 6 or 7, wherein scaffold includes microbubbles and the applying is effective to cause air filled microbubbles to cause the ultrasound energy to dissipate locally thereto and thereby cause the disruption of the liposomes.

16. A tissue engineering method comprising: forming a hydrogel scaffold having microbubbles incorporated therein, the hydrogel being sterilized to permit cells to be introduced and cultured therein; dissipating ultrasound energy in a focused beam so as to form channels within the scaffold. Attorney Docket No. T4356-1 8312WO01

17. The method of claim 16, where in the dissipating includes destroying the microbubbles to cause adjacent ones to fill with fluid thereby defining channels in the hydrogel.

18. The method of claim 16 or 17, wherein the microbubbles are gas-filled.

19. The method of claim 16, 17, or 1 8, wherein the microbubbles have a shell comprising a protein, lipid or polymer.

20. The method of any of claims 16 through 19, further comprising acoustically streaming a fluid through the channels.

21 . A method of mechanically stimulating cells in a hydrogel, the method comprising:

incorporating microbubbles into the hydrogel;

seeding the hydrogel with cells; and

applying an acoustic radiation force to the hydrogel.

22. The method of claim 21 , wherein the acoustic radiation force is periodic in amplitude.

23. The method of claim 21 or 22 further comprising acoustically streaming a liquid through the hydrogel.

24. The method of claim 21 , 22, or 23, wherein the microbubbles are gas- filled.

25. The method of any of claims 21 through 24, further comprising disrupting the microbubbles with ultrasonic energy to form channels in the hydrogel.

26. The method of claim 25, wherein the acoustically streaming is effective to force fluid through the channels.

27. The method of any of claims 21 through 26, wherein the microbubbles have a shell comprising a protein, lipid or polymer. Attorney Docket No. T4356-1 8312WO01

28. The method of any of claims 21 through 26, wherein the seeding includes seeding the hydrogel with chondrocyte cells.

29. The method any of claims 21 through 26, wherein the microbubbles are incorporated into the hydrogel before the hydrogel is polymerized.

30. The method of any of claims 21 through 26, wherein the applying acoustic radiation force is effectively to mechanically stimulate the cells.

31 . The method of any of claims 21 through 30, wherein the applying acoustic radiation force is amplitude modulated at less than 20000 Hz.

32. The method of any of claims 21 through 30, wherein the applying acoustic radiation force is amplitude modulated at less than 5000 Hz.

33. The method of any of claims 21 through 30, wherein the applying acoustic radiation force is amplitude modulated at less than 1 000 Hz.

34. The method of any of claims 21 through 30, wherein the applying acoustic radiation force is amplitude modulated at less than 500 Hz.

35. The method of any of claims 21 through 30, wherein the applying acoustic radiation force produces a mechanical stimulation by interference between the output of respective ultrasound transducers so as to produce a mechanical strain of the hydrogel that varies at a frequency of less than 20000 Hz.

36. The method of any of claims 21 through 30, wherein the applying acoustic radiation force produces a mechanical stimulation by interference between the output of respective ultrasound transducers so as to produce a mechanical strain of the hydrogel that varies at a frequency of less than 10000 Hz.

37. The method of any of claims 21 through 30, wherein the applying acoustic radiation force produces a mechanical stimulation by interference between Attorney Docket No. T4356-1 8312WO01 the output of respective ultrasound transducers so as to produce a mechanical strain of the hydrogel that varies at a frequency of less than 5000 Hz.

38. The method of any of claims 21 through 30, wherein the applying acoustic radiation force produces a mechanical stimulation by interference between the output of respective ultrasound transducers so as to produce a mechanical strain of the hydrogel that varies at a frequency of less than 500 Hz.

39. The method of any of claims 21 through 30, wherein the applying acoustic radiation force produces a mechanical stimulation by interference between the output of respective ultrasound transducers so as to produce a mechanical strain of the hydrogel that varies at a frequency of less than 100 Hz.

40. A tissue engineering system comprising:

a programmable controller; an actuator coupled to the controller; and a hydrogel having gas-filled microbubbles dispersed therein and being disposed adjacent to the actuator,

the programmable controller being programmed with software instructions to cause the controller to activate an ultrasound transducer to apply deformational loading to the hydrogel at a frequency of less than 1 000 Hz using acoustic radiation force.

41 . A tissue scaffold construct having incorporated therein live cells and agent depots configured to encapsulate an agent but being configured to release the agent when ultrasound energy of a predefined magnitude is applied to the agent depots.

42. The construct of claim 41 , wherein the agent includes a substance the influences the behavior of cells. Attorney Docket No. T4356-1 8312WO01

43. The construct of claim 42, wherein the agent includes any or all of a hormone, a nutrient, a growth factor, or an angiogenesis factor.

44. The construct of claim 41 , 42, or 43, wherein the agent depots comprise, in principal part, a polymer material.

45. The construct of claim 41 , 42, or 43, wherein the agent depots comprise, in substantial part, microbubbles.

46. A method of using the construct of any of claims 41 through 45, comprising: applying ultrasound to first selected regions of the construct to release an agent at the first selected regions in the construct at a first time and later applying ultrasound to second selected regions of the construct to release an agent at the second selected regions in the construct at a second time.

47. A method of using the construct of any of claims 41 through 45, comprising: applying ultrasound to selected regions of the construct to release an agent at the selected regions in the construct.

48. A tissue scaffold having an agent containing portion, a cell containing portion, and a barrier portion isolating the agent containing portion from the agent containing portion.

49. The tissue scaffold of claim 48, wherein the agent containing portion contains agent that influences the growth or behavior of the cells in the cell containing portion.

50. The tissue scaffold of claim 48 or 49, further comprising an ultrasound apparatus configured to disrupt the tissue scaffold barrier portion to release agent from the agent containing portion. Attorney Docket No. T4356-1 8312WO01

51 . A method of using the tissue scaffold of any of claims 48 through 50, comprising applying ultrasound to the barrier layer during a growth period of the cells in the cell containing portion.

52. The tissue scaffold of any of claims 48 to 50, wherein the cell containing portion, the barrier portion, and the agent containing portion are substantially of hydrogel.

53. The tissue scaffold of any of claims 48 to 50 or 52, wherein the barrier portion includes microbubbles.

54. The scaffold of claim 53, wherein the microbubbles include liposomes.

55. The scaffold of claim 53 or 54, wherein the microbubbles contain gas.