PORTILLO LUIS (CA)
AREIAS NICOLE (CA)
HUYER, LOCKE DAVENPORT AND ZHANG BOYANG, KOROLJ ANASTASIA, MONTGOMERY MILES, DRECUN STASJA, CONANT GENEVIEVE, ZHAO YIMU, REIS LEWI: "Highly Elastic and Moldable Polyester Biomaterial for Cardiac Tissue Engineering Applications", ACS BIOMAT SCI ENG, vol. 2, no. 5, 28 April 2016 (2016-04-28), pages 780 - 788, XP055766285, ISSN: 2373-9878, DOI: 10.1021/acsbiomaterials.5b00525
RAVICHANDRAN, R. ET AL.: "Minimally invasive injectable short nanofibers of poly(glycerol sebacate) for cardiac tissue engineering", NANOTECHNOLOGY, vol. 23, 5 September 2012 (2012-09-05), pages 385102, XP020228698, ISSN: 1361-6528, DOI: 10.1088/0957-4484/23/38/385102
MONTGOMERY, MILES, AHADIAN SAMAD, DAVENPORT HUYER LOCKE, LO RITO MAURO, CIVITARESE ROBERT A., VANDERLAAN RACHEL D., WU JUN, REIS L: "Flexible shape-memory scaffold for minimally invasive delivery of functional tissues", NATURE MATERIALS, vol. 16, no. 10, October 2017 (2017-10-01), pages 1038 - 1046, XP055766289, ISSN: 1476-4660, DOI: 10.1038/nmat4956
LOH, X. J. ET AL.: "Poly(glycerol sebacate) biomaterial : synthesis and biomedical applications", J MATER CHEM B, vol. 3, 21 October 2015 (2015-10-21), pages 7641 - 4652, XP055591187, ISSN: 2050-7518, DOI: 10.1039/C5TB01048A
SAPIR, Y. ET AL.: "Cardiac tissue engineering in magnetically actuated scaffolds", NANOTECHNOLOGY, vol. 25, 11 December 2013 (2013-12-11), pages 014009, XP020255899, ISSN: 1361-6528, DOI: 10.1088/0957-4484/25/1/014009
WAY, LOUISE, SCUTT NANETTE, SCUTT ANDREW: "Cytocentrifugation: a convenient and efficient method for seeding tendon-derived cells into monolayer cultures or 3-D tissue engineering scaffolds", CYTOTECHNOLOGY, vol. 63, no. 6, 25 September 2011 (2011-09-25), pages 567 - 579, XP055766294, ISSN: 1573-0778, DOI: 10.1007/s10616-011-9391-4
| CLAIMS: 1. A tissue construct comprising: a biocompatible and substantially linear scaffold; and a plurality of cells supported by and longitudinally aligned along the scaffold. 2. The construct of claim 1, wherein the scaffold is untethered. 3. The construct of claims 1 or 2, wherein the scaffold is tensioned to encourage elongation of the plurality of cells. 4. The construct of any one of claims 1 to 3, wherein the scaffold is relatively rigid yet compressible and resilient. 5. The construct of any one of claims 1 to 4, wherein the scaffold comprises a biocompatible polymer. 6. The construct of claim 5, wherein the biocompatible polymer is an elastomer. 7. The construct of claim 5, wherein the biocompatible polymer comprises poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate), poly(citric diol), poly(tri-methylene carbonate), polyester amide, polyester carbonate urethane urea, poly(octanediol-co-citrate), poly (lactic-co-glycolic acid), poly(glycolic acid), polylactic acid, poly(caprolactone), poly(hydroxyvalerate), polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl-methacrylate), poly(ethylene glycol), combinations thereof, or copolymers thereof. 8. The construct of claim 7, wherein the biocompatible polymer comprises poly(glycerol sebacate) acrylate. 9. The construct of any one of claims 1 to 8, wherein the scaffold is biodegradable. 10. The construct of any one of claims 1 to 9, wherein the scaffold comprises a zig zag shape. 11. The construct of claim 10, wherein the scaffold comprises a strut diameter of about 0.1 pm to about 1000 pm 12. The construct of any one of claims 1 to 11, wherein the scaffold further comprises magnetic particles. 13. The construct of claim 12, wherein the magnetic particles are embedded within the scaffold. 14. The construct of any one of claims 1 to 13, wherein the plurality of cells comprises mammalian cells. 15. The construct of any one of claims 1 to 14, wherein the plurality of cells comprises hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, smooth or skeletal muscle cells, myocytes, adipocytes, ectodermal cells, ductile cells, kidney cells, intestinal cells, parathyroid and thyroid cells, nerve cells, ocular cells, integumentary cells, pluripotent cells and stem cells, or combinations thereof. 16. The construct of any one of claims 1 to 15, wherein the plurality of cells comprises cardiomyocytes, fibroblasts or a combination thereof. 17. The construct of any one of claims 1 to 16, wherein the construct is for use in cell therapy. 18. The construct of any one of claims 1 to 17, wherein the construct is for delivery into or onto an organ via injection for cell therapy. 19. The construct of any one of claims 1 to 18, wherein the construct is for delivery into an organ via intramuscular injection for cell therapy. 20. The construct of any one of claims 1 to 19, wherein the construct does not include a hydrogel. 21. A delivery device, such as a syringe, for delivering the construct of any one of claims 1 to 20. 22. The delivery device of claim 21, wherein the syringe comprises a needle and wherein the needle bore is substantially blocked near the syringe end in order to retain the construct in the needle of the syringe. 23. The delivery device of claim 22, wherein the needle bore is blocked by a smaller coaxial needle. 24. The delivery device of claim 22, wherein the needle bore is blocked with a plug, such as a sponge plug. 25. The delivery device of any one of claims 21 to 24, comprising the construct. 26. A substantially linear biocompatible scaffold for supporting a plurality of cells. 27. A spring-like biocompatible scaffold for supporting a plurality of cells for elongate growth. 28. The scaffold of claim 26 or 27, wherein the scaffold is untethered. 29. The scaffold of any one of claims 26 to 28, wherein the scaffold is tensioned to encourage elongation of the plurality of cells. 30. The scaffold of any one of claims 26 to 29, wherein the scaffold is relatively rigid yet compressible and resilient. 31. The scaffold of any one of claims 26 to 30, wherein the scaffold comprises a biocompatible polymer. 32. The scaffold of claim 31, wherein the biocompatible polymer is an elastomer. 33. The scaffold of claim 31, wherein the biocompatible polymer comprises poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate), poly(citric diol), poly(tri-methylene carbonate), polyester amide, polyester carbonate urethane urea, poly(octanedio 1-co-citrate), poly (lactic-co-glycolic acid), poly(glycolic acid), polylactic acid, poly(caprolactone), poly(hydroxyvalerate), polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl-methacrylate), poly(ethylene glycol), combinations thereof, or copolymers thereof. 34. The scaffold of claim 33, wherein the biocompatible polymer comprises poly(glycerol sebacate) acrylate. 35. The scaffold of any one of claims 26 to 34, wherein the scaffold is biodegradable. 36. The scaffold of any one of claims 26 to 35, wherein the scaffold comprises a zig zag shape. 37. The scaffold of claim 36, wherein the scaffold comprises a strut diameter of about 0.1 pm to about 1000 pm. 38. The scaffold of any one of claims 26 to 37, wherein the scaffold further comprises magnetic particles. 39. The scaffold of claim 38, wherein the magnetic particles are embedded within the scaffold. 40. The scaffold of any one of claims 26 to 39, seeded with the plurality of cells, wherein the plurality of cells comprises mammalian cells. 41. The scaffold of claim 40, wherein the plurality of cells comprises hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, smooth or skeletal muscle cells, myocytes, adipocytes, ectodermal cells, ductile cells, kidney cells, intestinal cells, parathyroid and thyroid cells, nerve cells, ocular cells, integumentary cells, pluripotent cells and stem cells, or combinations thereof. 42. The scaffold of 41, wherein the plurality of cells comprises cardiomyocytes, fibroblasts or a combination thereof. 43. The scaffold of any one of claims 26 to 42, wherein the scaffold does not include a hydrogel. 44. A method of making a tissue construct, the method comprising seeding a plurality of cells onto the scaffold of any one of claims 26 to 43. 45. A culture plate for seeding cells onto a scaffold, the plate comprising an elongate bottom wall and side walls that are at a greater than 90 degree angle relative to the bottom wall. 46. The culture plate of claim 45, wherein the bottom wall is substantially rectangular and the side walls funnel towards the bottom wall. 47. The culture plate of claim 45 or 46, wherein the culture plate is a 384 well plate. 48. The culture plate of any one of claims 45 to 47 comprising the scaffold of any one of claims 26 to 42. 49. The culture plate of any one of claims 45 to 47, comprising the construct of any one of claims 1 to 25. 50. A method for making the scaffold of any one of claims 26 to 43, the method comprising polymerizing a biocompatible polymer to produce the substantially linear scaffold. 51. A method for making a tissue construct, the method comprising seeding a plurality of cells onto a spring-like biocompatible scaffold for elongate growth. 52. The method of claim 51, further comprising forming the scaffold by polymerization, such as photo-patterning polymerization. 53. The method of claim 51 or 52, further comprising placing the scaffold in a funnel- shaped culture well for seeding the cells. 54. The method of claim 53, wherein placing the scaffold in the well comprises centrifuging the scaffold and well to funnel the scaffold to the bottom of the well. 55. The method of any one of claims 51 to 54, wherein seeding the cells comprises centrifuging the cells and scaffold to aggregate the cells around the scaffold. |
FIELD
The present application relates to tissue engineering, and in particular, to tissue constructs with biocompatible linear scaffolds, and methods of making and uses thereof.
BACKGROUND
Tissue engineering applies the principles of biology and engineering to create lunctional tissues from human cells and scaffolds to replace damaged tissues in patients. Thus far, engineering of almost all tissues of the human body (e.g., cardiac 1 , vascular 2 , cartilage 3 , bone 4 , brain 5 , eye 6 , etc.) has been reported. However, to perform therapeutic repairs, lab-grown tissues, while preserving tissue structural organization, need to be delivered in vivo to repair damaged tissues in patients, which often makes the implantation surgery invasive. Although cells can be injected with hydrogels in a minimally invasive manner, the lack of tissue-level organization
impedes the immediate functionality and retention of cells upon delivery . Alternatively, expandable scaffolds can be used to deliver tissues via injection, but this approach is limited by the tissue size as well as the location of delivery as the folded tissue can only unfold on the surface of an organ, for example on the surface of the heart 13 . For cardiac repairs, epicardial implantation of engineered cardiac patches, leads to significant lunctional improvements but lacks physical integration with host tissues, hindered by the presence of the epicardium layer on the surface of the heart 14 .
Tissue engineering can also be applied to the equally challenging field of drug development. Nearly 2.5 billion dollars are used for research and development to develop one drug over an average of 5 years, of which $1.46 billion is spent on clinical trials and $1 098 billion on animal and preclinical studies. 15 However, this significant increase in expenditure does not necessarily correspond with an increase in approval success rates. In fact, roughly half of investigational drugs that enter late-stage clinical development fail during or after pivotal clinical trials due to concerns over safety and efficacy. 16 Some of these drugs include anthracyclines, anticancer, and antidiabetic drugs, which have been shown to cause direct myocardial toxicity, exacerbate underlying myocardial dysfimction, or induce heart failure. 17 Not only do these trials necessitate substantial investment on the part of investigators, but they also subject a large numbers of participants and patients to an increased risk of adverse events or death. 18
There is then the case of developing drugs for the treatment of cardiovascular disease. Despite the increasing global burden of cardiovascular disease, investment in cardiovascular drug development has remained stagnant over the past two decades. 19 This is due, in part, to the significant regulatory uncertainty associated with current biomarkers and putative surrogates in cardiovascular drug research. As such, the development of these drugs demand a direct assessment of risks and benefits, using clinically-evident cardiovascular endpoints via cardiovascular outcome trials. 19 The high costs associated with these trials have contributed to the development challenges faced by both novel cardiovascular therapies and other drugs where cardiovascular health is implicated.
There is a need for novel tissue constructs that overcome one or more of the deficiencies of existing tissue constructs.
SUMMARY
In accordance with an aspect, there is provided a tissue construct comprising:
a biocompatible and substantially linear scaffold; and
a plurality of cells supported by and longitudinally aligned along the scaffold.
In an aspect, the scaffold is untethered.
In an aspect, the scaffold is tensioned to encourage elongation of the plurality of cells.
In an aspect, the scaffold is relatively rigid yet compressible and resilient.
In an aspect, the scaffold comprises a biocompatible polymer.
In an aspect, the biocompatible polymer is an elastomer.
In an aspect, the biocompatible polymer comprises poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate), poly(citric diol), poly(tri-methylene carbonate), polyester amide, polyester carbonate urethane urea, poly(octanediol-co-citrate), poly (lactic-co-glycolic acid), poly(glycolic acid), polylactic acid, poly(caprolactone), poly(hydroxyvalerate), polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl-methacrylate), poly(ethylene glycol), combinations thereof, or copolymers thereof. In an aspect, the biocompatible polymer comprises poly(glycerol sebacate) acrylate.
In an aspect, the scaffold is biodegradable.
In an aspect, the scaffold comprises a zig zag shape.
In an aspect, the scaffold comprises a strut diameter of about 0.1 pm to about 1000 pm.
In an aspect, the scaffold lurther comprises magnetic particles.
In an aspect, the magnetic particles are embedded within the scaffold.
In an aspect, the plurality of cells comprises mammalian cells.
In an aspect, the plurality of cells comprises hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, smooth or skeletal muscle cells, myocytes, adipocytes, ectodermal cells, ductile cells, kidney cells, intestinal cells, parathyroid and thyroid cells, nerve cells, ocular cells, integumentary cells, pluripotent cells and stem cells, or combinations thereof.
In an aspect, the plurality of cells comprises cardiomyocytes, fibroblasts or a combination thereof.
In an aspect, the construct is for use in cell therapy.
In an aspect, the construct is for delivery into or onto an organ via injection for cell therapy.
In an aspect, the construct is for delivery into an organ via intramuscular injection for cell therapy.
In an aspect, the construct does not include a hydrogel.
In accordance with an aspect, there is provided a delivery device, such as a syringe, for delivering the construct described herein.
In an aspect, the syringe comprises a needle and wherein the needle bore is substantially blocked near the syringe end in order to retain the construct in the needle of the syringe.
In an aspect, the needle bore is blocked by a smaller coaxial needle.
In an aspect, the needle bore is blocked with a plug, such as a sponge plug.
In an aspect, the delivery device comprises the construct. In accordance with an aspect, there is provided a substantially linear biocompatible scaffold for supporting a plurality of cells.
In accordance with an aspect, there is provided a spring-like biocompatible scaffold for supporting a plurality of cells for elongate growth.
In an aspect, the scaffold is untethered.
In an aspect, the scaffold is tensioned to encourage elongation of the plurality of cells.
In an aspect, the scaffold is relatively rigid yet compressible and resilient.
In an aspect, the scaffold comprises a biocompatible polymer.
In an aspect, the biocompatible polymer is an elastomer.
In an aspect, the biocompatible polymer comprises poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate), poly(citric diol), poly(tri-methylene carbonate), polyester amide, polyester carbonate urethane urea, poly(octanediol-co-citrate), poly (lactic-co-glycolic acid), poly(glycolic acid), polylactic acid, poly(caprolactone), poly(hydroxyvalerate), polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl-methacrylate), poly(ethylene glycol), combinations thereof, or copolymers thereof.
In an aspect, the biocompatible polymer comprises poly(glycerol sebacate) acrylate.
In an aspect, the scaffold is biodegradable.
In an aspect, the scaffold comprises a zig zag shape.
In an aspect, the scaffold comprises a strut diameter of about 0.1 pm to about 1000 pm.
In an aspect, the scaffold further comprises magnetic particles.
In an aspect, the magnetic particles are embedded within the scaffold.
In an aspect, the scaffold is seeded with the plurality of cells, wherein the plurality of cells comprises mammalian cells.
In an aspect, the plurality of cells comprises hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, smooth or skeletal muscle cells, myocytes, adipocytes, ectodermal cells, ductile cells, kidney cells, intestinal cells, parathyroid and thyroid cells, nerve cells, ocular cells, integumentary cells, pluripotent cells and stem cells, or combinations thereof.
In an aspect, the plurality of cells comprises cardiomyocytes, fibroblasts or a combination thereof.
In an aspect, the scaffold does not include a hydrogel.
In accordance with an aspect, there is provided a method of making a tissue construct, the method comprising seeding a plurality of cells onto the scaffold described herein.
In accordance with an aspect, there is provided a culture plate for seeding cells onto a scaffold, the plate comprising an elongate bottom wall and side walls that are at a greater than 90 degree angle relative to the bottom wall.
In an aspect, the bottom wall is substantially rectangular and the side walls funnel towards the bottom wall.
In an aspect, the culture plate is a 384 well plate.
In an aspect, the culture plate comprises the scaffold described herein.
In an aspect, the culture plate comprises the construct described herein.
In accordance with an aspect, there is provided a method for making the scaffold described herein, the method comprising polymerizing a biocompatible polymer to produce the substantially linear scaffold.
In accordance with an aspect, there is provided a method for making a tissue construct, the method comprising seeding a plurality of cells onto a spring-like biocompatible scaffold for elongate growth.
In an aspect, the method further comprises forming the scaffold by polymerization, such as photo-patterning polymerization.
In an aspect, the method further comprises placing the scaffold in a funnel-shaped culture well for seeding the cells.
In an aspect, wherein placing the scaffold in the well comprises centrifuging the scaffold and well to funnel the scaffold to the bottom of the well. In an aspect, seeding the cells comprises centrifuging the cells and scaffold to aggregate the cells around the scaffold.
In accordance with an aspect, there is provided a tissue construct comprising:
a) a linear scaffold comprised of a biocompatible polymer and
b) a plurality of connected cells embedded onto the linear scaffold,
c) wherein the plurality of connected cells is assembled longitudinally along the axis of the linear scaffold.
In an aspect, tension exerted by the linear scaffold structurally supports an elongated shape of the tissue construct.
In an aspect, the linear scaffold facilitates compression of the tissue construct.
In an aspect, the biocompatible polymer is an elastomer.
In an aspect, the linear scaffold comprises a polymer material selected from the group consisting of poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate), poly(citric diol), poly(tri-methylene carbonate), polyester amide, polyester carbonate urethane urea, poly(octanediol-co-citrate), poly (lactic-co-glycolic acid), poly(glycolic acid), polylactic acid, poly(caprolactone), poly(hydroxyvalerate), polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl-methacrylate), poly(ethylene glycol) or combinations and copolymers thereof.
In an aspect, the linear scaffold is biodegradable.
In an aspect, the linear scaffold comprises poly(glycerol sebacate) acrylate.
In an aspect, the linear scaffold comprises a zig zag shape.
In an aspect, the linear scaffold comprises a strut diameter of about 0.1 pm to about 1000 pm.
In an aspect, the linear scaffold further comprises magnetic particles.
In an aspect, the magnetic particles are embedded within the linear scaffold.
In an aspect, the plurality of connected cells comprises mammalian cells.
In an aspect, the plurality of connected cells comprises hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, smooth or skeletal muscle cells, myocytes, adipocytes, ectodermal cells, ductile cells, kidney cells, intestinal cells, parathyroid and thyroid cells, nerve cells, ocular cells, integumentary cells, pluripotent cells and stem cells, or combinations thereof.
In an aspect, the plurality of connected cells comprises cardiomyocytes, fibroblasts or a combination thereof.
In an aspect, the construct is used for cell therapy.
In an aspect, the construct is delivered into or onto an organ via injection for cell therapy.
In an aspect, the construct is delivered into an organ via intramuscular injection for cell therapy.
In accordance with an aspect, there is provided a method for assembling a tissue construct comprising:
a) fabricating a linear scaffold comprised of a biocompatible polymer using photo- patterning polymerization,
b) transferring the linear scaffold to a fimnel-shaped well of a micro well plate, and c) allowing a plurality of cells to adhere to the linear scaffold and continue to grow to form a tissue construct comprised of a connected cellular aggregate.
In an aspect, the method fiirther comprises removing the tissue construct from the well of the micro well plate.
Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating aspects of the invention, are given by way of illustration only and the scope of the claims should not be limited by these aspects, but should be given the broadest interpretation consistent with the description as a whole.
BRIEF DESCRIPTION OF THE DRAWINGS
Certain aspects will now be described in greater detail with reference to the attached drawings in which: FIGURE 1 shows the chemical composition and degradation of PGSA micro-scaffolds in exemplary aspects: a) NMR of PGSA prepolymer shows the chemical structure and composition of the synthetic polymer. Molecular structure of the R group is shown in the dotted box; b) E elastic modulus of UV+Heat crosslinked PGSA (n=8) and UV crosslinked PGSA (n=7). *p<0.05. data displayed as mean ± SD and statistically assessed with one-way ANOVA; c) repeatability of elastic modulus quantification in between different batches of PGSA at different UV exposure times; d) comparison of elastic modulus of PGSA with 2% and 5% (m/m) photoinitiator (PI); e) brightfield images of the degradation process of PGSA micro- scaffolds with different concentrations of photoinitiator (PI) in 0.1 M NaOH solution (micro- scaffolds degraded to completion in all cases within 6 h).
FIGURE 2 shows a schematic of the photo-polymerization microfabrication technique used for tissue scaffold fabrication in an exemplary aspect.
FIGURE 3 shows PGSA micro-scaffolds in customized 384-well plate in exemplary aspects: a) image of the scaffold from side view and top view; b) image of an entire customized 384-well plate loaded with micro-scaffolds and zoomed-in image of the plate.
FIGURE 4 shows the high-throughput production of tissue constructs with (magnetic) micro-scaffolds in exemplary aspects: a) a customized 3D printed 384-well plate with firnnel- shaped wells to facilitate tissue assembly; b) a schematic outlining the process of scaffold loading, cell seeding, tissue formation, and tissue release; c-e) brightfield images of (c) an empty fimnel-shaped well, (d) a well loaded with a micro-scaffold, and (e) a well seeded with a cardiac tissue.
FIGURE 5 shows seeding density optimization for cardiac tissues in exemplary aspects: brightfield images of cardiac tissues seeded with 100% cardiac fibroblasts at various seeding densities.
FIGURE 6 shows the high-throughput assembly of cardiac micro-tissues in exemplary aspects: a) brightfield image of micro-tissues assembled with or without micro-scaffolds (scale bar, 1mm); b) quantification of the tissue length of micro-tissues assembled with (n=5) or without (n=3) micro-scaffolds over 4 days; c-d) fluorescent images of a micro-tissue assembled without a micro-scaffold and stained for F-actin and DAPI; e-f) fluorescent images of a micro tissue assembled with a micro-scaffold and stained for F-actin and DAPI, showing the collective alignment of the cellular structure; g-i) high magnification fluorescent images of a human cardiac micro-tissue assembled with a micro-scaffold and stained for F-actin, sarco meric a- actinin and DAPI, showing the presence and alignment of human cardiomyocytes within the micro-tissue (scale bars are 30 pm in d) and f); 20 pm in g), h) and i)).
FIGURE 7 shows the quantification of tissue alignment in exemplary aspects: a) visual directional analysis of cardiac tissue stained for F-actin with or without scaffolds; b) histogram of orientation distribution of F-actin staining for cardiac tissue with or without scaffolds (presence of a peak shows that the scaffold group had a higher alignment in comparison to the no scaffold group); c) coherency analysis shows a higher orientation consistency in the scaffold group (n=3, * p<0.05; sata displayed as mean ± SD and statistically assessed with one-way ANOVA).
FIGURE 8 shows the scalable production of vascularized neonatal rat cardiac scaffolded tissues in exemplary aspects: a) brightfield image overlaid with fluorescent images of a tissue assembled with 30% GFP-endothelial cells, 50% rat cardiomyocytes, and 20% rat fibroblasts over time (scale bar, 1mm); b) confocal fluorescent image of vascularized cardiac tissue stained for F-actin and GFP-endothelial cells (high magnification images show regions of the tissue labeled by white dotted boxes); c) brightfield image of tissue shown with overlay outlines in relaxation and contraction state; d) representative tissue contraction traces (scale bar shows the amplitude of contraction and time); e) summary of fimctional parameters from tissue contraction (n=3; data displayed as mean ± SD); f) brightfield image showing an array of cardiac tissues; g) brightfield image showing a cluster of cardiac tissues released from the plate.
FIGURE 9 shows magnetic guided anisotropic tissue assembly in vitro in exemplary aspects: a) magnetic alignment of scaffolds; b) correlation of magnetic field strength to distance from the magnet (the grey region is the cut off distance within which the scaffolds can respond to the magnet); c) brightfield images show the self-alignment and physical integration of a cluster of neonatal rat tissues in response to an external magnetic field and after 3 days in culture post assembly (ruler shown is in mm scale); d) fluorescent image of assembled neonatal rat tissues containing GPF-endothelial cells 3 days post assembly (scale bar, 2mm; high magnification images show regions of the tissue labeled by white dotted boxes and highlights the integration between tissues). FIGURE 10 shows magnetic alignment of the scaffolds in vitro in exemplary aspects: a- b) magnetic alignment of scaffolds in response to magnetic field of various direction; arrows indicate the direct of the magnetic alignment.
FIGURE 11 shows a customized surgical delivery tool from modified syringe for tissue injection using a 22-gauge needle that serves as a plunger fitted inside a 19-gauge needle to push out the loaded tissues during injection (tissues are loaded to the needle section of the syringe; the luminal space inside the needle is used as a reservoir for the tissues) in an exemplary aspect.
FIGURE 12 shows tissue injection and delivery in exemplary aspects: a) manipulation of iPSC-derived human cardiac tissue with or without scaffold shows scaffold-free tissue lack mechanical strength to support weight of the tissue for surgical manipulation and injection (black dotted circles indicate the tissue located at the tip of a tweezer); b) live and dead staining with CFDA and PI on cardiac tissues with or without surgical injection in vitro (scale bar; lmm); c) surgical injection of iPSC-derived human cardiac tissues into a gelatin-based transparent heart model; d) high magnification images showing the release and alignment of an iPSC-derived human micro-tissue in the orientation of the needle; e) image of a cluster of tissues all oriented in the same direction after multiple injections; f) image of multiple tissues delivered and aligned in a single injection (dotted white circle outline the injected tissues; black arrow indicates the orientation of alignment).
FIGURE 13 shows the surgical delivery of scaffolds (top) or tissue constructs (bottom) and magnetic guided self-assembly on muscles (white dotted circle outlines the injected scaffolds or micro-tissues; black arrows indicate direction of alignment) in exemplary aspects.
FIGURE 14 shows the ex vivo intra-muscular tissue delivery and in vivo tissue biocompatibility of constructs in exemplary aspects: a) a slice of heart chamber before and after tissue clearing; b-c) brightfield image of a heart slice with an injected neonatal rat cardiac tissue in the myocardium (scale bar, lmm); d) fluorescent image of an injected neonatal rat cardiac tissue in the myocardium showing implanted GFP -endothelial cells around the scaffold (scale bar, lmm); e) image of neonatal rat tissue immediately after implantation; f-h) histological section of subcutaneously implanted neonatal rat tissue stained for (f) H&E and (g-h) Masson’s Trichrome after 1 month (scale bar, lOOpm). DETAILED DESCRIPTION
Described herein are smart scaffolds with more advanced functionalities in addition to basic structural support for automatic assembly of complex biological tissues from individual tissue modules. Also described herein are more realistic models with the capacity to mimic the physical and chemical properties of physiological environments, in order to improve the predictive power of cardiovascular biomarkers that are used in preclinical research, thereby reducing the number of unsuccessfiil candidates that proceed to clinical trials. In the case of both targeted cardiac cell therapy and drug development, a smart scaffold as described herein will help achieve the higher complexity and lunctionalities that these applications demand.
Further, a new therapeutic solution is described for the intramyocardial delivery of fimctional cardiac tissues in a minimally invasive manner to facilitate cell retention/survival, re- muscularization, fimctional coupling, and reduced arrhythmias. Novel solutions that address the shortcomings of preclinical research (i.e. translational failures of traditional animal models and in vitro cell cultures) are also described.
More specifically, described herein, in aspects, is the fabrication of a three-dimensional (3D) tissue construct with a linear scaffold that provides structural support and guidance to tissue organization to impart an elongated shape to the construct. In aspects, the linear scaffold has a zig-zag shape, which functions similarly to a spring to facilitate scaffold compression while providing tension to induce longitudinal alignment of the assembled cells along the linear scaffold. This is particularly helpful for the assembly of cardiomyocytes to produce engineered cardiac tissues that compress (not stretch) the scaffolds when contracting, which can be used to mimic myocardial contractility.
The linear scaffold can be fabricated using a biocompatible and bioresorbable polymer such that assembled micro-tissue constructs may be delivered via injection for tissue repair and therapy to guide biological growth in a step-wise fashion at multiple length and time scales but also degrade as the tissue heals. The linear scaffold can also be synthesized to contain magnetic nanoparticles such that the tissue constructs may be remotely controlled and delivered to the therapeutic site with magnetic guidance. This approach demonstrates the potential to deliver fimctional tissue directly into host muscles, such as the intra-myocardium delivery of fimctional cardiac muscle tissues that bypass the cardiac epithelium to enhance tissue integration in cardiac repairs. Alternatively, these elongated tissue constructs can be used as a miniaturized 3D models in drug research and development, such as a cardiac micro-tissue that can better represent the functional and biomechanical properties of myocardial tissues and replace existing pre-clinical models.
The microwell plate-based fabrication method used to produce the tissue constructs described herein allows for cell seeding and micro-tissue assembly, without the use of any hydrogels to maintain an elongated shape, in a high-throughput manner. Additionally, this fabrication method allows for efficient quality control using high-content imaging of each individual construct and can be easily used to create microwell plate-based high-throughput drug testing platforms.
I Definitions
Unless otherwise indicated, the definitions and aspects described in this and other sections are intended to be applicable to all aspects herein described for which they are suitable as would be understood by a person skilled in the art.
The term“linear” is used herein to describe the scaffolds upon which cells are seeded and grow to provide a tissue construct. This term is used to indicate that the scaffold is longer than it is wide and does not necessarily imply that the scaffold is straight. Typically, the scaffold is spring-like and may be z-shaped, zig-zagged, serpentine, snake-like, helical, or undulating for example. In aspects, the scaffolds appear straight when viewed from one perspective and are zig-zagged when viewed from another perspective.
The term“longitudinally aligned” means that the cells growing on the scaffold tend to grow along the length of the linear scaffold, substantially retaining the linearity of the scaffold, rather than growing outward into a spheroid shape.
In understanding the scope of the present application, the articles“a”,“an”,“the”, and “said” are intended to mean that there are one or more of the elements. Additionally, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. It will be understood that any aspects described as“comprising” certain components may also“consist of’ or“consist essentially of,” (or vice versa ) wherein“consisting of’ has a closed-ended or restrictive meaning and “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effects described herein. For example, a composition defined using the phrase“consisting essentially of’ encompasses any known pharmaceutically acceptable additive, excipient, diluent, carrier, and the like. Typically, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight of non-specified components.
It will be understood that any component defined herein as being included may be explicitly excluded by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein. For example, in aspects, the use of a hydrogel is explicitly excluded from the constructs and methods described herein. In other aspects, the tissue construct is non-spheroidal.
In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.
The term“and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that“at least one of’ or“one or more” of the listed items is used or present.
Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
II. Constructs
In one aspect, provided herein is a tissue construct comprising a biocompatible and substantially linear scaffold. The tissue construct further comprises a plurality of cells supported by and longitudinally aligned along the scaffold. Typically, the scaffold and/or tissue construct is untethered, meaning it is free-floating in culture medium and/or it is not attached to any substrate. The scaffold is typically of a shape that provides some compressibility and the scaffold is resilient, in that it returns substantially to its original shape when compression forces are removed. In this way, the scaffold is tensioned in a substantially linear shape but can be compressed when cells growing on the scaffold contract. The compressibility of the scaffold is relatively rigid, with a Young’s modulus that is similar to that of the native tissue that the tissue construct is designed to simulate, such as muscle tissue, including cardiac tissue. For example, the scaffold may have a Young’s modulus from about 0.1 to about 2 MPa, such as from about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, aboutl.4, about 1.5, about 1.6, about 1.7, about 1.8, or about 1.9 to about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, aboutl.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 MPa. Typically, the scaffold has a Young’s modulus of about 0.5 to about 1.0 MPa
In some aspects, tension exerted by the linear scaffold structurally supports an elongated shape of the tissue construct as cells seeded on the linear scaffold assemble longitudinally along the axis of the linear scaffold. The linear scaffold also provides sufficient structural support to reduce tears or ruptures in the formed tissue construct. Longitudinal alignment of the assembled cells along the axis of the linear scaffold is particularly helpfiil for forming muscle fiber tissue constructs. Without the structural support from the linear scaffold, the tissue quickly forms a spheroid, which is not desirable for muscle tissue. In some aspects, the tissue construct is assembled without the use of hydrogels.
In some aspects, the linear scaffold facilitates compression of the tissue construct. In some aspects, the linear scaffold comprises a zig zag shape. The linear scaffold with a zig-zag shape fimctions similarly to a spring to facilitate scaffold compression. This is particularly helpfiil for the assembly of cardiomyocytes to produce engineered cardiac tissues that compress the scaffolds when contracting. In some aspects, the tissue constructs described herein can be used to mimic myocardial contractility.
In some aspects, the linear scaffold comprises a strut diameter of about 0.1 pm to about 1000 pm For example, the linear scaffold may comprise a strut diameter of from about 0.1, about 1, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, or about 900 to about 1, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 mhi. For example, typically the scaffold has a strut diameter of about 0.1 pm, about 10 pm, or about 100 pm.
In aspects, the linear scaffold is from about 0.1 mm to about 10 mm in length, such as from about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 to about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mm in length. For example, typically the scaffold is about 1 mm or 3.3 mm in length.
In some aspects, the linear scaffold is about 3.3 mm in length with a strut diameter of about 100 pm. In some aspects, the linear scaffold is about 1.0 mm in length with a strut diameter of about 0.1 pm.
It will be understood that the scaffold size may be described in terms of a ratio of the strut diameter to the length of the scaffold. For example, the ratio of strut diameter: length may be from about 1: 10 to about 1 : 100000, such as from about 1 : 10, about 1:50, about 1 : 100, about 1 :500, about 1 : 1000, about 1:5000, about 1 : 10000, or about 1 :50000 to about 1 :50, about 1 : 100, about 1 :500, about 1 : 1000, about 1 :5000, about 1 : 10000, about 1 :50000, or about 1 : 100000. For example, the ratio of strut diameter: length may be 1:33 or 1 : 10000.
In some aspects, the linear scaffold comprises a polymer material selected from, but not limited to, the group consisting of poly(glycerol sebacate) acrylate, poly(octamethylene maleate (anhydride) citrate), poly(citric diol), poly(tri-methylene carbonate), polyester amide, polyester carbonate urethane urea, poly(octanediol-co-citrate), poly (lactic-co-glycolic acid), poly(glycolic acid), polylactic acid, poly(caprolactone), poly(hydroxyvalerate), polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2- hydroxyethyl-methacrylate), poly(ethylene glycol), combinations thereof, and copolymers thereof.
In some aspects, the biocompatible polymer comprising the linear scaffold is an elastomer. In some aspects, the biocompatible polymer comprising the linear scaffold is a biodegradable elastomer, such as poly(glycerol sebacate) acrylate (PGSA). In some aspects, the construct is used for cell therapy. In some aspects, the construct is delivered into or onto an organ via injection for cell therapy. Using a biodegradable elastomer, such as PGSA, allows for assembled micro-tissue constructs (functional tissues that have a physical size of less than about 1 mm 3 ) to be delivered via injection for tissue repair and therapy and guide biological growth in a step-wise fashion at multiple length and time scales but also degrade as the tissue heals.
In some aspects, the construct is delivered into an organ via intramuscular injection for cell therapy. In some aspects, the construct comprises a cardiac micro-tissue and may be delivered via intra-myocardium injection that bypasses the cardiac epithelium to enhance tissue integration in cardiac repairs. In some aspects, orientation of delivered tissue constructs may be directed by the injection path to allow for repeated delivery of multiple constructs with uniform alignment.
In some aspects, the linear scaffold further comprises magnetic particles. In some aspects, the magnetic particles are embedded within the linear scaffold and are trapped inside the polymer until polymer degradation occurs. In some aspects, the magnetic particles are nanoparticles. In some aspects the nanoparticles are sized for ready excretion from the body, such as smaller than about 10. In some aspects, a magnetic field may be used to remotely guide the alignment of individual tissue constructs and assembly with one another. In some aspects, tissue constructs align in the direction of the magnetic field and multiple constructs may assemble closer to one another to form a more densely-spaced cluster. In some aspects, magnetic guidance may be used to remotely control and deliver tissue constructs to a therapeutic site.
In some aspects, the construct is used as a miniaturized 3D model in drug research and development. In some aspects, the tissue construct comprises a cardiac micro-tissue that can simulate the functional and biomechanical properties of myocardial tissues in order to replace existing pre-clinical models. In aspects, the performance of the tissue constructs described herein is at least equal to or improved as compared to existing pre-clinical models.
In some aspects, the cells in the tissue construct comprise mammalian cells. In aspects, the mammalian cells comprise hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, smooth or skeletal muscle cells, myocytes, adipocytes, ectodermal cells, ductile cells, kidney cells, intestinal cells, parathyroid and thyroid cells, nerve cells, ocular cells, integumentary cells, pluripotent cells and stem cells, or combinations thereof, such as cardiomyocytes, fibroblasts or a combination thereof. In some aspects, the cardiomyocytes are human cardiomyocytes. In some aspects, the fibroblasts are human cardiac fibroblasts.
The constructs described herein may be provided in a delivery device, such as a syringe. Thus, liirther described herein is a delivery device for delivering the constructs. The delivery device is typically a syringe comprising a modified injection needle. The needle typically comprises a needle bore that is substantially blocked near the syringe end in order to retain the construct in the needle of the syringe. The needle bore may be blocked by any method, however, typically it is blocked by a smaller coaxial needle or a plug, such as a sponge plug. By using the delivery device described herein, multiple constructs can be injected in a substantially aligned manner because the shape of the constructs prevents them from being delivered widthwise. In aspects, the constructs will only fit through the needle in a lengthwise or longitudinal manner.
Also described herein are scaffolds as described above, without cells seeded onto the scaffolds. It will be understood that naked scaffolds without cells may be provided so that an end user can seed the scaffolds as desired with any cell type or combination of cell types.
Also described herein are culture plates for seeding cells onto a scaffold. The plates are as described herein and comprise one or more wells with an elongate bottom wall and side walls that are at a greater than 90 degree angle relative to the bottom wall. In other words, the sides walls angle into the bottom wall from the top wall and act to firnnel the scaffold and/or any cells or medium towards the bottom of the well. In this way, the wells can be generally described as fimnel-shaped. Typically, the bottom wall is substantially rectangular, in which case the wells can be described as wedge-shaped. Typically, the culture plate is used for high throughput production of the tissue constructs so the plate will comprise multiple wells. For example, the plate may contain 384 wells. The plate may be provided with or without the naked scaffold or with or without the scaffold seeded with cells. The plate may be provided with or without a frilly formed tissue construct as described herein.
III. Methods
In aspects, there is provided a method for making the scaffold described herein.
Typically, the method comprises polymerizing a biocompatible polymer to produce the substantially linear scaffold. Further, there is provided a method for making a tissue construct, the method comprising seeding a plurality of cells onto a spring-like biocompatible scaffold for elongate growth. As described above, the scaffold is typically untethered throughout the seeding and growth of the cells.
The method typically further comprises forming the scaffold by polymerization, such as photo-patterning polymerization. In aspects, the scaffold is placed in a funnel-shaped culture well for seeding the cells, as described above. Typically, placing the scaffold in the well comprises centrifuging the scaffold and well to funnel the scaffold to the bottom of the well. Further, seeding the cells in aspects comprises centrifuging the cells and scaffold to aggregate the cells around the scaffold.
In another aspect, provided herein is a method for assembling a tissue construct comprising fabricating a linear scaffold comprised of a biocompatible polymer using photo- patterning polymerization, transferring the linear scaffold to a funnel-shaped well of a microwell plate and allowing a plurality of cells to adhere to the linear scaffold and continue to grow to form a tissue construct comprised of a connected cellular aggregate. For example, the well may be wedge shaped, or it may be described as having a substantially rectangular bottom wall, with side walls that angle in towards the bottom wall.
In some aspects, a microfabrication technique, utilizing photo-polymerization of the scaffold polymer material in custom-designed molds, is used to produce linear scaffolds in a high-throughput manner. In some aspects, the custom-designed mold matches the layout of a microwell plate. In some aspects, the custom-designed mold may be placed onto a microwell plate to insert an individual linear scaffold into a single well of the microwell plate.
In some aspects, the microwell plate format allows for cell seeding and tissue construct assembly in a high-throughput manner. In some aspects, the micro well plate is a 384-well plate. In some aspects, the microwell plate format allows for efficient quality control using high- content imaging of each individual construct in an individual well. In some aspects, the microwell plate containing tissue constructs may be used as a high-throughput drug testing platform.
In some aspects, the method for assembling a tissue construct further comprises removing the tissue construct from the well of the microwell plate. As the tissue construct is assembled unanchored in the well, the construct may be easily removed for subsequent use (e.g. for in vivo delivery or implantation). In some aspects, tissue constructs may be collected from the plate with a micro-pipette. In some aspects, the pipette is a multi-channel micro-pipette. In some aspects, tissue constructs may be collected from the plate with tweezers.
EXAMPLES
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
Example 1. Biodegradable linear scaffold fabrication
Polymer synthesis. A biodegradable polymer, poly(glycerol sebacate) acrylate (PGSA), was used as it is a biocompatible and bioresorbable elastomer that has already received market approval in Europe for clinical use as a surgical sealant 25 ’ 26 . PGSA is one of the first synthetic biodegradable polymeric elastomers that has received market approval by regulatory agencies. Other U.S. Food and Drug Agency (FDA) approved synthetic polymers, such as polylactones, poly(L-lactide), poly(glycolide) and their copolymers, are too stiff and incompliant when compared to elastic tissues such as the heart 27 . PGSA 26 has been extensively tested in animal models as vascular grafts 28 , tissue scaffolds 29 and adhesives 25 , etc. It can be rapidly crosslinked by UV light, hence is suitable for microfabrication. PGSA was synthesized according to protocol used previously 30 . Briefly, the prepolymer was first synthesized with a polycondensation reaction between equimolar amounts of glycerol (Sigma, G5516-1L) and sebacic acid (Sigma, 283258-250G). The two reagents were combined and allowed to react at 120°C under nitrogen purge for 1 h and then under vacuum for 24 h. The resulting prepolymer (20g) was then dissolved in 200mL of anhydrous dichloromethane with 20mg of 4- (dimethylamino) pyridine (DMAP; Sigma, 107700-25G). The reaction flask was cooled to 0°C under a nitrogen purge. 3.05mL Acryloyl Chloride was slowly added to the reaction flask parallel to an equimolar amount of trimethylamine. The reaction was allowed to reach room temperature and was stirred for an additional 24 h. The resulting mixture was dissolved in ethyl acetate, filtered, and dried at 45 °C and 5 Pa. Finally, 0.1% (w/w) photoinitiator (PI), 2- hydroxy-l-[4(hydroxyethoxy)phenyl]-2- methyl-1 propanone (Irgacure 2959), was added and thoroughly mixed with the polymer solution. The resulted PGSA polymer mixture was stored at 4°C and protected from light. The polymer structures were verified using 'H NMR spectroscopy as shown in Figure la.
Scaffold fabrication technique. A SU-8 master mold containing an array of 384 3.3 mm- long zig-zag scaffold structures matching the standard 384-well plate was designed in AutoCAD and fabricated according to standard photolithography. A poly(dimethylsiloxane) (PDMS) mold was then replicated from the SU-8 master mold. PGSA polymer mixture was spread onto the PDMS mold with a glass slide to fill the structural features on the PDMS mold. Excessive PGSA polymer was scraped off. The PDMS mold filled with PGSA polymer was then place inside a transparent bag filled with nitrogen and placed under a UV lamp. To crosslink the PGSA polymer a total UV energy output of 15mW/cm 2 was applied for a duration of 30 to 120 min. The crosslinked PGSA scaffolds were immersed in 70% ethanol to sterilize as well as release the scaffolds from the PDMS mold.
Micro-scaffold fabrication technique. A fabrication method that leverages capillary action was used to produce a miniaturized (1.0 mm- long) version of the linear scaffold using a UV photomask (custom designed in AutoCAD) to selectively crosslink regions of a micro- channel and photo-pattern an array of micro-scaffolds. Using a standard microfabrication technique 31 , a PDMS microfluidic mold was created with an embossed micro structure that is one continuous channel with six injection points per row for the fabrication of 1.0 mm micro - scaffolds. The polystyrene sheet was prepared by plasma treatment and a subsequent 5% Pluronic acid soak (20 min). The PDMS mold was placed onto the polystyrene sheet and loaded with PGSA prepolymer at the injection points. The sheet was then centrifuged (1000 RPM for 1 min) and placed in the oven at 70°C (4 hours). The sheet was fitted with the photomask and photo-polymerized under UV light (15mW/cm 2 , 30 min). Following crosslinking and washing with 70% ethanol, scaffolds (also referred to in the drawings as“wires” or“z-wires”) were released from the polystyrene sheet with a single edge blade and placed in a customized 384- well plate (Figure 2).
Scaffolds have a zig-zag shape with a strut diameter of lOOpm, which functions like a spring to facilitate scaffold compression (Figure 3a). This design encourages functional tissue contraction; the bulk structures of synthetic elastomers are nearly incompressible, but a spring like design can harness structural elasticity to support cyclic scaffold compression in cardiac tissue contraction. Moreover, the scaffold functions as a structural support to facilitate tissue compaction and remodeling, but also provides tension to induce alignment and elongation of the assembled cardiomyocytes along the scaffold.
Mechanical testing. For the mechanical testing of the PGSA material, PGSA discs with a thickness of 0.5mm and a radius of 1mm were fabricated at 30, 60, 90 and 120 min of UV exposure. Discs were washed with 70% ethanol in order to remove the non-crosslinked polymer. Compression tests were performed on a CELLSCALE ® UniVert™ mechanical tester with a 3mm cylindrical compression probe at room temperature. Young’s modulus was calculated from the stress and strain curve. By controlling UV exposure time, photo initiator concentration, and post-molding heat-crosslinking, the Young's Modulus of PGSA was fine- tuned to the range of 0.5-lMPa, which is close to the Young’s modulus of the native myocardium (Figure lb-d).
Scaffold degradation. PGSA degrades through a hydrolysis reaction as water molecules hydrolyze the ester bonds on the polymer chain over time. This process was confirmed through accelerated degradation by immersing the microfabricated scaffolds in a concentrated sodium hydroxide solution (0.1 M) where complete material degradation was confirmed within 6 hours (Figure le). Briefly, scaffolds were placed in 200pL of 0.1M NaOH solution at room temperature for 6 h to accelerate the hydrolytic degradation process. A brightfield image was acquired every 2 h until the scaffolds visibly disappeared, confirming complete degradation. Five to six linear scaffolds were used for each experimental condition.
Example 2 High-throughput tissue assembly platform
The microfabricated scaffolds, released from the molds, can then be cast into customized 384-well plates to facilitate high-throughput cell seeding and tissue assembly. The customized 384-well plate containing funnel-shaped wells and rounded rectangular bottoms (0.5x3.5 mm) was first designed in AutoCAD and the design was inverted to create a mold. The inverted mold for the customized 384-well plate was 3D printed with Stratasys 3D printing using Tangoblack plus ink. The printed mold was washed in NaOH solution for 2 days, dried and then baked at 80°C for 1 day. The mold was then coated with Trichloroperfluorooctyl silane (Sigma, 448931-lOg). A customized PDMS plate was replicated from the 3D printed mold. The PDMS plate was hard-baked at 300°C for 15 min before it is glued onto a CELLSTAR ® OneWell™ polystyrene plate (VWR, 30617-592) with additional PDMS glues. Since the array of 384 micro-scalfolds was designed to match the layout of the 384-well plate, this array of micro-scaffolds was dropped into the customized 384-well plate in one step: by capping the PDMS mold containing the UV-crosslinked PGSA scaffolds onto the plate so that each individual scaffold is placed onto one well and pressing the PDMS mold against the plate filled with 70% ethanol to release the scaffolds into their corresponding well. The plate was then centrifuged for 30 sec at 1000RPM to push the scaffolds into the bottom of each well. The funnel-shaped well design automatically directed and positioned the micro -scaffolds to the bottom of the wells (Figure 3b). The plate was left in 70% ethanol to sterilize for 2 h at room temperature. Lastly, the ethanol in the plate was slowly switched to culture media after 2-3 media changes. The plate is now ready for cell seeding (Figure 4).
Example 3 Elongated tissue constructs using linear scaffolds
Assembled tissue production. Fresh cardiomyocytes were isolated from 2-day-old neonatal Long Evans rat pups as has been previously reported with minor modifications. Briefly, rat pups were euthanized by decapitation and their hearts were collected and placed in ice-cold PBS. The hearts were then quartered into small pieces and digested overnight in a 0.5% (w/v) solution of porcine trypsin (Sigma, T4799) in PBS at 4°C overnight. After, the hearts were further digested in a 50% (w/v) solution of Collagenase II (Worthington, LS004176) in PBS at 37°C in a series of five 8min digestions. The collected cells were pre-plated in T75 flasks for 60min at 37°C and 5% CO2. The supernatant was collected, and the nonadherent cells were classified as the enriched CM population and the adherent cells were classified as the enriched fibroblast population. To assemble the micro-tissues, primary human cardiac fibroblasts or a mixture of 50% hESC-derived human cardiomyocytes and 50% primary human cardiac fibroblasts were used for seeding. HiPSC-CMs were purchased from Cell Dynamics or differentiated from a healthy hiPSC line using a 2D monolayer protocol in a chemically defined medium. Human cardiomyocytes were used wherever possible. But due to the limited supplies of human cardiomyocytes, rat cardiomyocytes were used to demonstrate high-throughput tissue production and tissue assembly where large quantities of cells are needed. Rat cardiomyocytes were also used for the ex vivo tissue injection into rat hearts and for in vivo implantation in immuno-competent rats.
Cells were cultured in cardiac culture media that is composed of Dulbecco’ s modified Eagle’s medium (Gibco) containing glucose (4.5 g /liter). 10% (v/V) fetal bovine serum (FBS; Gibco), 1% (v/V) Hepes (100 U/ml; Gibco), and 1% (v/V) penicillin-streptomycin (100 mg/ml; Gibco). Human cardiac cells were cultured in plating media purchased from Cellular Dynamics (R1132). Cells were suspended in culture media at 20 million cells per mL. Next. 5 to 10 pL of cell suspension were pipetted into each well, which is equivalent to 0.1 to 0.2 million cells per well (Figure 5] However, 7.5 pL of cell suspensions at 40 million cells per mL was later used and pipetted into each well which is equivalent to 0.3 million cells per well. The plate was then centrifuged for 1 min at 1000 RPM to aggregate the cells to the bottom of the well and completely aggregate the cells around the scaffolds. At least 10 mL of culture media was then applied onto the plate to fill the entire plate. The plate was placed in an incubator and media was changed once every day for 1 week. The cells quickly self-aggregated, starting to contract around the scaffolds in 24h, and completely compacted around the scaffolds over 3 to 4 days (Figure 6a-b). The entire cell seeding process was carried out with standard tissue culture equipment (e.g., pipettes and centrifuge], which can be readily scaled up (e.g., multi-channel pipettes].
Importantly, using this seeding process, cells can naturally assemble on their own without needing a hydrogel matrix (e.g., Collagen I, Fibrin or Matrigel™ , etc.], which can be expensive and introduce animal-derived growth factors not yet approved for clinical use and prone to batch-to-batch variation (e.g., Matrigel™). With the support of the scaffold, it was found that the cells can naturally assemble on their own without a hydrogel matrix. The length of the tissue was measured in Photoshop from the brightfield images.
For in vivo delivery or implantation, tissues were removed and collected from the plate with a 1 mL pipette or sterile tweezers and suspended in a separate tissue culture flask filled with a mixture of PBS and FBS (1: 1000] A mixture of 50% primary rat cardiomyocytes, 20% primary rat cardiac fibroblasts, and 30% green fluorescent protein human umbilical vein endothelial cells were used for the fabrication of vascularized microtissues. Cells were cultured in endothelial cell growth medium 2 (Cedarlane Labs, C-22011) containing 10% (v/v) fetal bovine serum (FBS; Gibco). Cells were suspended in culture media at 100 million cells per mL. Next 10pL of cell suspension was pipetted into each well which is equivalent to 1 million cells per well. The plate was then centrifuged for lmin at 150Gto aggregate the cells to the bottom of the well and aggregate the cells around the scaffold. Tissues were cultured for nine days prior to analysis. The GFP-endothelial cells in the scaffolded tissues were images with confocal microscope (Nikon A1 confocal with ECLIPSE Ti microscope), as well as a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc. Montreal, Canada).
Immunofluo rescent staining and imaging. To assess the morphology of the engineered cardiac tissues, the tissues were first fixed in 4% (w/v) para- formaldehyde in PBS for 15 min at room temperature, then permeated and blocked in 5% FBS and 0.25% Triton X-100 in PBS for 1 hour. Next, the tissues were incubated in primary antibody against sarcomeric a-actinin (mouse, 1:200, Abeam, ab9465), overnight at 4°C, followed by incubation with a secondary antibody, Alexa 488-conjugated anti-mouse immunoglobulin G (IgG) (1:200, Life Technologies, A21202) and a phalloidin 66-conjugated anti-F-actin (1:300, Life Technologies, A22285). Tissues were then washed and imaged with confocal microscopy (Nikon A1 confocal with ECLIPSE Ti microscope). DAPI was used to visualize cell nuclei.
Tissue contraction analysis. Video analysis of contracting tissues was made using ImageJ plugin MuscleMotion following previously published procedures. Frequency was calculated dividing the number of recorded beats by the length of video sample multiplied by 60 to obtain beats per minute. Contraction time was calculated by subtracting the time point at which the contraction amplitude peak was highest minus the time point at which the contraction curve started rising from baseline. Relaxation time was calculated by subtracting the time point at which the contraction curve returned to baseline minus the time point at which the contraction amplitude peak was highest. Amplitude of contraction was calculated by measuring the tissue contraction (relaxed tissue length minus contracted tissue length). Rising slope of contraction curve was calculated by subtracting the highest amplitude of contraction minus the baseline amplitude of contraction before that peak and then dividing by the contraction time. In order to determine the difference of cell alignment between the scaffold and no scaffold groups, tissues coherency analysis was made using ImageJ plugin OrientationJ as has been previously shown. Coherency is a measurement of coherent directionality that is calculated to indicate whether local image features are oriented or not. Higher coherency values indicate a strongly coherent orientation of the local fibers whereas low values indicate no preferential orientation. This parameter has been used to measure elastin and collagen architecture and orientation in arterial and scar tissue.
Functional elongated tissue constructs. The scaffolds provided sufficient structural support for the formation of an elongated tissue, without hindering contraction, by allowing for structural compression. As seeded cells connect with each other and aggregate around the scaffold, the cells collectively exert a force onto the scaffold, attempting to compress the scaffold. However, the relatively rigid scaffold, instead of being compressed, exert a counter force onto the tissue, forcing the tissue to elongate along the scaffold. The micro-scaffolds provided sufficient structural support to prevent tears or ruptures in the formed tissues, while not hindering tissue contraction by allowing structural compression. This scaffold-induced tension within the tissue readily aligns the cardiac cells along the scaffold, leading to the formation of a muscle fiber (Figure 6a,e,f). Without the structural support from the scaffold, the tissue quickly forms a spheroid, which is not ideal for muscle alignment (Figure 6a,c,d). F-actin staining shows uniform cellular alignment along the scaffold while cell orientation appears to be random in the scaffold-free cell aggregates (Figure 6c-f and Figure 7). Sarcomeric-a-actinin staining reveals the presence of elongated cardiomyocytes with visible cross-striation intertwined with cardiac fibroblasts (Figure 6g-i). Scaffold-induced tensile force also presented a synergistic improvement of endothelial cell assembly and alignment along the scaffold struts (Figure 8a-b). In three days, a rudimentary vasculature network was visible throughout the entire tissue. Network integrity was highlighted by vessel interconnectivity and high density, with maximum vessel separation of 100-200pm. The assembled tissues can contract macroscopically and compress the scaffold. The contraction of the tissue can be traced (Figure 8c-d). The amplitude and frequency along with other contraction parameters were extracted (Figure 8e). If needed for drug testing applications, the amplitude of tissue contraction could be translated into force of contraction. This can be done by simulating the correlation between contraction force and the mechanical compression of the scaffolds. With this approach, a large array of tissue can be seeded and cultivated at a time (Figure 8f).
Because the scaffolds and tissues were not anchored onto the plate for cultivation, assembled tissues were easily released and collected from the plate (e.g. with a multi-channel pipette) for further manipulation or transplantation (Figure 8g). Due to the small size of these micro-tissues (300 pm in diameter and ~2 mm in length), they can be easily loaded and injected with a syringe without disrupting the tissues. To fill an entire 384-well plate will require 115 million cells according to the current seeding density. Thus, in considering scale up for clinical studies, only 9 plates are needed to produce the nearly 1 billion cells required for transplantation. While it is possible to create thousands of tissue spheroids with a stirring bioreactor, described herein is the first feasible approach to assemble elongated functional tissues at a large scale. Furthermore, since no expensive hydrogel materials are required, this process reduces the production costs. Importantly, in contrast to a bioreactor-based tissue production process, this multi-well plate tissue assembly method allows each tissue to be routinely imaged with a high-content imaging system to track and monitor the culture condition of every single tissue over time. This capability allows for efficient quality control by easily identifying and eliminating low-quality tissues (e.g., tissues that do not form properly or do not function at an expected level) by quantifying cell compaction and assessing the quality of cardiac tissue contraction. Currently, there are no other methods that allow quality control in a high-throughput manner with such precision.
Example 4 Magnetically responsive tissue self-assembly
To impart magnetic capability to the scaffolds, magnetic iron oxide (II, III) nanoparticles (<10 nm in diameter, Sigma, 544884-5G) were dispersed in the PGSA prepolymer solution at concentrations of 0.2-1 wt% with sonication. The polymer nanocomposite was then cast onto the PDMS mold and crosslinked under UV light (15 mW/cm 2 for 60 min). The additional crosslinking time was required to achieve the desirable mechanical properties as the dispersed magnetic nanoparticles deflect light and reduce UV penetration efficiency. After photopolymerization, the nanoparticles were permanently trapped inside the polymer until polymer degradation. Magnetic fields offer an attractive opportunity to externally guide the assembly of individual functional tissues for three reasons: 1) magnetic fields are easy to generate and can penetrate thick materials and biological tissues to provide guidance in a remote fashion 32 , 2) commercially available magnetic nanoparticles are widely used in bioimaging, and have been shown to be nontoxic and can be excreted from the body over time 33,34 and 3) magnetic fields can be easily directed with magnets into various paterns to guide organ-specific tissue organization.
For tissue assembly experiments, micro-tissues were extracted and assembled with magnetic guidance from commercially available Neodymium magnets with a magnetic induction of 434 mT and cultured for an additional three days under the magnetic field to allow tissue integration. Magnetic induction was measured at multiple distance points from the magnet using a teslameter (Weite Magnetic Technology Co. WT10A). Nanoparticles smaller than 10 nm were chosen as it has been shown that these can be readily excreted from the body 33 . The nanoparticles also did not interfere with the degradation of the scaffolds. To guide the organization of the micro-scaffolds and the assembled micro-tissues, the Neodymium magnets were used to generate a magnetic field with a local strength up to 0.5T (Figure 9a-b). It was observed that the orientation of a cluster of micro-scaffolds embedded with magnetic nanoparticles can be easily controlled on demand with a magnetic field strength above 0.13T using a hand-held magnet (Figure 9a-b and Figure 10). Using the same procedure, a cluster of micro-tissues can be assembled. The micro-tissues first align in the direction of the magnetic field. As the magnet was moved closer to the tissue, the micro-scaffolds and the micro-tissues then assemble closer to each other to form a more densely-spaced cluster (Figure 9c). The cluster of micro-tissues was then cultured for another 3 days in the presence of the magnetic field. During this time, cells at areas of contact between the micro -tissues formed intercellular junctions connecting the micro-tissues to form an integrated tissue (Figure 9d).
Example 5 Surgical tissue delivery and assembly
To demonstrate the feasibility of delivering the micro-tissues surgically with syringe injection, a syringe was customized as a delivery tool to improve tissue loading and injection. For the fabrication of the delivery tool, a 22G needle was glued with epoxy to the piston of a lmL syringe and was left to dry overnight. The barrel of the lmL syringe was assembled with a 19G needle. The piston with the 22G needle was placed into the syringe’s barrel with the 19G needle. The 22G needle was trimmed to the length of the 19G needle so that they both aligned as shown in Figure 11. Alternatively, a 1 ml syringe with a 14Gx2” needle can be assembled with a sponge plug in between the syringe and needle in order to trap the tissues inside the needle. The assembled syringe can be preloaded with 0.1 ml of the PBS/FBS mixture with the purpose of preventing the adhesion of tissues to the needle.
Collected micro-tissues were loaded into the delivery system from the needle side using a fine tweezer. After loading, tissues were stored inside the needle prior to injection to ensure a smooth delivery by the plunger. Scaffold presence provides important structural stability to the delivery process; elongated tissues without scaffolds fold and crumble with handling, and cannot be easily manipulated, loaded, or injected while maintaining any predictable structural orientation (Figure 12a). Given the small size of scaffold-supported micro-tissues (300pm in diameter and ~3mm in length), they were easily loaded to, and injected with, a syringe without disrupting organized tissue structure. In order to assess the viability of the tissues after the injection process, an assay to detect living and dead tissue was performed before and after injection. The tissue was first loaded into the delivery tool and then injected into a six well plate. The tissues were then stained with 5- Carboxyfluorescein diacetate 95% (CFDA) (Sigma, C4916-25MG) at a working concentration of 0.0025mg/mL, as well as propidium iodine (PI) (Sigma, P4864) at a working concentration of 0.001 mg/mL in PBS. Live and dead images were captured with the Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc. Montreal, Canada). With the scaffold, the injection process was quick and gentle; injected tissues showed comparable viability (according to live and dead staining) to tissues pre-injection (Figure 12b).
To demonstrate tissue delivery on the surface of an organ, such as muscle, an animal cadaver was used as a model in vitro by delivering the cardiac tissues on top of animal muscular tissue and further aligned and assembled with a IT neodymium magnet. A cluster of scaffolds and fimctional micro-tissues were first delivered with injection to the surface of the delivery site, then a magnetic field was applied by waving a magnet over the delivery site to aggregate and align the delivered scaffolds and tissues in a uniform direction (Figure 13). Multiple injections can be applied to achieve a large tissue coverage. To secure the tissues for in vivo studies, an FDA- approved fibrin glue spray (EVICEL® Fibrin Sealant) can be used after magnetic alignment.
To demonstrate the intramuscular delivery of the micro-tissues, the same cadaver cannot be used because it was impossible to visualize the micro-tissues once after delivery inside the muscle. However, it was found that the mechanical properties of native tissues can be modeled with a transparent gelatin hydrogel matrix for the purpose of demonstrating intra-organ delivery. Therefore, a transparent heart model was made by heating a mixture of 7% (w/v) gelatin from bovine skin (Sigma, G9391-100G) in distilled water at 90°C for 15min and then cooling it on a plastic heart model at room temperature for lhr as shown in Figure 12c. Collected micro-tissues were injected into the gelatin heart with either multiple injections of single tissue or a single injection of multiple tissues. In this model, the syringe needle carrying the micro-tissues precisely delivered the tissues with ease at the site of injection (Figure 12d). It was found that injected tissues consistently self-aligned in the direction of injection without the need of magnetic guidance. This is likely because the orientation of the delivered tissues is constrained by the open space created along the injection path, presenting an opportunity for localized tissue organization. The same injection can be repeated multiple times, delivering an array of micro - tissues with uniform alignment without the use of magnets (Figure 12e). Alternatively, multiple micro-tissues can be injected at the same location with one injection (Figure 121). The ability to repeatedly provide the same injection multiple times for delivering an array of micro-tissues with uniform alignment without the use of magnets will simplify the surgical procedure. Alignment of delivered micro-tissues will be pre-determined by the injection path during surgery.
Translation of this process was demonstrated with the delivery of micro-tissue to the myocardium of a heart cadaver using the same procedure. After injection, the heart was sliced into 1mm thickness, and individual tissue slices were cleared in 70% (v/V) T 2,2'-Thiodiethanol (TDE, Sigma, 166782-500G) solution for 7 days prior to imaging. Although the injection process could not be seen directly as it is taking place, visualization was achieved with a tissue clearing technique (Figure 14a) which allowed visualization of the injected tissue in its entirety, completely embedded inside the myocardium wall and oriented in the general direction along the myocardium wall (Figure 14b-c). The boundary of the GFP labelled micro-tissue following injection was visible, confirming maintenance of in vivo tissue micro environmental structure. (Figure 14d).
Next, tissue was subcutaneously implanted to the dorsal region of an adult Long Evans rat (Figure 14e). For in vivo implantation, scaffolded rat cardiac micro-tissue was delivered into the subcutaneous space located on the dorsal region of adult Long Evans rats. The animal was first anesthetized with 3% isoflurane at a flow rate of 1 L/min. 5 mg/kg Carprofen analgesic was administered subcutaneously, and the dorsal pouch was prepared for surgery. A 1cm incision was made on the dorsal region and the scaffolded micro-tissues (n=5) were delivered into the subcutaneous space. The incision was sutured using absorbable 4-0 vicryl sutures. For post operative pain management, carprofen was administered subcutaneously for two days. The animals were monitored for four weeks, followed by euthanization with CO2 inhalation. The site of injection was later isolated and processed for histological studies. Briefly, tissue explants from the site of injection were fixed in 10% formalin, processed and sectioned. Tissue sections (4pm thick) were stained with Masson’s trichrome and Hematoxylin and Eosin.
One month after implantation, the micro-tissue began to integrate with the host tissue, where the infiltration of host cells to the implant, including fibroblasts and blood vessels, was clearly visible (Figure 14f-h). A moderate host response was observed with the infiltration of giant cells and macrophages near the implantation borders which is consistent with previous studies 25 .
Following this protocol, lunctional cardiac tissues could be delivered with intramyo cardial injection, bypassing the epicardium, while maintaining a high-level tissue organization with respect to the host tissues. Given that injection is a simple delivery method that requires minimal surgical manipulation in vivo, the delivery process could be guided by ultrasound imaging in both animal and human surgeries. The same strategy can be applied to repair skeletal muscles or neuronal tissue where intra-tissue delivery is required, and tissue structural organization is important.
REFERENCES:
1. Radisic, M. et al. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proceedings of the National Academy of Sciences of the United States of America 101, 18129-18134 (2004).
2. Kurobe, H., Maxfield, M. W., Breuer, C. K. & Shinoka, T. Concise review: Tissue- engineered vascular grafts for cardiac surgery: Past, present, and fiiture. Stem cells translational medicine 1, 566-571 (2012).
3. Moreira-Teixeira, L., Georgi, N., Leijten, I, Wu, L. & Karperien, M. Cartilage tissue
engineering. (2011).
4. Reichert, J. C. & Hutmacher, D. W. in Tissue engineering 431-456 (Springer, 2011).
5. Lancaster, M. A. el al. Cerebral organoids model human brain development and
microcephaly. Nature 501, 373-379 (2013).
6. Iso, Y. et al. Multipotent human stromal cells improve cardiac fimction after myocardial infarction in mice without long-term engraftment. Biochemical and biophysical research communications 354, 700-706, doi: 10.1016/j.bbrc.2007.01.045 (2007).
7. Zeng, L. et al. Bioenergetic and lunctional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling. Circulation 115, 1866-1875, doi: 10.1161/CIRCULATIONAHA.106.659730 (2007).
8. Noiseux, N. el al. Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac fimction despite infrequent cellular fiision or differentiation. Molecular therapy : the journal of the American Society of Gene Therapy 14, 840-850, doi: 10.1016/j.ymthe.2006.05.016 (2006).
9. Leor, J. et al. Bioengineered cardiac grafts: A new approach to repair the infarcted
myocardium? Circulation 102, III56-61 (2000).
10. Reinecke, H. & Murry, C. E. Taking the death toll after cardio myocyte grafting: a reminder of the importance of quantitative biology. JMol Cell Cardiol 34, 251-253,
doi: 10.1006/jmcc.2001.1494 (2002).
11. Muller-Ehmsen, J. et al. Survival and development of neonatal rat cardio myocytes
transplanted into adult myocardium. Journal of molecular and cellular cardiology 34, 107-116 (2002).
12. Laflamme, M. A. et al. Cardio myocytes derived from human embryonic stem cells in pro survival factors enhance fimction of infarcted rat hearts. Nat Biotechnol 25, 1015- 1024, doi: 10.1038/nbtl327 (2007). 13. Montgomery, M. et al. Flexible shape-memory scaffold for minimally invasive delivery of fimctional tissues. Nature Materials 16, 1038-1046 (2017).
14. Holmes, J. W., Borg, T. K. & Coveil, J. W. STRUCTURE AND MECHANICS OF
HEALING MYOCARDIAL INFARCTS. Annual Review of Biomedical Engineering 7, 223-253, dokdoi: 10.1146/annurev.bioeng.7.060804.100453 (2005).
15. DiMasi, J. A., Grabowski, H. G. & Hansen, R. W. Innovation in the pharmaceutical
industry: new estimates of R&D costs. Journal of health economics, 47: pp. 20-33 (2012).
16. Adams, C. P. and Brantner, V. V. Spending on new drug development 1. Health Economics,
19: pp.130-141 (2010).
17. Page, R, O’Bryant, C.L., Cheng, D., Dow, T.J., Ky, B., Stein, C.M., Spencer, A.P., Trupp,
R. J., and Lindenfeld, J. Drugs That May Cause or Exacerbate Heart Failure. Circulation, 134: pp.e32-e69 (2016).
18. Hwang, T., et al. Failure of Investigational Drugs in Late-Stage Clinical Development and
Publication of Trial Results. JAMA International Medicine, 176(12): pp.1826-1833 (2016).
19. Fordyce, C.B., Roe, M.T., Ahmad, T., Libby, P., Borer, J.S., Hiatt, W.R, Bristow, M.R.,
Packer, M., Wasserman, S.M., Braunstein, N., Pitt, B., DeMets, D.L., Cooper-Amold, C., Armstrong, P.W., Berkowitz, S.D., Scott, R., Prats, J., Gabs, Z.S., and Califf, RM. Cardiovascular Drug Development: Is it Dead or Just Hibernating? Journal of the American College of Cardiology, 65(15): pp. 1567-1582 (2015).
20. Engelmayr, G. C., Jr. et al. Accordion-like honeycombs for tissue engineering of cardiac anisotropy. Nat Mater 7, 1003-1010 (2008).
21. Chuong, C., Sacks, M., Templeton, G, Schwiep, F. & Johnson, R. Regional deformation and contractile function in canine right ventricular free wall. American Journal of Physiology-Heart and Circulatory Physiology 260, H1224-H1235 (1991).
22. Rappaport, D., Adam, D., Lysyansky, P. & Riesner, S. Assessment of myocardial regional strain and strain rate by tissue tracking in B-mode echocardiograms. Ultrasound in Medicine & Biology 32, 1181-1192, doi:
http://dx.doi.Org/10.1016/j.ultrasmedbio.2006.05.005 (2006).
23. Demer, L. L. & Yin, F. C. Passive biaxial mechanical properties of isolated canine
myocardium. The Journal of physiology 339, 615-630 (1983).
24. Jawad, H., Lyon, A. R, Harding, S. E., Ali, N. N. & Boccaccini, A. R. Myocardial tissue engineering. British medical bulletin 87, 31-47, doi: 10.1093/bmb/ldn026 (2008).
25. Lang, N. etal. A blood-resistant surgical glue for minimally invasive repair of vessels and heart defects. Science translational medicine 6, 218ra216-218ra216 (2014).
26. Nijst, C. L. E. etal. Synthesis and characterization of photocurable elastomers from
poly(glycerol-co-sebacate). Biomacromolecules 8, 3067-3073, doi: 10.1021/bm070423u (2007).
27. Liu, Q., Jiang, L., Shi, R. & Zhang, L. Synthesis, preparation, in vitro degradation, and
application of novel degradable bioelastomers— A review. Progress in Polymer Science 37, 715-765 (2012).
28. Wu, W., Allen, R. A. & Wang, Y. Fast-degrading elastomer enables rapid remodeling of a cell-free synthetic graft into a neoartery. Nature medicine 18, 1148-1153,
doi: 10.1038/nm.2821 (2012).
29. Engelmayr, G. C. et al. Accordion-like honeycombs for tissue engineering of cardiac
anisotropy. Nature Materials 7, 1003-1010, doi: 10.1038/nmat2316 (2008). 30. Ifkovits, J. L, Padera, R. F. & Burdick, J. A. Biodegradable and radically polymerized elastomers with enhanced processing capabilities. Biomedical materials 3, 034104 (2008).
31. Anderson, J. R. et al. Fabrication of topologically complex three-dimensional micro fluidic systems in PDMS by rapid prototyping. Analytical chemistry 72, 3158- 3164 (2000).
32. McBain, S. C., Yiu, H. H. & Dobson, J. Magnetic nanoparticles for gene and drug delivery.
International journal of nanomedicine 3, 169 (2008).
33. Frey, N. A, Peng, S., Cheng, K. & Sun, S. Magnetic nanoparticles: synthesis,
functionalization, and applications in bio imaging and magnetic energy storage.
Chemical Society Reviews 38, 2532-2542 (2009).
34. Pankhurst, Q. A, Connolly, J., Jones, S. & Dobson, J. Applications of magnetic
nanoparticles in biomedicine. Journal of physics D: Applied physics 36, R167 (2003).
The above disclosure generally describes the present invention. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
All publications, patents and patent applications cited above are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Although preferred aspects of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.