Patent Application
20170281828
The invention relates to 3D bioprinting of artificial tissue and more specifically to an artificial cornea produced using 3D bioprinting.
Disease or damage to one or more layers of the cornea can lead to blindness that is commonly treated by corneal transplant. Approximately 40,000 patients undergo corneal transplant surgery in the United States every year. The vast majority of these people receive a replacement cornea from a human donor. Although the surgery has a high success rate, the supply of donor tissue is limited, and wait lists can be long. In the developing world, access to donor tissue is even more difficult. Further, while human donor transplants are the standard treatment for corneal blindness, the complications and limitations inherent in them have prompted development of synthetic corneal substitutes. Existing synthetic corneas can be categorized into: 1) fully synthetic prostheses (e.g., keratoprostheses) and 2) hydrogels that permit regeneration of the host tissue.
Keratoprostheses, or Kpros, the best-known artificial corneas, perform the refractive function of the cornea. Although Kpros have been available for many years in various forms, the fabrication of synthetic stromal equivalents with the transparency, biomechanics, and regenerative capacity of human donor corneas remain a formidable challenge. Further, the application of keratoprostheses is impeded by the complicated implantation procedures and major post-surgical complications, including infection, calcification, retroprosthetic membrane formation and glaucoma. In some cases, due to their propensity for infection, patients must take a lifelong course of antibiotics. As a result, the artificial cornea is used only as a last resort in patients who have repeatedly rejected natural donor tissue or who are otherwise not eligible for such transplant surgery.
The second type of engineered corneas are synthetic hydrogel-based, cell-free implants, which are designed to recruit host cells to grow an epithelial layer on the implant's surface and restore functionality. Many of these hydrogel implants resemble organic tissue and have a high elastic modulus with desirable optical properties. However, in most cases, mechanical or biological fixation is problematic—integration of the implanted scaffold with the host tissue is an extremely time-consuming process. This slow time-course is further exacerbated by the limited cell repopulation activity in patients who are older and/or severely injured. In addition, some of these hydrogel implants have reportedly become partially biodegraded after long-term implantation, leading to loss of transparency and failure of the grafting. Attempts to address some of the problems with cell-free implants include incorporation of glucosaminoglycans in the hydrogel matrix, which are believed to be necessary for cell adhesion and modulation of degradability.
One of the transformative applications of bionanotechnology is to create revolutionary approaches for the reconstruction and regeneration of human tissues and organs. This promise is based on the powerful capability that nanotechnology provides in a biological context: unique modalities of control over cellular machinery at the nanoscale. Due to their special surface characteristics, subcellular length scales, and precisely directed modular architectures, nanostructures and their incorporation within tissue engineering constructs serve new paradigms for regenerative medicine. 3D bioprinting—which uses biomaterials, cells, proteins, and other biological compounds as building blocks to fabricate 3D structures through additive manufacturing processes—offers novel approaches that can accelerate the realization of anatomically correct tissue constructs for transplantation. This collection of emerging technologies and their synergistic integration—by providing nanotechnology-enabled 3D tissue models that mimic normal and pathological physiology—can not only redefine the clinical capabilities of regenerative medicine but also transform the toolsets available for drug discovery and fundamental research in the biological sciences.
An approach to overcome drawbacks that are being experienced with existing artificial cornea technologies would be to provide a tissue-engineered cell-based corneal substitute that resists rejection and is easily integrated with host tissue. The present invention is directed to such an approach.
In an exemplary embodiment, a method and system are provided for fabrication of cell-laden corneal substitutes using a 3D bioprinting platform. Such artificial corneas provide a new approach that avoids many of the complications involved in existing methods for treatment of corneal epithelial disease. According to an embodiment of the invention, 3D bioprinters allow for cell encapsulation within a printed network, enabling live printing of tissue structures with micro- and nanometer scale resolution. The cell-laden corneal substitutes can shorten the time for transplants to integrate with host tissue. Further, the digital (i.e., customizable) nature of 3D printing allows one to develop patient-specific tissue models with designed shape and curvature. Such 3D-printed cornea tissues will have immediate applications in clinical transplantation, human ocular surface disease modeling (e.g., for dry eye diseases), early drug screening to replace or reduce the need for animal testing, and in drug efficacy testing for wound healing.
According to an exemplary embodiment, an artificial cornea is fabricated by separately culturing live stromal cells, live corneal endothelial cells (CECs) and live corneal epithelial cells (CEpCs), and 3D bioprinting separate stromal, CEC and CEpC layers to encapsulate the live cells into separate hydrogel nanomeshes. The CEC layer is attached to a first side of the stromal layer and the CEpC layer to a second side of the stromal layer to define the artificial cornea.
In one aspect of the invention, a method for fabricating an artificial cornea, comprises culturing live stromal cells; 3D bioprinting a stromal layer encapsulating the live stromal cells into a first hydrogel nanomesh; culturing live corneal endothelial cells (CECs); 3D bioprinting a CEC layer encapsulating the live CECs into a second hydrogel nanomesh; culturing live corneal epithelial cells (CEpCs); 3D bioprinting a CEpC layer encapsulating the live CEpCs into a third hydrogel nanomesh; and attaching the CEC layer to a first side of the stromal layer and the CEpC layer to a second side of the stromal layer. In some embodiments the steps of culturing are performed in parallel. The steps of 3D bioprinting the CEC layer and the CEpC layers may be performed in parallel. The CEC layer may be attached to the first side of the stromal layer by sequentially printing the stromal layer and the CEC layer. Alternatively, the CEC layer may be attached to the first side of the stromal layer by applying a thin film of hydrogel between each of the layers and curing via UV exposure. The CEpC layer may be attached to the second side of the stromal layer by applying a thin film of hydrogel between each of the layers and curing via UV exposure. In a preferred embodiment, prior to 3D bioprinting the CEC layer, the CECs are mixed with a prepolymer solution of acryloyl-polyethylene glycol (PEG)-collagen. The prepolymer solution may further include methacrylated hyaluronic acid (MA-HA). In another preferred embodiment, prior to 3D bioprinting the CEpC layer, the CEpCs are mixed with a prepolymer solution of acryloyl-PEG-collagen. The prepolymer solution may further include MA-HA. In another preferred embodiment, prior to 3D bioprinting the stromal layer, encapsulating the stromal cells in an acryloyl-PEG-collagen hydrogel, which may further include MA-HA. The stromal cells may be encapsulated at a cell density in the range of around 5 million/ml to 25 million/ml stromal cells.
In some embodiments, the live CEpCs are cultured and differentiated from limbal stem cells (LSCs). The LSCs may be obtained from autologous tissue. The live CECs may be cultured and differentiated from CEC progenitors from a human donor. The CEC progenitors may be obtained from autologous tissue.
In another aspect of the invention, an artificial cornea comprises a layered structure comprising a 3D bioprinted stromal layer comprising live stromal cells encapsulated into a first hydrogel nanomesh, the stromal layer having a first side and a second side; a 3D bioprinted CEC layer comprising live CECs encapsulated into a second hydrogel nanomesh; and a 3D bioprinted CEpC layer comprising live CEpCs encapsulated into a third hydrogel nanomesh; wherein the CEC layer is attached to the first side of the stromal layer and the CEpC layer is attached to the second side of the stromal layer. In some embodiments of the artificial cornea, one or more of the CEC layer and the CEpC layer is attached by a thin film of hydrogel applied between the layers and cured via UV exposure.
The live stromal cells are preferably encapsulated into a hydrogel prior to bioprinting the stromal layer. The hydrogel may be acryloyl-PEG-collagen, and may further include MA-HA. The live CECs are also encapsulated into a hydrogel prior to bioprinting the CEC layer. The hydrogel may be acryloyl-PEG-collagen, and may further include MA-HA. The live CEpCs are also encapsulated into a hydrogel prior to bioprinting the CEpC layer. The hydrogel may be acryloyl-PEG-collagen, and may further include MA-HA. The live CEpCs may be obtained from cultured and differentiated LSCs.
By integrating the emerging technologies in the multidisciplinary domains of nanotechnology, 3D bioprinting, and regenerative medicine, we have developed artificial corneas to change the clinical landscape by eliminating the current dependency on corneal donor tissue and by providing a new strategy for restoring vision that would otherwise be lost in human patients with severe corneal blindness. The native, multilaminar anatomy of the cornea is well suited as an initial application of our layer-by-layer nanomesh integrated 3D printing approach.
By integrating the emerging technologies in the multidisciplinary domains of nanotechnology, 3D bioprinting, and regenerative medicine, we have developed artificial corneas to change the clinical landscape by eliminating the current dependency on corneal donor tissue and by providing a new strategy for restoring vision that would otherwise be lost in human patients with severe corneal blindness. The inventive approach utilizes nano-based 3D printing for corneal regeneration. The native, multilaminar anatomy of the cornea is well suited as an initial application of our layer-by-layer nanomesh integrated 3D printing approach.
The 3D live printing (“3dLP”) technology utilizes continuous 3D printing of a series of layers by way of digital micromirror device (DMD) projection and an automated stage. Similar 3D printing systems have been previously disclosed for different applications. (See, e.g., International Publication No. WO2014/197622, and International Publication No. WO2012/071477, which are incorporated herein by reference).
Fabrication of an artificial cornea using a 3D hydrogel matrix employs digital mask (i.e., “maskless”) projection printing in which a digital micro-mirror device (DMD) found in conventional computer projectors to polymerize and solidify a photosensitive liquid prepolymer using ultraviolet (UV) or other light sources appropriate for the selected polymer.
The computer controller 10 may display an image of the desired structure 8 for a given layer, as shown, and/or may display the desired parameters of the matrix. A quartz window or other light transmissive material 15, spacers 18, and base 19, all supported on the translation stage 16, define a printing volume or “vat” containing the prepolymer solution 13. Additional solution 13 may be introduced into the printing volume as needed using a syringe pump (not shown.) Based on commands generated by controller 10, the system spatially modulates collimated UV light using DMD chip 12 (1920×1080 resolution) to project custom-defined optical patterns onto the photocurable prepolymer solution 13.
To generate 3D structures, projection stereolithography platforms such as DPsL employ a layer-by-layer fabrication procedure. In an exemplary approach, a 3D computer rendering (made with CAD software or CT scans) is deconstructed into a series of evenly spaced planes, or layers. For purposes of illustration, a simple honeycomb pattern representing one layer of a desired mesh-like structure is displayed on display 8 of computer controller 10. The pattern for each layer is input to the DMD chip 12, exposing UV light onto the photocurable (pre-polymer) material 13 to create a polymer structure 17. After one layer is patterned, the computer controller 10 lowers the automated stage 16 and the next pattern is displayed to build the height of the polymer structure 17. Through programming of the computer controller 10, the user can control the stage speed, light intensity, and height of the structure 17, allowing for the fabrication of a variety of complex structures 20. It should be noted that while a single honeycomb structure is illustrated, any combination of patterns, may be used to construct multi-layer structures of different patterns overlying each other.
As an alternative to the DMD chip, a galvanometer optical scanner or a polygon scanning mirror, may be used. Both of these technologies, which are commercially available, are known in their application to high speed scanning confocal microscopy. Selection of an appropriate scanning mechanism for use in conjunction with the inventive system and method will be within the level of skill in the art.
According to an exemplary embodiment, the process for fabricating a cell-based artificial cornea follows a 3-step strategy. Referring to
The following examples provide details of steps of used in an embodiment of the invention:
Cornea epithelial cells (CECs) undergo continuous renewal from limbal stem or progenitor cells (LSCs), and deficiency in LSCs or corneal epithelium, which turns cornea into a non-transparent, keratinized skin-like epithelium, causes corneal surface disease that leads to blindness. How LSCs are maintained and differentiated into corneal epithelium in healthy individuals, and which molecular events are defective in patients have been largely unknown.
Traditionally, the LSC growth and expansion process requires mouse 3T3 feeder cells, which carry the risk of contamination from animal products, thereby rendering it unsuitable for creating clinically-viable 3D bioprinted corneas. To overcome these obstacles, an in vitro feeder-cell-free, chemically-defined cell culture system to grow LSCs from rabbit and human donors, was developed to enable generation and expansion of a homogeneous population of LSCs, and subsequent differentiation into corneal epithelial cells (CEpCs). This culture system is based on the determination that the transcription factors p63 (tumor protein 63) and PAX6(paired box protein PAX6) act together to specify LSCs, and WNT7A controls corneal epithelium differentiation through PAX6. In the limbal stem cells, WNT7A acts upstream of PAX6 and stimulates its expression via frizzled homolog 5 (FZDS), a receptor for WNT proteins. WNT7A is a secreted morphogen involved in developmental and pathogenic WNT signaling. PAX6 is a transcription factor that controls the fate and differentiation of various eye tissues. RNAi-mediated knockdown of WNT7A or PAX6 induced human limbal stem cells to transition from a corneal to a skin epithelial morphology, a critical defect tightly linked to common human corneal diseases. The WNT7A and PAX6 knockdown cells also had lower expression of corneal keratin 3 (KRT3; CK3) and KRT12 and greater expression of skin epithelial KRT1 and KRT10 than wild-type limbal cells.
Notably, transduction of PAX6 in skin epithelial stem cells is sufficient to convert them to LSC-like cells, and upon transplantation onto eyes in a rabbit corneal injury model, these reprogrammed cells are able to replenish CECs and repair damaged corneal surface. Further details of this process are described in a letter published in Nature, “WNT7A and PAX6 define corneal epithelium homeostatis and pathogenesis”, Nature (2014) doi:10.1038/nature13465), published on-line 2 Jul. 2014, which is incorporated herein by reference. Proliferating LSCs were characterized by expression of P63 and K19, with a high percentage staining positive for the mitotic marker Ki67. We established a 3D LSC differentiation system in which stratified CEpC layers were grown in a basement membrane resembling the Bowman's membrane. Small molecule-ROCK inhibitor Y27632 was used to direct differentiation of LSCs to CEpCs, as evidenced by strong expression of CEpC-specific marker K3/K12.
In parallel, we developed a feeder-cell-free, chemically defined cell culture system containing fibroblast growth factor 2 (FGF2) to grow CEC progenitor cells from human donors. These CEC progenitor cells were then expanded into a homogeneous population of CEC progenitors that were subsequently differentiated into CECs. We observed the hexagonal shape morphology present in native anatomy with strong expression of typical CEC marker ZO-1.
Further, we tested the potential that LSCs cultured on gelatin methacrylate (GelMA) based matrix might be used to treat and repair corneal epithelial defects on a rabbit LSC deficiency model, which mimics a common corneal disease condition in humans. In this test, rabbit GFP-labeled LSCs transplants formed a continuous sheet of epithelial cells with positive staining of corneal specific K3/12 and successfully repaired epithelium defect of the entire corneal surface, and restored and maintained cornea clarity and transparency for over 5 months.
Corneal stromal cells were also cultured and expanded in vitro. These stromal cells shared similar markers of fibroblast, such as Fibronectin, FSP1 and Vimentin.
The 3D bioprinting platform offers a rapid biofabrication approach for constructing cell-laden hydrogel scaffolds that 1) have complex user-defined 3D geometries composed of a naturally derived biomaterial; 2) allow for consistent 3D distribution of cells encapsulated within the hydrogel; 3) support cell viability and proliferation; and 4) feature dynamic, multi-scale mechanical cell-scaffold interactions. Importantly, these constructs enable control and integration of complex 3D geometries while providing a physiologically-relevant internal 3D distribution of encapsulated cells. Through such precise control of spatial and temporal distributions of biological factors in 3D scaffolds, we are able to evaluate the interactions of cells with extracellular matrix (ECM) proteins at the nanometer length scale, with the ultimate goal of creating advanced, clinically translatable biomimetic scaffolds.
Using 3D bioprinting, artificial corneas are fabricated using the same dimension and curvature of the native cornea to replicate the patient's cornea. The naturally derived material can support cell growth within the construct and recruit host cells for better integration of the constructs. Due to the high efficiency of the 3D printing technology, a few seconds is sufficient for one layer. Therefore, it is possible to maintain a highly homogenous cell distribution within each layer. In addition, spatial localization of different cell types can be precisely controlled, which is critical for corneal function. For example, we can fabricate small features around 5 microns, i.e., smaller than a cell. With this resolution, we can control the spatial localization of very small cell population, even single cell. By using materials of different degradation profile, we can guide the cell migration and thus control their temporal distribution. By patterning growth factors within the constructs, we can also modulate the cell proliferation/differentiation, and manage the cell distribution.
Collagen has been used extensively as a biomaterial for corneal tissue engineering, as it comprises the main component of corneal extracellular matrix (ECM). Collagen, as a matrix constituent, has been demonstrated to support epithelial cells in forming a protective layer and to promote re-innervation by neurons. A chemically-crosslinked biosynthetic collagen matrix has shown significant promise in a phase I clinical trial. In order to modulate the degradation and mechanical properties of a collagen matrix, most studies have used chemical crosslinking approaches, which are largely incompatible with cell encapsulation. Acryloyl-PEG-collagen (Ac-Col) offers an excellent alternative for corneal tissue engineering due to its biocompatibility, optical properties, and ability for photopolymerization. Preliminary tests have been performed to assess the optical properties of a stromal cell-laden film made of GelMA, which is an Ac-Col analogue.
Evaluation of the impact on optical transparency of varied hybrid hydrogel combinations and exposure times was performed.
Several material compositions have been tested and the optical property of most of the material choices is very good. In one example, with 7.5 wt % GelMA or Ac-Col and 25 wt % PEGDA plus 0.075 wt % LAP (lithium phenyl-2,3,6-trimethylbenzoylphosphinate) as photoinitiator, produced a transparent film that exhibited comparable absorbance to that of PBS solution in the range of 280 nm to 1000 nm. The UV exposure time does not appear to affect the transparency of this film. In terms of MA-HA, 7.5 wt % GelMA with 2.5 wt % MA-HA and 2.5% PEGDA provides excellent optical properties as well after 30 seconds of UV exposure.
As is known in the art, because most photoinitiators are cytotoxic. Selection of the type and concentration of photoinitiator to obtain the desired film properties while maintaining cell viability will be within the level of skill in the art.
Three corneal layers were fabricated using 3D live printing as described above. Specifically, a PEGDA nanomesh was embedded in acryloyl-PEG-collagen to support the corneal stroma. The CEpC layer and CEC layer were built on each side of the stroma layer. The resulting bioprinted cornea was transplanted onto a rabbit recipient eye.
New Zealand white rabbits were anaesthetized with intramuscular injection of xylazine hydrochloride (2.5 mg/ml) and ketamine hydrochloride (37.5 mg/ml). A corneal recipient stromal bed with a reverse-button like structure was created in the recipient eye using a femtosecond laser machine (Zeiss). The bioprinted corneal donor tissue was cut into a button-shape structure to fit onto the prepared recipient stromal bed. The surface was then covered by a human amniotic membrane (Bio-tissue), which was secured with 10.0 VICRYL sutures (Ethicon) to the recipient conjunctiva.
According to the embodiments described herein, the use of 3D bioprinting technology allows for cell encapsulation, enabling live printing of tissue structures with micro and nanometer resolution. The cell-laden corneal substitutes can reduce the amount of time required for the transplants to integrate with the host tissue. In addition, the digital (i.e., customizable) nature of 3D printing allows development of patient-specific tissue models with designed shape and curvature. The custom shape and curvature can be designed according to the patient's native cornea.
Using procedures that are known in the art, corneal topography measurements can be obtained for the patient prior the transplant procedure. For example, instruments used in clinical practice most often are based on Placido reflective image analysis, which uses the analysis of reflected images of multiple concentric rings projected on the cornea to obtain keratometric dioptric range and surface curvature. Using the clinical data generated by such testing, computer software can be used to generate patient specific corneal design, which will then be fabricated using the 3D printing platform. A layer by layer printing approach may be used. In some cases, in order to generate highly complex corneal geometries, it may be appropriate to utilize a non-linear 3D printing scheme such as that disclosed in PCT Application No. PCT/US2015/050522, filed Sep. 16, 2015, which is incorporated herein by reference.
3D-printed cornea tissues fabricated according to the procedures described herein will have immediate applications in clinical transplantation, human ocular surface disease modeling (e.g., for dry eye diseases), early drug screening to replace or reduce the need for animal testing, and in drug efficacy testing for wound healing. This technology provides a strong basis for the development of temporary or permanent cornea replacements. The embodiments described herein could lead to readily available, complex engineered tissues that recapitulate the functionality of their natural human counterparts and are suitable for clinical adoption as well as emerging biomedical research.
1. Fagerholm P, Lagali NS, Merrett K, Jackson WB, Munger R, Liu Y, Polarek JW, Soderqvist M, and Griffith M. A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study. Sci Transl Med. 2010;2(46):46ra61
2. Myung D, Duhamel PE, Cochran JR, Noolandi J, Ta CN, and Frank CW. Development of hydrogel-based keratoprostheses: a materials perspective. Biotechnol Prog. 2008;24(3):735-41
3. Crabb RA, Chau EP, Evans MC, Barocas VH, and Hubel A. Biomechanical and microstructural characteristics of a collagen film-based corneal stroma equivalent. Tissue Eng. 2006;12(6):1565-75.
This application claims the benefit of the priority of U.S. Provisional Application No. 62/054,924, filed Sep. 24, 2014, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/51999 | 9/24/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62054924 | Sep 2014 | US |
