The present disclosure relates to biomaterials and methods for use in tissue regeneration applications.
The rational design of biomaterials for regenerative applications has long sought to approximate the biological composition of the native extracellular matrix (ECM). The main components of the human ECM are collagens, proteoglycans, laminin, fibronectin, and elastin, which, along with other matrix macromolecules and growth factors, link together to form a structurally stable network that contributes to the mechanical properties of different tissues. The ECM, however, is tissue-specific, where cells secrete matrix molecules based on their local conditions, such as biological function, mechanical loading, hypoxia, and variability in nutrient concentration. Furthermore, the composition of the ECM varies dynamically through life to regulate various processes of development, differentiation, and remodeling. The bioactive nature of the native ECM of various tissues has been well established. Therefore, researchers have capitalized on the cell instructive and regenerative potential of these naturally bioactive materials to develop matrix-derived scaffolds for regenerative applications, examples of which include repair of bone, cartilage, brain and spinal cord, pancreas and a host of other tissues.
Despite the remarkable capacity of ECM-derived proteins to facilitate tissue regeneration and healing and their inherent capacity to self-assemble into structured hydrogels, their primary disadvantage remains the lack of control over their material properties, such as stiffness, degradability, microporosity, etc; all of which have been shown to significantly influence cell behavior. Additionally, their weak polymerization chemistry and resultant material properties do not usually support the fabrication of 3D constructs with the complex microarchitectural features that are typically associated with native tissues.
For example, autologous bone grafting has a long history as the gold standard in bone repair and regeneration, due to its high osteoconductivity, osteogenicity, and osteoinductivity. However, the limitations associated with this approach (e.g. potential scarcity, impact on the patient of harvesting the bone) have spurred the continued search for xenogenic materials, such as allografts and synthetic materials, that may be effective alternatives for promoting regeneration. One such material is demineralized bone matrix (DBM), which is an established tool for bone repair in the clinical setting. As native collagen is the principal component of the bone ECM, demineralized bone matrix materials share the drawback of a lack of control over mechanical properties. From an engineering point of view, this is a constraint in regulating tissue mechanics, which is an important factor in determining cell behavior and differentiation.
In order to address the limitations of current materials used in tissue regeneration and engineering, a bone-derived biomaterial has been developed that comprises bone ECM proteins functionalized with (meth)acrylates, which render the biomaterial crosslinkable in the presence of a crosslinking agent while maintaining the biological advantages associated with the composition of the native ECM.
In an embodiment, a biomaterial having a tunable material property comprises a demineralized, decellularized bone extracellular matrix material (bECM) functionalized with a crosslinkable (meth)acrylate monomer. As used herein, “(meth)acrylate” encompasses acrylates and methacrylates as well as alkyl esters thereof, i.e., alkyl acrylates and alkyl methacrylates. As the bECM is derived from bone extracellular matrix, in a particular aspect the bECM can comprise elements of the organic component of bone extracellular matrix, including but not limited to collagen, non-collagenous proteins, and proteoglycans. In a particular embodiment, one or more side chains of one or more of the proteinaceous components in the bECM are functionalized with the crosslinkable (meth)acrylate monomer. In accordance with the present disclosure, the biomaterial comprises bone extracellular matrix material that is demineralized and decellularized. Therefore, in an aspect inorganic components of bone extracellular matrix, including but not limited to hydroxyapatite and other salts of calcium and/or phosphate, are substantially absent from the biomaterial or are present in very small amounts. In another aspect, cells and cellular material that were native to the bone extracellular matrix from which the bECM is derived are also substantially absent from the biomaterial or are present in very small amounts.
In accordance with the present disclosure, protein (meth)acrylation is employed to endow the matrix with crosslinkable moieties that allow for the control of the final mechanical properties of the biomaterial. In a particular embodiment, the biomaterial further comprises a crosslinking agent. It will be recognized by those of skill in the art with the aid of the present disclosure that a number of approaches and agents may be suitably employed to initiate crosslinking in the biomaterial. Crosslinking initiators include, but are not limited to, light, pH, temperature, hydration, and various chemical agents. Initiators that arise from the physical or chemical environment of the material to be crosslinked (e.g. pH or temperature) can allow for self-assembly of the crosslinked material.
It is contemplated that photopolymerization of bone extracellular matrix proteins using light sensitive moieties enables formation of hydrogel scaffolds with precisely tuned and reproducible microscale architectures and physico-mechanical properties, through modulation of light exposure. Furthermore, the photocrosslinkable nature of this material allows for straightforward bioprinting of cell-laden micro-tissue constructs using a digital light processing (DLP) based approach, which is advantageous over conventional methods of fabrication of demineralized and de-cellularized bone. Accordingly, in an embodiment, the crosslinking agent is a photoinitiator. In a specific embodiment, the photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate, lithium acylphosphinate, 2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone2-Benzyl-2-(dimethylamino)-4′-morpholinobutyrophenone, 4′-tert-Butyl-2′,6′-dimethylacetophenone, 2,2-Diethoxyacetophenone, 2,2-Dimethoxy-2-phenylacetophenone, a blend of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide and 2-hydroxy-2-methylpropiophenone, 4′-Ethoxyacetophenone, 3′-Hydroxyacetophenone, 4′-Hydroxyacetophenone, 1-Hydroxycyclohexyl phenyl ketone, 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, 2-Hydroxy-2-methylpropiophenone, 2-Methyl-4′-(methylthio)-2-morpholinopropiophenone, 4′-Phenoxyacetophenone, or combinations thereof. As will be recognized by those of skill in the art with the aid of the present disclosure, the amount of photoinitiator or other crosslinking agent included in the biomaterial can be selected based on the identity of the crosslinking agent and the desired results. In a more specific embodiment, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphinate and is present in an amount of from about 0.05% w/v to about 5% w/v. As given photoinitiator may be activated by light having wavelengths within a particular range. In a particular embodiment, the biomaterial comprises a photoinitiator having an activation wavelength in the ultraviolet range. In another particular embodiment, the biomaterial comprises a photoinitiator having an activation wavelength in the visible range. In a specific exemplary embodiment, the photoinitiator has an activation wavelength of from about 200 nm to about 500 nm. In another specific exemplary embodiment, the photoinitiator has an activation wavelength of from about 400 nm to about 700 nm.
As the primary component of the biomaterial, the amount of bECM contained in the biomaterial can be selected according to the desired material properties or to be suited for particular uses. In an embodiment, the bECM is present in an amount of from about 0.5% w/v to about 40% w/v. The biomaterial can also include biological material that promotes biological processes involved in tissue regeneration, such as vasculogenesis or the invasion of appropriate progenitor cell types. Accordingly, in an embodiment, the biomaterial comprises a population of living cells that are not autologous to the ECM from which the biomaterial is derived. In specific embodiments, the population can comprise vascular endothelial cells and/or stem cells. In another embodiment, the biomaterial includes microvascular fragments. In addition, the components of the biomaterial can be combined in a carrier that is suitable for the intended use. In a particular embodiment, the carrier is a pharmaceutically acceptable medium. Nonlimiting examples include bioinert solvents such as saline and water.
Initiation of crosslinking in the biomaterial results in formation of a hydrogel having properties that result at least in part from the particular composition of the biomaterial, the degree of crosslinking, the crosslinking agent, and the parameters of crosslinking initiation. Accordingly, a hydrogel construct can comprise a demineralized, decellularized bone extracellular matrix material (bECM) functionalized with a crosslinkable (meth)acrylate monomer, wherein the bECM is at least partially crosslinked. As one use of such a construct is to promote tissue regeneration in the site where it is placed, the hydrogel construct can include biological materials selected to promote regeneration. In this way the construct can serve as a device for delivering such material to a tissue to be regenerated. Accordingly, in an embodiment, the hydrogel construct comprises a population of living cells that are not autologous to the ECM from which the biomaterial is derived. In specific embodiments, the population can comprise vascular endothelial cells and/or stem cells. In another embodiment, the hydrogel construct includes microvascular fragments. The hydrogel construct can be formed in situ, e.g., uncrosslinked biomaterial can first be delivered to a tissue site to be regenerated, after which crosslinking is initiated. In other applications the hydrogel construct can be formed before delivery to the site. Accordingly, in embodiments the hydrogel construct can be processed to facilitate storage, handling, and delivery. In a specific embodiment, the hydrogel construct is lyophilized.
An aspect of the embodiments described herein is that the composition of the crosslinkable biomaterial provides for tunability of material properties of the resultant hydrogel. These material properties include stiffness, microporosity, degradability, and other properties that can influence the behavior of cells involved in regenerating a particular tissue. The biomaterial of the present disclosure provides a number of avenues for tuning these mechanical properties, including changing the (meth)acrylate concentration, crosslinker concentration, curing time and/or combination of these parameters, which is advantageous in controlling the stem cell microenvironment. For photocrosslinkable biomaterial in particular, in situ photopolymerization is possible using light sources that are used clinically, such as UV or visible light sources. It should also be noted that the present biomaterial provides a multi-component hydrogel that carries the proteolytic by-products of pepsinized collagen and resulting peptides of a wide range of non-collagenous proteins. This is in contrast to single component hydrogels, such as methacrylated gelatin (GelMA), methacrylated hyaluronic acid (meHA) or methacrylated tropoelastin (MeTro), which have only one major component like gelatin, HA, and tropoelastin, respectively. While not being bound to a particular theory, it is surmised that differences in physical properties and tunability thereof between biomaterials of the present disclosure and other hydrogel materials may be attributed to the heterogeneity in the molecular network that is formed in the cross-linked bone peptides (of different lengths and types) versus the more homogenous and controlled one-component matrices of other hydrogels.
With the foregoing in mind, in an embodiment, a method of making a hydrogel construct, comprises introducing an amount of a biomaterial into a shaping device, and curing the biomaterial to form a hydrogel having a material property, where the biomaterial comprises a demineralized, decellularized bone extracellular matrix material (bECM) functionalized with a crosslinkable (meth)acrylate monomer; and a crosslinking agent. In a particular embodiment, the crosslinking agent is a photoinitiator, and the curing step comprises exposing the biomaterial to light for an exposure time so as to achieve the material property. Without being bound to a particular theory, it is surmised that the ability of a hydrogel scaffold to promote certain regenerative processes, e.g., microvascular network formation, is linked to the stiffness of the hydrogel. Accordingly, in a specific embodiment, the material property is an elastic modulus. It should be noted however, that other aspects of the biomaterial of the present disclosure may also contribute to enhanced regenerative processes. For example, the biomaterial can include pro-angiogenic peptides that are found in bone extracellular matrix and that persist in the biomaterial when it is made.
In accordance with the method, the shaping device is any device, structure, or system that can be used to impart a particular shape or dimension to the hydrogel construct, either before or during curing. In one embodiment, the shaping device is a mold into which a quantity of the biomaterial is placed and then cured. In an alternative embodiment, the shaping device is a microfluidic channel. In another embodiment, the shaping device is a three-dimensional (3D) printing platform. As noted above, biomaterial of the present disclosure can be used as a bioink for DLP bioprinting, where photocrosslinkability of the biomaterial is a key element. One of the highlights of the printed structures is the printability of the biomaterial in microdimensions leading to the formation of particular hydrogel constructs termed microgels. Microgels are a special class of materials that have gained attention in fabricating complex tissues. Microgels are crosslinked polymer networks in the micron range, which has considerable advantages over bulk hydrogels in the area of controlled release of drugs and protein, hydrolytic degradation, personalized medicine screening and microscale tissue engineering. In microscale tissue engineering, microgels are used as building blocks for the bottom-up building of hetero-architecture to mimic complex tissues. Accordingly, in a particular embodiment making a hydrogel construct comprises assembling a plurality of hydrogels to form such a structure.
Shape can be a significant factor in microgel geometry to create centimeter-scale tissue-like structures. For example, tightly packed cell-laden tissue-like structures can be created through bottom-up self-assembly in a multiphase environment (liquid-air system). Hexagonal cell-laden microgels form tissue-like structures through an interface-directed assembly process, while more complex building blocks like lock-and-key microgels allowed greater control over self-assembly. Since cell-laden biomaterial of the present disclosure allows printing in multiple complex shapes, similar lock-and-key shaped cell-laden microgels can be printed to create complex shapes and tissues. Also, culturing the hydrogels described herein with two or more cells can further address the complexity of building tissues. Therefore, these hydrogel constructs can be an effective ECM-based building block for bottom-up manufacturing of complex tissues. Injectability of microgels is another parameter that plays a significant role in therapeutic delivery for treating site-specific tissue damage like myocardial infarction, enhancing neovascularization and cellular differentiation. The injectability of the biomaterial of the present disclosure opens up venues for investigations in basic biology and clinically-oriented minimally invasive implantation procedures.
In an embodiment, a kit for use in tissue engineering comprises a biomaterial in a container, where the biomaterial comprises bECM functionalized with a crosslinkable (meth)acrylate monomer and a crosslinking agent. In a more specific embodiment, the the crosslinking agent is a photoinitiator. In a particular embodiment, the kit further comprises instructions for delivering an amount of the biomaterial and curing the bECM to form a hydrogel. In other specific embodiments, the kit further comprises a delivery device for delivering an amount of the biomaterial and said delivery device can specifically be configured to be operably connected to the container for delivering the biomaterial directly from the container. In other particular embodiments, the biomaterial is formulated as a paste or alternatively as a lyophilized powder.
In GelMA synthesis, pH is typically maintained at 7.4 by phosphate buffer, which introduces methacryloyl groups to the reactive amine and hydroxyl groups of the amino acid residues. The main limitation in this process is that the referred pH limits the reactivity of amine and hydroxyl groups. In accordance with the present disclosure, this problem is addressed by improved synthesis protocol, where a higher pH is maintained during the reaction using a buffer (e.g. carbonate bi-carbonate) to keep the isoelectric point (IEP) of the bone proteins high, keeping the free amino groups of lysine neutral and allowing for it to react with a (meth)acrylic reagent. Accordingly, in an embodiment, a method of making a biomaterial having a tunable material property, comprises the steps of demineralizing powdered bone by treating the powdered bone with acid to produce demineralized bone material; extracting lipids from the demineralized bone material; decellularizing the demineralized bone material to produce demineralized bone extracellular matrix material; solubilizing collagen in the demineralized bone extracellular matrix material; and reacting the demineralized bone extracellular matrix material with a (meth)acrylic reagent to produce a crosslinkable bone extracellular matrix material, wherein said reacting is performed at a pH of from about 8 to about 10. In another specific embodiment, the reacting step is performed with an amount of (meth)acrylic reagent of from about 0.1 ml to about 0.3 ml per gram of demineralized bone extracellular matrix material. In another embodiment, the reacting step is performed at a reaction temperature of from about 30° C. to about 55° C. In still another embodiment, the reacting step is performed for a reaction time of from about 1 to about 5 hours.
In a specific embodiment, the (meth)acrylic reagent is methacrylic anhydride. However, it will be recognized by those of skill in the art with the aid of the present disclosure, that (meth)acrylate reagents having reactive (meth)acrylate groups can be used to functionalize the proteins of the bECM, including without limitation, salts and copolymers.
The inventors have also found that ECM protein yield from this process can be enhanced by centrifugation and filtering of intermediate products and by-products of certain steps. For example, in an embodiment, the method of making the biomaterial further comprises centrifuging a solution produced by any one of the above steps to produce a precipitate and a supernatant; filtering solids of a size from the supernatant; and then performing the subsequent step on the solids.
Demineralized and decellularized bone ECM bone was extracted as summarized in
Following this, the processed bone matrix was subjected to enzymatic digestion by pepsin (1 mg/ml in 0.01 N HCl), where a suspension of 10 mg of matrix per ml of pepsin was stirred under agitation for 96 hours until the matrix was solubilized. Finally, the solubilized matrix was centrifuged for 30 min at 4° C., filtered under vacuum and stored at −20° C.
For the synthesis of methacrylated bECM (also referred hereinafter in both its uncrosslinked and crosslinked forms, as applicable, as “BoneMA”), the frozen demineralized and decellularized bone matrix was lyophilized for two days. Next, 10% (w/v) of the lyophilized matrix was dissolved in 0.25M carbonate-bicarbonate buffer, and the pH was adjusted to 9 using 4M NaOH or 6M HCl under constant stirring at 50° C. Once the bone matrix was dissolved, methacrylic anhydride (0.2 ml/g of bone matrix) was added to the solution in a dropwise manner under constant stirring while maintaining the temperature. The reaction was allowed to proceed for 3 hours, and the pH was maintained at 9 by adding 4M NaOH. After the completion of the reaction, the modified bone matrix solution was diluted 4× times with warm deionized water. Unreacted methacrylic anhydride was removed from the solution by dialyzing it against deionized water for 24 hours, following which the solution was filtered, lyophilized and stored at −80 ° C. until further use (see
The spectra of unmodified bone matrix showed the expected peaks corresponding to the amino acids containing primary amines and aromatic groups (
Gelatin methacryloyl (GelMA) was used as a control to compare against the biological properties of BoneMA. GelMA was synthesized as per the protocol described by Nichol et al. Porcine skin type A gelatin (10% w/v) (Sigma, St Louis, Mo., USA) was dissolved in Dulbecco's phosphate buffered saline (DPBS, Sigma) warmed to 50° C. to which, 8% (v/v) methacrylic anhydride (Sigma) was added dropwise and allowed to react for 2 h. Next, the solution was diluted 5× times with DPBS and dialyzed with 12-14 kDa dialysis tubing against warm distilled water (45±5° C.) for 5 days. The warm water was changed two times a day for 5 days. The resulting methacrylated prepolymer was lyophilized and stored at room temperature until further use. For GelMA sample synthesis, GelMA was crosslinked for 15 sec, 30 sec, and 45 sec using the bioprinter as described previously, and the resultant constructs are identified as GelMA15, GelMA30, and GelMA45 respectively. The GelMA samples were treated similarly to BoneMA for SEM and live/dead analysis
The freeze-dried methacrylated bECM was dissolved in DPBS (5% (w/v)) with 0.15 w/v% Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, Tokyo Chemical Industry, L0290) photoinitiator. A digital light processing (DLP) 3D printer (Ember, Autodesk) was used to prepare hydrogel samples for different experiments by exposing the hydrogel prepolymer to 20 mW/cm2 of light (405±5 nm) for 15, 30, and 45 sec. For physical and mechanical characterization, samples were printed with 5 mm in diameter and 1.5 mm in thickness, whereas biological characterization tests used 450 thick specimens printed with geometrical shapes described below (N=5).
The hydrogel mechanical properties were measured using a DHR-1 rheometer (TA Instruments) fitted with a UV curing accessory containing a bandpass filter for 405±5 nm. The intensity of the light from an Acticure® 4000 (EXFO) coupled with the rheometer was matched to the DLP printer at 20 mW/cm2 using a power meter (Power Max5200, Molectron). Shear modulus measurements were performed as a function of crosslinking time under 0.1% strain and frequency of 10 Hz and were converted to elastic modulus using a previously reported equation.
The tunability of the mechanical properties of the newly formed methacrylated material was examined as a function of light exposure by comparing the elastic moduli of hydrogels polymerized for 15, 30 and 45 seconds, using the DLP 3D printer as a light source. The elastic modulus of the material was found to increase significantly as a function of the duration of light exposure, starting at 0.9 kPa when crosslinked for 15 s, and increasing gradually to 1.3 (p<0.05) and 1.5 (p<0.001), at 30 and 45 s respectively (
The rheological studies revealed relevant information regarding the photocrosslinking process of the methacrylated bECM (
The hydrogel samples were prepared according to the above procedure for all time points. For SEM, the BoneMA hydrogel was immersed in 5 mL DPBS at 37° C. for 24 h (n=5). Next, samples were fixed with 2.5% glutaraldehyde solution for 1 hour. After fixing, the samples were dehydrated for 10 minutes in a series of ethanol solutions of concentration 25%, 50%, 75%, 90%, and 100%, respectively. After dehydration, samples were critical point dried for 3 hours. These dried samples were coated with gold/palladium and imaged with FEI Quanta 200 SEM at 15 kV.
SEM images showed the presence of the typical pore-like microstructures that are commonly observed in covalently crosslinked hydrogels with BoneMA crosslinked for 15 seconds having distinctly larger pores than its more crosslinked counterparts (
Since different aspects and applications of BoneMA were being tested, samples were prepared using different cell lineages for various experiments. To that end, human dental pulp stem cells (HDPSC) and human mesenchymal stem cells (HMSC) were used to assess the general bioink properties, such as cytocompatibility and bioprintability, while green fluorescent protein (GFP)-expressing human umbilical vein endothelial cells (HUVECs) (cAP0001GFP, Angio-proteomie, USA) were employed to investigate the vasculogenic potential of the synthesized biomaterial. HDPSCs (Lonza, USA) and HMSCs were each cultured in α-MEM medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin. HUVECs were cultured in Endothelial Cell culture medium (Vasculife-VEGF, Lifeline Cell Technologies) on 0.1% gelatin-coated substrates. Cells were maintained in an incubator at 37° C., 5% CO2 incubator, and the medium was replaced every 2 days.
In order to assess the cytocompatibility of BoneMA, HDPSCs were trypsinized and resuspended in BoneMA (5% (w/v)) containing LAP (0.15% (w/v)) at a cell density of 5×105 cells/mL. This cell-laden prepolymer solution was photopolymerized as described above for 15, 30, and 45 sec. Cell viability was measured on days 1, 3, and 7 post-encapsulation using a membrane permeability based fluorescent live/dead staining kit (Molecular Probes), and the fraction of live cells was estimated using ImageJ software from at least 3 distinct regions per sample (N=5).
HDPSCs remained highly viable through at least 7 days in culture within all BoneMA hydrogel constructs, with average viability of 91, 94 and 90% for BoneMA crosslinked for 15, 30 and 45 seconds on day 7, respectively (
The vasculogenic potential of BoneMA was examined in comparison to GelMA, which was synthesized with a comparable degree of methacrylation and photo-crosslinked for the same amounts of time. To that end, HUVECs were encapsulated in BoneMA hydrogels (5% (w/v)) containing LAP (0.15% (w/v)) at a cell density of 1×107 cells/ml, crosslinked for 15, 30 and 45 s as described previously. Vascular capillary network formation in these hydrogels was characterized daily for 7 days and quantified for the number of segments, number of end points and junctions, as well as the total length of the network using an ImageJ.
In order to assess the vasculogenic potential of BoneMA, we compared vascular network formation in HUVEC-laden hydrogels crosslinked for 15, 30, and 45 sec (
Quantification of vascular network formation (
To bioprint BoneMA in various geometrical shapes, specific print patterns were designed using CAD (Fusion 360, AutoDesk) and converted into image slices with the accompanying 3D printing software (Print Studio, Autodesk). The print patterns were designed arbitrarily with star, square, triangle, and rhombus shapes, as well as flower, spiral, concentric circles, and the OHSU logo. Shapes were printed with length and width dimensions that ranged from 600 μm (square) to 2.5 mm (OHSU logo) with a thickness of 450 μm, under printing exposures of 25 sec. For bioprinting of the said geometries, the bioink was formulated by mixing HMSCs with the BoneMA (5% (w/v)) pre-polymer containing LAP (0.15% (w/v)) at a concentration of 5×105 cells/ml. The HMSO-laden bioprinted BoneMA was fixed with 4% (v/v) paraformaldehyde and permeabilized using 0.1% (v/v) Triton X-100. Next, the samples were treated with 1.5% (w/v) bovine serum albumin (Sigma Aldrich) in DPBS for 1 h, followed by Image-iT FX signal enhancer (Invitrogen, Calif.) for 30 min, after which they were immunostained with ActinGreen™ 488 ReadyProbes™ (Invitrogen).
Fluorescence microscopy images of bioprinted patterns using BoneMA encapsulating HMSCs and immunostained for F-Actin (
All above data are presented as mean±standard deviation. Data were compared using one way ANOVA followed by Tukey posthoc test (α=0.05) with Graphpad Prism 8.
It will be apparent to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.
This application claims priority to U.S. Provisional Patent Application No. 62/983,482 filed on Feb. 28, 2020, entitled “BIOMATERIALS AND RELATED METHODS AND KITS,” which is hereby incorporated herein by reference in its entirety.
This invention was made with government support under Grant Nos. R01DE026170 and 3R01DE026170-03S1 awarded by the National Institute for Dental and Craniofacial Research (National Institutes of Health). The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US2021/020014 | 2/26/2021 | WO |
Number | Date | Country | |
---|---|---|---|
62983482 | Feb 2020 | US |