Patent Application
20230399605
The present disclosure relates to a scaffold biomaterial to produce a tissue or other cell-based product.
Plant-derived biomaterials have been reported for tissue engineering applications. In particular, previous work from the inventors demonstrates that the decellularization of plant tissues resulted in cellulose-rich three-dimensional (3D) scaffolds (WO2020/227835 and WO2017/136950). In addition, the inventors have demonstrated that these scaffolds also perform well after implantation into animal models, resulting in a high degree of tissue integration and vascularization. Other groups have reported similar findings with plant tissues and mammalian cell types to demonstrate the utility of plant-derived biomaterials for biomedical and food-based tissue engineering applications. However, such approaches are reliant on the structure and mechanical properties of the natural starting material.
Plant-derived proteins have been examined for creating supports for mammalian cells in tissue engineering applications. Proteins such as soy, zein and camelina have been studied, as well as gluten proteins derived from wheat, such as gliadin and glutenin. These wheat derived proteins can be purified and made into films suitable to culture mammalian cells. Reports have shown that such gluten films are acceptable substrates for osteoblasts. Further, a gluten film was shown to support the growth of osteoblasts but with less efficiency due to the cytotoxicity of gliadin. In another approach, wheat protein-based scaffolds have been prepared by electrospinning, in which ultrafine fibrous structures are obtained, creating a polymer melt film of wheat glutenin. Such scaffolds have been shown to support the culture of adipose derived mesenchymal stem cells. However, these methods are labor and resource intensive, requiring two days to purify the proteins and seven days for them to be electro-spun.
Beyond bio-medicine application, the development of naturally-derived 3D biomaterials has gained considerable interest in recent years due to their potential for use in the food industry [5,12,16]. Although still emerging, the broader goal of cellular agriculture is to replace products produced by traditional agricultural methods with biotechnological approaches, notably through synthetic biology and tissue engineering [17,18]. One specific area of interest within the field is the cultivation of mammalian cells in vitro for the preparation of meat-like products. Although a number of challenges remain to achieve this goal, a significant body of work has begun to address issues such as the large-scale production of relevant cell types, creating sustainable and ethically sourced media and developing suitable scaffolds. While exciting, it is important to also recognize that the future potential of these methods is still debated as efforts to address the continued dependence on animal products in traditional cell culture (for example, fetal bovine serum), water/electricity use and any potential health benefits in the final meat products are still ongoing. With that said, there remains an intense interest in developing solutions which address these problems, and others, potentially opening up a new future in which foods can be more sustainably created and distributed globally. Plant-derived scaffolds are of particular interest due to the potential of creating edible scaffolds. There is a need in the art for methods to prepare scaffolds for tissue culture which are simple and straightforward to implement.
Scaffolds containing starch and other materials such as ethylene vinyl alcohol have been fabricated for biomedical engineering applications. A primary area of interest is the use of starch blended with polymers or other biomaterials to serve as a bone tissue scaffold. Such scaffolds may be prepared by extrusion. (Salgado et al., 2004, Tissue Engineering, 10(3/4):465-474). Starch has been described as a polymer for wound healing applications. Investigators have fabricated starch-based nanofibrous scaffolds by electrospinning for wound healing applications. (Waghmare et al., 2018, Bioactive Materials, 3:255-266).
Fiume et al., 2019, Molecules, 24:2954 describes the use of bread as a template to make an inorganic scaffold. To prepare the inorganic scaffold, the bread was coated with a glass powder and then subjected to elevated temperature conditions to “burn off” the bread, leaving only the inorganic material comprising silica. The process described is limited to providing a material that served as an inorganic scaffold for bone engineering. Further, such process is reliant on a number of complicated steps to prepare the silica-containing scaffold.
The present disclosure provides one or more improvements and/or useful alternatives to providing scaffolds for producing a tissue or other cell-based product for use in a variety of applications.
The present disclosure relates to a bread-derived scaffold biomaterial to produce a tissue or other cell-based product for use in a variety of applications.
According to one aspect of the disclosure, there is provided a bread-derived scaffold biomaterial for supporting cells, the scaffold comprising a bread crumb and wherein the bread crumb has a three-dimensional porous structure to support the cells.
According to another aspect of the disclosure, there is provided a tissue or cell-based product having a three-dimensional structure comprising the bread-derived scaffold biomaterial, the bread-derived scaffold biomaterial having pores supporting a population of the cells.
According to a further embodiment, the tissue or cell-based product is a food product.
In one embodiment, the food product is a meat product. In such embodiment, the population of cells comprise myocytes and/or adipocytes.
According to another aspect of the disclosure, there is provided a process for producing a tissue or cell-based product comprising growing cells on the bread-derived scaffold biomaterial.
According to another aspect of the disclosure, there is provided a use of a bread-derived scaffold biomaterial to produce a tissue or a cell-based product.
In a further aspect, there is provided a method for producing a bread-derived scaffold biomaterial, comprising:
According to one embodiment of any of the foregoing aspects, the bread crumb is derived from a bread that is yeast-free.
According to any one of the foregoing aspects or the embodiment, the bread crumb is leavened with a bicarbonate salt, such as sodium bicarbonate.
According to any one of the foregoing aspects or embodiments, the crumb comprises one or more gluten proteins.
According to any one of the foregoing aspects or embodiments, the crumb comprises one or more non-gluten proteins.
According to any one of the foregoing aspects or embodiments, the bread crumb is cross-linked with a cross-linking agent.
According to any one of the foregoing aspects or embodiments, the cross-linking agent is glutaraldehyde or transglutaminase.
According to any one of the foregoing aspects or embodiments, the crumb is for supporting the growth of cells that are selected from mammalian cells, fish cells, avian cells, reptile cells, amphibian cells, crustacean cells, plant cells, invertebrate cells, algae cells, bacteria cells, archaea cells or fungal cells. In certain embodiments, the cell is a fibroblast, satellite cell, myoblast, myocyte, smooth muscle cell, myofibroblast, myotube, cardiomyocytes, neutrophil, macrophages, lymphocytes, monocytes, platelets, pre-osteoblast, osteoblast, osteoclast, pre-adipocyte, adipocyte, periodontal ligament stem cells, fibrocytes, chondrocyte, tenocyte, keratinocytes, hepatocytes, neuron, neural precursor cells, dorsal root ganglion cells, glial cells, astrocytes, epithelial cells, endothelial cells, stem, mesenchymal stem or induced pluripotent stem cell or any combination thereof.
According to any one of the above aspects or embodiments, the tissue includes skeletal muscle, smooth muscle, cardiac muscle, bone, fat/adipose, kidney, liver, lung, skin, neural, vascular tissues or any combination thereof.
According to any one of the foregoing aspects or embodiments, the cells are grown on the scaffold in vitro and/or in vivo.
In a further embodiment, the method described above further comprises a step of sterilizing the bread-derived scaffold biomaterial.
In a further embodiment, the method described above further comprises cross-linking the bread-derived scaffold biomaterial with a cross-linking agent.
In a further embodiment, the method described above comprises using a leavening agent that is a chemical agent. The chemical agent may be a salt of a bicarbonate ion.
Described herein is a scaffold biomaterial comprising a bread crumb that has a three-dimensional porous structure that has been shown to support the growth of a variety of cell types. Advantageously, the inventors have found that bread-derived scaffolds can be used as an alternative to synthetic or animal-derived scaffolds and may be used in a number of applications, such as, but not limited to biomedical engineering, cosmetics, agriculture, for preparing edible product, and other applications known to those of skill in the art.
By the term “crumb”, it is meant a portion of a bread that has a three-dimensional porous structure resulting from the use of a leavening agent during its production. Generally, the crumb is the internal portion of a bread product, such as a loaf. For example, for breads comprising a crust, the crumb portion is the internal part of the bread that excludes the crust.
By the term “cell-based product”, it is meant any product comprising a plurality of cells that are differentiated or undifferentiated, including a tissue, and that is produced either in vitro or in vivo. This includes, without limitation, any product for human or animal use in biomedical or food applications.
By the term “microcarrier”, it is meant a support for cells in any form in liquid tissue culture in a vessel.
The crumb is derived from any suitable type of bread that allows for the growth of cells. This includes bread derived from leavening a dough with a leavening agent that generates a gas, which is typically carbon dioxide. In some embodiments, the porous structure allows for the infiltration, growth and/or migration of cells within the crumb structure.
The pore sizes in the crumb can vary significantly over a range of micrometers to millimeters. The leavening agent and/or its concentration may be selected to provide a desired and/or consistent pore size to the scaffold. The pore size may be selected to optimize the growth and/or migration of the cells in the scaffold. In one embodiment, a high porosity scaffold is employed to avoid an anoxic environment within the scaffold structure.
A suitable pore size can be assessed by a variety of known techniques including image analysis. Scaffolds may be imaged by optical microscopy or by scanning electron microscopy (SEM). The pores in the scaffold may be partially or completely interconnected as determined by microscopy. A scaffold having suitable pore sizes and interconnectivity thereof can be readily selected by those of skill in the art. Assessment of pore size and interconnectivity can be carried out by techniques described in Ashworth and Cameron, 2014, Materials Technology, Advanced Performance Materials, 29(5):281-295, relevant sections being incorporated herein by reference.
In some embodiments, the bread crumb for the scaffold is most advantageously prepared from a bread that is leavened without yeast. A non-limiting example of a suitable leavening agent is sodium bicarbonate, although other non-yeast leavening agents may be used as required. For example, the scaffold may be prepared from bread crumb derived from a soda bread. Soda bread is generally prepared with ingredients comprising flour, salt and sodium bicarbonate and does not contain biological cultures, such as yeast. However, the crumb may be prepared from other types of breads, including in some embodiments, those leavened with yeast or other biocatalysts.
By way of example, a soda bread for use in preparation of the crumb may be prepared using appropriate amounts of flour, salt and a leavening agent, such as baking power as dry ingredients, which are admixed together or separately with water. The resultant mixture may be kneaded and then baked at an appropriate temperature to facilitate rising. The crumb may be obtained from the baked product by removing an internal portion thereof using any mechanical implement suitable for such use. In one example, a biopsy punch is used, although other methods for removing the internal crumb portion may be utilized as would be appreciated by those of skill in the art.
In one non-limiting embodiment, the crumb is sterilized prior to its use as a scaffold. This may be carried out by any suitable method. For example, the crumb may be contacted with a sterilizing agent, such as an alcohol or other chemical that is capable of destroying or reducing the concentration of unwanted microorganisms in the crumb structure. For in vitro applications, to facilitate the growth of cells within the scaffold, the bread crumb may be treated with a liquid culturing media prior to seeding with cells to promote adherence thereof. The culture media may be exchanged as required during the culturing process.
In one embodiment, the bread crumb comprises one or more proteins. For example, the crumb may comprise one or more gluten proteins. Advantageously, a gluten protein may impart stability to the scaffold. In another embodiment, the scaffold may comprise one or more non-gluten proteins, examples of which include albumin and globulins. Such proteins have utility in the creation of biomaterials. The bread crumb will typically comprise starch as well.
In certain embodiments, the scaffold may be chemically modified to introduce cross-linking. An example of a non-limiting cross-linking agent is glutaraldehyde (GA), although other cross-linking agents are encompassed by the present disclosure. In another embodiment, the cross-linking agent is transglutaminase. A suitable cross-linking agent may be selected based on the particular application for which the scaffold is used.
The bread crumb scaffold is cultured under conditions effective to produce a scaffold that supports a desired population of cells.
The bread crumb scaffold may be used to support a variety of different types of cells, or combinations of such cells. This includes, but is not limited to mammalian cells, fish cells, avian cells, reptile cells, amphibian cells, crustacean cells, plant cells, invertebrate cells, algae cells, bacteria cells, archaea cells or fungal cells. In certain embodiments, the cell is a fibroblast, satellite cell, myoblast, myocyte, smooth muscle cell, myofibroblast, myotube, cardiomyocytes, neutrophil, macrophages, lymphocytes, monocytes, platelets, pre-osteoblast, osteoblast, osteoclast, pre-adipocyte, adipocyte, periodontal ligament stem cells, fibrocytes, chondrocyte, tenocyte, keratinocytes, hepatocytes, neuron, neural precursor cells, dorsal root ganglion cells, glial cells, astrocytes, epithelial cells, endothelial cells, stem, mesenchymal stem or induced pluripotent stem cell or any combination thereof.
Culturing of the bread crumb scaffold with cells comprises introducing cells to the scaffold under conditions that promote growth and proliferation of the cells. In some embodiments, prior to seeding the scaffold, the cells are cultured in vitro under suitable conditions known to those of skill in the art.
To prepare bread scaffolds for seeding, the scaffold may be sterilized. The sterilization includes any suitable technique. In one embodiment, the bread scaffold is placed in a sterilizing solution that reduces or eliminates the concentration of unwanted microbes. In one embodiment, the sterilizing solution comprises an alcohol, such as ethanol. Subsequent to sterilization, the scaffold may be rinsed with a suitable solution, such as but not limited to a buffer to remove a chemical used for sterilizing.
In one embodiment, bread scaffolds are most advantageously soaked in culture media prior to seeding with cells to encourage adherence of the cells to the scaffold structure. The culture media may include serum, such as fetal calf serum or horse serum. The serum content may vary from 0.5% to 20% depending on the type of cells being cultured.
In those embodiments in which the bread scaffold is soaked, the cells are typically added after the soaking. The scaffold which is seeded with cells may be incubated for any period of time at a temperature effective to allow the cells to adhere to the scaffold. In one embodiment, the culture media is exchanged during incubation. The cells may be maintained on scaffolds for any desired period of time to facilitate their adherence, growth and/or proliferation within the three-dimensional structure of the scaffold. In some embodiments, the seeding with cells is repeated after a period of time.
The crumb scaffold or portions thereof finds use in microcarrier culturing used in industrial applications. According to such embodiments, the crumb scaffold may be introduced to a vessel, such as a bioreactor, and may function in a similar manner as a “microcarrier”, which is a support matrix (e.g., beads or other matrices) used to facilitate dense cell growth thereon and improve the yield of the tissue or cell-based product during manufacture. Often the support matrix is maintained in suspension in a culture medium in a vessel as particles with stirring, although the bread scaffold may be present in the vessel in any suitable solid form. Microcarrier culturing has broad applicability but may be particularly suitable for cells that rely on adherence to a support (e.g., adherent cells). Many conventional microcarrier culturing processes use beads, which have the limitation of low surface area. The crumb scaffold described herein is porous, thereby increasing surface area for cell adherence and growth. Accordingly, in some embodiments, the crumb scaffold is introduced into a vessel and functions as a microcarrier to increase available surface area for cell growth during proliferation in suspension culture. In addition to improved surface area offered by the scaffold of the disclosure, micro-carrier-based processes are advantageous in that they allow for more precise cell growth control, reduced bioreactor volume (thereby reducing space otherwise used to accommodate large bioreactors in an operation) and/or decreased labour costs.
Microcarrier-based processes may be carried out in a variety of vessels, including but not limited to spinner flasks, rotating wall microgravity bioreactors or fluidized bed bioreactors. In one non-limiting embodiment, a stirred bioreactor is most suitable for microcarrier culturing.
The crumb scaffold is for use to produce any tissue or cell-based product for use in an in vitro or in vivo application. Non-limiting examples are set forth below.
Examples of tissue include skeletal muscle, smooth muscle, cardiac muscle, bone, fat/adipose, kidney, liver, lung, skin, neural, vascular tissues or any combination thereof.
In one non-limiting embodiment, the crumb scaffold is used as a scaffold to prepare products for human or animal consumption. An example of an edible product for human or animal consumption is a meat product produced by tissue engineering. The meat may be used for human consumption or for pet food. Other edible products for human or animal consumption are encompassed by the present disclosure. In one embodiment, the crumb is used to produce a vegetarian food product.
In those embodiments in which the edible product is a meat product, the crumb scaffold may comprise myocytes, including precursors thereof. A myocyte includes those cells typically found in muscle tissue, including smooth muscle cells, cardiac muscle cells, skeletal muscle cells and combinations thereof. The myocyte includes a mammalian, avian or fish myocyte. The myocyte may be a myocyte substitute, which is a cell that can differentiate into myocytes or muscle cells under suitable conditions. The scaffold may also comprise adipocytes, including precursors or substitutes thereof.
Further embodiments include use of the bread crumb scaffold in therapy and/or biomedical applications. This includes the use of an implantable scaffold for supporting cell growth, for promoting tissue regeneration, for promoting angiogenesis, for a tissue replacement procedure and/or as a structural implant for cosmetic surgery. Further embodiments encompass therapeutic treatment and/or cosmetic methods employing such scaffolds, as well as other applications which may include veterinary uses. In one embodiment, the tissue is a soft tissue.
Further specific therapeutic or biomedical applications include tissue regeneration including soft tissue repair, neuro-regeneration, skin reconstruction, artificial corneas and skeletal/cardiac muscle regeneration.
In certain embodiments, scaffold biomaterials as described herein may be used as a structural implant for repair or regeneration following spinal cord injury; as a structural implant for tissue replacement surgery and/or for tissue regeneration following surgery; as a structural implant for skin graft and/or skin regeneration surgery; as a structural implant for regeneration of blood vasculature in a target tissue or region; as a tissue replacement for skin, spinal cord, heart, muscle, nerve, blood vessel, or other damaged or malformed tissue; as a vitreous humour replacement (in hydrogel form); as an artificial bursae, wherein the scaffold biomaterial forms a sac-like structure containing scaffold biomaterial in hydrogel form; and/or as a structural implant for cosmetic surgery.
In another embodiment, the scaffold biomaterial described herein is for use as a microcarrier in a vessel, such as a bioreactor, in order to support the growth and adherence of cells. The use of scaffold biomaterial as a microcarrier has wide ranging applications in therapy, biomedical applications and in the food industry.
Soda bread was prepared by adding, in a ceramic bowl, 120 g of all purpose flour (Five Roses™), 2 g of iodized table salt (Windsor™) and 10 g of baking power (Kraft™) and mixing. Subsequently, 70 mL of water was added to the dry ingredients. The water was previously heated for 30 seconds in a microwave until its temperature was about 75 degrees Celsius. The mixture was combined to form a dough and shaped into a ball. The dough was kneaded for 3 minutes with the addition of flour as needed to reduce sticking. Once flattened into a circular disk with a height of approximately 2.5 cm, the dough was place in a glass bread pan lined with parchment paper. The dough was baked for 30 minutes at 205 degrees Celsius in a preheated oven. The cooled bread was stored in a resealable plastic bag (Ziploc™) at −20 degrees Celsius until use.
When ready for use, the bread was thawed to room temperature. A 6 mm biopsy punch was used to extract cylindrical shapes from the internal portion of the loaf (also referred to herein as the “crumb”). The cylinders were cut with a blade (Leica™) to form circular scaffolds, which were about 2.5 mm in thickness. Two formulations were tested: the native untreated scaffolds as well as a group of chemically crosslinked scaffolds. To crosslink the samples with glutaraldehyde (GA), an adapted approach was used for similar protein-based scaffolds (R. Hickey, A. E. Pelling, The rotation of mouse myoblast nuclei is dependent on substrate elasticity, Cytoskeleton. 74 (2017) 184-194 and Z. Al-Rekabi, A. E. Pelling, Cross talk between matrix elasticity and mechanical force regulates myoblast traction dynamics, Phys. Biol. 10 (2013) 066003, each of which is incorporated herein by reference). A 0.5% GA solution was prepared from a 50% electron microscopy grade glutaraldehyde stock (Sigma™), which was diluted with PBS (Fisher™). The scaffolds were incubated in the GA solution overnight in the fridge. Afterwards, the scaffolds were rinsed 3 times with PBS. To reduce any remaining traces of unreacted glutaraldehyde, the scaffolds were incubated in a 1 mg/mL NaBH4 (Acros Organics™) solution on ice, made immediately before use. Once the formation of bubbles ceased, the samples were rinsed 3 times with PBS. In some cases, the bread scaffolds were also crosslinked with transglutaminase (TG; Modernist Pantry™). TG is a well-known enzyme that catalyzes protein crosslinking by forming covalent links between the carboxamide and amino groups of glycine and lysine respectively. TG was mixed with the dry ingredients at a concentration of 1% (w/w) in advance of baking.
NIH3T3 mouse cells stably expressing GFP were used in this study (ATCC). Cells were cultured in high glucose Dulbecco's Modified Eagle medium (MDEM) (HyClone™), supplemented with 10% fetal bovine serum (HyClone™) and 1% penicillin/streptomycin (HyClone™) at 37 degrees Celsius and 5% CO2. The culture media was exchanged every second day and the cells were passaged at 70% confluence. To test the suitability of the scaffold to support the proliferation of other cell types, C2C12 mouse myoblasts and MC-3T3 mouse pre-osteoblasts were also cultured on the scaffolds according to the protocols above. In the case of MC-3T3 cells, the DMEM was replaced with Minimum Essential Medium (ME) (ThermoFisher™).
To prepare bread scaffolds for seeding, they were placed in 70% ethanol for 30 minutes in order to sterilize them and subsequently rinsed twice with PBS. Bread scaffolds were additionally soaked in complete media prior to seeding to encourage adherence. A droplet containing 1.0×105 cells was then gently placed on top of each of scaffolds, which were contained in 12-well plates. The samples were placed in the incubator for 3-4 hours to allow the cells to adhere to the scaffolds. The culture media was exchanged every 48-72 hours. Cells were maintained on scaffolds for two weeks in a standard cell culture incubator.
Differentiation of C2C12 and MC-3T3 cells was also carried out. C2C12 differentiation was initiated after first allowing the cells to grow to confluence over a period of two weeks. At this point, cells were cultured in myogenic differentiation media (DMEM, 2% Horse Serum, 1% penicillin/streptomycin) for up to two weeks in order to stimulate cell fusion and myogenesis. MC-3T3 cells were differentiated following a similar protocol but with osteogenic differentiation media (MEM, 10% fetal bovine serum, 1% penicillin/streptomycin, 50 μg/mL ascorbic acid and 10 mM p-glycerophosphate) for up to four weeks.
Before staining, the scaffolds were fixed in 4% paraformaldehyde for 10-15 minutes. Following 3 rinses with a duration of 5 minutes each in PBS, the samples were stained using 200 μL of a DAPI solution (1:500 in PBS) for 15 minutes to label nuclei. In cases where C2C12 and MC-3T3 cells were cultured, after fixation with paraformaldehyde, the cells were permeabilized with Triton X-100. Phalloidin alexa fluor 488 (ThermoFisher™) stock solution (1:100 in PBS) was incubated on the samples for 20 min at room temperature to label actin. In cases of antibody staining, samples were first washed with an ice-cold wash buffer (PBS, 5% FBS, 0.05% sodium azide) and placed on ice. C2C12 myotubes were labeled by incubating with an MF-20 myosin heavy chain primary antibody at a 1:200 dilution (DSHB Hybridoma Product) for 30 min followed by a rat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 at a 1:100 dilution for 30 min. Between each stain the sample was incubated with the wash buffer for 30 min and the entire process was carried out on ice. In cases where deposited fibronectin was labelled the process was similar to the above. However, samples were incubated with a primary anti-fibronectin antibody at a 1:200 dilution (Abcam) for 30 min, followed by a rabbit anti-mouse IgG secondary antibody conjugated to Alexa Fluor 546 at a 1:100 dilution for 30 min. After staining, all scaffolds were rinsed for 2 minutes with PBS. The scaffolds were then stained with a 0.2% congo Red solution for 15 minutes, which was followed by 5-10 washes with PBS prior to mounting and imaging.
Confocal images were obtained using an A1R high speed laser scanning confocal system on a TiE inverted optical microscope platform (Nikon™, Canada) with appropriate laser lines and filter sets. Images were analyzed using ImageJ™ open access software. Brightness and contrast adjustments were the only manipulations performed to images. The ImageJ™ software was also used to count the number of cells in different areas of the scaffolds. Image analysis was conducted for quantifying pore size and volume fraction by collecting confocal Z-stacks, applying a threshold to obtain binary images at each optical plane, denoising and image quantification of pore area and volume.
The preparation of the samples for SEM began with a fixation in paraformaldehyde. This was followed by a dehydration through successive washes of ethanol with increasing concentration (35%-99%). The samples were dried using a critical point dryer and gold-coated at a current of 15 mA for three minutes with a Hitachi™ E-1010 ion sputter device. SEM images were acquired at a voltage of 2.00 kV on a JEOL JSM-7500F FESEM. In the case of scaffolds cultured with MC-3T3 cells, energy-dispersive spectroscopy (EDS) was performed on three different areas of each scaffold surface and analyzed for mineral aggregates.
Cell viability was assessed with the Alamar blue assay (Invitrogen™). Cells were seeded onto scaffolds and assessed after 1 and 13 days in culture. In each case, samples were incubated with 10% (v/v) Alamar blue solution standard culture media for 2 hours in an incubator. Following incubation, the fluorescence was measured in a microplate reader at 570 nM against reference wavelength at 600 nM. The results are expressed in arbitrary units (AU) and normalized against the initial reads after 1 day in culture of the respective cell types.
A Glutathione Assay (Cayman™ Chem) was conducted to evaluate the abundance of antioxidants within cells following incubation. Following two weeks of incubation the NIH3T3, C2C12 and MC-3T3 cultures were evaluated for glutathione content according to the manufacturer's guidelines. In brief, both 2D cell culture and 3D bread samples were collected and lysed in 2-(N-morpholino)ethanesulfonic acid (MES) buffer and centrifuged at 10,000×g for 15 minutes and then deproteinated with metaphosphoric acid (MPA). The resulting lysates were quantified against a standard curve as described by the supplier and normalized against the protein content of each sample by bradford assay.
Cytotoxicity was evaluated using the CyQuant™ LDH Cytotoxicity Assay (ThermoFisher™) to evaluate cell health. Samples of NIH3T3, C2C12 and MC-3T3 cells were incubated for two weeks in culture as described previously and compared against 3D TCP controls. Fractions referred to as “Spontaneous” (what is released in culture), and “Maximum” (the maximal value following lysis) were both collected to provide a %-Cytotoxicity value as described by the manufacturer. Results are expressed as the difference between the spontaneous and max reported values comparing the 2D TCP and 3D bread experimental conditions.
The Young's modulus of the scaffolds was determined by compressing the material to a maximum 20% strain, at a rate of 3 mm/min, using a custom-built mechanical tester controlled with LabVIEW™ software. The force-compression curves were converted to stress-strain curves and the slope of the linear regime between 10-20% compression was fit to extract the Young's modulus.
For the comparison of time series data, a one-way ANOVA™ with Tukey's post-hoc analysis was used to determine the statistical difference between sample populations. To compare between two distinct populations, a student's t-test was employed. In all cases alpha=0.05. Where indicated, all values are presented as the mean±standard deviation. Analysis and statistical tests were conducted using the OriginLab™ software package.
Scaffolds were prepared as described in the method section. As an initial step, dry ingredients were combined, followed by mixing in warm water and kneading (
This example examines cell proliferation and infiltration of bread crumb over the course of two weeks in culture. The results below show that the BB and cross-linked BB (xBB) scaffolds were stable over time in cell culture conditions and media.
The baked bread (BB) scaffolds were continuously and completely submerged in cell culture media at 37 degrees Celsius for the entire length of time. Due to concerns that the native structure of the scaffold may begin to soften significantly and/or decompose over this time course, scaffolds cross-linked with glutaraldehyde (GA) were prepared to create a more stable structure. The mechanical properties of BB and xBB scaffolds were then measured after initially submerging in cell culture media (Day 1), 24 hr (Day 2) and 288 hr (Day 13) in culture media at 37 degrees Celsius with no mammalian cells (
Regardless, in both cases the BB and xBB scaffolds maintain their highly porous morphology and structure after immersion in cell culture media as evidenced by both SEM and confocal imaging (
Transglutaminase (TG) is an alternative cross-linking agent that is compatible for use in food processing. To validate the use of TG for preparing food, the enzyme was added to the original formulation at a concentration of 1% (w/w) prior to baking. Once prepared, the crosslinked scaffolds (tgBB) generally appeared more hydrated and robust. Mechanical testing confirms that the addition of TG significantly (p=5.32719×10−4) increased the Young's modulus of the scaffold in its dry form (161.7±18.3 kPa, n=12) compared to the dry BB scaffold (82.1±7.1 kPa, n=12). (
To further characterize the BB and xBB scaffolds, 1.3×1.3 mm confocal volumes were analyzed to quantify the porosity of the scaffolds. Depth coded confocal image stacks of the BB scaffold reveals a highly complex structure with a number of shallow pits and large pores which extend through the entire imaging volume (
The pore structure of the BB and xBB scaffolds was largely composed of individual isolated pores and surface pits, as well as networks of interconnected pores underneath the outer surface (
A depth coded confocal image of an xBB scaffold is also shown for comparison (
In order to assess the ability of the cells to penetrate into the scaffolds, they were cross sectioned as described previously (Hickey et al., 2018, Customizing the shape and microenvironment biochemistry of biocompatible macroscopic plant-derived cellulose scaffolds”, 4, 11, 3726-3736). Briefly, on Day 13, a 6 mm diameter, 2.5 mm thick xBB cylindrical scaffold was cut longitudinally with a microtome blade to produce two half cylinders. Subsequently, the cut side of the half cylinder could then be washed, fixed and prepared for imaging as described above. The results reveal that cells are indeed able to infiltrate into the deeper portions of the scaffold (
In this example, to assess cell proliferation, the scaffolds were subsequently seeded with NIH3T3 cells stably expressing green fluorescent protein (GFP).
After seeding, scaffolds (n=3 at each time point) were subsequently imaged using confocal microscopy at Day 2, 5, 7, 9, 11 and 13. The presence of cells after two days reveals that they adhere to both formulations and tend to initially invade inside of the scaffold pores (
To demonstrate the utility of these scaffolds for multiple cell types, cultured C2C12 muscle myoblasts (
In the case of C2C12 cells, they were able to proliferate on both scaffold formulations in a manner consistent with the NIH3T3 cells. As they do not express GFP the actin cytoskeleton was stained in addition to the scaffold and nuclei (
In the case of the MC-3T3 pre-osteoblasts, the highly porous nature of the substrate is consistent with various other scaffolds encountered in bone tissue engineering. In these cases, pre-osteoblasts are differentiated into osteoblasts which can mineralize porous 3D microenvironments. Cells were first cultured for two weeks in proliferation media followed by switching into osteogenic media (OM) for an additional two weeks to differentiate. During differentiation with OM, or during biomaterial induced osteoinduction, this model cell line results in the formation of calcium and phosphorus rich mineral deposits on the underlying scaffold. In this case, after two weeks in proliferation media, cells were observed attached and proliferating on BB scaffolds in a manner consistent with the other cell types. As already noted, the scaffolds are highly porous and the cells are observed in the pores (
In this study, our goal was to establish the possibility of utilizing highly available and accessible materials to create scaffolding capable of supporting mammalian cell growth. Taken together, data presented here demonstrates that these scaffolds are generally compatible with a multitude of cell types.
To assess the viability and metabolic activity of the cells at Day 1 and approximately 2 weeks after seeding (Day 13/14), an Alamar blue assay was employed. Such technique has been successfully used on 3D scaffolds and is described in Baino, F. et al., Processing methods for making porous bioactive glass-based scaffolds—A state-of-the-art review. Int. J. Appl. Ceram. Tech. 2019, 16, 1762-1796, which is incorporated herein by reference. Cell viability was determined by normalizing the measured values against the Day 1 controls to determine a relative fold-increase. As shown in
To assess the relative health of NIH3T3, C2C12, MC-3T3 cells when cultured on the scaffolds in comparison to traditional 2D tissue culture plastic, both a lactate dehydrogenase (LDH) and glutathione (GSH) assay were conducted. The LDH assay is a method for determining cytotoxicity and involves the measurement of certain enzymes released by damaged or necrotic cells. LDH is a cytoplasmic enzyme found in all cells and is released into the media when the plasma membrane is damaged or during cell death. Likewise, it has been well established that glutathione levels are a strong indicator of the ability of cells to detoxify and buffer against oxidative stress. When total glutathione levels are observed to decrease, this change can be measured to assess the degree of oxidative stress in a population of cells.
For the three cell types in question (NIH3T3, C2C12, MC-3T3) both LDH and GSH in populations of cells cultured on BB scaffolds were examined after two weeks in culture (
Consistent with these observations, the GSH assay supports a similar conclusion. In this case, NIH3T3 (n=16 in both conditions), C2C12 (n=16 in both conditions), MC-3T3 were compared (n=15 and n=16, on tissue culture plastic and BB scaffolds respectively). In all cases, total GSH was observed to increase by a factor of two to four when cultured on BB scaffolds as opposed to tissue culture plastic (p=4.09462×10-20, p=1.54964×10−13, p=3.53×10−3 for NIH3T3, C2C12 and MC-3T3 cells respectively) (
The above description and examples are merely illustrative and additional embodiments and alternatives are included within the scope of the present disclosure.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051595 | 11/9/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63112712 | Nov 2020 | US |
