Porous glucomannan scaffold and method for producing the scaffold

Abstract

The present invention provides a method for producing a glucomannan scaffold with uniform porosity and interconnectivity. The scaffold is prepared by maintaining a glucomannan gel under specified conditions for a sufficient length of time to meet the curing phase. This method improves product consistency while reducing manufacturing waste. The resulting glucomannan scaffold promotes cell growth and is suitable for three-dimensional tissue culture and engineering.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 927,207, filed October 29, 2019, which is incorporated herein by reference in its entirety. Background of the Invention

[0004] Tissue engineering requires functional cells, appropriate biochemical factors, and biomaterial scaffolds to regenerate, improve, replace, or repair damaged, diseased, or missing tissues and / or organs in an effort to improve clinical outcomes for patients. Tissue engineering can also be used to develop bioconstructs to study and screen toxins, drugs, proteins, and various compounds. While cells are the fundamental building blocks for generating tissues and bioconstructs, biomaterial scaffolds play a key role in establishing an environment conducive to the constructed tissue, either in vivo or in vitro.

[0005] Scaffolds can be constructed from naturally derived materials (e.g., carbohydrate- or protein-based biomaterials) or synthetic materials (e.g., polymer- or ceramic-based materials). Scaffold materials should be biocompatible and biodegradable, and they should have mechanical properties consistent with the tissue / site of interest. Scaffold architecture should also exhibit sufficient pore size and density to allow for the diffusion of cells and nutrients (O'Brien et al., 2001). Materials Today 2011 14:88-95).

[0006] Carbohydrate-based biomaterials that have been used as scaffolds for tissue engineering include, but are not limited to, alginate, chitosan, glycosaminoglycans, and hybrid composites. Glucomannan (GM) polymers are hypercross-linked to produce highly porous, thermally stable, biocompatible, and biodegradable structures that are beneficial for biological applications.

[0007] Carbohydrate-based scaffolds have also been used as vehicles for osteoinductive factors, such as recombinant human BMP-2. rhBMP-2 has been used off-label in anterior cervical discectomy and fusion (ACDF), but at concentrations far exceeding naturally occurring BMP-2 levels to compensate for leakage beyond the implant site into adjacent tissues. Such leakage and high doses are associated with adverse events. Therefore, a vehicle that effectively localizes BMP-2 to the implant site and reduces the effective dose is desirable.

[0008] Current methods for producing glucomannan scaffolds result in structural collapse and uneven porosity. This observation has not been investigated, and to date, no efforts have been made to prevent structural collapse or uneven porosity. Instead, portions of the scaffold construct with the appropriate structure and porosity are simply selected and excised from the rest to provide the optimal structure for tissue engineering. Unselected, unsuitable portions of the scaffold construct are discarded, resulting in significant waste and increased overall production costs. Furthermore, equipment and laboratory irregularities contribute to structural collapse and uneven porosity. Equipment variations (due to freezer age, make, model, or configuration), location variations (due to gel height or depth within the freezer), operational variations (due to power fluctuations or interruptions), and user variations (due to opening / entering the freezer) all lead to temperature differences even within a single freezing system.

[0009] Therefore, methods for improving scaffold properties are necessary. The method for manufacturing porous glucomannan scaffolds disclosed in US Pat. No. 9,359,591 results in structural collapse of the glucomannan scaffold. The present invention provides an improved method for achieving better results in the structural properties of glucomannan scaffolds. SUMMARY OF THE INVENTION

[0011] It has been found that by controlling the variable length of the curing phase, uniformly porous and interconnected scaffolds can be produced. In one embodiment, the present invention provides a method for preparing a glucomannan scaffold comprising freezing a glucomannan gel under conditions that result in a curing phase of about 10-2000 minutes, or in some embodiments, 50-1500 minutes. The method may include monitoring the temperature of the gel.

[0012] In another embodiment, the present invention provides a method for preparing a glucomannan scaffold, comprising the steps of: a) cooling a glucomannan gel from a temperature of about 25° C. to a temperature of about 4° C.; b) maintaining the glucomannan gel in a solidification stage for at least about 10 minutes; c) thawing the glucomannan gel to a temperature of about 25° C.; d) optionally repeating steps a), b), and / or c). In some embodiments, the preparation method comprises at least 1, 2, 3, or 4 freeze-thaw cycles.

[0013] The present invention also provides a stent made by the manufacturing method described herein.

[0014] In another embodiment, the present invention provides a scaffold having a porosity of at least about 50% and / or an interconnectivity of at least about 50%. In one embodiment, the porosity and / or interconnectivity are uniform throughout the scaffold. In another embodiment, the volume of the scaffold having the porosity and / or interconnectivity characteristics is at least 10% of the volume of the glucomannan gel. In one embodiment, the scaffold has a uniform pore size of about 100-500 μm.

[0015] In one embodiment, the method of the invention comprises the step of neutralizing the pH of the scaffold. Similarly, in one embodiment, the invention provides a scaffold having a pH of about 7.

[0016] The scaffolds of the present invention have a backbone comprising at least 50% w / w carbohydrate mixtures, and in some embodiments, at least 50% glucomannan. The backbone may comprise a composite material. In some embodiments, the scaffolds include one or more bone components (e.g., calcium and phosphate) and / or one or more morphogenetic proteins (e.g., BMP-2).

[0017] In another embodiment, the stent is radiolucent.

[0018] In another embodiment, the scaffold can be used for experimental modeling and therapeutic applications. Such experimental and therapeutic uses include, but are not limited to, neovascularization, orthopedics, cardiovascular, neurology, wound healing, hemostasis, drug screening and drug delivery, tissue regeneration, organoids, tissue (including soft tissue) and bone (re)generation, dermatology, and dentistry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A A glucomannan scaffold made using prior art techniques is shown. It exhibits significant structural collapse, resulting in non-uniform porosity. Figure 1B Shown is a GM scaffold made using the present method as described.

[0021] Figure 2 A stage diagram of a glucomannan (GM) system is shown. The temperature of the GM system is plotted along the y-axis, and time is plotted along the x-axis. The GM gel first undergoes a temperature change until it reaches a freezing (cooling) temperature, followed by a constant temperature period, which we define as the time length of the solidification stage (LTSP). Supercooling occurs when the gel reaches a temperature below freezing. LTSP is the stage in which all the liquid in the gel freezes, forming ice crystals. The temperature then cools further to reach the final temperature of the system.

[0022] Figure 3 Showing a highly porous glucomannan scaffold with interconnected pores. Figure 3A glucomannan scaffold is shown immersed in a colored dye. Figure 3 B shows the dye penetrating the scaffold through the interconnected pores. Figure 3 C shows a scaffold completely saturated with dye.

[0023] Figure 4 Shown is the time course of rhBMP-2 retention in HCCP, as determined by indirect detection. HCCP scaffolds were incubated in a BMP-2 solution for 5 minutes (loading) and then washed for 30 minutes, 16, 40, and 64 hours. The residual rhBMP-2 concentration in the solution was measured by ELISA (black bars). Scaffold retention of rhBMP-2 was calculated by subtracting the cumulative residual rhBMP-2 concentration from the baseline (dashed line) stock concentration. Error bars represent the standard error of the mean (N = 5 replicates).

[0024] Figure 5 Indirect measurement of rhBMP-2 retention by HCCP is shown. HCCP scaffolds were placed in an rhBMP-2 solution and incubated for 30 minutes. The rhBMP-2 residual (black bar) represents the concentration of rhBMP-2 remaining in the solution after incubation with HCCP. The baseline control was an rhBMP-2 stock solution incubated without HCCP; PBS served as a negative control (error bars represent the standard error of the mean; N = 12 replicates in the ELISA assay). The gray bar represents BMP-2 retention in the scaffolds calculated by subtracting the mean "residual" from the mean "baseline" concentration (N = 3 replicates, labeled HCCP 1, 2, and 3).

[0025] Figure 6 shows direct detection of rhBMP-2 retention by HCCP. HCCP discs were incubated overnight in rhBMP-2 solution (rhBMP-2; 1 μg / ml), bone marrow (BM), bone marrow + rhBMP-2 (BM + rhBMP-2), or PBS, then rinsed and stained with an anti-BMP-2 antibody. Secondary DAB staining revealed the presence of rhBMP-2 as a dark brown color (A). Three replicates were performed for each condition (i, ii, iii). Densitometric analysis revealed significantly higher DAB signals in the rhBMP-2-treated group compared to all other groups (B).

[0026] Figure 7Demonstrating the binding of endogenous BMP-2 to HCCP in scaffold explants. For colorimetric visualization of endogenous BMP-2, 5 μm paraffin sections were stained with anti-BMP-2 antibodies and 3,3'-diaminobenzidine. The images show positive brown precipitates at 2-week (a, b) and 4-week (d, e) intervals. Rabbit IgG controls were used in sections at 2-week (c) and 4-week intervals (f). Black triangles (a) indicate HCCP. Solid black arrows (a, b, e) indicate positive brown staining along HCCP pores. White arrows with black borders (b, e) indicate unfilled HCCP pores with negative brown staining. The dash-dotted black arrow (a) indicates osteoblasts with punctate morphology. Detailed Description of the Invention

[0028] I. Definitions

[0029] As used herein, the term "glucomannan" refers to a naturally derived oligosaccharide composed of β-1,4-linked D-glucose and D-mannose in a ratio of about 1:1.6 with branches approximately every 11 residues (Alonso-Sande et al. Eur J Pharm Biopharm. 2009 72:453-462) and its derivatives. Glucomannan has a backbone with approximately 5-10% substituted acetyl groups, which participate in hydrogen bonding and hydrophobic interactions to impart solubility. Exemplary glucomannan derivatives include, but are not limited to, water-soluble derivatives, such as O-alkyl derivatives and O-carboxyalkyl derivatives, derivatives with various degrees of substitution (for example, without limitation, greater than or less than 5-10% substituted acetyl groups), derivatives with various degrees of oxidation, graft copolymers (for example, without limitation, acrylate and acrylamide copolymers), and salts thereof (for example, quaternary ammonium salts thereof).

[0030] As used herein, the term "glucomannan gel" refers to a heat-stable, homogeneous suspension of cross-linked carbohydrates. Glucomannan gel can be formed in various ways, including, but not limited to, by hydrolyzing the acetyl groups of glucomannan in the presence of a base.

[0031] As used herein, the term "glucomannan scaffold" refers to a three-dimensional porous matrix formed by sublimating a glucomannan gel. The glucomannan scaffold provides an environment suitable for cell culture and tissue engineering, including tissue regeneration.

[0032] The following methods and compositions are described using glucomannan gels and glucomannan scaffolds as examples, but the present invention encompasses gels and scaffolds that are not entirely comprised of glucomannan or that are combined with glucomannan. Other naturally derived materials (e.g., carbohydrate- or protein-based biomaterials) or synthetic materials (e.g., polymer- and ceramic-based materials) may be included or combined in the gel and / or scaffold. In embodiments of the present invention, the scaffold comprises at least about 50% (w / w) glucomannan, and in some embodiments, at least 60%, 70%, 80%, 90%, or 95% glucomannan (w / w).

[0033] The "backbone" of a scaffold refers to the structural components that define and maintain the porous structure. The components that comprise the scaffold's backbone are the materials used in the formation of the glucomannan gel. After the glucomannan scaffold is formed, additional components can be embedded into the backbone so that they are embedded in the backbone but not integral to it. Furthermore, these components can be embedded within the backbone to allow for slow and sustained release of the components over time in vivo as the glucomannan scaffold degrades.

[0034] As used herein, the term "solidification phase" (SP) refers to the phase transition from liquid to solid. The solidification phase begins when the system (initially a gel) first reaches the freezing temperature and ends when the temperature of the (now solid) system drops below the freezing temperature. The "length of time in solidification phase" (LTSP) is the time that the system remains at a substantially constant temperature during the freezing phase transition. If the system exhibits a supercooling phase, the solidification phase includes the supercooling phase. That is, the length of time in solidification phase begins at the moment the system first reaches the freezing temperature and ends only after the phase transition is complete. For the glucomannan gel composition of the present invention, the freezing point is in the range of -0.01°C to -3.00°C (slightly below 0°C, the freezing point of water). Therefore, for a gel composed of glucomannan as the backbone carbohydrate, the solidification phase will be graphically represented as a plateau at about -0.8°C, see Figure 2 .

[0035] As used herein, the term "freezing conditions" refers to subjecting the glucomannan system to a temperature equal to or lower than 0°C to induce and maintain SP to achieve uniform porosity. Freezing conditions include temperatures in the range of -80 to -20°C, -20 to -10°C, -10 to -5°C, -5 to 0°C, or a gradually decreasing temperature range using a controlled rate freezing program. In one embodiment, the freezing conditions include a temperature equal to or lower than -0.33°C.

[0036] The term "thawing conditions" refers to subjecting the glucomannan system to a temperature above 0°C. Thawing conditions include temperatures of 0°C to 5°C, 0°C to 10°C, 0°C to 20°C, room temperature conditions (20°C to 25°C, or about 23°C), or temperatures above 25°C.

[0037] As used herein, "controlled rate" conditions refer to subjecting the system to a preselected rate of temperature change (e.g., in a laboratory-scale controlled rate freezer) to induce and maintain SP. In contrast, "constant temperature" conditions refer to subjecting the system to a constant, preselected temperature. In both cases, "controlled rate" and "constant temperature" freezing conditions refer to the temperature settings of the equipment (e.g., freezer) and / or the environment (physical, chemical), rather than the temperature of the glucomannan system, which can be separately monitored and recorded (i.e., to measure the length of the solidification phase).

[0038] As used herein, a "freeze-thaw cycle" is a two-part procedure in which a glucomannan system at or below the glucomannan freezing point (i.e., a frozen or partially frozen glucomannan system) is subsequently a) subjected to thawing conditions, i.e., a temperature above the freezing point of the glucomannan system, and then 2) subjected to freezing conditions. In each freeze-thaw cycle, the thawing conditions and / or freezing conditions may be the same as or different from the previous thawing or freezing conditions, or a combination thereof. Each thawing step and each freezing step may be independently selected from controlled rate and constant temperature conditions.

[0039] As used herein, porosity refers to a ratio of at least 50% of the pore volume to the total volume of a glucomannan scaffold, as measured by mercury intrusion porosimetry. In some embodiments, the porosity may be at least 50%, 60%, 70%, or 80%. Furthermore, "uniform" porosity is achieved when the scaffold exhibits a uniform porosity throughout (e.g., from the periphery to the center) after being prepared from the gel. Previous methods for making porous glucomannan scaffolds (U.S. Patent No. 9,359,591) subject the glucomannan gel to a specific temperature to promote freezing. However, this method results in structural collapse and uneven porosity within the scaffold. In contrast, the present invention discloses that controlling different and multiple variables—the SP—will produce more accurate and consistent results. More specifically, adjusting the LTSP will produce improved product outcomes.

[0040] As used herein, "interconnected" pores refer to a network of pores that are permeable and allow substances to flow from one pore to another ( Figure 3 In some embodiments, the degree of interconnectivity can be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the scaffold. The degree of interconnectivity can be determined by, for example, Figure 3 Furthermore, "homogeneous" interconnectivity is achieved when the scaffold, after being prepared from the gel, exhibits a degree of interconnectivity that is consistent throughout (eg, from the periphery to the center).

[0041] Interconnected pores are important for cell penetration, nutrient delivery, waste removal, and the flow of cell signaling factors. Interconnected pores provide a unique opportunity to establish a new vasculature within the newly formed tissue throughout the scaffold / tissue construct. Often, in clinical settings, the size of the tissue defect or injury requiring regeneration may exceed the capacity for nutrient delivery or waste removal via diffusion. Therefore, neovascularization is essential not only at the periphery of the scaffold / tissue construct but also throughout the newly formed tissue to prevent cell apoptosis and subsequent necrosis. Therefore, in addition to high porosity, interconnectivity within the scaffold is crucial for successful tissue engineering and regeneration. Furthermore, in the context of blood coagulation, insufficient penetration of blood components involved in clotting can lead to persistent bleeding. Highly porous scaffolds containing interconnected pores can serve as hemostatic devices, where fibrin fibers (or platelet aggregates) crosslink / form throughout the scaffold. This provides a stable blood clot that aids in hemostasis. Furthermore, such scaffolds can be used in extraction sockets without the need for sutures, as the blood clot formed throughout the scaffold construct prevents the scaffold from dislodging from the socket ("dry socket"). This method allows the dentist to conveniently place the bracket in the extraction socket without sending the patient to the oral surgery department.

[0042] In one embodiment, the initial volume of the glucomannan gel, prior to being subjected to any freezing conditions, can range from 100-5000 mL, 250-2500 mL, 100-1000 mL, or approximately 450 mL. For example, in one embodiment, the initial gel is cylindrical with dimensions of 1-5 cm in thickness and 10-20 cm in diameter. In other shapes, configurations, and sizes, the initial gel volume can be the same as or different from the exemplary cylindrical volume and dimensions. The inventors have discovered that by monitoring the LTSP, appropriate freezing conditions can be adjusted to accommodate a variety of gel shapes and sizes for diverse tissue engineering applications. For example, the initial gel can be poured into any structure, or the resulting scaffold can be cut into a variety of shapes, including cubes, sheet cylinders, tubules, or rings (donuts). In a specific embodiment, the aforementioned cylindrical diameter ranges have a surface area to volume ratio (SA:V) in the range of 0.5 to 2.5, 0.6 to 2.4, or 0.75 to 1 / cm. In a specific embodiment, the SA:V ratio is from 0.8 to 0.9 / cm.

[0043] Yield is measured as the volume percentage of the initial glucomannan gel converted to a glucomannan scaffold having a uniform porosity of at least 50%. Yield measurements exclude solidified glucomannan that does not fall within the specified porosity range (which is cut from the scaffold product by the previous method and discarded as manufacturing waste). In the present invention, the method produces a suitable porous scaffold of at least 10%, at least 25%, at least 35%, or preferably at least 50% or more from the initial gel material.

[0044] II. Curing stage

[0045] The present invention provides an improved glucomannan scaffold. The glucomannan scaffold of the present invention is suitable for use in three-dimensional cell culture and tissue engineering or organoids. The glucomannan scaffold provides a highly porous structure and pore size suitable for culturing cells. Furthermore, the scaffold is uniform, thermally stable, elastic, biocompatible, and biodegradable, and can be formed into any shape and size suitable for 3D tissue culture and engineering, or organoids, for example, by molding or cutting.

[0046] According to the manufacturing technique disclosed in U.S. Patent No. 9,359,591, the glucomannan gel is frozen by placing it in a petri dish and then freezing it in a blast freezer at a temperature of -50°C or less for 30 minutes. See U.S. Patent No. 9,359,591. In contrast, the improved freezing temperature or freezing rate of the present method provides a specific LTSP to produce a glucomannan scaffold with uniform porosity. In one embodiment, the glucomannan gel is at a temperature below 50°C at the beginning of the freezing step. In some embodiments, the glucomannan gel is at a temperature above 0°C and below: 40°C, 35°C, 30°C, 25°C, 10°C, or 5°C. In one embodiment, the glucomannan gel is at approximately room temperature (20°C to 25°C, or approximately 23°C) at the beginning of the freezing step. In another embodiment, the glucomannan gel is at approximately 4°C at the beginning of the freezing step.

[0047] In one embodiment, the method comprises freezing the glucomannan gel, wherein the freezing conditions result in a LTSP of about 10-2000 minutes. In some embodiments, the LTSP is at least about 10-30, 60, 100, 200, 270, 280, 300, 350, 360, 375, 400, 425, 440, 450, 460, 470, 475, 480, 490, 500, 525, 550, or 600 minutes. In some embodiments, the LTSP is about 10-60 minutes, 250-2000 minutes, 250-1500 minutes, 250-1000 minutes, 250-750 minutes, 500-1500 minutes, 500-1000 minutes, 250-750 minutes, 400-500 minutes, or 450-500 minutes.

[0048] In one embodiment, the SP includes a supercooling phase. In this case, the LTSP begins when the system first reaches a freezing temperature (e.g., about -0.8°C), includes a supercooling peak below freezing temperature, continues through the plateau at about freezing temperature, and ends only when the system begins its descent toward a final freezing temperature or ambient freezing temperature (e.g., freezer temperature).

[0049] In another embodiment, there is no supercooling phase. In this case, the LTSP begins when the system first reaches a freezing temperature (e.g., about -0.8°C); it continues through the plateau at about freezing temperatures and ends when the system begins its descent toward the final freezing temperature or ambient freezing temperature (e.g., freezer temperature).

[0050] In some embodiments, the method includes monitoring the temperature of the glucomannan system during one or more freezing steps. Temperature monitoring can be accomplished by inserting a temperature probe (e.g., Cooper Atkins Digital Thermometer, LCD, Immersion Probe model DTT361-01) to measure the internal temperature of the glucomannan system. In one embodiment, a laboratory temperature probe can remotely report and / or record temperature changes of the glucomannan system within the freezer. Monitoring temperature can help define specific freezing conditions for a predetermined glucomannan gel volume and / or size. It can also be useful to account for equipment and laboratory variability. Monitoring temperature allows the user to achieve a desired LTSP while accounting for equipment variations (due to freezer age, make, model, or configuration), location variations (due to gel height or depth within the freezer), operational variations (due to power fluctuations or outages), and user variations (due to opening / entering the freezer).

[0051] The LTSP parameter can be achieved by subjecting the glucomannan system to freezing conditions comprising controlled rate cooling and / or constant temperature. In some embodiments, the freezing conditions comprise controlled rate cooling. Controlled rate cooling is achieved by using a laboratory, food, or clinical grade controlled rate freezer. The end temperature of the controlled rate freezing step conditions can be selected from a temperature at or below 0°C, such as a temperature of about -78°C to -20°C, -20°C to -10°C, -10°C to -5°C, or -5°C to 0°C.

[0052] In some embodiments, the controlled rate is about -0.05°C / min to -1°C / min, -0.1 to -10°C / min, -0.5°C / min to -0.005°C / min, -0.05°C / min to -0.005°C / min, or -0.06°C / min to -0.04°C / min. In one embodiment, the controlled rate is about -0.05°C / min.

[0053] In some embodiments, freezing conditions include exposure to a constant temperature. Constant temperature freezing conditions include any temperature equal to or lower than about 0°C. Constant temperature freezing conditions include temperatures of about -78 to -20°C, -20 to -10°C, -10 to -5°C, -5 to -1°C. The duration of exposure can be equal to or greater than about 5 min, 15 min, 30 min, 1 hr, 2 hr, 2.5 hr, 3 hr, 4 hr, or 5 hr. In some embodiments, the duration of exposure at a constant freezing temperature is about 1-5 hours. In other embodiments, the duration of exposure can be at least about 5, 8, 10, 12, or 24 hours.

[0054] In another embodiment, the freezing conditions comprise both a controlled rate period and a constant temperature period.In one embodiment, the freezing conditions comprise a controlled rate period followed by a constant temperature period.

[0055] III. Freeze-thaw cycles

[0056] In some embodiments, the method comprises one or more freeze-thaw cycles. A freeze-thaw cycle is a period of time in which the glucomannan system used to induce, adjust, and maintain SP is subjected to thawing conditions at a temperature equal to or below about 0°C, followed by freezing conditions (which may be the same as or different from one or more previous freezing steps, or a combination thereof). The method may comprise 0, 1, 2, 3, 4, or more freeze-thaw cycles. In one embodiment, the method comprises one freeze-thaw cycle.

[0057] "Thawed conditions" include temperatures of about 0° C. to 5° C., 0° C. to 10° C., 0° C. to 20° C., room temperature conditions (about 20° C. to 25° C., or about 23° C.), or temperatures above about 25° C. In one embodiment, the thawing conditions bring the glucomannan system to a temperature of about room temperature.

[0058] When the thawing step causes the glucomannan system to reach a temperature above 0°C, the next cycle may include an intermediate cooling step prior to the subsequent freezing step. For example, in some embodiments, the thawing step comprises warming the glucomannan system from a temperature below about 0°C (e.g., allowing it to warm at room temperature) to a temperature above about 0°C, 5°C, 10°C, 25°C, 30°C, or 35°C. The thawed glucomannan system is then subjected to a cooling step, wherein the system temperature is lowered to a temperature below the thawing temperature but above about 0°C, for example, about 4°C. The glucomannan system is then subjected to a freezing step as described above. Similar to the freezing conditions, the thawing and / or cooling conditions can be achieved by controlled rate and / or constant temperature conditions.

[0059] IV. Glucomannan Gel Preparation

[0060] The methods of the present invention may include various additional steps. In some embodiments, the method further includes forming a reaction mixture comprising a carbohydrate mixture having at least about 50% (w / w) glucomannan, an alkaline solution, and water; and heating the reaction mixture at a temperature of about 50° C. to about 130° C. to form a glucomannan gel and / or increasing the pressure of the glucomannan gel to about 0.1 psi to 50 psi above atmospheric pressure to form the glucomannan gel.

[0061] The glucomannan gel used in the methods of the present invention is a mixture of a carbohydrate mixture having at least about 50% (w / w) glucomannan and an aqueous solution. The glucomannan can be provided as a glucomannan powder. In some embodiments, the glucomannan powder is dissolved in water to provide a glucomannan solution containing about 1% to about 5% w / v glucomannan in water. The glucomannan powder can be dissolved in the aqueous solution under any suitable temperature and pressure conditions. Examples of carbohydrates that can be included include, but are not limited to, alginate, chitosan, starch, and plant or bacterial-based polysaccharides.

[0062] To induce the glucomannan gel to the SP, the method may include cooling the reaction mixture (previously heated to promote dissolution) before applying the GM gel to the freezing system. In some embodiments, as described herein, the temperature of the glucomannan gel is cooled to below about 80°C before applying the GM gel to the freezing system to maintain the SP. In certain embodiments, the temperature of the glucomannan gel is cooled to below about 50°C before applying the GM gel to the freezing system. The cooling step can be performed at a controlled rate or at a constant temperature. In one embodiment, the cooling step is performed by returning the heated glucomannan mixture to room temperature.

[0063] The aqueous solution may have any suitable composition. The aqueous solution may be a mixture of water or water and one or more agents that do not degrade or digest the neutral glucomannan system. Examples of suitable aqueous solutions include, but are not limited to, water, buffer solutions, and cell culture media.

[0064] In one embodiment, the aqueous solution is a buffer solution. Examples of suitable buffer solutions include, but are not limited to, PBS, TAPS, BIS-TRIS propane, TRIS, HEPES, TES, MOPS, PIPES, and MES. In some embodiments, the buffer solution is PBS, HEPES, MES, MOPS, TRIS, or BIS-TRIS propane. In a preferred embodiment, the buffer solution is PBS.

[0065] In another embodiment, the aqueous solution is a cell culture medium. Examples of suitable cell culture media include, but are not limited to, Roswell Park Memory Institute medium (RPMI), Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 Medium (DMEM / F12), Iscove's Modified Dulbecco's Medium (IMDM), National Collection of Type Cultures Medium (NCTC), and Osteogenic Induction Medium (OIM). In some embodiments, the cell culture medium is Roswell Park Memory Institute medium (RPMI), Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 Medium (DMEM / F12), Iscove's Modified Dulbecco's Medium (IMDM), or National Collection of Type Cultures Medium (NCTC).

[0066] In another embodiment, the glucomannan gel may include an acidic solution. Examples of suitable acidic solutions include, but are not limited to, hydrochloric acid, acetic acid, tartaric acid, malic acid, and citric acid.

[0067] In another embodiment, the glucomannan gel can include an alkaline solution. The alkaline solution can be any solution containing an alkali metal salt or an alkaline earth metal salt. Representative alkali metal salts and alkaline earth metal salts include, but are not limited to, sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate, magnesium hydroxide, magnesium carbonate, calcium hydroxide, and calcium carbonate. In some embodiments, the alkaline solution contains calcium hydroxide.

[0068] V. Glucomannan Scaffold Preparation and Modification

[0069] The glucomannan scaffolds of the present invention may be basic (having a pH greater than about 8), or they may be neutralized (having a pH of about 7) according to the methods described in US Patent 9,359,591, which is incorporated herein by reference in its entirety.

[0070] The glucomannan scaffolds of the present invention can then be sterilized by autoclaving to render them ready for implantation and other in vivo applications.

[0071] In some embodiments, the method further comprises removing water from the glucomannan system. Water can be removed by any suitable method known in the art. In some embodiments, the removal step is performed by freeze drying, sublimation, or thermally induced phase separation. In certain embodiments, the removal step is performed by sublimation.

[0072] In one aspect of the present invention, a glucomannan scaffold comprises one or more bone components. A bone component is defined as any substance that contributes to osteogenesis or bone formation. Bone components include, but are not limited to, organic components such as extracellular matrix and / or bone matrix or ground bone, and inorganic components such as calcium, phosphate, potassium, magnesium, and hydroxyapatite. In one embodiment, the scaffold comprises calcium and / or phosphate as bone components. In another embodiment, the scaffold comprises calcium. The bone component(s) are integrally bonded to the scaffold's backbone. The total concentration of the bone components relative to the glucomannan system ranges from approximately 0.1 to 95% (w / w). For example, the scaffold can be manufactured using 1-20%, 1-10%, or approximately 5% CaOH:glucomannan powder (w / w).

[0073] This concentration is desirable because it makes the scaffold radiolucent. At higher concentrations, the scaffold would be radiopaque, thereby hindering the visualization and monitoring of stem cell adhesion and bone formation. At lower concentrations, the scaffold is less effective in causing new bone formation. Most products available in the orthopedic market are radiopaque, meaning that even if osteogenesis does not occur, these products can exhibit radiographic densities similar to those of developing or mature bone. Therefore, radiolucent products will provide surgeons, doctors, and patients with the ability to visualize and monitor bone regeneration over time using radiography. CT provides excellent trabecular and cortical bone resolution and a quantitative method for assessing mineral density using Hounsfield units (HU). HU is the standard linear attenuation coefficient of tissue and easily provides information about bone mass. Typically, HU values ​​for bone range from 300 to 3,000. Therefore, "radiolucency" for bone applications is defined as HU below 300. Consistent with this observation, the scaffolds of the present invention measured HU less than 300 and provide a unique way to monitor and quantify bone repair and regeneration, starting at HU below 300 at baseline and increasing HU over time as the bone repair and regeneration process occurs.

[0074] In another aspect of the present invention, the glucomannan gel and / or glucomannan scaffold are modified to promote cell adhesion and proliferation. Exemplary modifications include, but are not limited to, incorporation of cell adhesion promoters, chemical crosslinking, surface coatings, and introduction of functional groups. All such molecules and structures known in the art are included in the disclosure of the present invention.

[0075] The glucomannan scaffolds of the present invention may include one or more cell adhesion promoters. As used herein, the term "cell adhesion promoter" refers to natural or synthetic agents that enhance cell adhesion or attachment to a culture substrate, for example, by modifying the substrate surface and / or altering the surface charge. Cell adhesion promoters may also promote cell growth and cell differentiation. Cell adhesion promoters may also enhance the adsorption of serum or extracellular matrix proteins to the culture substrate. In some embodiments, the cell adhesion promoter may be poly-L-lysine (PLL), poly-D-lysine (PDL), RGD peptide (RGD), KQAGDV, VAPG, FGL, an amine group, fibronectin, elastin, collagen, or laminin. The extracellular matrix protein may be derived from any suitable source, including but not limited to mammalian cells. In some embodiments, the cell adhesion promoter is PLL or RGD. In certain embodiments, the cell adhesion promoter is PLL.

[0076] In another embodiment, the glucomannan scaffold includes a suitable chemotactic molecule. Exemplary chemotactic molecules include, but are not limited to, serum, chemokines, morphogenetic proteins, growth factors, and hyaluronic acid. In one embodiment, the glucomannan scaffold includes one or more bone morphogenetic proteins. Bone morphogenetic proteins include, but are not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, and BMP-15. In one embodiment, the scaffold includes BMP-2. The BMP can be recombinant or naturally derived. The concentration of the bone morphogenetic protein solution in the scaffold is approximately 0.001 to 1.5 mg / mL.

[0077] The scaffold may include other osteoinductive factors such as fibroblast growth factor-2 (FGF-2) and / or platelet-derived growth factor (PDGF). In humans, the concentration of BMP-2 measured in the supernatant of fractured bones is approximately 23.2 pg / mL (Glass et al. Proceedings of the National Academy of Sciences 2011 108:1585-1590). However, recombinant human BMP-2 (rhBMP-2) is delivered clinically at a dose of 1.5 mg / mL, significantly higher than the native concentration of BMP-2 after injury. rhBMP-2 is used in off-label anterior cervical discectomy and fusion (ACDF) at concentrations as high as 2.5 mg / mL, a concentration 3.5 times higher than that used in the pilot study (Shields et al. Spine 200631:542-547). This higher dose is associated with increased complications, including hematoma, neck swelling, dysphagia, and excessive edema (Shields et al. Spine200631:542-547). Other adverse events traced to high-dose rhBMP-2 include dysphagia and abnormal adipose tissue formation (Shields et al. Spine 2006 31:542-547). Although the use of BMP-2 has become increasingly popular, 85% of its use between 2003 and 2007 was off-label (Ong et al. Spine 201035:1794-1800). This has led to a significant focus on BMP-2 due to complications caused by high clinical doses (Lykissas et al. World Journal of Orthopedics 2017 8:531-555). In addition, it is important to localize rhBMP-2 to the injury site to allow osteoblast precursors to proliferate and differentiate into mature osteocytes (Sandhu et al. Spine 2003 28:64-73) without leaking into adjacent tissues. However, rhBMP-2 has been shown to produce unintended side effects in surrounding areas when its action extends beyond the target area, leading to ectopic bone formation in addition to the side effects listed above (Tannoury et al. The Spine Journal 2014 14:552-559, Shields et al. Spine 2006 31:542-547). A common commercial carrier for rhBMP-2 is absorbable collagen sponge (ACS). Although ACS has been shown to be an effective carrier for rhBMP-2, the rapid release rate, possibly due to collagen degradation, requires that rhBMP-2 be implanted at a high concentration to deliver an effective dose, further increasing the risk of complications (Mariner et al. Journal of Orthopaedic Research 201231:401-406, Winn et al. Clinical Orthopaedics and Related Research 1999 367:95-106). Therefore, a carrier that effectively localizes rhBMP-2 to the implantation site and reduces the effective dose is desirable.

[0078] In another embodiment, the glucomannan scaffold includes a suitable cell signaling molecule. Exemplary cell signaling molecules include, but are not limited to, extracellular matrix proteins, peptide motifs, and growth factors, as well as other molecules known in the art.

[0079] VI. Instructions for Use

[0080] The scaffolds of the present invention can be used in various in vitro and in vivo methods. The scaffolds can be used for experimental modeling as well as therapeutic applications. Such experimental and therapeutic uses include, but are not limited to, neovascularization, orthopedics, cardiovascular, neuroscience, wound healing, hemostasis, drug screening and drug delivery, tissue regeneration, organoids, tissue (including soft tissue) and bone (re)generation, dermatology, and dentistry.

[0081] In some embodiments, the method further comprises growing cells on the glucomannan scaffold. Suitable cell types and culture conditions are known in the art. Example

[0082] Example 1: Freeze / Thaw Cycle

[0083] Glucomannan powder (1-5 g) was dissolved in 100 ml of water along with 0.15 g of calcium hydroxide (Sigma-Aldrich, St. Louis, MO, USA) in a beaker, mixed thoroughly, and incubated at room temperature for 30 minutes. The beaker containing the GM solution was covered with aluminum foil and incubated in a water bath maintained at a temperature above 80°C for at least 30 minutes. After cooling to room temperature, the resulting GM gel was cut into smaller pieces and soaked in water at room temperature overnight.

[0084] To produce a porous glucomannan scaffold with structural consistency and uniform porosity, a glucomannan gel was placed on a metal mesh in a temperature-controlled chamber. The temperature in the chamber was lowered from room temperature to 4°C at a rate of -0.05°C / min. Once the temperature reached 4°C, the gel was incubated for 1-5 hours. Subsequently, the temperature was lowered again to -20°C at a rate of -0.05°C / min and held at -20°C for 5 hours. The glucomannan gel was subjected to these specific temperature and time conditions to ensure that the LTSP was within the range required to produce structural consistency and uniform porosity. After 5 hours, the glucomannan gel was thawed in water at 25°C / min to room temperature for 3-7 hours or until completely thawed. The water was squeezed out before the next temperature cycle. In this example, this temperature cycle was performed four times.

[0085] After the final cycle, water was sublimed for up to 72 hours with the shelf temperature reaching 120°C and the vacuum maintained at 100–300 mTorr (13 to 40 Pa). Following the drying cycle, the glucomannan scaffolds were neutralized by boiling in phosphate-buffered saline (pH 7.4) in a pressurized chamber for 30 minutes, followed by two cycles of washing and boiling in distilled water in a pressurized chamber for 30 minutes each. After the final wash, water was sublimated from the glucomannan scaffolds as described above. The resulting porous GM product was cut into custom shapes and sizes, packaged in polyethylene bags containing desiccant, sealed, and stored at room temperature until use.

[0086] Example 2: Length of curing phase

[0087] Glucomannan gels were prepared using the same method as in Example 1. After preparation, the glucomannan gels were placed in a constant-temperature freezer at selected temperatures (e.g., -78°C, -20°C, -10°C, -5°C, and -1°C). To determine the LTSP, the temperature of the gel was measured. To obtain the gel temperature, a temperature probe (Cooper Atkins Digital Thermometer, LCD, Immersion Probe) was placed in the center of the gel. The temperature was then measured at selected intervals (e.g., 30 seconds, 1 minute, 5 minutes, and 10 minutes) and plotted.

[0088] Example 3: Induction of Osteogenesis by Ultra-Highly Cross-linked Carbohydrate Polymers Containing Calcium

[0089] We are investigating the osteogenesis-inducing properties of calcium-containing Osteo-P® BGS. In contrast to orthotopic bone formation (i.e., bone formation adjacent to existing bone), studies of heterotopic bone formation investigate the ability of a test article to form bone in the absence of appropriate biochemical, biomechanical, and osteogenic or endogenous stem cells. Thus, it eliminates potential extraneous experimental variables associated with osteogenesis induction, allowing researchers to investigate the ability of a test article to induce bone formation. In this ongoing study, a well-characterized rat model was utilized to investigate whether HCCP, combined with calcium from the bone component, could induce osteogenesis when implanted subcutaneously in an avascular environment.

[0090] Materials and methods

[0091] Rats (Wistar IGS male rats, Charles River, Wilmington, MA) were subcutaneously implanted with either HCCP containing calcium integrated into the pore microstructure (HCCP-Ca, N = 6) or a control HCCP construct containing sodium (HCCP-Na ["Control"], N = 6) in an avascular location at least 1 cm from the incision site (to avoid potential mixing with blood). CT images were acquired at baseline and weekly until the implants were harvested 1 month after implantation. Individual implants were collected and processed for histology (hematoxylin and eosin staining), immunohistochemistry (IHC), and von Kossa staining.

[0092] CT manifestations

[0093] CT images showed an increase in radiodensity at the implantation site at 4 weeks compared to baseline. Quantitative assessment of Hounsfield Units (HU), an indicator of ossification, was used to compare HCCP-Ca to controls at all time points. For comparison, the amount of HU observed 4 weeks after implantation is equivalent to the amount of developing fetal bone at mid-gestation.

[0094] Gross and histological findings

[0095] The implants were harvested and grossly imaged. HCCP-Ca showed evidence of neovascularization containing fresh blood. However, limited neovascularization was observed with the control construct. Consistent with the gross appearance, histological evaluation also revealed infiltration of newly formed blood vessels into the HCCP-Ca, with the control construct displaying a limited number of vessels. Notably, the control construct was observed to have more cells exhibiting a fibroblastic morphology, while the HCCP-Ca was observed to have cells exhibiting an osteoblast-like morphology. Additional sections revealed some morphological differences between the control construct and the HCCP-Ca.

[0096] IHC and Von Kossa findings

[0097] Implants were further evaluated by IHC for expression of bone sialoprotein (BSP), a key component of mineralized tissues such as bone. Red staining confirmed the observation of numerous BSP-expressing cells in the HCCP-Ca. All other conditions (test control, control construct) showed background staining, exhibiting characteristically different staining patterns. Von Kossa staining is intended for histological visualization of calcium deposits. Consistent with all other findings (CT, histology, and IHC), von Kossa staining demonstrated minimal signal in the control construct, while HCCP-Ca showed evidence of calcium deposition.

[0098] in conclusion

[0099] The results of these studies indicate that HCCP-Ca was observed to have: (1) elevated radiodensity levels similar to those observed in the bone of a developing fetus (most likely proximal to cancellous bone, ribs) during mid-gestation, based on CT observations; (2) evidence of neovascularization when implanted in an avascular environment; (3) expression of bone sialoprotein in the cytoplasm of cells infiltrating the product; and (4) von Kossa staining in cells localized in the product when implanted subcutaneously in rats.

[0100] Example 4: Binding affinity of ultra-high cross-linked carbohydrate polymers to bone morphogenetic protein-2

[0101] Hypercross-linked carbohydrate polymers (HCCPs) have been shown to effectively bridge and repair critical-sized bone defects. This study was conducted to investigate the interaction of HCCP with recombinant and endogenous bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. HCCPs were incubated in buffer and bone marrow spiked with recombinant human BMP-2 (rhBMP-2), washed extensively, and BMP-2 binding affinity was assessed using qualitative and quantitative immunoassays. HCCPs were also implanted into critical-sized defects in the femoral condyles of New Zealand White rabbits to correlate the in vitro and in vivo findings. Results demonstrated that rhBMP-2 bound to and was retained within HCCP, as confirmed by antibody staining and ELISA. Implantation of HCCPs into critical-sized bone defects revealed that endogenous BMP-2 was localized to the surface of the HCCP constructs, surrounded by BMP-2-expressing cells. These findings suggest that the binding affinity of HCCP for BMP-2 could play an important role in the repair and bridging of critical-sized bone defects and support HCCP as a novel carrier for rhBMP-2. This study supports the hypothesis that the binding affinity of ultrahighly cross-linked carbohydrate polymer (HCCP) for BMP-2 plays a key role in early osteogenesis in critical-sized bone defects. This study also provides evidence that HCCP may be a suitable alternative to ACS. When investigated as a bone graft substitute for repairing critical-sized defects in rabbit femoral condyles, HCCP exhibited a mean degradation profile of 16 weeks, compared with 2–4 weeks for ACS, resulting in significant bone regeneration. Therefore, the binding affinity of HCCP for both rhBMP-2 and endogenous BMP-2 may provide a valuable means of establishing early osteogenesis at the implant site and provides evidence for its clinical application as a novel carrier.

[0102] Materials and methods

[0103] Characterization and Preparation of HCCP: HCCP is composed of synthetic cross-linked carbohydrate chains. HCCP has been shown to be biocompatible and non-pyrogenic, immunogenic, cytotoxic, or carcinogenic in in vitro and in vivo studies. The microstructure of HCCP was characterized using mercury intrusion porosimetry (Micromeritics Instrument Corporation, Norcross, GA, USA) and exhibited an average pore size range of 50–500 μm. HCCP discs (7 mm x 5 mm), granules (2 mm x 5 mm), and cubes (1 cm 3 For in vivo applications, HCCP was sterilized by autoclaving before use.

[0104] Quantitative rhBMP-2 binding affinity determination: HCCP cubes (1 cm 3 , N = 5) were incubated in PBS containing 1 μg / mL rhBMP-2 (R & D Systems, Minneapolis, MN, USA). A sample of the HCCP cube surrounded by the solution (the "loaded" sample) was collected at 5 minutes. The HCCP was transferred and submerged in fresh phosphate-buffered saline (PBS, Gibco, Life Technologies, Carlsbad, CA, USA), and a sample of the HCCP surrounded by PBS was collected 30 minutes later. Similarly, for consecutive time points (30 minutes, 16 hours, 40 hours, and 64 hours), the HCCP was transferred and submerged in fresh PBS, and samples were collected at each time point.

[0105] Samples were diluted 50-fold in PBS, and residual rhBMP-2 filtered from each wash was analyzed by ELISA. PBS and a 1 μg / mL rhBMP-2 solution (not incubated with HCCP) served as controls. Two independent ELISA assays were performed to assess the ability of HCCP to retain rhBMP-2. The first assay utilized a sandwich (2,2'-azino-bis)ABTS ELISA kit (Peprotech, Rocky Hill, NJ, USA) for absorbance readings. ELISA plates were primed overnight with the capture antibody according to the kit manufacturer's protocol. The following day, the plates were washed four times in 0.05% Tween-20 in PBS (wash buffer) and blocked for 1 hour with 1% bovine serum albumin (BSA, Gibco, Life Technologies, Carlsbad, CA, USA) in PBS to prevent nonspecific binding. The plates were then washed four times with wash buffer.

[0106] The second ELISA assay detected the retention of rhBMP-2 on HCCP after 30 minutes. 3 , N = 3) were incubated in 1 ml of PBS containing 1 μg / ml rhBMP-2. After 30 minutes, the HCCP cubes were removed from the rhBMP-2 solution, the excess solution was wrung out, and the residual concentration of the rhBMP-2 solution was diluted 500-fold and quantified using ELISA according to the manufacturer's instructions (ThermoFisher, Waltham, MA, USA). PBS and a 1 μg / mL rhBMP-2 solution (not incubated with HCCP) served as controls.

[0107] Visualization of rhBMP-2 Retention: Bone marrow aspirates (BMA) were collected from male New Zealand White rabbits (4.0 kg, >6 months of age) under sedated and aseptic conditions. All surgical and animal care procedures were approved by the Institutional Animal Care and Use Committee (Protocol# MM-007102). A 22-gauge spinal needle primed with a sterile 10% solution of 0.5 M ethylenediaminetetraacetic acid (EDTA, Gibco, Life Technologies, Carlsbad, CA, USA) was used to aspirate up to 3 mL of bone marrow from the femur. BMA was collected in a sterile 5 mL tube containing tripotassium EDTA. HCCP discs (7 mm x 5 mm, N = 3) were cultured overnight in 24-well plates containing PBS and 1 μg / mL rhBMP-2. HCCP discs were also cultured in bone marrow spiked with 1 μg / mL rhBMP-2. PBS and bone marrow without rhBMP-2 served as controls. The HCCP discs were then removed from the wells, rinsed three times with 1 mL of PBS, and incubated with primary mouse anti-human BMP-2 antibody (1 μg / mL) (Abeam, Cambridge, MA, USA). After one hour, the HCCP discs were washed extensively in PBS and stained using a 3,3-diaminobenzidine (DAB) kit (R&D Systems, Minneapolis, MN, USA). Images of the stained HCCP discs were uploaded to ImageJ (National Institute of Health, Bethesda, MD, USA), and the rhBMP-2 signal was determined by measuring pixel intensity. RGB images of the stained HCCP discs were captured against a white background. Images were converted to 32-bit grayscale and inverted in ImageJ. For each image, a region of interest (ROI) was drawn around the disc, excluding surrounding shadows. The mean pixel intensity value was measured for each selection. To account for illumination differences between images, the mean pixel intensity value of the background was subtracted from the mean intensity of the HCCP discs.

[0108] Endogenous BMP-2 Binding Affinity for HCCP: We investigated whether endogenous BMP-2 exhibits in vivo binding affinity for HCCP using an established critical-size bone defect model in New Zealand White (NZW) rabbits. All surgical and animal care procedures were approved by the Institutional Animal Care and Use Committee (Protocol# MM-003828). Male NZW rabbits (N = 6, 4.0 kg (±0.5), 13 months of age) were sedated with a ketamine-midazolam mixture (20 mg / kg; 2 mg / kg) and anesthetized with isoflurane (>5%). The femurs were clamped and disinfected with povidone-iodine and 70% ethanol solution. The lateral femoral condyle was accessed by incising and dissecting the skin, superficial fascia, and deep fascia. Cylindrical defects measuring 7 mm in diameter and 10 mm in depth were created bilaterally using a 2 mm high-speed burr (Medtronic, Minneapolis, MN, USA), and approximately 1 cc of HCCP pellets (2 mm x 5 mm pellets) were implanted in each defect. The implant site was sealed with bone wax, sutured with 4-0 absorbable PDS-II sutures (Ethicon, Somerville, NJ, USA), and repaired with surgical staples. Rabbits were treated with antibiotics and analgesics for three days after surgery and underwent daily clinical observation for the duration of the study. HCCP was harvested from the animals using a sodium pentobarbital and sodium phenytoin solution 2 weeks (N = 3) and 4 weeks (N = 3) after euthanasia.

[0109] Immunostaining: HCCP explants were fixed in 10% formalin for at least 24 hours and then transferred to 70% ethanol for embedding in paraffin (VDX Veterinary Diagnostics, Davis, CA USA). Sections (5 μm) were deparaffinized in Preparation 83 (CBG Biotech, Solon, OH, USA) and rehydrated in decreasing concentrations of absolute ethanol before mounting in PBS. Slides were incubated with the serum blocker provided in the DAB staining kit (R&D Systems, Minneapolis, MN, USA), incubated with 1 μg / mL primary mouse BMP-2 antibody (Abeam, Cambridge, MA, USA) for 1 hour, washed in PBS, and then stained using the same DAB staining kit to determine the presence of endogenous BMP-2. Finally, the slides were mounted with MM83 mounting medium (CBG Biotech, Solon, OH, USA).

[0110] Statistical Analysis: Results are reported as mean ± standard error of the mean and were calculated using Microsoft Excel (Microsoft, Redmond, WA, USA). Statistical significance was determined by analysis of variance or two-sided Student's t-test. Statistics for image densitometric data were performed using the Welch t-test in Excel.

[0111] result

[0112] Retention of rhBMP-2 by HCCP: To indirectly analyze the retention of rhBMP-2 loaded HCCP, ELISA was performed in two independent assays. The first ELISA assay analyzed the retention of rhBMP-2 by HCCP over a duration of 64 hours ( Figure 4 The second ELISA assay tested 3 separate rhBMP-2 loaded HCCP cubes; retention was determined after 30 minutes ( Figure 5 Both ELISA assays showed a decrease in the amount of rhBMP-2 detectable in the rhBMP-2 solution after incubation with HCCP. HCCP was found to retain 286 ng of rhBMP-2 / g HCCP, with 90% confidence intervals of [159, 418] and 95% confidence intervals of [129, 449].

[0113] Figure 4 The results showed that after incubation in a BMP-2 solution, HCCP scaffolds retained an average of 44.91 ng / mL of rhBMP-2. After 30 minutes of PBS washing, the rhBMP-2-loaded HCCP scaffolds retained an average of 34.22 ng / mL of rhBMP-2. After 16 hours of PBS washing, the average amount of rhBMP-2 retained in the scaffolds was 32.11 ng / mL. After 40 hours of PBS washing, the average amount of rhBMP-2 retained in the scaffolds was 31.65 ng / mL. After 60 hours of PBS washing, the average amount of rhBMP-2 retained in the scaffolds was 31.61 ng / mL. This data provides evidence that HCCP scaffolds do retain rhBMP-2.

[0114] also, Figure 5Results showed that after HCCP incubation, the baseline control had 1.08 μg / mL of rhBMP-2 remaining in the solution, with no rhBMP-2 remaining in the scaffold. The negative control, PBS solution, contained neither rhBMP-2 in the solution nor in the scaffold. After HCCP incubation, HCCP 1 had 0.17 μg / mL of rhBMP-2 remaining in the solution, with 0.91 μg / mL remaining in the scaffold. After HCCP incubation, HCCP 2 had 0.31 μg / mL of rhBMP-2 remaining in the solution, with 0.77 μg / mL remaining in the scaffold. After HCCP incubation, HCCP 3 had 0.21 μg / mL of rhBMP-2 remaining in the solution, with 0.87 μg / mL remaining in the scaffold.

[0115] Direct Detection of rhBMP-2 Retention by HCCP: To provide direct evidence of HCCP's ability to bind and retain rhBMP-2, HCCP discs were loaded with rhBMP-2, then washed three times in PBS and immunostained with an anti-BMP-2 antibody. The presence of rhBMP-2 was detected using a DAB secondary colorimetric assay. HCCP soaked in rhBMP-2 showed a high DAB signal, while control discs showed minimal detection (Figure 6). HCCP discs incubated overnight in rhBMP-2 solution had an adjusted intensity of 42.73 pixel intensities. HCCP discs incubated overnight in bone marrow and rhBMP-2 had an adjusted intensity of 24.93 pixel intensities; HCCP in bone marrow alone had an intensity of 16.02 pixel intensities; and HCCP in PBS had an intensity of 17.67 pixel intensities. This data provides direct evidence of a statistically significant difference in image intensity between PBS control vs. rhBMP-2 loaded HCCP (17.7 ± 5.37 vs. 42.7 ± 2.74 mean pixel intensity, p-value 0.003).

[0116] Detection of endogenous BMP-2 in HCCP: Next, the ability of HCCP to bind endogenous BMP-2 was evaluated by implanting HCCP into rabbit femoral condyles. Scaffolds were removed at 2 and 4 weeks, processed, and stained for endogenous BMP-2 ( Figure 7 Serial 5 μm sections taken from multiple locations throughout the tissue block sample revealed endogenous BMP-2 deposition along the pore walls. Numerous BMP-2-expressing cells were observed throughout the HCCP pores. A rabbit IgG isotype control showed minimal to no BMP-2 signal. Figure 7 Positive brown staining in the scaffold explants showed evidence of endogenous BMP-2 binding to HCCP in addition to osteoblast formation.

[0117] discuss

[0118] The safety profile of HCCP and its diverse applications as an osteoconductive bone graft substitute provide a promising vehicle for delivering rhBMP-2 for bone regeneration and repair in various clinical settings. The localization of BMP-2 on the implant device may be important for promoting the recruitment, proliferation, and differentiation of endogenous osteogenic stem and progenitor cells at the implant site. In our previous study, HCCP was shown to bridge critical defects in the femoral condyle as early as 10 weeks in rabbits (Koleva et al. BioResearch Open Access 2019 8:111-120). This study was conducted to investigate the interaction between HCCP and BMP-2 and to propose potential mechanisms of action. The results demonstrated stable in vitro retention of rhBMP-2 in HCCP and in vivo deposition of endogenous BMP-2 on the surface of the three-dimensional porous HCCP construct. Furthermore, a significant number of BMP-2-expressing cells were observed within the pores of HCCP where BMP-2 was localized, suggesting that HCCP acquires osteoinductive properties after implantation through the binding of endogenous BMP-2 to the microstructured surface of HCCP.

[0119] During endochondral healing, lamellar bone formation, or intramembranous healing following mechanical trauma or stimulation through autocrine and paracrine regulatory mechanisms, BMP-2 expression is upregulated particularly in osteoblasts and vascular cells. However, it has also been shown to cause a number of serious side effects, including ectopic bone formation and inflammation of surrounding tissues (Robin et al. Spine 2010 35:1350-1354, Wong et al. The Spine Journal 2008 8:1011-1018), suggesting that rhBMP-2 leaks from the implant site. Interestingly, earlier studies published by Wyeth et al. showed that a significant amount of 125I-rhBMP-2 delivered using collagen sponges left the implant site (~50% at 24 hours, 68% at 7 days, and >90% at 2 weeks) (Geiger et al. Advanced Drug Delivery Reviews 2003 55:1613-1629), indicating poor retention of 125I-rhBMP-2 in collagen and / or rapid degradation of the collagen carrier. Interestingly, in a recent study (Gunnella et al. The Spine Journal 2017 17: 1699-1711), when rhBMP-2 was delivered with poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced brushite-forming cement (CPC), doses below 100 μg (~1 / 100 times the current clinical dose) have been shown to significantly enhance bone formation. This observation may be due to better retention of rhBMP-2 at the implant site and / or slower degradation of the carrier.

[0120] Other synthetic polymers have also been investigated as carriers of rhBMP-2 due to their chemical and mechanical properties and ease of fabrication and sterilization ( Wang et al. Journal of Biomedical Nanotechnology 2017 13:1446-1456). Examples of synthetic polymers include poly-L-lactic acid (PLA), polyglycolic acid (PGA), and a combination of the two as PLGA. However, these polymers are known to potentially cause inflammatory responses when used in clinical settings (Anderson et al. Advanced Drug Delivery Reviews 199728:5-24, Ceonzo et al. Tissue Engineering 200712:301-308). Other disadvantages of using synthetic polymers include local pH reduction due to accumulation of by-products and limited biological functionality (Ceonzo et al. Tissue Engineering 2007 12:301-308). One alternative material to circumvent the limitations of other synthetics may be polysaccharide polymers, which have gained popularity due to their biocompatibility, sustainability, and renewable properties (Wahab et al. Composites from Renewable and Sustainable Materials 2016). Polysaccharides are composed of carbohydrate chains, which simplifies the composition of HCCP. Our studies on HCCP have demonstrated its promising potential as a novel polysaccharide delivery system for rhBMP-2, as demonstrated by its ability to localize endogenous BMP-2 throughout its structure. Furthermore, HCCP has been shown to bridge critical bone defects significantly more efficiently than PLGA (Koleva et al. BioResearch Open Access 2019 8:111-120), while maintaining radiolucency (radiopaque in the case of bone formation), unlike mineralized / cement-based carriers.

[0121] In summary, BMP-2 localization to HCCP may partially contribute to the repair and regeneration of critical-sized bone defects. It is possible that other mechanical and chemical properties of HCCP may provide additional contributions to osteogenesis at the implant site. The binding affinity of BMP-2 to HCCP provides additional opportunities for the development of more effective and safer alternatives to absorbable collagen sponges.

[0122] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will recognize that certain changes and modifications may be practiced within the scope of the appended claims. Although particular features may be described herein with respect to certain embodiments, these features may be applied to any embodiment of the present invention. In addition, each reference provided herein is hereby incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference.

Claims

1. A method for preparing a porous glucomannan scaffold, the method comprising freezing a glucomannan gel having an initial volume in the range of 100-5000 mL and a surface area / volume ratio (SA:V) in the range of 0.75 to 1 / cm2 before being subjected to any freezing conditions, wherein the freezing conditions result in a curing phase of 10-2000 minutes to produce the glucomannan scaffold, wherein the curing phase begins when the glucomannan gel first reaches a freezing temperature and ends when the temperature drops below the freezing temperature, and wherein the freezing point is in the range of -0.01°C to -3.00°C, and the method further comprises monitoring the temperature of the glucomannan gel by inserting a temperature probe.

2. The method according to claim 1, wherein the curing stage is 50-1500 minutes.

3. The method of claim 1, further comprising at least one freeze-thaw cycle.

4. The method of claim 3, comprising 2 to 4 freeze-thaw cycles.

5. The method of claim 1, wherein the glucomannan scaffold comprises a porous portion exhibiting a uniform pore size of 100-500 μm.

6. The method of claim 1, wherein the glucomannan scaffold has a porosity of at least 50% and an interconnectivity of at least 50%.

7. The method of claim 6, wherein the volume of the glucomannan scaffold having a porosity of at least 50% and an interconnectivity of at least 50% is at least 10% of the volume of the glucomannan gel.

8. The method of claim 1, further comprising neutralizing the glucomannan scaffold to a pH of about 7.

9. The method of claim 1, further comprising contacting the glucomannan scaffold with a cell culture medium and / or a cell adhesion promoter.

10. The method of claim 1, further comprising: a) cooling the glucomannan gel from a temperature of about 25° C. to a temperature of about 4° C.; b) maintaining the glucomannan gel at the solidification stage for at least 10 minutes; c) thawing the glucomannan gel to a temperature of about 25° C.; d) optionally repeating steps a), b) and / or c).

11. A scaffold prepared according to the method of any one of claims 1 to 10, wherein the scaffold comprises a backbone of at least 60% (w / w) glucomannan, and the scaffold has a porosity of at least 50% and an interconnectivity of at least 50%, wherein the scaffold comprises one or more morphogenetic proteins, and the one or more morphogenetic proteins comprise BMP-2.

12. The stent of claim 11, wherein the framework comprises one or more composite materials.

13. The scaffold of claim 11, further comprising one or more bone components.

14. The scaffold of claim 13, wherein the one or more bone components are selected from the group consisting of calcium, phosphate, and magnesium.

15. The stent of claim 13, wherein the stent is radiolucent.

16. Use of a glucomannan scaffold prepared according to the method of any one of claims 1 to 10 or a scaffold according to any one of claims 11 to 15 in the preparation of an implant for treating a patient in need thereof.

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