Wound dressing assembly

Abstract

Wound dressing assemblies and methods of processing are described in which a hydrogel mat can be formed by introducing a first amount of pullulan, a salt, and a crosslinker, and introducing a second amount of collagen such that the weight ratio of pullulan to collagen is between 20: 1 and 22: 1. Pulullan and collagen may be mixed to form a pulullan mixture, and a third amount of an initiator solution may be introduced into the pulullan mixture to form a component mixture. The component mixture may be placed for a predetermined period of time, whereby the pullulan polysaccharides are cross-linked with each other and the collagen remains uncross-linked to form a hydrogel. The hydrogel may be cooled from room temperature to a first cooling temperature at a predetermined cooling rate, and then dried from the first cooling temperature to a second drying temperature at a predetermined heating rate while exposing the hydrogel to a predetermined negative pressure.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of priority of U.S. Provisional Application 63 / 486,928, filed February 24, 2023, which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0002] The present invention relates generally to medical devices and methods. More particularly, the present invention relates to equipment and methods for producing wound dressings made from pullulan and collagen. BACKGROUND

[0003] Compositions and methods for manufacturing pullulan-collagen pads that exhibit superior handling properties, durability, and a porous dermal-like ultrastructure that is maintained in vitro are provided. These pullulan-collagen pads can be used as wound dressings, drug delivery platforms, skin substitutes, and cell delivery scaffolds.

[0004] Conventional wound dressings utilizing collagen are manufactured using a variety of techniques, but such techniques are often limited by factors such as cost, ability to maintain an optimal humidity environment, short shelf life, and the like.

[0005] Accordingly, there is a need for a manufacturing process that can efficiently manufacture effective pullulan-based collagen hydrogel pads for wound dressings. SUMMARY

[0006] Pullulan-based collagen hydrogel pads can be processed by varying a variety of production process parameters such as wash duration, salt content, and lyophilization parameters such as heating ramp time, temperature, and duration to improve their uniformity and performance in wound remodeling. A mixture of pullulan, potassium chloride (KCl), and sodium trimetaphosphate (STMP) can be first prepared. A collagen suspension can also be prepared in a dilute acid solution, for example in HC1, and an HC1-KCl buffer can also be added to the collagen suspension and further mixed for a period of time to provide a uniform collagen suspension. The mixture of pullulan, KCl, and STMP can then be mixed with the collagen suspension. This step facilitates the precipitation of collagen fibers at a relatively high salt concentration (e.g., by KCl) and a neutral pH (e.g., a pH of 6-7). Finally, an initiator, sodium hydroxide (NaOH), can be introduced and mixed with the component mixture.

[0007] Upon completion of the component mixing, aliquots of the reaction mixture can be separated into patch form by pouring the reaction mixture into mold forms, and the tray can be shaken for a period of time to ensure the mixture spreads evenly within each mold. The mixture can then be left in the tray at a specified temperature (e.g., room temperature) for a specified period of time (e.g., 8 hours or more) for the pullulan crosslinking.

[0008] The mixture can undergo gel formation via STMP crosslinking of the pullulan polymer in the presence of NaOH. However, the collagen fibers can remain in a native state without crosslinking due to the fact that the collagen molecules precipitate and are prevented from the crosslinking reaction.

[0009] Once the reaction is stopped, the individual pads can be washed for a period of time. This process can optionally be repeated an additional number of times, after which a final volume of deionized water can be introduced into the tray, and various parameters of the decanted water can be measured (e.g., pH, conductivity, etc.).

[0010] Once the hydrogels have been washed, they can undergo a freeze-drying process in which the hydrogels are first slowly cooled and frozen to a lower temperature, and then dried under high vacuum levels.

[0011] The manufacturing steps result in the formation of pores within the hydrogel pad such that the pore diameter can range between, for example, 100 pm - 250 pm in diameter, which is shown to be within an optimal pore diameter range for fibroblast infiltration and tissue regeneration when the hydrogel pad is applied to a patient as a wound dressing. In one variation, the average pore diameter can be, for example, 250 pm. Furthermore, the rate of cooling during freeze-drying as described results in the desired pore diameter and consistency in the pad formation. Upon completion of the freeze-drying, the individual pads can be removed from the tray and trimmed to size according to the desired application.

[0012] A method of forming a wound dressing can generally include placing a collagen suspension for a predetermined period of time; introducing a first amount of pullulan; introducing a second amount of collagen such that a weight ratio of the pullulan to the collagen is between 20: 1 and 22: 1; mixing the first amount of component mixture and the second amount of collagen to form a final component mixture; introducing a third amount of an initiator solution into the final mixture to form a gel, whereby the pullulan crosslinks to one another and the collagen remains uncrosslinked; cooling the hydrogel pad from room temperature to a first cooling temperature at a predetermined cooling rate; and drying the hydrogel pad from the first cooling temperature to a second drying temperature at a predetermined heating rate while the hydrogel pad is exposed to a predetermined negative pressure.

[0013] One embodiment of a wound dressing can generally include a pullulan-collagen hydrogel, where the hydrogel includes a first amount of a pullulan, potassium chloride (KCl) and sodium trimetaphosphate (STMP) mixture, a second amount of a collagen suspension, and a third amount of a NaOH initiator. The weight ratio of pullulan to collagen can be between 20: 1 and 22: 1, and the weight ratio of KCl to STMP can be about 0.96: 1. The pore size of the hydrogel pad can be between 100 μιη and 250 μιη.

[0014] One embodiment of a wound dressing can generally represent a cohesive hydrogel after hydration with saline. The hydrogel can generally include a cross-linked network of pullulan and embedded collagen fibers. The collagen fibers can desirably be organized in a network within the network of pullulan and can provide the hydrogel with mechanical strength as well as a biocompatible support for cells. While the collagen can generally be arranged in a reticulated, dispersed, or interwound manner, the collagen can be arranged in alternative configurations as desired. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A flow chart illustrating one variation of a process for manufacturing a pullulan-collagen hydrogel pad is shown.

[0016] Figure 2A and Figure 2B A perspective view and a top view of an assembly that can be used to form a uniform mixture of components is shown.

[0017] Figure 3 A perspective view of one variation of a manufacturing tray used to produce a pad is shown.

[0018] Figure 4 A perspective view of one variation of an assembly used to wash a pad using deionized water is shown. DETAILED DESCRIPTION

[0019] In processing wound dressings, pullulan-collagen pads can be processed by varying a number of production process parameters such as wash duration, salt content, and lyophilization parameters such as heating ramp times, temperature, and duration to improve their uniformity and performance. Such processes also help to prevent cross-linking of collagen in the wound dressing and thereby preserve the native form of the collagen.

[0020] Figure 1An example of a flow chart 10 is illustrated for one variation for manufacturing a collagen sponge based on pullulan. First, a mixture of pullulan, potassium chloride (KCl), and sodium trimetaphosphate (STMP) can be prepared 12, for example, in a volume of water. In one example, an amount of KCl, for example, 24.00 g ± 0.20 g, can be combined with an amount of STMP, for example, 25.00 g ± 0.20 g, along with an amount of pullulan, for example, 25.00 g ± 0.20 g, in a container such as a glass beaker. The weight ratio of KCl to STMP can be about, for example, 0.96: 1.

[0021] A collagen suspension can also be prepared 14 by first weighing an amount, for example, 1.2 g ± 0.05 g, of collagen, for example, insoluble collagen fiber powder derived from bovine hide. The weight ratio of pullulan to collagen can accordingly be in a range between, for example, 20: 1 and 22: 1, or the weight ratio of pullulan to collagen can accordingly be in a range between, for example, 20.2: 1 and 21.5: 1, or the weight ratio of pullulan to collagen can be, for example, 20.8: 1. A volume, for example, 60 mL, of WFI can be added to the collagen and mixed, and a volume, for example, 60 mL, of an acid such as an HC1 solution or HC1-KCl buffer having a pH of 2.20 can also be added to the collagen suspension and further mixed for a period of time, for example, greater than or equal to 12 hours or more, to provide a uniform collagen suspension.

[0022] In mixing the collagen suspension, the pullulan, KCl, and STMP preparation 12 and the collagen suspension 14 can be mixed by applying mechanical energy to form a uniform viscous suspension. Figure 2A and Figure 2BA perspective view and a top view of one example of a syringe mixing assembly 40 that can be used to form a uniform mixture of components via turbulent plug flow mixing is illustrated. The assembly 40 can include one, two, three, four, or more syringes or containers 42A, 42B, 42C, 42D that can be fluidly coupled to one another via tubing couplings while being supported by a support structure. Each syringe can then be caused to pass the suspension between each syringe by pressing the appropriate plunger in a corresponding manner, such that the application of this mechanical energy can pass the components 18 from one syringe to another a specified number of times, such as 1 to 16 or more. The component mixing 18 can be repeated, such as 1 to 2 or more times, and each component can be mixed with one another by passing each component from one syringe to another, such as 1 to 16 or more times, until the suspension has completed mixing such that a gel-type slurry is formed. Other forms of mechanical energy can also be used to mix the suspension, such as a rotary mixer, a blender, etc.

[0023] After initial mixing, an initiator 16, such as 15 mL ± 0.1 mL of 1 N NaOH, can be introduced into a syringe and mixed with the suspension by passing the components 18 from one syringe to another a specified number of times, such as 1 to 16 or more.

[0024] Where the component mixing 18 is complete, an aliquot of the reaction mixture can be separated into a patch form 20 by pouring the reaction mixture into a mold. Figure 3 A perspective view of one variation of a manufacturing tray 50 used to produce pads is illustrated. The tray 50 is shown in this example as having a base 52, and a plurality of molds 54 are defined on the surface of the tray 50, where a mixture can be introduced into each mold 54 to form an individual hydrogel 56. The example shown illustrates individual square molds (e.g., 42 mm x 42 mm x 2 mm), each mold containing a volume of suspension (e.g., 3.5 mL). Once the mixture has been introduced into one or more molds 54, the tray 52 can be shaken for a period of time (e.g., 200 rpm for about 10 seconds) to ensure the mixture is evenly spread within each mold 54. Thereafter, the mixture can be left in the tray 52 under a gas-tight lid at a specified temperature (e.g., room temperature) for a specified period of time (e.g., 8 hours or more) to form a gel.

[0025] The mixture can undergo gel formation via cross-linking of the pullulan polymer. However, the collagen fibers can remain in a native state and not cross-link due to the fact that the collagen fibers precipitate at relatively high salt concentrations (e.g., by KCI) and at neutral pH (e.g., a pH of 6-7). The change in physical state protects the collagen from cross-linking by the STMP.

[0026] Once the reaction is stopped, the individual gels within each mold 54 can be washed 24 using a volume of deionized water. The tray 50 including the individual hydrogel pads 56 can be placed within a container 60, as shown in the perspective view of Figure 4 A volume, e.g., 3 L, of deionized water can be introduced into the tray 50 and the entire assembly can be exposed to a vibrational force, e.g., an orbital shaker, for a predetermined period of time, e.g., 30 minutes at 25 rpm, to wash the hydrogels 56. After the wash is complete, the water can be decanted from the container 60 and another volume, e.g., 3 L, of deionized water can be introduced into the tray 50, after which the assembly can again be exposed to a vibrational force, e.g., 30 minutes at 25 rpm. This process can optionally be repeated an additional number of times, after which a final volume, e.g., 1 L, of deionized water can be introduced into the tray 50 and, after a residence period of time, e.g., 10 minutes, a volume of the final wash liquid can be decanted and a variety of parameters of the decanted water can be measured (e.g., pH, conductivity, etc.). For example, verifying a neutral pH level (e.g., 7.0-8.0) 26 of the decanted water from the final wash liquid can indicate that the hydrogels 56 are washed and have a relatively low salt concentration.

[0027] This can also be an indication that the collagen fibers within the hydrogel pads 56 are desirably organized in a networked fashion within the hydrogel network, as the collagen fibers provide mechanical strength to the hydrogel as well as a biocompatible support for cells. Such an arrangement allows the collagen to target skin cells, particularly with increased mechanical strength. While the collagen can generally be arranged in a networked, dispersed, or interwoven fashion, the collagen can be arranged in alternative configurations as desired.

[0028] Once the hydrogels 56 have been washed, they can undergo a lyophilization process 28, in which the hydrogels are first slowly cooled and frozen to a lower temperature, followed by drying under high vacuum levels during the lyophilization process. The hydrogel pads 56 can be cooled at a controlled temperature rate and over a controlled period of time. For example, as the tray is cooled, the tray can be cooled from room temperature to -20°C over a first period of time, e.g., 175 minutes, at 1 minute intervals, and cooled to -40°C over a second period of time, e.g., 40 minutes, with a hold period of 20 minutes at the -40°C temperature.

[0029] With cooling complete, the trays can then be returned to temperature and simultaneously dried under high vacuum levels. For example, the temperature of the hydrogel pads 56 can be gradually increased to, for example, -5°C over a first time period, for example, 120 minutes, and held for 600 minutes while the pressure is held between, for example, 20 to 23 mTorr. The temperature can be further increased to up to 25°C over a second time period, for example, 60 minutes, and held for 500 minutes while the pressure is held between, for example, 20 to 23 mTorr.

[0030] These lyophilization settings can result in the formation of pores within the hydrogel 56 such that the pore diameter is between, for example, 150 to 250 μιη, or such that the average pore diameter is a diameter of, for example, 160 μιη, which has been shown to be within an optimal pore diameter range for fibroblast infiltration and tissue regeneration when the hydrogel 56 is applied as a wound dressing to a patient. Further, the rate of cooling during lyophilization as described results in the desired pore diameter and consistency in the formation of the pad 56.

[0031] With lyophilization complete, the individual pads 56 can be removed from the trays 50 and trimmed 30 to size according to the desired application. The individual pads 56 can be placed on a cutting fixture and the pads 56 are then cut to size, for example, 50 mm x 50 mm, or any other size as desired or required.

[0032] The applications of the disclosed invention discussed above are not limited to certain processes or applications of the pads on any particular area of the body, but can include any number of other treatments and areas of the body. Modifications of the above-described methods and devices for carrying out the application, and variations of aspects of the application that are obvious to those of skill in the art are intended to be within the scope of the disclosure. Further, a variety of combinations of aspects between examples are also contemplated and are deemed to be within the scope of the disclosure.

Claims

1. A method of forming a wound dressing, comprising: introducing a first amount of pullulan; introducing a second amount of collagen such that a weight ratio of pullulan to collagen is between 20: 1 and 22: 1; mixing the first amount of pullulan and the second amount of collagen to form a component mixture; introducing a third amount of an initiator solution to the component mixture to form a reaction mixture; placing the reaction mixture for a predetermined period of time, whereby the pullulan crosslinks to one another and the collagen remains uncrosslinked, to form a hydrogel; cooling the hydrogel at a predetermined cooling rate from room temperature to a first cooling temperature; and drying the hydrogel at a predetermined warming rate from the first cooling temperature to a second drying temperature while the hydrogel is exposed to a predetermined negative pressure.

2. The method of claim 1, wherein introducing the first amount of pullulan comprises introducing a mixture of the pullulan, potassium chloride (KCI), and sodium trimetaphosphate (STMP).

3. The method of claim 2, wherein a weight ratio of the KCI to the STMP is about 0.96:

1.

4. The method of claim 1, wherein introducing the second amount of collagen, wherein the weight ratio of the pullulan to the collagen is between 20: 1 and 22:

1.

5. The method of claim 1, wherein mixing the first amount of pullulan and the second amount of collagen comprises mechanical mixing.

6. The method of claim 1, wherein introducing the third amount of initiator solution comprises introducing a volume of 1 N NaOH.

7. The method of claim 1, wherein placing the collagen suspension further comprises shaking the collagen suspension for a period of time.

8. The method of claim 1, wherein placing the collagen suspension further comprises placing the collagen suspension at room temperature for 8 hours or more.

9. The method of claim 1, further comprising washing the hydrogel with water prior to cooling the hydrogel.

10. The method of claim 9, further comprising washing the hydrogel until a pH level of the water is neutral.

11. The method of claim 1, wherein cooling the hydrogel comprises cooling from room temperature to an intermediate cooling temperature at a first cooling rate, and further cooling from the intermediate cooling temperature to the first cooling temperature at a second cooling rate.

12. The method of claim 1, wherein drying the hydrogel comprises warming from the first cooling temperature to an intermediate drying temperature at a first warming rate, and further warming from the intermediate drying temperature to the second drying temperature at a second warming rate.

13. The method of claim 12, wherein the predetermined negative pressure.

14. The method of claim 12, further comprising controlling a pore size of the hydrogel to have a diameter of 100 pm to 250 pm.

15. The method of claim 1, further comprising trimming the hydrogel.

16. A wound dressing, comprising: a pullulan-collagen hydrogel, wherein the hydrogel comprises: a first amount of a mixture of pullulan, potassium chloride (KCl), and sodium trimetaphosphate (STMP), a second amount of a collagen suspension, and a third amount of a NaOH initiator; wherein the weight ratio of pullulan to collagen is between 20: 1 and 22: 1, and the weight ratio of the KCl to the STMP is about 0.96: 1, and wherein the pore size of the hydrogel is between 100 μm and 250 μm.

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