Wound healing model

Abstract

This invention generally relates to the field of wound healing. One embodiment is a method for generating a delayed wound model in animals, the method comprising contacting the wound with a composition comprising an electrospun scaffold, wherein the scaffold is made of 80% w / w PCL and 20% w / w rat tail collagen and has been immersed in a biofilm conditioned medium derived from Staphylococcus aureus or the small molecule drug FK866.

Description

Technical Field

[0001] This invention generally relates to the field of wound healing. In particular, this specification teaches a method and composition for generating a delayed wound healing model in animals. Background Technology

[0002] Wound healing is the process of repairing or replacing damaged tissue. It typically involves four stages: hemostasis, inflammation, proliferation, and remodeling. Acute wound healing usually occurs rapidly in healthy individuals and requires little prompting. In older adults or those with diabetes, healing is often slower and may become chronic. This represents a significant unmet clinical need.

[0003] Most animal wounds heal rapidly without intervention, unlike chronic human wounds, which can remain open for months or years. While diabetic animals exhibit delayed wound healing, the slowdown is only 1 to 2 days in rats and 3 to 4 days in pigs. Therefore, there are no good animal models to test new therapies for chronic wounds. This lack of good models is considered a reason why many wound healing therapies have failed in human trials.

[0004] Therefore, it is usually necessary to overcome or improve one or more of the above difficulties. Summary of the Invention

[0005] This article discloses a method for generating a wound model in animals, which involves contacting the wound with a composition comprising a scaffold and an aging inducer.

[0006] This paper discloses animal models of wounds generated according to the methods defined in this paper.

[0007] This article discloses a method for identifying candidate therapeutic agents for treating wounds, the method comprising a) generating a wound model in an animal according to a method defined herein, and b) contacting the wound model with a candidate therapeutic agent.

[0008] This article discloses a composition for inducing wound model formation in animals, the composition comprising a scaffold and an aging inducer.

[0009] This article discloses a dermal substitute for inducing wound formation in animals, the dermal substitute comprising a scaffold and an aging inducer. Attached Figure Description

[0010] Embodiments of the invention are described below by way of non-limiting example only, with reference to the accompanying drawings, wherein:

[0011] Figure 1 Technical illustration - Creation of a poor wound healing model.

[0012] Figure 2Timeline comparison between rat wound models. When comparing diabetic wound models with poor wound models, the time to model generation was significantly reduced, which means less animal use, less animal suffering, and less human / research time.

[0013] Figure 3 : The generation of a rat model of poor healing.

[0014] Figure 4 Macroscopic analysis of rat wounds to compare three models: acute wound healing, diabetic wound healing, and poor wound healing.

[0015] Figure 5 Histological analysis of rat wounds to compare three models: acute wound healing, diabetic wound healing, and poor wound healing.

[0016] Figure 6 The development of a poor healing model in pigs.

[0017] Figure 7 Macroscopic analysis of wounds generated in acute and poor healing models in pigs.

[0018] Figure 8 Histological analysis of wounds generated in acute and poor healing models in pigs.

[0019] Figure 9 Histological image of senescent cells (β-galactosidase) present in an undesirable wound on day 20 after stent removal. Senescent cells are visible at the wound boundary, throughout the granulation tissue, and along the vessel walls.

[0020] Figure 10. Images showing wound progression 5 or 10 days after in situ scaffold placement. B) Schematic diagram showing the measurement of neoepithelial tongue. C) A diagram depicting representative H&E images of acute and malformed wounds. Reepithelialization from the wound edge is traced by black lines. Scale bar = 2000 μm. D) Quantitative assessment of the degree of reepithelialization (n = 1, n represents 1 biological replicate). Reepithelialization was analyzed by expressing the total reepithelialization distance as a percentage of the wound bed width.

[0021] Figure 11 Epidermal tongue thickness analysis. A) Schematic diagram showing the area and length measurements of the newly formed epidermal tongue. B) A diagram depicting representative H&E images of acute and malformed wounds. The epidermal tongue region is outlined by black dots. The migration of the epidermal tongue from the wound edge is traced by black lines. Scale bar = 1000 μm. C) Quantitative assessment of the mean thickness of the epidermal tongue (n = 1, n represents 1 biological replicate). The thickness of the epidermal tongue was analyzed by taking the area over the reepithelialization distance.

[0022] Figure 12Analysis of inflammatory status in acute and malformed wounds. A) Schematic diagram showing the regions of interest selected for quantitative measurement. B) A diagram depicting representative H&E images of acute and malformed wounds (selected regions are outlined in black in A). Scale bar = 50 μm. C) Quantitative assessment of histological inflammation scores for acute and malformed wound beds (n = 1, n represents 1 biological replicate).

[0023] Figure 13. Macroscopic and histological assessment of acute and malformed wounds. A) Macroscopic image showing the wound closure progression of acute and malformed wounds. B) Schematic diagram showing the results of neoepithelialization tongue measurements. C) A diagram depicting representative H&E images of acute and malformed wounds. Reepithelialization from the wound edge is marked by a black line. Scale bar = 2000 μm. D) Quantitative assessment of the degree of reepithelialization (n = 3, n represents 1 biological replicate). Reepithelialization was analyzed by expressing the total reepithelialization distance as a percentage of the wound bed width.

[0024] Figure 14 Analysis of epidermal tongue thickness and Cx43 expression. A) Schematic diagram showing the area and length measurements of the neonatal epidermal tongue. B) Graph depicting representative H&E and immunofluorescence staining (Cx43 is shown by dot staining) images from acute and lesions. The epidermal tongue region is outlined by black dots. Migration of the epidermal tongue from the wound edge is marked by black lines. Black dashed squares depict the areas captured in the corresponding confocal images. The epidermal tongue is marked by white dashed lines. Black scale bar = 1000 μm, white scale bar = 40 μm. C) Quantitative assessment of the mean thickness of the epidermal tongue (n = 3, n represents 1 biological replicate). The thickness of the epidermal tongue was analyzed by taking the area over the re-epithelialization distance. D) Quantification of Cx43 expression in the anterior epidermal tongue (n = 3, n represents 1 biological replicate).

[0025] Figure 15 Analysis of inflammatory status in acute and malformed wounds. A) Schematic diagram showing the selected regions of interest for quantitative measurement. B) Graph depicting representative H&E images of acute and malformed wound beds (selected areas are outlined in black in A). Scale bar = 50 μm. C) Quantitative assessment of histological inflammation scores for acute and malformed wound beds (n = 3, n represents 1 biological replicate). D) Graph depicting representative H&E images of acute and malformed wound margins (selected areas are outlined in black in A). Scale bar = 50 μm. E) Quantitative assessment of histological inflammation scores for acute and malformed wound margins (n ​​= 3, n represents 1 biological replicate).

[0026] Figure 16Analysis of collagen content in acute and malformed wounds. A) Schematic diagram showing the selected region of interest for quantitative measurement. B) Graph depicting representative Masson's tricolor images of acute and malformed wound beds (selected areas are outlined in black in A). Scale bar = 50 μm. C) Quantitative assessment of collagen deposition in acute and malformed wound beds (n = 3, n represents 1 biological replicate). D) Graph depicting representative H&E images of acute and malformed wound margins (selected areas are outlined in black dots in A). Scale bar = 50 μm. E) Quantitative assessment of collagen loss at the edges of acute and malformed wounds (n = 3, n represents 1 biological replicate).

[0027] Figure 17 Analysis of senescent cell populations in acute and malformed wounds. A) Schematic diagram showing the regions of interest selected for quantitative measurements. B) Graph depicting representative X-gal images of the acute and malformed wound beds (selected regions are outlined in black in A). Scale bar = 50 μm. C) Quantitative assessment of X-gal-positive cells in the acute and malformed wound beds (n = 3, n represents 1 biological replicate). D) Graph depicting representative H&E images of the edges of acute and malformed wounds (selected regions are outlined in black in A). Scale bar = 50 μm. E) Quantitative assessment of X-gal-positive cells at the edges of acute and malformed wounds (n = 3, n represents 1 biological replicate).

[0028] Figure 18. Summary of features observed in the poor wound model. Detailed Implementation

[0029] This disclosure teaches a method for generating a wound model in animals.

[0030] This article discloses a method for generating a wound model in animals, the method comprising a) contacting the wound with a composition comprising a scaffold and an aging inducer.

[0031] This method may include exposure under sufficient time and conditions to generate a wound model in an animal.

[0032] In one embodiment, a method for generating a wound model in an animal is provided, the method comprising a) contacting the wound with a composition comprising one or more polymers and an aging inducer.

[0033] Unbound by theoretical constraints, the inventors developed a poor wound healing model in rats and pigs. This model encapsulates several characteristics of chronic wounds in humans, such as chronic inflammation, excessively thickened non-migratory epidermis, senescent fibroblasts, and endothelial cells. Unlike diabetic rats and pigs, where healing is delayed by a maximum of 2–4 days, these wounds remained unclosed after 10 days and 30–35 days, respectively. More importantly, this model represents an improvement over standard practice compared to the currently FDA-approved streptozotocin-induced diabetic wound healing model. In particular, the animals experienced minimal pain and distress while achieving the characteristics of poor wound healing compared to inducing diabetes and related side effects.

[0034] As used herein, a “wound” is an area of ​​damaged tissue. Damaged tissue may be caused by external trauma (e.g., mechanical wounds, such as those resulting from surgical procedures (including incisions, tooth extractions, or other surgical procedures), or accidental wounds), infection, and / or inflammation. Examples of wounds include: wounds caused by incisions (e.g., cutting instruments, such as surgical incisions), lacerations (often caused by blunt or broken instruments), punctures, abrasions, burns caused by exposure to heat, electricity, or radiation (used for example, sunburn and laser surgery); surgical wounds such as those resulting from tooth extractions; corrosive chemicals; skin wounds caused by aging or environmental factors, including, for example, cracked or dry skin; ulcers (lesions on the surface of the skin or mucous membranes); wounds in subjects with diabetes or other conditions (e.g., localized ischemia), including foot injuries due to nerve damage (diabetic neuropathy) and numbness caused by insufficient blood flow to the legs and feet, foot ulcers, pressure sores, and bedsores.

[0035] In one embodiment, the method includes creating a wound in the animal prior to step (a). The wound may be a fresh wound. The wound may be a skin wound. The wound may be a full-thickness skin wound. In one embodiment, the wound is a skin puncture wound or an excision wound. The wound may be created by a puncture biopsy. The puncture biopsy may be a full-thickness puncture biopsy. The puncture biopsy may be, for example, a round puncture biopsy with a diameter of about 6 mm in rats or about 10 mm to 12 mm in pigs.

[0036] In one implementation, the wound model is a poor wound model. A poor wound model can possess many characteristics of human chronic wounds.

[0037] As used herein, the term “malnourished wound” can refer to a wound that has failed to heal in an orderly and timely manner during the normal stages of wound healing. A “malnourished wound model” or “malnourished wound healing model” attempts to generalize several characteristics of chronic wounds in animal models. These characteristics may include, for example, chronic inflammation, excessively thickened non-migratory epidermis, senescent fibroblasts and endothelial cells, overexpression of the gap junction protein Cx43, elevated ROS levels, and high pH (see, for example, Figure 18).

[0038] In one implementation, the wound model is a non-human model. Non-human models can be used to understand chronic wound healing in humans.

[0039] In one implementation, the wound model is a poor wound model. In another implementation, the wound model is a poor wound healing model.

[0040] The term "healing" in relation to wounds or skin damage refers to the process of repairing wounds or skin damage.

[0041] As used herein, the term "contact" can refer to direct contact between the composition and an animal's wound, such that they are in close proximity or associated with each other. Contact can occur as a result of, for example, the application or implantation of the composition.

[0042] In one embodiment, the composition is capable of inducing a foreign body reaction (FBR) within an animal's wound.

[0043] The term "scaffold" refers to a three-dimensional structure formed by a network of one or more polymers that can be inserted into an animal's wound. A scaffold provides sufficient mechanical strength or stiffness to resist wound contraction and prevent it from being extruded from the wound. It also maintains structural integrity so that it can be cleanly removed from the wound.

[0044] In one embodiment, the composition comprises a scaffold formed of one or more polymers (e.g., one, two, three, four, or more). For example, the scaffold can be inserted into a wound (e.g., a wound created by a perforated biopsy). The scaffold may be an electrospun scaffold. Alternatively, the scaffold can be produced by other manufacturing methods, such as by 3D printing, solvent casting, and freeze-drying. In one embodiment, the composition comprises a solid scaffold, such as a solid electrospun scaffold. The composition (or scaffold) may be a disc or rod that can be inserted into a wound.

[0045] The term "electrospinning" generally refers to a technique that uses a high-voltage power supply, a spinneret (e.g., a hypodermic needle), an injection pump, and a conductive collecting plate or liquid bath (e.g., aluminum foil or 100% ethanol) that can be used to prepare an electrospinning scaffold. To perform the electrospinning process, the electrospinning liquid (i.e., a melt or solution of the desired material dissolved in a solvent to form fibers) is typically first loaded into a syringe and then fed by the injection pump at a set, specific rate.

[0046] When liquid is fed by an injection pump with a sufficiently high voltage, the repulsion between the charges fixed on the surface of the resulting droplets overcomes the limitations of surface tension and causes a liquid jet to be ejected from the orifice. The charged jet then undergoes a stirring and stretching process, subsequently forming uniform fibers. Furthermore, as the jet stretches and the solvent evaporates, the diameter of the fibers can be continuously reduced to the desired scale (e.g., micrometers or nanometers), and under the influence of the electric field, the fibers can then be forced to move towards the collector.

[0047] In one embodiment, one or more polymers are selected from polycaprolactone (PCL), collagen, silk protein, gelatin, a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), a copolymer of lactic acid and glycolic acid (PLGA), polylactic acid (PLA), and poly(glycolic acid) (PGA). In one embodiment, one or more polymers comprise or are composed of collagen. In one embodiment, one or more polymers comprise or are composed of PCL.

[0048] In one embodiment, one or more polymers comprise collagen and a synthetic polymer, or consist of collagen and a synthetic polymer. The synthetic polymer is selected from the group consisting of PCL, PLGA, PLA, and PGA, as well as any other synthetic polymers known in the art.

[0049] In one embodiment, one or more polymers comprise or consist of PLC and collagen. In one embodiment, one or more polymers comprise or consist of PLGA and collagen. In one embodiment, one or more polymers comprise or consist of PLA and collagen. In one embodiment, one or more polymers comprise or consist of PGA and collagen.

[0050] The composition may contain about 70% to 90% w / w of synthetic polymers. For example, the composition may contain about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% w / w of synthetic polymers. The composition may contain about 10% to 30% w / w of collagen. For example, the composition may contain about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% collagen. In one embodiment, the composition comprises about 80% w / w of a synthetic polymer and about 20% w / w of collagen.

[0051] The composition may contain about 70% to 90% w / w PCL. For example, the composition may contain about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% w / w PCL. The composition may contain about 10% to 30% w / w collagen. For example, the composition may contain about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% collagen. In one embodiment, the composition comprises about 80% w / w PCL and about 20% w / w collagen.

[0052] In one embodiment, the collagen is type I collagen. In one embodiment, the collagen is rat collagen. The rat collagen may be rat tail collagen. In one embodiment, the collagen is rat tail type I collagen.

[0053] The term "senescence inducer" can refer to any agent capable of inducing senescence in animal cells, the process by which cells age and permanently cease dividing without dying. Senescence inducers can include DNA damaging agents, reactive oxygen species (ROS) generators, differentiation agents, or ionizing radiation. Senescence inducers can be biofilm conditioned medium (BCM) or nicotinamide phosphoribosyltransferase inhibitors. Senescence inducers can also be, for example, afedipine, bleomycin, cisplatin, doxorubicin, etoposide, mitoxantrone, retinol, hydroxyurea, carboplatin, or docetaxel. Senescence inducers can also be those described in Ewald JA et al. (2010) or... - Any aging inducers disclosed in Pietrasik, J. et al. 2020.

[0054] In one embodiment, the aging inducer is a biofilm conditioned medium (BCM). The biofilm conditioned medium can be a bacterial biofilm conditioned medium. The bacteria can be any bacteria that can be found in a wound. The biofilm conditioned medium can be a biofilm conditioned medium derived from Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, or Enterococcus faecalis.

[0055] In one embodiment, the aging inducer is a nicotinamide phosphoribosyltransferase inhibitor. The term "inhibitor" can refer to any molecule (e.g., a small molecule or peptide) capable of reducing the activity of nicotinamide phosphoribosyltransferase or reducing the protein level of nicotinamide phosphoribosyltransferase. An inhibitor can also be a molecule (e.g., a nucleic acid or ribozyme) that reduces the expression of the gene encoding nicotinamide phosphoribosyltransferase.

[0056] Nicotinamide phosphoribosyltransferase inhibitors can be, for example, Daporinad (FK866) or CHS-828 (or GMX-1778). Nicotinamide phosphoribosyltransferase inhibitors can be present at concentrations of approximately 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or 1000 μM.

[0057] Nicotinamide phosphoribosyltransferase inhibitors can also be nucleic acids, such as antisense RNA or shRNA. Inhibitors can also be gene-editing compositions for knocking out or reducing nicotinamide phosphoribosyltransferase. Such gene-editing compositions are well known in the art and include, for example, the CRISPR-Cas gene-editing system, the Talen gene-editing system, and the ZincFinger gene-editing system.

[0058] In one embodiment, the method includes providing an animal. The animal may be a mammal. The animal may be, for example, a mouse, a rat, or a pig.

[0059] In one embodiment, the method includes sealing the wound after step a). The method may include sealing the wound with an airtight material. The wound may be covered with a paraffin membrane, Tegaderm, Opsite film, and / or adhesive crepe bandage. Wound sealing may help prevent contamination.

[0060] In one embodiment, the method includes contacting the wound with the composition and / or removing the composition once a day for two, three, four, five or more days.

[0061] In one implementation, the method further includes exposing the wound to the aging inducer every other day, two days, three days, four days, five days, or more.

[0062] In one embodiment, the method includes removing the composition from the wound to allow the creation of a wound model in an animal.

[0063] This article discloses a method for inducing a foreign body reaction (FBR) in animals, the method comprising a) contacting a wound with a composition as defined herein.

[0064] This paper discloses animal models of wounds generated according to the methods defined in this paper.

[0065] In one embodiment, a wound animal model is provided. The wound animal model may include a wound that has been in contact with a composition comprising a scaffold and an aging inducer. The wound animal model may be suitable for evaluating wound healing, such as poor / delayed wound healing.

[0066] This document discloses a method for identifying candidate therapeutic agents for treating wounds, the method comprising a) generating a wound model in an animal according to a method defined herein, and b) contacting the wound model with a candidate therapeutic agent. The method may include testing the candidate therapeutic agent on the wound to determine whether the candidate therapeutic agent is safe and / or effective for treating wounds, such as in humans.

[0067] This document also provides compositions for inducing wound model formation in animals. This document discloses compositions comprising a scaffold and an aging inducer. This document discloses compositions comprising one or more polymers and an aging inducer. This document discloses a composition for inducing wound model formation in animals, the composition comprising one or more polymers and an aging inducer.

[0068] This document also provides a method for preparing compositions as defined herein. The method may include mixing (or soaking) one or more polymers with an aging inducer. The method may also include mixing (or soaking) one or more polymers in a solution containing an aging inducer. The solution may, for example, be a Pluronic gel (e.g., 30% Pluronic gel). The solution may contain BCM or about 100 μM of FK866.

[0069] This article discloses a dermal substitute for inducing wound formation in animals, the dermal substitute comprising one or more polymers and an aging inducer.

[0070] As used herein, when referring to values ​​or amounts of mass, weight, time, volume, concentration, or percentage, the term “about” is intended to cover variations of ±20% from a specified amount in some embodiments, ±10% from a specified amount in some embodiments, ±5% from a specified amount in some embodiments, ±1% from a specified amount in some embodiments, ±0.5% from a specified amount in some embodiments, and ±0.1% from a specified amount in some embodiments, because such variations are suitable for performing the disclosed methods.

[0071] Throughout this specification and the following description, unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” shall be understood to imply inclusion of the specified integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0072] References to any prior publications (or information derived therefrom) or any known matter in this specification are not, and should not be construed as, an acknowledgment or endorsement or any implication in any way that such prior publications (or information derived therefrom) or known matter constitute part of the general knowledge in the relevant field of this specification.

[0073] Those skilled in the art will understand that variations and modifications can be made to the invention described herein, in addition to the specific descriptions. It should be understood that the invention includes all such variations and modifications falling within its spirit and scope. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of any two or more of the stated steps or features.

[0074] Some embodiments of the present invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the general scope of the above description.

[0075] Example

[0076] method

[0077] An incision was made in the back of a rat or pig, and a biopsy was performed (6 mm for rats, 10-12 mm for pigs). An electrospun scaffold (80% PCL and 20% rat tail collagen) was then inserted into the wound. The scaffold was soaked in an aging-inducing agent. This could be Staphylococcus aureus biofilm conditioned medium (BCM) or a small molecule drug (FK866). The wound was then covered with a paraffin-embedded dressing, followed by Opsite, and the scaffold was secured in place with an adhesive crepe bandage. The aging-inducing agent was reapplied to the wound for the first three days. After five or ten days, the scaffold was removed to allow the wound to heal.

[0078] Acute wounds of this size in rats and pigs typically heal completely within ten days. However, the poorly healed wounds generated in our model healed much more slowly and exhibited known characteristics of chronic wounds in humans. Granulation tissue formed very rapidly in these wounds, but the epidermis remained excessively thickened over a considerable distance around the wound. Epithelial cells at the wound margins failed to migrate, and these wounds remained open even 10 days (for rats) or 30–35 days (for pigs) after scaffold removal. These cells also showed significant upregulation of Cx43, which is seen in chronic wounds in humans and is known to inhibit cell migration. Inflammation remained high in the granulation tissue and in the surrounding intact tissue, which was filled with neutrophils and macrophages. Giant cells were observed where macrophages fused together. The upper dermal vessels were highly inflamed, with numerous neutrophil extravasations visible. Extracellular matrix degradation was observed in these areas. Many cells within the granulation tissue and surrounding intact tissue were positive for senescence markers.

[0079] in conclusion

[0080] The inventors unexpectedly discovered that rat tail collagen induces a severe inflammatory response in rat wounds. They believe that rat tail collagen has high biocompatibility because it is derived from rats. This also leads to excessive thickening of the epidermis at the wound edge, preventing migration, and expressing high levels of Cx43, a known inhibitor of migration (as seen in chronic human wounds).

[0081] Subsequent studies examined the effects of biofilm conditioned medium (BCM) on wound healing. The medium can be toxic, but if cells survive, they may change appearance, becoming larger, flatter, and accumulating granules in the cytoplasm. Cellular senescence has been observed. Adding BCM to the scaffold induced senescence in and around the wound, affecting fibroblasts, endothelial cells, and leukocytes. Adding senescence-inducing drugs had similar effects.

[0082] In one set of experiments, wounds were covered with an airtight paraffin membrane to keep the medication in place. This had the unexpected effect of inducing excessive epidermal thickening and cell migration failure. Placing the paraffin membrane on the wound scaffold exacerbated the excessive thickening.

[0083] All these treatments combined mimic several characteristics of chronic human wounds. The inventors did not anticipate that the impact on wound closure would be as significant as the impact it would have on wounds that remained open for 30-35 days after stent removal.

[0084] References

[0085] Ewald JA et al., Therapy-induced senescence in cancer, J Natl Cancer Inst 102(20):1536–1546 (2010).

[0086] -Pietrasik, J. et al., Mechanisms and significance of therapy-induced and spontaneous senescence of cancer cells. Cell. Mol. Life Sci. 77, 213–229 (2020).

Claims

1. A method for generating a wound model in animals, the method comprising a) contacting the wound with a composition comprising: i) an electrospun scaffold comprising collagen and a synthetic polymer; and ii) an aging inducer. in, The synthetic polymer is selected from one or more of polycaprolactone, copolymers of lactic acid and glycolic acid, polylactic acid, and poly(glycolic acid), and The aging inducer is a biofilm conditioned medium or a nicotinamide phosphoribosyltransferase inhibitor.

2. The method of claim 1, wherein, The wound model described is a poor wound model.

3. The method of claim 1 or 2, wherein, The scaffold comprises polycaprolactone and collagen.

4. The method of claim 3, wherein, The scaffold contains 80% w / w polycaprolactone and 20% w / w collagen.

5. The method of claim 1 or 2, wherein, The collagen in question is rat collagen.

6. The method according to claim 1, wherein, The nicotinamide phosphoribosyltransferase inhibitor is Daporinad.

7. The method according to claim 1 or 2, wherein, The method includes creating a wound in the animal prior to step (a).

8. The method according to claim 7, wherein, The wound was created through a puncture biopsy.

9. The method according to claim 1 or 2, wherein, The method includes sealing the wound.

10. The method according to claim 1 or 2, wherein, The method involves contacting the wound with the aging inducer once a day for three days.

11. Use of a composition in the preparation of an animal model of a wound for identifying candidate therapeutic agents for treating wounds, wherein, The composition comprises: i) an electrospun scaffold comprising collagen and a synthetic polymer; and ii) an aging inducer. The synthetic polymer is selected from one or more of polycaprolactone, copolymers of lactic acid and glycolic acid, polylactic acid, and poly(glycolic acid). The aging inducer is a biofilm conditioned medium or a nicotinamide phosphoribosyltransferase inhibitor.

12. A composition for inducing wound formation in animals, the composition comprising: i) an electrospun scaffold comprising collagen and a synthetic polymer; and ii) an aging inducer. in, The synthetic polymer is selected from one or more of polycaprolactone, copolymers of lactic acid and glycolic acid, polylactic acid, and poly(glycolic acid), and The aging inducer is a biofilm conditioned medium or a nicotinamide phosphoribosyltransferase inhibitor.

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