Method of treating a wound and method of manufacturing a skin graft

Abstract

Synthetic skin constructs are produced using 3D bioprinting and a cleaning process involving supercritical carbon dioxide (ScCO2). The ScCO2 process effectively removes all cellular materials, minimizing the risk of immune rejection and ensuring the skin constructs are safe and optimally effective.

Description

PATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT)METHOD OF TREATING A WOUND AND METHOD OF MANUFACTURING A SKIN GRAFTCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present International Application claims priority to U.S. Provisional Patent Application No. 63 / 685,448, filed on August 21, 2024, and U.S. Provisional Patent Application No. 63 / 768,242, filed on March 7, 2025, the entire disclosures of each of which are incorporated by reference herein.FIELD

[0002] The present disclosure relates to novel synthetic skin grafts, methods for making such skin grafts, and methods for treating patients with such skin grafts.BACKGROUND

[0003] The present disclosure is directed to treatments for severe skin injuries, including bums, acute and chronic wounds (e.g., diabetic ulcers) and surgical wounds as well as skin diseases that are subject to ongoing skin loss and wounding such as epidermolysis bullosa. Each year, millions of patients suffer from these conditions, leading to prolonged recovery times, significant scarring, and substantial healthcare costs, estimated to be as high as $96.8 billion annually in the U.S. alone. Traditional treatments, such as autologous skin grafts, are limited by donor site availability and can result in complications like poor graft incorporation, infection at the recipient as well as donor site and poor healing and scarring at the donor site.

[0004] Improved grafts for treating these skin injuries, leading to reduced scarring and possessing improved integration and non-rejection by the patient, remain desirable.SUMMARY

[0005] In accordance with the present disclosure, novel synthetic skin grafts are provided, methods for preparation thereof, and their use. In embodiments, a method of preparing a synthetic skin construct of the present disclosure includes fomiing a synthetic bottom vasculature layer, forming a synthetic middle dermal layer on the bottom vasculature layer, forming a synthetic top epidermal layer on the synthetic middle dermal layer, treating the synthetic skinPATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT) construct with supercritical carbon dioxide to decellularize the synthetic skin construct, and sterilizing the synthetic skin construct for use.

[0006] In embodiments, the synthetic bottom vasculature layer, the synthetic middle dermal layer, and the synthetic top epidermal layer are formed of a biocompatible polymer in combination with cells selected from fibroblasts, keratinocytes, vascular cells, mural cells, dental pulp stem cells, human umbilical vein endothelial cells, or combinations thereof.

[0007] In some embodiments, the biocompatible polymer includes a natural polymer selected from gelatin, collagen, alginate, chitosan, fibrin, fibrinogen, or combinations thereof.

[0008] In other embodiments, the biocompatible polymer includes a synthetic polymer selected from polyethylene glycol, poly (lactic-co-glycolic acid), poly(e-caprolactone), or combinations thereof.

[0009] In embodiments, the synthetic bottom vasculature layer includes a combination of collagen, fibrinogen, and fibrin, embedded with dental pulp stem cells, dermal fibroblasts, pericytes, or dermal pre-adipocytes, and human endothelial cells, the synthetic middle dermal layer includes collagen embedded with human fibroblasts, and the synthetic top epidermal layer includes keratinocytes.

[0010] In embodiments, 3D bioprinting is used to apply the biocompatible polymer in combination with the cells to form the synthetic bottom vasculature layer, the synthetic middle dermal layer, the synthetic top epidermal layer, or any combination thereof.

[0011] In some embodiments, treating the synthetic skin construct with supercritical carbon dioxide to decellularize the synthetic skin construct occurs under pressures from about 1500 psi to about 5000 psi, at a temperature from about 25°C to about 55°C, for a period of time from about 1 hour to about 6 hours.

[0012] In other embodiments, treating the synthetic skin construct with supercritical carbon dioxide to decellularize the synthetic skin construct occurs in the presence of ethanol as a cosolvent.

[0013] In embodiments, the methods of the present disclosure further include applying a sacrificial biomaterial embedded within the synthetic bottom vasculature layer, the synthetic middle dermal layer, the synthetic top epidermal layer, or any combination thereof, to mimic vasculature architecture within the synthetic skin construct.PATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT)

[0014] In some embodiments, treating the synthetic skin construct with supercritical carbon dioxide removes the sacrificial biomaterial from the synthetic skin construct.

[0015] In other embodiments, the method of the present disclosure further includes lyophilizing the synthetic skin construct prior to treating the synthetic skin construct with supercritical carbon dioxide.

[0016] In some embodiments, lyophilizing the synthetic skin construct occurs at temperatures from about -10°C to about -110°C, for a period of time from about 4 hours to about 36 hours, under pressures from about 0.001 mBar to about 0.005 mBar.

[0017] In embodiments, the method of the present disclosure further includes applying a patient’s autologous cells to the synthetic skin construct prior to grafting the synthetic skin construct to the patient.

[0018] In embodiments, the autologous cells are selected from keratinocytes, fibroblasts, melanocytes, vascular cells, mural cells, or combinations thereof.

[0019] In other embodiments, a method of preparing a synthetic skin construct includes forming a synthetic bottom vasculature layer, forming a synthetic middle dermal layer on the bottom vasculature layer, forming a synthetic top epidermal layer on the synthetic middle dermal layer, lyophilizing the synthetic skin construct; treating the synthetic skin construct with supercritical carbon dioxide under pressures from about 1500 psi to about 5000 psi, at a temperature from about 25°C to about 55°C, for a period of time from about 1 hour to about 6 hours to decellularize the synthetic skin construct, and sterilizing the synthetic skin construct for use, wherein the synthetic bottom vasculature layer, the synthetic middle dermal layer, the synthetic top epidermal layer, or any combination thereof are formed using 3D bioprinting of a biocompatible polymer in combination with cells selected from fibroblasts, keratinocytes, vascular cells, mural cells, dental pulp stem cells, human umbilical vein endothelial cells, or combinations thereof.

[0020] In embodiments, the biocompatible polymer is selected from gelatin, collagen, alginate, chitosan, fibrin, fibrinogen, polyethylene glycol, poly(lactic-co-glycolic acid), poly(e- caprolactone), or combinations thereof.

[0021] In some embodiments, the method further includes applying a sacrificial biomaterial embedded within the synthetic bottom vasculature layer, the synthetic middle dermal layer, thePATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT) synthetic top epidermal layer, or any combination thereof to mimic vasculature architecture within the synthetic skin construct, wherein treating the synthetic skin construct with supercritical carbon dioxide removes the sacrificial biomaterial from the synthetic skin construct.

[0022] In some embodiments, the method of the present disclosure includes applying a patient’s autologous cells selected from keratinocytes, fibroblasts, melanocytes, vascular cells, mural cells, or combinations thereof to the synthetic skin construct prior to grafting the synthetic skin construct to the patient.

[0023] The present disclosure also provides synthetic skin constructs prepared by the methods disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures A and IB depict collagen fiber architecture of skin constructs before ScCCh treatment; and

[0025] Figures 2 A and 2B depict collagen fiber architecture of the skin constructs after the ScCCh treatment.DETAILED DESCRIPTION

[0026] The following detailed description of embodiments of the present disclosure will be made in reference to the disclosure in elements within the purview of the skilled artisan. In describing the embodiments of the present disclosure, explanation about related functions or constructions known in the art are omitted for the sake of clearness in understanding the concept of the disclosed invention to avoid obscuring the invention with unnecessary detail.

[0027] Moreover, technical features described herein according to one exemplary configuration of the disclosure may be applicable to other exemplary configurations of the disclosure, and thus duplicative descriptions may be omitted herein.

[0028] In accordance with the present disclosure, synthetic, ready-to-use, prevascularized skin constructs are provided. As used herein, the terms “skin construct” and “skin graft” are used interchangeably, with the term “skin graft” encompassing the synthetic “skin construct” of the present disclosure. These advanced skin constructs are engineered with a network of tiny blood vessels using two groundbreaking technologies: three-dimensional printing with biological materials (“3D bioprinting”) and a special cleaning process involving supercritical carbonPATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT) dioxide (ScCO2). The ScCO2 process effectively removes all cellular materials, minimizing the risk of immune rejection and ensuring the skin constructs are safe and optimally effective.

[0029] In accordance with the present disclosure, a skin construct is first prepared from non- autologous human cells. Suitable cells for use in preparing the skin construct of the present disclosure include fibroblasts, keratinocytes, vascular cells, mural cells, dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs), combinations thereof, and the like. Such cells are readily available from commercial sources.

[0030] Once obtained, the non-autologous human cells may be cultured to expand the cell population and permit the use of these cells in forming the skin construct of the present disclosure. Methods for culturing these cells to increase the cell population are readily apparent and known. In general, the cells are cultured in a suitable growth medium at a suitable temperature for a suitable period of time and obtained from the culture medium by centrifugation, filtration, combinations thereof, and the like. For example, in some embodiments, the cells may be cultured in a medium such as Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO BRL / Life Technologies). In some cases, the culture media may be supplemented with other biologic additives, such as Ham’s F-12 medium, fetal bovine serum (FBS), adenine, antibiotics such as penicillin and / or streptomycin, growth factors such as epidermal growth factor, combinations thereof, and the like, with additional additives that are acceptable for use in culture media. The cells may be incubated in the culture media for a suitable time, at a suitable temperature, in embodiments at about 37°C for several days, under humid conditions.

[0031] Exemplary culture media for the above cells include:

[0032] Human Fibroblasts: DMEM (10567-014) with 10% FBS and 1% penStrep (penicillinstreptomycin).

[0033] HUVEC : Endothelial Growth Medium From Lonza . Cat (CC-4176, CC-3156).

[0034] DPSCs: MEM a (cat: 12561049) with 10% FBS and 1% penStrep.

[0035] Keratinocyte: 46% DMEM (10567-014), 46% DMEM-F12(11320033), 0.1% epidermal growth factor, 0.1% cholera toxin, 0.1% insulin, 0.1% Adenine, 0.4% Hydrocortisone, 5% FBS, 1% penStrep.PATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT)

[0036] Once obtained, the cells to be utilized to form the skin construct of the present disclosure are utilized to prepare bioinks. These bioinks, in turn, are used in conjunction with a 3D bioprinting device to fomi the skin constructs of the present disclosure.

[0037] To prepare the bio ink, the cells described above are combined with a suitable carrier medium and placed into a printer cartridge which, in turn, is placed into the 3D printer used to form the skin construct of the present disclosure. Suitable carrier materials are within the purview of those skilled in the art and include both natural and synthetic polymers. Natural polymers which may be used as the carrier medium include those found in the extracellular matrix (ECM), as well as those extracted from suitable host organisms. In aspects, natural polymers which may be used as the carrier material in forming the bioink include gelatin, collagen, alginate, chitosan, fibrin, fibrinogen, combinations thereof, and the like. These natural polymers generally have a high water content, which is similar to native ECM. They also are biocompatible, degradable and possess low immunogenicity, making them good candidates for use in 3D bioprinting.

[0038] In some cases, especially where enhanced mechanical properties, for example strength, are desired, synthetic polymers may be used as the carrier material to form the bioink. Suitable synthetic polymers which may be used as the carrier medium include both nonbiodegradable and biodegradable polymers. Nonbiodegradable synthetic polymers which may be utilized include polyethylene glycol (PEG). Biodegradable synthetic polymers which may be used include poly(lactic-co-glycolic acid) or poly(e-caprolactone). The above synthetic polymers may, in some embodiments, be combined with other materials to enhance their properties.

[0039] Suitable concentrations of cells in the carrier medium may be from about 0.25e4 cells / mL to about 6e6 cells / mL, in embodiments from about le6 cells / mL to about 3e6 cells / mL

[0040] Suitable 3D printing systems for use in forming the skin construct of the present disclosure are commercially available and within the purview of those skilled in the art. Such 3D printing systems include droplet-based bioprinting, which originates from inkjet printing technology, and extrusion-based bioprinting.

[0041] Droplet-based bioprinting includes three major categories: inkjet bioprinting; laser- assisted bioprinting; and electrohydrodynamic jet bioprinting. Inkjet bioprinting can be further divided into two categories: continuous-inkjet bioprinting and drop-on-demand inkjetPATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT) bioprinting. Continuous- inkjet bioprinting relies on the tendency of a bioink solution to flow through a nozzle under pressure and subsequently shatter into continuous droplets. Electrically conductive drops of bioink can be directed to a precise location using electric and / or magnetic forces. For drop-on-demand inkjet bioprinting, pressure pulses are introduced through an actuator to dispense the bio ink. Continuous- inkjet bioprinters generate droplets at a relatively faster rate, while drop-on-demand inkjet bioprinters generate droplets when required.

[0042] Laser-assisted bioprinting relies on a laser-induced force to apply a bioink onto a substrate, including laser-guided direct writing and laser-induced forward transfer (LIFT) as well as emerging technologies derived mainly from LIFT (absorbing film-assisted LIFT, biological laser processing, etc.).

[0043] Electrohydrodynamic jet printing employs an electric field to pull the bioink through an orifice and does not require the use of high pressure to expel the bioink from the 3D printer. Bioink droplets are ejected when the electrostatic stresses overcome the surface tension at the orifice.

[0044] Extrusion-based bioprinting involves a bioink dispersing system with one or more printheads, a temperature control module and a program control platform. Extrusion-based bioprinting employs pneumatic pressure or mechanical (usually from a piston or screw) forces to disperse a premixed cell- laden bioink from the nozzle to a collector substrate, such as a plate, or a previous biological substrate, such as a sheet formed of collagen, fibrinogen, fibrin, or the like. The printhead, together with printing cartridge, provides movement in x-, y- and z- axes. Filaments are generated according to pre-designed 3D CAD models layer-by-layer to form the desired construct.

[0045] Other bioengineering technologies, e.g., coaxial bioprinting, multi-material bioprinting and microfabrication technologies, can be combined with extrusion-based bioprinting for skin bioengineering, including the formation of the vascularized skin constructs of the present disclosure.

[0046] As noted above, in aspects of the present disclosure, the skin construct is prepared to possess a vascularized architecture. The vascularized architecture is formed, in embodiments, using sacrificial materials. For example, a sacrificial biomaterial may be administered alongPATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT) with the cells and carrier medium used to form the bioink, to embed the sacrificial biomaterial within the printed structure, followed by dissolving the sacrificial biomaterials from the skin construct of the present disclosure to form hollow channels therein, which results in the vascularized architecture of the skin construct of the present disclosure. In some cases, gelatin may be used as the sacrificial biomaterial.

[0044] In other embodiments, the skin construct of the present disclosure may be perfused with vascular endothelial cells (ECs), which can adhere to the channels formed within the 3D printed skin construct and further assist in the formation of vascular- like structures.

[0047] Alternatively, coaxial bioprinting may be used to form vascularized skin constructs.

[0048] To further enhance the vascularization of the resulting skin construct, as well as the fomiation of blood vessels in vivo, growth factors may be added to the skin construct. The physical structure of the skin construct may also be adjusted to promote vascularization. For example, in some embodiments, vascular endothelial growth factor, basic fibroblast growth factor, platelet derived growth factor, epidermal growth factor, insulin-like growth factor, and / or stem cell-derived exosomes may be added to the skin construct for delivery to a marginally vascularized tissue bed and / or to promote vascularization, tissue healing, and skin construct incorporation. Moreover, as ECs can sense mechanical signals and micro topography of the 3D environment, a stiffer matrix may inhibit migration of ECs and formation of microvascular assembly. Alternatively, uniaxially aligned scaffolds can improve vascularization using porous freeze-dried collagen scaffolds compared with randomly oriented pores.

[0049] Upon application in vivo, vascular cells from the patient will migrate into the hollow channels of the decellularized skin construct of the present disclosure, further promoting the vascularization of the skin construct described herein and angiogenesis.

[0050] The bioinks described above, including the cultured cells in the carrier medium, are respectively filled into cartridges of a 3D printer, and printing cartridges used by the respective are prepared.

[0051] In some embodiments, decellularized pre -vascularized skin constructs of the present disclosure include a multi-layered structure to mimic native skin architecture. As noted above,PATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT) the skin construct is prepared using 3D bioprinting, with a synthetic bottom vasculature layer first formed of a collagen-fibrin extracellular matrix (ECM) embedded with dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) to promote vasculogenesis and angiogenesis. A synthetic middle dermal layer is then applied thereto, the middle layer including a collagen ECM embedded with human fibroblasts, providing structural integrity and supporting epidermal development. Finally, a synthetic top epidermal layer is formed by printing a keratinocyte cell slurry onto the construct, which is then cultured for 21 days to allow maturation.

[0052] In other embodiments, as noted above, the skin construct of the present disclosure may be prepared with a sacrificial material, such as gelatin, to form channels therein which mimic the vascular architecture of skin. In embodiments, such a skin construct may include a synthetic bottom vasculature layer first formed of a collagen-fibrin ECM embedded with dental pulp stem cells DPSCs. Gelatin in combination with HUVECs may be used as a sacrificial bioink to facilitate precise vascular architecture.

[0053] In some embodiments, horizontal channels may be formed by depositing the gelatin / HUVEC bioink along the synthetic bottom vasculature layer, followed by deposition of an upper vasculature layer, formed of the same materials used to form the synthetic bottom vasculature layer, i.e., a collagen-fibrin ECM embedded with dental pulp stem cells DPSCs. The upper vasculature layer is allowed to cross-link at room temperature, with the sacrificial bioink remaining in place in the pattern of the applied vasculature architecture.

[0054] Alternatively, vertical channels may be formed in the lower vasculature layer to form a vertical vasculature architecture by injecting the gelatin / HUVEC bioink under sufficient pressure into the collagen-fibrin ECM embedded with DPSCs vasculature layer, thereby forming vertical vascular patterns therein. In embodiments, combinations of these techniques may be utilized to form an artificial vascular architecture that mimics the vasculature found in skin.

[0055] The cellular and nuclear components of the cells used in forming the skin constructs of the present disclosure may lead to immune reactions in vivo, impacting the repair and therapy effects desired when using the skin constructs of the present disclosure. Accordingly, inPATENT APPLICATIONAtty. Docket: 050-9472 (51801-1 PCT) aspects, the skin construct of the present disclosure is subjected to a decellularization process to remove as many immunogenic components as possible from the skin construct. Suitable decellularization techniques include physical, chemical, biological, and combined procedures. In embodiments, the skin construct is subjected to a treatment with supercritical carbon dioxide (ScCO2) to decellularize the skin construct, which will enhance integration of the skin construct and minimize host rejection, while maintaining the architecture of the skin construct of the present disclosure.

[0056] Moreover, as noted above, in some embodiments sacrificial materials may be included in one of the bioink compositions used to form the skin construct of the present disclosure. Where a sacrificial biomaterial is administered along with the cells and carrier medium bioink to embed the sacrificial biomaterial within the printed structure, the treatment with ScCO2, in addition to decellularizing the skin construct, may also remove the sacrificial biomaterials from the skin construct to form hollow channels therein, which results in the vascularized architecture of the skin construct of the present disclosure. Moreover, the resulting skin construct, in embodiments, maintains growth factors therein.

[0057] In embodiments, decellularization by treatment with ScCO2 may occur under pressures from about 1500 pounds per square inch (psi) to about 5000 psi, in embodiments from about 2000 psi to about 3000 psi, in embodiments about 2455 psi, at a temperature from about 25°C to about 55°C , in embodiments from about 35°C to about 45°C , in embodiments about 40°C, for a period of time from about 1 hour to about 6 hours, in embodiments from about 2 hours to 4 hours in embodiments for about 3 hours. In some cases, the decellularization may occur in the presence of a co-solvent, such as ethanol.

[0058] In some embodiments, the skin construct of the present disclosure may be exposed to ScCO2 followed by rapid depressurization (RDP) in order to facilitate enhanced removal of DNA and cellular debris.

[0059] Suitable equipment for use in carrying out the ScCO2 treatment include the Helix SFE Version R3U from (Applied Separations Inc., Allentown, PA, USA), as well as ScCO2 equipment sold by the Parr Instrument Company.PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)

[0060] In some embodiments, prior to treating the skin construct of the present disclosure with ScCO2, the skin construct may be subjected to a lyophilization step. Lyophilization methods and equipment used for such lyophilization are within the purview of those skilled in the art. The lyophilization step results in dehydration of the skin construct of the present disclosure prior to its treatment with ScCO2. Suitable temperatures for lyophilization are from about - 10°C to about

[0061] -110°C, in embodiments from about -25°C to about -75°C , in embodiments about - 50°C, for a period of time from about 4 hours to about 36 hours, in embodiments from about 18 hours to about 30 hours, in embodiments about 24 hours. Lyophilization optimally occurs under low pressure, in embodiments from about 0.001 mBar to about 0.005 mBar.

[0062] It has been surprisingly been found the lyophilizing the skin construct of the present disclosure prior to decellularization of the skin construct with ScCO2 enhances the decellularization of the skin construct.

[0063] After the decellularization step, the skin construct may then be sterilized for use. For example, in some embodiments, the skin construct may be placed in ScCO2 with peracetic acid (PAA) for sterilization. Other sterilization techniques known to those skilled in the art may be used to sterilize the skin constructs of the present disclosure.

[0064] The resulting skin construct is a decellularized pre-vascularized skin substitute that serves as a biomimetic scaffold, promoting revascularization, minimizing immune response, and facilitating skin regeneration for advanced wound healing applications.

[0065] In some embodiments, autologous cells from the patient to which the skin construct of the present disclosure is to be applied may be added to the skin construct prior to grafting. For example, in embodiments keratinocytes (KCs), fibroblasts (FBs), melanocytes, vascular cells, and / or mural cells may be obtained from the patient, cultured, where necessary to increase their number, and then administered to the skin construct prior to grafting through the use of cell spray technology. Suitable cell spray apparatuses and methods for using same to apply cells to tissue and / or other substrates, including the skin constructs disclosed herein, are within the purview of those skilled in the art. For example, in some embodiments, the RECELL® Autologous Cell Harvesting Device (ACHD), which can expand a small biopsy up to 80 timesPATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT) its original area, can be used to apply a cell suspension derived from cells from the patient onto the skin construct of the present disclosure. The use of the spray-cell technology may not only provide extensive wound coverage but also promote regenerative healing processes, reducing scarring.

[0066] The application of autologous cells to the skin construct of the present disclosure may enhance integration and non-rejection of the skin construct of the present disclosure.

[0067] In embodiments, Digital Image Speckle Correlation (DISC), may be used in both the preparation and application of the skin construct of the present disclosure. DISC is a non-invasive method for quantifying wound healing progress through spatially resolved imaging of mechanical properties. Unlike conventional imaging techniques, DISC provides a real-time, quantitative assessment of tissue stiffness and strain dynamics, offering valuable insights into wound healing and skin construct integration without the need for invasive biopsies.

[0068] In general, the DISC approach utilizes a controlled mechanical deformation applied to the skin, using a tensiometer modified for direct imaging. Consecutive video images may be captured, allowing for digital image correlation algorithms to track natural surface features (e g., pores, hair follicles) as speckles to measure displacement and strain. Differences in mechanical properties distinguish healthy, healing, and fully integrated tissue.

[0069] The DISC approach provides various advantages when used in the preparation and application of the skin construct of the present disclosure to a patient. Such advantages include:• Non-invasive assessment of scaffold integration and wound healing;• Biomechanical optimization of engineered skin constructs;• Real-time monitoring to reduce reliance on invasive biopsies; and,• Long-term tracking of tissue remodeling and scarring prevention.

[0070] The methods of the present disclosure address a significant unmet need in the treatment of severe skin injuries, including burns, chronic wounds (e.g., diabetic ulcers) and surgical wounds. Each year, millions of patients suffer from these conditions, leading to prolonged recovery times, significant scarring, and substantial healthcare costs, estimated to be as high as $96.8 billion annually in the U.S. alone. Traditional treatments, such as autologous skin grafts,PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT) are limited by donor site availability and often result in complications like poor vascularization and immune rejection.

[0071] The method provides an advanced alternative to traditional skin grafts by creating prevascularized, biocompatible skin constructs using 3D bioprinting and ScCO2 decellularization. These skin constructs are engineered to promote faster and more effective wound healing, particularly in patients with compromised blood circulation, such as those with diabetes or peripheral arterial disease. The integration of autologous cell-spray technology further enhances the grafts' ability to reduce scarring and improve cosmetic and functional outcomes. This technology has the potential to revolutionize wound care by reducing recovery times, minimizing complications, and improving patient quality of life, thereby addressing a critical gap in the current market for advanced wound healing solutions.

[0072] In some embodiments, the skin construct described herein may be turned into a free flap (vascularized tissue with a source artery and vein) by placing it under a surgically created arteriovenous fistula to create a vascularized skin flap.

[0073] Embodiments of the invention described herein include the creation of pre-vascularized skin constructs using advanced 3D bioprinting and supercritical carbon dioxide (ScCO2) decellularization. The 3D bioprinting process constructs intricate vascular networks within the skin constructs, enhancing integration and healing. ScCO2 decellularization removes cellular material while preserving the extracellular matrix, minimizing immune rejection. The technology also integrates autologous cell-spray methods, promoting faster, scar-free healing, particularly in poorly vascularized wounds.

[0074] In certain embodiments, the method provides a ready-to-use, pre-vascularized skin constructs for treating severe burns, surgical wounds, and acute and chronic ulcers, offering faster healing and reduced scarring compared to traditional grafts.

[0075] In certain embodiments, the method provides a customizable kit containing prevascularized skin constructs and autologous cell-spray devices tailored to individual patient needs for use in home care settings or outpatient clinics.

[0076] Embodiments of the invention described herein provide a method of treating a wound and method of manufacturing a skin construct. The methods described herein offer innovativePATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT) solutions for advanced wound care, particularly in the treatment of severe bums, acute traumatic wounds, chronic wounds like diabetic ulcers, skin conditions such as epidermolysis bullosa, and surgical wounds. By creating pre-vascularized, biocompatible skin constructs through cutting-edge 3D bioprinting and specialized decellularization techniques, it accelerates healing, reduces scarring, and enhances patient outcomes. The technology is versatile and can be applied in various healthcare settings, including hospitals, bum centers, and outpatient clinics, as well as in emergency and military medical scenarios. It has the potential to significantly improve the quality of life for patients with challenging wounds and reduce overall healthcare costs by promoting faster recovery and minimizing complications.

[0077] In certain embodiments, the method provides a specialized skin construct designed to treat diabetic foot ulcers, featuring enhanced vascularization to promote healing in patients with poor circulation.

[0078] In certain embodiments, the method provides a skin construct aimed at reducing scarring and improving aesthetic outcomes in reconstructive surgery, including facial reconstruction and scar revision procedures.

[0079] In certain embodiments, the method provides a portable kit containing pre-vascularized skin constructs for immediate application in emergency situations, such as burn injuries in conflict zones or disaster areas.

[0080] In certain embodiments, the method provides a skin construct designed for use in veterinary medicine to treat severe wounds in pets, livestock, and wildlife, promoting faster healing and reducing the need for more invasive procedures.

[0081] In certain embodiments, the method provides a research-grade skin construct and scaffolds for use in preclinical studies and drug testing, allowing researchers to study wound healing and tissue regeneration in controlled environments.

[0082] In certain embodiments, the method provides on-demand custom skin constructs and cell- spray treatments for patients with chronic, non-healing wounds, with regular monitoring and adjustments to the treatment plan.PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)

[0083] In certain embodiments, the method provides on-demand custom skin constructs and cell- spray treatments for patients with epidermolysis bullosa that are subject to lifelong recurrent wounds, with regular monitoring and adjustments to the treatment plan.

[0084] In certain embodiments, the method provides an advanced wound care solution designed for use in extreme environments, such as on the battlefield or in space, where rapid and effective treatment of severe injuries is critical.

[0085] In certain embodiments, the method provides a training module and workshop focused on the use of advanced skin constructs and wound healing technologies, helping to educate the next generation of surgeons and healthcare professionals.

[0086] In certain embodiments, the method provides an over-the-counter product, such as advanced bandages or topical treatments, incorporating elements of the technology to enhance wound healing for minor cuts, abrasions, and burns.

[0087] In certain embodiments, use of 3D bioprinting to create intricate vascular networks within the skin constructs offers superior vascularization compared to traditional grafts and commercial skin constructs. This enhances the integration of the graft with the host tissue, particularly in poorly vascularized wound beds, leading to faster and more effective healing.

[0088] In certain embodiments, a supercritical carbon dioxide (ScCO2) decellularization process effectively removes all cellular materials while preserving the extracellular matrix (ECM). This minimizes the risk of immune rejection, a common issue with many current skin constmcts and tissue- engineered products.

[0089] In certain embodiments, by optimizing the mechanical properties and biochemical cues within the scaffold, the method promotes regenerative healing rather than fibrotic healing. This leads to significantly reduced scarring, improving both cosmetic and functional outcomes for patients.

[0090] In certain embodiments, the integration of bioprinting and autologous cell-spray technology allows for the creation of personalized skin constructs tailored to the specific wound characteristics of each patient. This level of customization is not offered by most existing products, providing a more precise and effective treatment option.PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)

[0091] In certain embodiments, the method can be used for a wide range of applications, including treating severe bums, acute and chronic wounds (e.g., diabetic ulcers), and surgical wounds. Its adaptability makes it suitable for various medical conditions and patient profdes, unlike some competing products that are more narrowly focused.

[0092] In certain embodiments, pre-vascularization and use of a patient’s own cells contribute to more rapid wound healing, reducing recovery times compared to traditional grafts or nonvascularized skin constructs. This can lead to shorter hospital stays and quicker return to normal activities for patients

[0093] In certain embodiments, the combination of advanced vascularization and minimized immune rejection results in a lower risk of complications such as graft failure or infection. This enhances patient outcomes and reduces the need for additional interventions.

[0094] In certain embodiments, although the technology involves advanced techniques, the potential for faster healing, fewer complications, and reduced scarring can lead to overall cost savings in healthcare. This includes lower hospitalization costs, fewer follow-up procedures, and reduced need for long-term wound care.

[0095] In certain embodiments, by reducing scarring and promoting better functional outcomes, your technology significantly enhances the quality of life for patients. This is particularly important for individuals with severe burns, acute or chronic wounds, where the psychological impact of scarring and prolonged healing can be substantial

[0096] In certain embodiments, the use of 3D bioprinting allows for the scalable production of skin constructs, making it easier to meet demand and offer a consistent product. This scalability is an advantage over more labor-intensive or limited-supply graft options.

[0097] In certain embodiments, the use of supercritical CO2 for decellularization is a more environmentally friendly alternative to traditional chemical methods. It reduces the use of harsh chemicals and minimizes waste, making the process safer and more sustainable.

[0098] In certain embodiments, the method is versatile enough to be used in various clinical settings, from emergency bum care to routine surgical procedures. This flexibility enhances its appeal to a wide range of medical professionals and institutions

[0099] Traditional methods of treating severe skin injuries often rely on autologous skin grafts, which, while highly effective due to their integration capabilities derived from the patient's own tissue, arePATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT) limited by donor site availability and patient morbidity. Accordingly, tire present disclosure provides several advantages:

[0100] I Advanced Vascularization Techniques. The recited method transcends the limitations of both autologous grafts and commercially available skin grafts by leveraging state-of-the-art 3D bioprinting technology to pre-form intricate vascular networks within the skin constructs before implantation. The 3D printing capabilities permits the creation of a vascularized skin construct with more vascular channels than native skin, potentially offering better incorporation in marginally vascularized wound beds. The disclosed tissue-engineered constructs as a viable alternative to autologous grafts, particularly in scenarios where traditional grafts are less likely to succeed due to inadequate vascularization at the wound site. By providing a scalable, highly vascularized option, the disclosed skin constructs aim to dramatically improve outcomes for patients with severe skin injuries, offering enhanced integration capabilities that could rival or even exceed those of autologous skin grafts in marginally vascularized wounds.

[0101] II. Supercritical CO2 Decellularization. The use of supercritical carbon dioxide (ScCO2) in decellularization is a pioneering method in the field of regenerative medicine. Unlike traditional cellularization techniques that often involve harsh chemicals or enzymatic treatments which can damage the structural and functional integrity of the extracellular matrix (ECM), ScCO2 provides a gentle yet effective alternative. This method ensures the complete removal of cellular material to minimize immune response after grafting without compromising the biomechanical and biochemical cues that are critical for tissue regeneration. ScCO2 decellularization preserves essential proteins and growth factors in the ECM, w hich are pivotal for successful recellularization and integration of the skin construct with the host tissue.

[0102] III. Integration of Bioprinting with ScCO2 Decellularization. The dual approach of combining 3D bioprinting with ScCO2 decellularization represents a significant advancement in skin tissue engineering. This integration results in highly customized, biocompatible, and prevascularized skin constructs that are ready for clinical use. The ability to tailor these constructs specifically to the needs of individual patients based on wound characteristics could revolutionize the treatment of acute and chronic wounds resulting from bums, trauma, or skin diseases.

[0103] IV. Minimization of Scarring. The disclosed method includes the use of optimized scaffold materials and bioactive compounds designed specifically to minimize scarring. By manipulating the mechanical properties and biochemical cues within the scaffold, cellular behaviors can be influenced toPATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT) promote regenerative rather than fibrotic healing. This not only enhances tire cosmetic and functional outcomes of the skin constructs but also significantly improves the quality of life for patients by reducing the physical such us hypertrophic scarring and scar contracture and psychological burden of scarring.

[0104] V. Autologous Cell-Spray Technology. Hie combination of the above described 3D printing and ScCO2 decellularization may also include the use of cell-spray technology, which uses a patient’s own cells. This method significantly improves the regenerative capabilities of the skin constructs, promoting healing and reducing scarring in a way that is not achievable with standard grafting techniques.

[0105] VI. Customization for Individual Needs. The disclosed methods permit tailoring treatments to individual patient needs. By combining bioprinting with ScCO2 decellularization, the investigation enables the production of customized, ready-to-use skin constructs that are both biocompatible and immediately functional.

[0106] The following Examples are being submitted to illustrate embodiments of the present disclosure. These Examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure. Also, parts and percentages are by weight unless otherwise indicated. As used herein, ‘Toom temperature” refers to a temperature of from about 20° C. to about 30° C.EXAMPLE 1

[0107] A variety of materials are tested for use in forming the skin construct of tire present disclosure, including collagen, fibrinogen, and / or fibrin, and the addition of growth factors (e.g., VEGF, EGF) to the skin construct are tested to optimize the vascular network formation.

[0108] 3D Bioprinting is utilized to fabricate skin constructs with designed vascular patterns, ensuring precise material and cell deposition.

[0109] The constructs are cultured under optimized in vitro conditions to promote the development and maturation of vascular networks.

[0110] In vitro analysis of the skin constructs is conducted to determine growth factors secretion. The secretion of growth factors such as VEGF and EGF is conducted using ELISA and Immunohistochemistry (IHC).

[0111] Capillary Network Density is analyzed using confocal microscopy.

[0112] Cell proliferation is quantified using DAPI and proliferation markers (Ki67).PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)

[0113] Animal model testing is conducted to detennine the integration of these constructs on nude rodent models with full-thickness wounds, using digital image speckle correlation (DISC) analysis. [001 1 1] Histological assessment is conducted by staining excised tissues with H&E, Trichrome, and CD31, anti-eGFP (to differentiate between rodent and human blood vessels) to evaluate integration and anastomosis.

[0114] Outcome Measures: the effectiveness of each vascular configuration confinns robust vascular networks, faster inosculation in animal models; and the examples will determine optimal biomaterial mixtures and growth factor concentrations resulting in minimal wound contraction and enhancing integration.EXAMPLE 2

[0115]

[0113] Bio-printed vascularized skin constructs prepared and evaluated as described above in Example 1 are subjected to ScCO2 decellularization to remove cellular components while aiming to preserve the extracellular matrix (ECM) and embedded growth factors. The bio-printed constructs described above in Example 1 are treated with ScCO2 using optimized parameters to balance cellular removal with ECM preservation.

[0116] The resulting treated skin constructs are tested in vivo using immunocompetent rodent models. Cellular residue testing is performed using DAPI staining and DNA assays to confirm the absence of any cellular material post-decellularization.

[0117] Growth Factor Analysis is performed using ELISA and IHC to detect the presence and concentration of key growth factors like VEGF and EGF.

[0118] Assessment of collagen's structural alignment, physical morphology, and biomechanical Stability is determined using techniques such as Picrosirius Red Staining, Scanning electron Microscopy (SEM), and Atomic Force Microscopy (AFM).

[0119] The decellularized constructs are implanted into regular rodent models (mice) to evaluate immune response and integration. The constructs are monitored for signs of immune rejection (swelling, redness), and integration.

[0120] Outcome Measures: Successful decellularization is determined as indicated by the absence of cellular material and preserved growth factors; integrity and functionality of the ECM as determined by mechanical and structural assessments; biocompatibility and integration of the constructs is determinedPATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT) in immunocompetent mouse models reviewing immune response and effectiveness in promoting tissue regeneration.EXAMPLE 3

[0121] Male and female Yorkshire pigs (3-5 months old; 30-60 kgs) with six full-thickness wounds (4 cm x 4 cm) each, created on the dorsal surface, are subjected to the following treatments.

[0122] Assign wounds to four treatment protocols: Cell Spray technology of autologous cells onto the wound (using RECELL® Autologous Cell Harvesting Device (ACHD)) alone (R), decellularized Tissue Engineered Vascularized skin construct (TEV) of Example 2 alone, R followed by TEV, and TEV followed by R.

[0123] Harvest a 12 cm2skin sample using a dermatome, process with ACHD, and spray the resultant cell suspension onto wounds. Assess wounds on Days 7, 14, 21, and 28 post-treatments. Scar fonnation is assessed using scoring systems (Vancouver Scar Scale (VSS) / Manchester Scar Scale (MSS)). histological analysis, and mechanical testing with durometers / cutometers to assess hardness and elasticity. Coverage Assessment is assessed by measuring wound closure rates and tissue quality and integration is examined through macroscopic and microscopic evaluations.

[0124] Outcome Measures: efficacy in scar minimization and wound coverage is observed, as is biological functionality of regenerated tissue.

[0125] Data Analysis. For Examples 1 and 2 above, quantitative data is analyzed using variance of mean and t-test analysis, while categorical data will be compared using the chi-square test of goodness of fit. Comparisons between different types of channels fonning the vascular architecture within the synthetic skin constructs are conducted in both in vivo and in vitro studies and, additionally, in vivo data is compared with in vitro results to evaluate consistency. Each mouse is tested with two constructs, and a sample size of 20 constructs is deemed sufficient based on preliminary data, resulting in ten mice used per group. For Example 3, which involves a pilot study with twelve pigs to test six skin constructs treatments, data will be analyzed using repeated measures ANOVA to assess the effects of each treatment across multiple time points. Each pig will serve as its own control, with post-hoc tests with Bonferroni correction applied to identify specific differences between treatment groups. Uris strategy aims to determine preliminary efficacy and optimize treatments.PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)EXAMPLE 4

[0126] Decellularization using Supercritical Carbon Dioxide (ScCCL).Cell Culture

[0127] Human umbilical vein endothelial cells (HUVECs; passage 7, ATCC) were cultured in Endothelial Growth Medium-2 (EGM-2: Lonza) and maintained at 37°C in a humidified 5% CO2 atmosphere. Dental pulp stem cells (DPSCs; passage 7. isolated under IRB protocol) were expanded in a-Minimum Essential Medium (a-MEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). Human dermal fibroblasts (passage 7, IRB-approved) were cultured in DMEM (Gibco, with 10% FBS and 1% penicillin-streptomycin.

[0128] Human kcratmocytcs (passage 4, IRB-approved) were cultured on mitomycin C-treated (irradiated) 3T3 fibroblast feeder layers in defined keratinocyte medium composed of DMEM / Glutamax and DMEM / F-12 (Gibco) supplemented with 5% FBS, 0.4 pg / mL hydrocortisone, 5 pg / mL insulin, 0.18 mM adenine, and 1% penicillin-streptomycin. Irradiated 3T3 cells were plated in advance to establish a supportive monolayer before keratinocyte seeding.

[0129] All cell types were maintained under standard culture conditions at 37°C with 5% CO2. Cells were passaged at approximately 80% confluency using TrypLE Express or 0.05% Trypsin-EDTA (Gibco). and media were replenished every 48 hours.Gel Preparation

[0130] Tire dermal compartment of the engineered skin construct was prepared in two sequential layers: a vascularized lower layer composed of HUVECs. DPSCs, and fibroblasts embedded in a collagen-fibrin matrix, and an upper dermal layer including fibroblasts suspended in a collagen-only matrix.

[0131] To fabricate the vascularized lower layer, type I collagen (Millipore; 4.6 mg / mL stock) was neutralized on ice using 10x DMEM (Gibco). sterile water. 7.5% NaHCCL. and L-glutamine to achieve a final collagen concentration of 1 mg / mL and physiological pH (7.0). Separately, fibrinogen (40 mg / mL) was dissolved in l x DMEM and mixed with thrombin (100 U / mL) at a final concentration of 0.5 U / mg fibrinogen to initiate enzymatic crosslinking into fibrin. The collagen and fibrin solutions were then combined on ice and immediately mixed with cells at the following final densities: 1 x 106HUVECs / mL, 2.5 x IQ5DPSCs / mL, and 2.5 x 105fibroblasts / mL (4: 1: 1 ratio). Tire 0.5 rnL gel-cellPATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT) mixture was dispensed into pre-chilled 24 mm Transwell inserts (Coming, Cat. 3450) and incubated at 37°C for 1 hour to allow full polymerization.

[0132] For the upper dermal layer, a collagen-only matrix (final concentration: 1.2 mg / mL) was prepared without fibrinogen or thrombin. Human fibroblasts were suspended in this solution at a density of 7.5 x 104cells per insert. The mixture was gently overlaid onto the solidified vascular gel and allowed to gel for 40 minutes at 37°C. To prevent matrix contraction and provide structural support, a sterile stainless-steel ring (Midwest Fastener, Cat. 05325) was placed on top of the constructs.

[0133] All steps were carried out under sterile conditions on ice. Thrombin was used exclusively in the vascularized layer to crosslink fibrinogen and was added immediately before casting to control the gelation timing.Construct Assembly

[0134] Skin constructs were assembled within 24 mm Transwell inserts (Coming. Cat. 3450) beginning with a thin acellular collagen base layer, which provided structural support. This was followed by sequential addition of the vascularized layer and the fibroblast-rich upper dermal layer, as previously described.

[0135] After polymerization, a sterile stainless-steel ring (Midwest Fastener. Cat. 05325) was placed atop the gel to prevent contraction and confine the area for keratinocyte seeding. Constructs were maintained in EGM-2 medium for 7 days to allow vasculature formation and demral maturation.

[0136] On Day 8. human keratinocytes (3 x 105cells per insert) were seeded as a slurry onto the construct surface. After 1 hour to allow attachment. 2 mb of JG1 medium was added to the insert and 3 mL to the well. Medium was transitioned to JG2 on Days 10-11. From Day 12 onward, constructs were cultured at the air-liquid interface using JG-AL medium (1.5 mL in the well) for an additional 6-10 days to promote keratinocyte stratification and fonnation of a multilayered epidermis.

[0137] Constructs were harvested at designated time points (6-10 days) and directly frozen in -80 °C for storage, for later ScCCL decellularization.ScCOz Treatment

[0138] The above skin constructs were treated with ScCCL for 2 hours at 37°C under 4000 psi, followed by DNase treatment (50 U / mL for 4 hours at 37°C).PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)

[0139] Staining with DAPI (4',6-diainidino-2-phenylindole) showed near-complete removal of nuclear content after treatment, confirming decellularization efficacy. Histological evaluation of the engineered skin constructs before and after ScCCE decellularization demonstrated preservation of extracellular matrix architecture. The pre-treatment (control) sections showed intact cellular content and dense nuclear staining, while the post-treatment sections exhibited absence of cellular nuclei and retention of the dermal collagen matrix. The structural integrity and organization of collagen fibers were maintained, as evidenced by continuous, undistorted fibrillar networks throughout the scaffold cross-sections.

[0140] SEM analysis of the skin constructs was also conducted before and after ScCCE decellularization. FIGS. 1A and IB depict collagen fiber architecture of tire skin constructs before the tScCOi treatment, while FIGS. 2A and 2B depict the collagen fiber architecture of the skin constructs after the ScCCE treatment. As is apparent from the Figures, the ScCCE treatment (4000 psi, 37°C, 2 hours) of the skin construct preserved scaffold ultrastructure, including the characteristic triple-helix collagen fibers. These findings support the compatibility of ScC'CE with engineered skin scaffold architecture.

[0141] In summary, the present disclosure introduces novel techniques and integrates advanced technologies to overcome longstanding barriers in the field of tissue engineering. Tire disclosed methods use of 3D bioprinting for vascular network formation and ScCO2 for gentle decellularization provides for the development of next-generation of tissue engineered skin constructs that promise better healing outcomes, reduced scarring, and higher patient satisfaction.

[0142] It will be understood that various modifications may be made to the aspects and features disclosed herein. Therefore, the above description should not be construed as limiting, but merely as exemplifications of various aspects and features. It will be understood by those skilled in tire art that various changes in from and details may be made therein without departing from the spirit and scope of the present invention and equivalents thereof.

Claims

PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)WHAT IS CLAIMED IS:

1. A method of preparing a synthetic skin construct, comprising: fonning a synthetic bottom vasculature layer; forming a synthetic middle dermal layer on the bottom vasculature layer; forming a synthetic top epidermal layer on the synthetic middle dermal layer; treating the synthetic skin construct with supercritical carbon dioxide to decellularize the synthetic skin construct; and sterilizing the synthetic skin construct for use.

2. Tire method of claim 1, wherein the synthetic bottom vasculature layer, the synthetic middle dermal layer, and the synthetic top epidermal layer are fonned of a biocompatible polymer in combination with cells selected from fibroblasts, keratinocytes, vascular cells, mural cells, dental pulp stem cells, human umbilical vein endothelial cells, or combinations thereof.

3. Tire method of claim 2, wherein the biocompatible polymer includes a natural polymer selected from gelatin, collagen, alginate, chitosan, fibrin, fibrinogen, or combinations thereof.

4. The method of claim 2, wherein the biocompatible polymer includes a synthetic polymer selected from polyethylene glycol, poly (lactic-co-glycolic acid), poly(e-caprolactone), or combinations thereof.

5. The method of claim 1, wherein the synthetic bottom vasculature layer includes a combination of collagen, fibrinogen, and fibrin, embedded with dental pulp stem cells, dermal fibroblasts, pericytes, or dermal pre-adipocytes, and human endothelial cells, the synthetic middle dermal layer includes collagen embedded with human fibroblasts, and the synthetic top epidennal layer includes keratinocytes.

6. The method of claim 2, wherein 3D bioprinting is used to apply the biocompatible polymer in combination with tire cells to form the synthetic bottom vasculature layer, the synthetic middle dermal layer, the synthetic top epidermal layer, or any combination thereof.PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)7. The method of claim 2, wherein treating the synthetic skin construct with supercritical carbon dioxide to decellularize the synthetic skin construct occurs under pressures from about 1500 psi to about 5000 psi, at a temperature from about 25 °C to about 55 °C, for a period of time from about 1 hour to about 6 hours.

8. The method of claim 1, wherein treating tire synthetic skin construct with supercritical carbon dioxide to decellularize the synthetic skin construct occurs in the presence of ethanol as a co-solvent.

9. The method of claim 2, further comprising applying a sacrificial biomaterial embedded within the synthetic bottom vasculature layer, the synthetic middle dermal layer, tire synthetic top epidermal layer, or any combination thereof, to mimic vasculature architecture within the synthetic skin construct.

10. The method of claim 9, wherein treating the synthetic skin construct with supercritical carbon dioxide removes the sacrificial biomaterial from the synthetic skin construct.11 . The method of claim 1, further comprising lyophilizing the synthetic skin construct prior to treating the synthetic skin construct with supercritical carbon dioxide.

12. The method of claim 11, wherein lyophilizing the synthetic skin construct occurs at temperatures from about -10°C to about -110°C, for a period of time from about 4 hours to about 36 hours, under pressures from about 0.001 mBarto about 0.005 mBar.

13. The method of claim 1, further comprising applying a patient’s autologous cells to the synthetic skin construct prior to grafting the synthetic skin construct to the patient.

14. The method of claim 13, wherein the patient’s autologous cells are selected from keratinocytes, fibroblasts, melanocytes, vascular cells, mural cells, or combinations thereof.PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)15. A synthetic skin construct prepared by the method of claim 1.

16. A method of preparing a synthetic skin construct, comprising: forming a synthetic bottom vasculature layer; forming a synthetic middle dermal layer on the bottom vasculature layer; forming a synthetic top epidermal layer on the synthetic middle dermal layer; lyophilizing the synthetic skin construct; treating the synthetic skin construct with supercritical carbon dioxide under pressures from about 1500 psi to about 5000 psi, at a temperature from about 25°C to about 55°C, for a period of time from about 1 hour to about 6 hours, to decellularize tire synthetic skin construct; and sterilizing the synthetic skin construct for use. wherein the synthetic bottom vasculature layer, the synthetic middle dermal layer, the synthetic top epidermal layer, or any combination thereof are formed using 3D bioprinting of a biocompatible polymer in combination with cells selected from fibroblasts, keratinocytes, vascular cells, mural cells, dental pulp stem cells, human umbilical vein endothelial cells, or combinations thereof.

17. The method of claim 16. wherein the biocompatible polymer is selected from gelatin, collagen, alginate, chitosan, fibrin, fibrinogen, polyethylene glycol, poly(lactic -co-glycolic acid), poly(e- caprolactone), or combinations thereof.

18. The method of claim 16, further comprising applying a sacrificial biomaterial embedded within the synthetic bottom vasculature layer, the synthetic middle dennal layer, the synthetic top epidermal layer, or any combination thereof to mimic vasculature architecture within the synthetic skin construct, wherein treating the synthetic skin constmct with supercritical carbon dioxide removes the sacrificial biomaterial from the synthetic skin construct.

19. The method of claim 16, further comprising applying a patient’s autologous cells selected from keratinocytes, fibroblasts, melanocytes, vascular cells, mural cells, or combinations thereof to the synthetic skin constmct prior to grafting the synthetic skin constmct to the patient.

20. A synthetic skin constmct prepared by the method of claim 16.PATENT APPLICATIONAtty Docket: 050-9472 (51801-1 PCT)21. A method of preparing a synthetic skin construct, comprising: forming a synthetic bottom vasculature layer; forming a synthetic middle dermal layer on the bottom vasculature layer; forming a synthetic top epidermal layer on the synthetic middle dennal layer; treating the synthetic skin construct with supercritical carbon dioxide to decellularize the synthetic skin construct; and sterilizing the synthetic skin construct for use, wherein the synthetic bottom vasculature layer, the synthetic middle dermal layer, and the synthetic top epidermal layer are formed of a biocompatible polymer in combination with dental pulp stem cells.

22. A method of decellularizing a synthetic skin construct comprising treating the construct with supercritical carbon dioxide under conditions sufficient to remove cellular components while preserving extracellular matrix architecture.

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