WO2024259424A2 - Biosorbable nanofibrous scaffolds - Google Patents

Abstract

A bioresorbable nanofibrous scaffold having nanofibers of silk fibroin and polyhydroxybutyrate (PHB). The silk fibroin and the PHB are arranged so that an area of the bioresorbable nanofibrous scaffold has concentrated amounts of the silk fibroin and another area of the bioresorbable nanofibrous scaffold has concentrated amounts of the PHB. The silk fibroin and the PHB may be coaxially arranged such that the PHB forms a center of the nanofibers and the silk fibroin surrounds the PHB concentrically, or the silk fibroin and the PHB may be arranged so that the silk fibroin and the PHB are layered.

Description

BIO SORB ABLE NANOFIBROUS SCAFFOLDS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and benefit of U.S. Provisional Patent Application No. 63/521,334, filed June 15, 2023, the entire disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with government support under NH BioMade EPSCoR Award 1757371 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

[0003] This disclosure relates to scaffolds used in medical applications.

BACKGROUND OF THE DISCLOSURE

[0004] Polypropylene mesh is a common type of synthetic mesh used for medical applications. Typically, it is made from a widely-used plastic that is inert. This mesh can be used as a surgical mesh that is typically a loosely-woven sheet used as either a permanent or temporary support for organs and other tissues during surgery. For example, surgical meshes may be used to aid in the repair of pelvic floor disorders.

[0005] Over 25% of adult women are reported to experience pelvic floor disorders including stress urinary incontinence (SUI). SUI is characterized by involuntary urination when intraabdominal pressure increases, such as when coughing, laughing, straining in exercise, or sneezing. SUI affects about one in three women during their lifetime. Older women and women who have undergone vaginal births are at higher risk of developing SUI. Incontinence can have a significant impact on a woman’s quality of life. While there are non-surgical interventions such as pelvic floor exercises and pessaries, conservative treatments have a failure rate of up to 50%. While this is already an extremely prevalent problem, this number is likely underreported due to embarrassment. The incidence of pelvic floor weakness and prolapse increases with age with parous and multiparous women at increased risk. With an aging population and increased life expectancies, SUI and its surgery with subsequent complications will continue to become more prevalent.

[0006] The most common surgical intervention is the surgical implantation of a permanent polypropylene sling mesh to support the mid-urethra (FIG. 1). However, the mesh can erode local tissue and become exposed over time. Typically, the soft tissue directly superior and anterior of the mesh will be eroded due to the high coefficient of friction, abrasion, and micromotion, as shown in FIGS. 2a-b. Approximately 5-20% of women who undergo this procedure develop complications, such as tissue erosion by the mesh. When in prolonged contact with soft tissue, polypropylene can irritate and erode surrounding tissue, so it may be an insufficient option for surgical procedures. For example, in 2010, over 250,000 women underwent surgery to repair their SUI. 80% of those procedures used polypropylene mesh. The failure rate of SUI meshes is about 5-20%.

[0007] Of the 300,000 patients who receive mid-urethral sling surgeries in the U.S. every year, 2-3% experience mesh erosion, and up to 20% remain incontinent which may require revision surgeries where more synthetic mesh is placed, compounding the problem. When a polypropylene mesh must be replaced in a patient, the exposed mesh near the urethra is removed, but the remaining mesh in the pelvic floor remains implanted with the potential to cause more erosion. Reported complication rates are heterogeneous primarily due to inconsistent definitions of mesh failure. However, for mesh-related complications prevalent to this proposal, erosion through the vaginal wall is the most commonly reported.

[0008] Due to the problems surrounding polypropylene mesh in prolapse, the FDA recalled and reclassified transvaginal mesh to a class III device. However, no additional modifications were required after premarket approval. While mechanically the mesh used in reconstructive surgeries are supportive, the biological interaction with the surrounding tissue causes many complications which has led to over 100,000 lawsuits filed in the US.

[0009] Autologous fascia grafts represent an alternative to polypropylene mesh, but they require an additional harvesting procedure, often resulting in discomfort, prolonged procedural time, donor site morbidity, and increased recovery time. Allograft and xenografts have also been used, but risk transmission of infection and can lead to inadequate relief of symptoms. While fascial slings were among the first SUI procedures performed, the minimally invasive artificial sling placements still overtook the market. However, after the FDA warnings and legal implications, the use of synthetic mesh has decreased and there has been an uptick in fascial sling procedures. While autologous fascia can provide adequate relief of SUI symptoms without risk of erosion and mesh exposure, it does require a second procedure, increasing the overall risk to the patient.

[0010] Women’s health overall requires an increase in research to help develop safe, productive, and effective devices to improve patient outcomes for SUI. Alternatives such as tissue engineered solutions should be targeted to fabricate an optimized, a minimally -invasive, and a biocompatible solution to SUI. Thus, improved scaffolds and methods of fabrication are needed.

BRIEF SUMMARY OF THE DISCLOSURE

[0011] The present disclosure provides a bioresorbable nanofibrous scaffold and method of making a bioresorbable nanofibrous scaffold using electrospinning techniques. A polymer dissolved in a solvent is ejected out of a syringe needle tip, where an applied high voltage evaporates the solvent and creates a polymer nanofiber. This nanofiber is then projected and spun by static forces towards a collecting drum, with or without rotation for the mechanical alignment of fibers.

[0012] The embodiments disclosed herein use a bioresorbable nanofibrous scaffold that facilitates scar tissue growth in vivo to create an autologous support without a second surgery. The bioresorbable nanofibrous scaffold may be fabricated out of two natural polymers: silk fibroin (SF) and polyhydroxybutyrate (PHB) that are electrospun together to create a mechanically stable and biocompatible implant with focal adhesion points.

[0013] Embodiments of the present disclosure combine PHB and SF in a scaffold that, upon the addition of fibroblasts, has shown promising in vitro and in vivo results. This combination of materials integrates: 1) combined natural polymers fabricated through coaxial electrospinning, 2) increased cellular focal adhesion points on the exterior of the scaffold with maintained mechanical properties, and 3) patient-specific cellular attachment for improved biocompatibility and enhanced integration. Embodiments of the PHB-SF scaffolds possess adequate mechanical properties and in vitro biocompatibility with suggested in vivo benefits.

[0014] In embodiments, the addition of PHB and SF improves mechanical properties and promoted fibroblastic growth, respectively. SF’s ability to promote healthy in vivo tissue formation in the vaginal and pelvic floor supports its combination with PHB for use in a bioresorbable nanofibrous scaffold. Embodiments of the present disclosure may improve the use of biopolymers in pelvic floor reconstructive surgery.

[0015] Embodiments of the present disclosure may mimic native tissue architecture in the pelvic floor, may include fibrous tissue growth, and may possess anisotropy and tensile strength to sufficiently support the urethra.

[0016] The present disclosure includes a bioresorbable nanofibrous scaffold that may have nanofibers of silk fibroin and polyhydroxybutyrate (PHB). The silk fibroin and the PHB may be arranged so that an area of the bioresorbable nanofibrous scaffold may have concentrated amounts of the silk fibroin and another area of the bioresorbable nanofibrous scaffold may have concentrated amounts of the PHB.

[0017] In embodiments of the present disclosure, the silk fibroin and the PHB may be coaxially arranged such that the PHB forms a center of the nanofibers and the silk fibroin surrounds the PHB concentrically.

[0018] In embodiments of the present disclosure, the silk fibroin and the PHB may be arranged such that the silk fibroin and the PHB are layered.

[0019] In embodiments of the present disclosure, a diameter of the nanofibers may be from 50 nm to 10 pm.

[0020] In embodiments of the present disclosure, a tensile strength of the nanofibers may be from 2MPa to 5MPa.

[0021] In embodiments of the present disclosure, the bioresorbable nanofibrous scaffold may include biological materials disposed on the nanofibers.

[0022] In embodiments of the present disclosure the biological materials may include. TGF- Beta, fibroblast growth factor (FGF), collagen, fibronectin, cognitive tissue growth factor (CTFG), integrin, proteoglycan, elastin, tenascin, and/or other small molecules.

[0023] In embodiments of the present disclosure, the biological materials may be coated onto the bioresorbable nanofibrous scaffold.

[0024] In embodiments of the present disclosure, the bioresorbable nanofibrous scaffold may include fibroblasts harvested and isolated from a patient disposed on the nanofibers.

[0025] In embodiments of the present disclosure, the fibroblasts may be seeded onto the bioresorbable nanofibrous scaffold. [0026] In embodiments of the present disclosure, the bioresorbable nanofibrous scaffold may include focal adhesion points.

[0027] In embodiments of the present disclosure, the bioresorbable nanofibrous scaffold may be configured to be implanted in a human body to provide temporary support for medical applications.

[0028] In embodiments of the present disclosure, the bioresorbable nanofibrous scaffold may be configured to dissolve and promote autonomous fibrous growth to replace the temporary support of the bioresorbable nanofibrous scaffold.

[0029] In embodiments of the present disclosure, the bioresorbable nanofibrous scaffold may dissolve over a period from 6 months to 3 years.

[0030] In embodiments of the present disclosure, the nanofibers may be electrospun.

[0031] In embodiments of the present disclosure, the nanofibers may be coaxially electrospun.

[0032] The present disclosure provides a bioresorbable nanofibrous scaffold that may include nanofibers of silk fibroin and co-polymers of polyhydroxybutyrate (PHB). The silk fibroin and the co-polymers of PHB may be arranged so that an area of the bioresorbable nanofibrous scaffold may have concentrated amounts of the silk fibroin and another area of the bioresorbable nanofibrous scaffold may have concentrated amounts of the co-polymers of PHB.

[0033] In embodiments of the present disclosure, the silk fibroin and the co-polymers of PHB may be coaxially arranged such that the co-polymers of PHB may form a center of the nanofibers and the silk fibroin may surround the co-polymers of PHB concentrically.

[0034] In embodiments of the present disclosure, the silk fibroin and the co-polymers of PHB may be arranged so that the silk fibroin and the co-polymers of PHB are layered.

[0035] In embodiments of the present disclosure, the co-polymers of PHB may include poly 3 -hydroxybutyrate (P3HB) or prolyl 4-hydroxylase (P4HB).

[0036] In embodiments of the present disclosure, a diameter of the nanofibers may be from 50 nm to 10 pm. DESCRIPTION OF THE DRAWINGS

[0037] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying drawings.

[0038] FIG. 1 displays a schematic of a mid-urethral sling placement.

[0039] FIG. 2a displays a mesh placement in the vaginal wall.

[0040] FIG. 2b displays a laparoscopic image of mesh erosion into a vaginal wall (left) and a laparoscopic image of mesh erosion through a distal urethra (right).

[0041] FIG. 3 displays the state of silk during steps of a silk extraction protocol.

[0042] FIG. 4a displays an exemplary schematic of silk fibroin extraction and electrospinning collection.

[0043] FIG. 4b displays an exemplary schematic of electrospinning and collection.

[0044] FIG. 5a displays an exemplary schematic of coaxial electrospinning and collection.

[0045] FIG. 5b displays a separated needle tip used in coaxial extrusion.

[0046] FIG. 6 displays SEM images of exemplary bioresorbable nanofibrous scaffolds that were fabricated, demonstrating fiber diameter and alignment differences between concentrations and ratios of polymers.

[0047] FIGS. 7 displays confocal images of nuclei attached to a 3 wt% 50/50 scaffold at 10X.

[0048] FIG. 8 displays confocal images of nuclei attached to a 3 wt% 50/50 scaffold at 20x.

[0049] FIG. 9 is a chart showing ultimate tensile strength of exemplary electrospun bioresorbable nanofibrous scaffolds.

[0050] FIGS. lOa-b displays charts of representative stress-strain curves for each scaffold type at 3 wt% (10(a)) and at 5 wt% (10(b)).

[0051] FIG. 11 displays a chart showing ultimate tensile strength of all electrospun scaffolds.

[0052] FIG. 12 displays SEM images at 1.5kx magnification of 3% wt/v 50/50 (left) and 72/25 (right) PHB/SF.

[0053] FIG. 13 displays confocal image of hVF nuclei stained with DAPI (blue) and collagen (red) on 3% wt/v 0/100 PHB/SF for 2 weeks. [0054] FIG. 14 displays UTS of 3% wt/v scaffolds cultured for 2 weeks with and without hVF cells.

[0055] FIG. 15 displays a SEM image of 3% wt/v 0/100 PHB/SF electrospun scaffold with hVFs cultured for 1 week.

[0056] FIG. 16 displays HVFs stained with DAPI and collagen on 3wt% 100/0 PHB/SF after 2 weeks of culture.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0057] Although claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0058] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include all values to the magnitude of the smallest value (either lower limit value or upper limit value) and ranges between the values of the stated range.

[0059] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0060] The present disclosure provides a bioresorbable nanofibrous scaffold fabricated out of at least two natural polymers, such as silk fibroin (silk or SF) and polyhydroxybutyrate (PHB). Embodiments of the present disclosure include a tunable protocol to electrospin scaffolds made of SF and PHB that may be inserted into the body. In embodiments of the present disclosure, the SF and PHB may be arranged so that some areas of the scaffold have concentrated amounts of SF and other areas of the scaffold have concentrated amounts of PHB. For example, in SF concentrated areas, the SF is more prevalent than the PHB, such that 51% to 100% of the area of the scaffold is concentrated with SF. Further, in PHB concentrated areas, the PHB is more prevalent than the SF, such that 51% to 100% of the area of the scaffold is concentrated with PHB. [0061] In an embodiment, solutions of SF and PHB may be mixed together and electrospun into a scaffold. In other embodiments, the solutions of SF and PHB may be separate and coaxially electrospun so that the scaffold is composed of an SF exterior surrounding a PHB core. In embodiments, the individual nanofibers forming the scaffolds may have an area concentrated with SF and another area concentrated with PHB, such that 51% to 100% of the area of the individual nanofiber is concentrated with SF and/or 51% to 100% of the area of the individual nanofiber is concentrated with PHB. For example, each individual nanofiber of the scaffold may have concentrated areas of SF and concentrated areas of PHB, such that each individual nanofiber includes both SF and PHB. After these individual nanofibers are spun together, the arrangement allows for the scaffold to have an area of the scaffold that is concentrated with SF, and another area of the scaffold that is concentrated with PHB. In an embodiment, the arrangement of the SF and PHB in the nanofibers that form the scaffold may be random. In other embodiments, the arrangement of the SF and the PHB in the nanofibers that form the scaffold may be coaxial, such that the nanofibers are arranged such that the concentrated areas of the PHB are arranged in an interior portion of the scaffold, and the concentrated areas of the SF are arranged in an exterior portion of the scaffold.

[0062] Even further, embodiments of the present disclosure may include a layered scaffold including concentrated areas of SF and PHB. For example, the scaffold may include individual nanofibers that are only concentrated with SF and other individual nanofibers that are only concentrated with PHB. These individually concentrated nanofibers may be spun together to form a scaffold. In an embodiment, the scaffold may include PHB nanofibers spun together into a structure only made of PHB. SF may be spun (or deposited) around the structure formed with the PHB nanofibers, for a layered approach. Numerous layers of the same or different materials may be spun/deposited around the already formed structure. In embodiments, the SF nanofibers may be the original structure, with the PHB being spun around. In an embodiment, the scaffold may be a cylindrical shape. In other embodiments, the scaffold may be a flat and rectangular shape. To form a flat and rectangular scaffold, a hollow cylindrical scaffold may be cut in half, to form a flat rectangular scaffold. In other embodiments, the layered scaffold may include a gradient of SF and PHB. For example, a portion of the scaffold may be formed of only SF and another portion of the scaffold may be PHB, such that the SF portion of the scaffold is next to the PHB portion of the scaffold. The present disclosure includes any layering combination of materials. After these individual nanofibers are spun together, the arrangement allows for the scaffold to have an area of the scaffold that is concentrated with SF, and another area of the scaffold that is concentrated with PHB.

[0063] Embodiments of the resulting scaffold disclosed herein may be made into any shape. For example, the scaffold may be three-dimensional, such as cylindrical in shape. Further, the scaffold may be a flat structure that is rectangular. The structure and shape of the scaffold may be dependent on the application of the scaffold and the ways in which it is being used and implanted in the body.

[0064] Nanofibrous scaffolds possess low coefficients of friction and improved biocompatibility, often initiating native tissue integration without irritation. Thus, embodiments of the nanofibrous scaffolds offer a suitable setting for cell attachment and growth due to their high aspect ratio and biomimetic resemblance to native extracellular matrix (ECM).

[0065] Embodiments of the present disclosure may be implanted into the body, for example, for the aid in pelvic floor reconstruction surgeries, SUI surgeries, hernia repair, or tendon repair for orthopedic applications. The scaffold may be implanted into the body for other applications, not mentioned here. The scaffold may promote autonomous fibrous growth to replace the temporary support the scaffold provides and may dissolve over time.

[0066] Both SF and PHB are approved for use in medical devices and implants and are biocompatible. Using two natural materials for a scaffold can promote cell adhesion while maintaining mechanical properties of the device. Embodiments of the bioabsorbable nanofibrous scaffold are a mechanically stable and biocompatible implant with focal adhesion points. Focal adhesion points are assemblies through which mechanical forces and regulatory signals may be transmitted between the interacting cells and the ECM. For example, in an embodiment, the focal adhesion points may be structures that mediate the regulatory effects, such as signaling events, of a cell in response to ECM adhesion.

[0067] In an embodiment, the scaffold may dissolve over time and promote autonomous fibrous growth to replace the temporary support the scaffold provides. In an instance, the scaffold may dissolve in the body after implantation. SF and PHB can leave scar tissue to act as a permanent support after these materials degrade. In an embodiment, degradation may take up to six months to three years, or longer. The target degradation time can be six months to one year, but other periods are possible.

[0068] Other delayed absorbable materials that dissolve over time and recruit fibroblasts for a permanent scar tissue support to be left in its place may be used instead of SF or PHB, or in combination with SF and PHB.

[0069] SF may be extracted from silk cocoons. In an instance, a five-day extraction process may be used. As shown in FIGS. 3 and 4a, this extraction process may include boiling the cocoons, dialyzing the dissolved silk, centrifuging, and lyophilizing. The silk may be concentrated. Polyhydroxybutyrate (PHB) is a bacterial byproduct.

[0070] Embodiments disclosed herein can recruit and promote a fibrous construct while providing the support necessary to address the SUI, pelvic organ prolapse, hernias, or other medical applications.

[0071] The present disclosure further provides a method of making a bioresorbable nanofibrous scaffold using electrospinning techniques. A polymer dissolved in a solvent is ejected out of a syringe needle tip, where an applied high voltage evaporates the solvent and creates a polymer nanofiber. This nanofiber is then projected and spun by static forces towards a collecting drum, with or without rotation for the mechanical alignment of fibers.

[0072] In an embodiment the elecrospinner can be used to make nanofibrous scaffolds made of natural polymers such as SF and polyhydroxybutyrate (PHB). The electrospinning, shown in FIGS. 4a-b, can run a positive voltage through a solution to evaporate the solvent (e.g. Hexafluoro- 2 -propanol) and separate polymers for deposition. The SF and PHB can be mixed by placing both components on a shaker table to mix and dissolve in solution, such as for approximately 24 hours, and then electrospun to form the scaffold.

[0073] Embodiments of the present disclosure further include a tunable protocol to coaxially electrospin coaxial scaffolds composed of a SF exterior surrounding a PHB interior (or PHB core), as shown in FIG. 5a. For example, coaxial electrospinning processes may be used to generate a scaffold with concentrated regions of SF and concentrated of PHB, for example, a SF exterior surrounding a PHB core. Coaxial electrospinning may use two syringe pumps, one pump containing the SF solution, and the other containing the PHB solution (dual syringe and pump shown in FIG. 5b). The two syringe pumps are fed into a nozzle, attached to an inner and outer needle. The inner needle is inside the outer needle. When a voltage is applied, the inner needle spins the PHB and the outer needle spins the SF, so that the SF is spun around the PHB core.

[0074] Under both the traditional electrospinning methods and the coaxial electrospinning methods as disclosed herein, the scaffolds may be spun at concentrations of 0.05 to 20 wt% for SF and PHB with both aligned and random orientation fibers.

[0075] In embodiments of the present disclosure, the average thickness of each nanofiber being spun together may be from 50 nm to 10 pm or from 200nm-900 nm. Further, in an embodiment, the average thickness of each SF or PHB region of the nanofibers may be from 5 nm to 9.75 pm. In embodiments, the fiber diameter may be less than 50 nm, for example, in applications in which the scaffold must have weaker mechanical properties. In applications in which the scaffold is implanted to address SUI, pelvic organ prolapse, hernia, or other medical applications, the fiber diameter may be approximately 500 nm.

[0076] In embodiments, the number of nanofibers in a scaffold varies, based on the quantity and concentration of the solution being electrospun. For example, adding more SF to the solution may allow for finer or thinner scaffold. Further, longer electrospinning processes may allow for thicker nanofibers and/or scaffold. Generally, in embodiments, electrospinning a greater amount of polymer solution will result in a scaffold with a greater number of nanofibers. Larger nanofiber thicknesses means that the scaffold may be too big or small for the fibroblasts to conform to their natural morphology. Smaller nanofiber thicknesses may not provide sufficient strength or support for certain applications.

[0077] The diameter of the nanofiber may be based on the application in which the nanofibrous scaffold is being used. The nanofibrous scaffold may mimic the structure of an extracellular matrix in tissue to promote cell growth and the fiber diameter may have properties that allow for cell adhesion. The size of the diameters of the nanofiber may facilitate fibroblast cell growth in vitro. The electrospinning parameters can be adjusted for the desired nanofiber diameter and nanofiber alignment. For example, positive and negative voltage, flow rate, working distance, solution concentration, and type of solvent can be adjusted, and the values for these parameters can vary. For example, adding greater amounts of weight/volume of solution to the syringe may increase the nanofiber diameter. [0078] In embodiments, different ratios of SF and PHB solution may be used, such as from 85/15 to 15/85. For example, ratios of SF to PHB in solution such as 85/15, 75/25, 50/50, 25/75, or 15/85 may be used. Other ratios are possible.

[0079] In embodiments of the present disclosure, other materials may be added to the nanofiber. For example, polylactic acid (PLA), polycaprolactone (PCL), polyethylene oxide (PEO), and/or poly(vinyl alcohol) (PVA) may be added to the nanofiber. Further, co-polymers of PHB, such as poly 3 -hydroxybutyrate (P3HB) and prolyl 4-hydroxylase (P4HB) may also can be used depending on the mechanical properties and cellular response. For example, in an embodiment, it has been demonstrated that fibroblasts attach to a biopolymer P4HB when spun into a nanofibrous scaffold. Even further, copolymers of PLA, such as poly(lactic-co-glycolic) acid (PLGA), poly-L- lactic acid (PLLA), poly(glycolic acid) (PGA), etc., may be used. The use and/or addition of PLA, PCL, PEO, PVA, or co-polymers of PLA or PHB may promote the attachment and proliferation of fibroblasts, such as vaginal fibroblasts, as compared to macro-fiber materials. Natural and unnatural fibers may be used in the formation of the scaffold. In embodiments, different ratios of solution may be used. For example, ratios of SF to the materials described above or PHB to the materials described above in solutions such as from 85/15 to 15/85. For example, ratios of 85/15, 75/25, 50/50, 25/75, or 15/85 may be used. In embodiments using these materials, the materials may be added to the solution at a range from 0.05 to 20 wt%.

[0080] In embodiments using PLA, PCL, PEO, PVA, or co-polymers of PLA or PHB, the average thickness of each nanofiber being spun together may be from 50 nm to 10 pm or from 200nm-900 nm. Further, in an embodiment, the average thickness of each SF or PLA, PCL, PEO, PVA, or co-polymers of PLA or PHB region of the nanofibers may be from 5 nm to 9.75 pm.

[0081] In embodiments using PLA, PCL, PEO, PVA, or co-polymers of PLA or PHB, the nanofibers and scaffold may be structured as explained above, for example through classic electrospinning, coaxial electrospinning, or through electrospinning in a layered or gradient approach.

[0082] The scaffold may further include biological molecules such as TGF-Beta, fibroblast growth factor (FGF), collagen, fibronectin, cognitive tissue growth factor (CTFG), integrin, proteoglycan, elastin, tenascin, or other small molecules to promote autonomous fibrous growth to replace the temporary support the scaffold provides. For example, the addition of these biological molecules to the scaffold may enhance extracellular matrix deposition. In an embodiment, the biological materials may be dissolved in the solution of SF and PHB prior to the electrospinning process. In other embodiments, the biological materials may be added in by post processing, such as coating the scaffold or soaking the scaffold in a bath, after formation of the scaffold. In embodiments of the present disclosure, the biological molecules may be added to the solution at a range from 0.05 to 20 wt%.

[0083] Embodiments of the present disclosure may include the addition of patient-specific fibroblasts to improve biocompatibility and trigger healthy fibrous tissue growth. Embodiments of the present disclosure include tunable manufacturing methods of a nanofibrous scaffold and seeding the nanofibrous scaffold with autologous fibroblasts.

[0084] In an embodiment, fibroblasts harvested and isolated from a patient may be seeded onto the scaffold and cultures to promote full integration before implantation of the nanofibrous scaffold into the patient. For example, in an embodiment the fibroblasts may be seeded onto the scaffold at a concentration of 50,000 cells/cm², but other concentrations are possible. Further, the fibroblasts may be seeded on a portion of the scaffold or on the entirety of the scaffold. For example, the fibroblasts may be seeded on at least one side of the scaffold. For example, the fibroblasts may be seeded on one or two sides of the scaffold.

[0085] In embodiments, utilizing the advantages of two biomaterials, fibroblast cell recruitment, and adhesion, may aid in the creation of permanent tissue-based support. Fibroblasts will deposit collagen during the beginning stages of healthy fibrous tissue formation, which will be facilitated through recruitment by the nanofibrous scaffold. For example, in an embodiment, the SF improves cell attachment points and the PHB maintains the mechanical properties.

[0086] Embodiments of the present disclosure include a scaffold that improves fibroblast attachment and ECM deposition. Embodiments of the present disclosure, including an autologous, bioresorbable alternative to polypropylene mesh slings, can be used in future SUI animal models, establishing biocompatibility and fibroblast infiltration of the scaffolds.

[0087] Embodiments of the present disclosure include fabricated scaffolds with strong mechanical properties that provide adequate mechanical support of the urethra. For example, in an embodiment the ultimate tensile stress and Young’s modulus may be greater than 0.8MPa and 9.45MPa respectively. In an embodiment, the tensile strength of the nanofibers may be from 2MPa to 5MPa. However, the tensile strength may be higher depending on the amount of material used in the fabrication of the nanofibrous scaffold.

[0088] Embodiments of the present disclosure include a device that is not made out of autologous material, but instead uses the chosen naturally derived, commercially available, FDA- approved materials to promote collagen deposition and the formation of fibrous tissue.

[0089] Embodiments of the present disclosure may be implanted for SUI repair through the same mechanisms as polypropylene mesh, as described in FIG. 1. For example, utilizing strings attached to the end of the slings that fits into the tunneller (i.e., insertion device) that is pulled through the transobturator or retropubic space. A loop of suture material can be attached to use the tunneller for insertion. The embodiments disclosed herein can provide sufficient tensile strength to ensure a scaffold can support a urethra.

[0090] Embodiments of the present disclosure made from electrospinning may be spun into a cylindrical scaffold. The cylindrical scaffold may be cut in half to form a flat rectangular scaffold. [0091] The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any matter.

EXAMPLE 1

[0092] This example provides a description of an embodiment of the nanofibrous scaffold described herein.

[0093] Over 25% of adult women experience pelvic floor disorders, including SUI. The most common surgical intervention for SUI is implantation of a mid-urethral sling composed of polypropylene (PP) mesh. Although highly effective for most patients, PP mesh carries a risk of erosion and exposure due to its host tissue-mesh incompatibility and friction in 3-7% of patients. Mesh erosion and exposure can cause an increased risk of infection, bleeding, and chronic pain. Autologous fascia, a biocompatible material harvested from the Iliotibial band, can be used as an alternative to PP mesh. However, it requires a second procedure for harvesting, resulting in donor site morbidity and prolonged surgery time.

[0094] Embodiments disclosed herein provide a biologically-derived manufactured scaffold to promote fibrous healing while the construct is degraded in vivo. In this example, the scaffolds were fabricated using an electrospinner (BIONICIA®) to create a nanofibrous mat out of biologic polymers. To electrospin materials, polymers are dissolved in a volatile solvent before being ejected out of a needle tip. As the solution is ejected, a high voltage is applied to polarize the liquid, and the solvent evaporates as the thin polymer fiber is drawn toward the collecting drum by electrostatic forces.

[0095] In this example, two natural polymers, silk fibroin (SF) and polyhydroxybutyrate (PHB), were combined at different weight percents (wt%) within the solvents and fractional mix: 3 and 5 wt% concentration with the ratio of PHB/SF: 100/0, 75/25, and 50/50. All scaffolds were spun at 15 cm, flow rate of approximately 7 mL/hour, positive voltage of approximately 7 kV, and a negative voltage on the drum of -4 kV. Imaging samples were cut into 1 cm² sections and gold sputter coated for scanning electron microscopy (SEM). Tensile samples were cut into 4x2.5 cm sections along the length of the scaffolds and tested at a rate of 10 mm/min on an Instron 68SC.

[0096] SEM showed a statistical difference in fiber size, especially between the 3 and 5 wt% scaffolds, as shown in FIG. 6. Fiber size may be relevant for subsequent fibroblast attachment during in vitro experiments. Fibers were more aligned in the 3 wt% scaffolds, while fibers in the 5 wt% scaffolds were observed to be more randomly aligned.

[0097] Results demonstrate that natural polymers can be combined and electrospun to form nanofibrous scaffolds with varying fiber diameter and alignment. The more aligned fibers had higher ultimate tensile strength, but shorter elongation at break (FIGS. 9a-b). Further, the failure modes were different for differing weight percentages, as evidenced by the shape of the stress-strain curve. Work-to-failure can be used to predict interactions with the biological system and likely in vivo failure modes.

[0098] As tested in this example, in an embodiment, the parameters may be 3 wt% 50/50 SF/PHB, a flow rate of 8 ml/hour, a working distance of 15 cm, a negative voltage of -4.0 kV, a solution concentration of 3 wt%, hexafluoroisopropanol (HFIP) as a solvent, and a positive voltage of 6.5 to 8.5 kV.

[0099] Scanning electron microscopy was used to image the fiber diameter and fiber alignment of six exemplary scaffolds, as shown in FIG. 6. Confocal imaging was used to show fibroblast adhesion and proliferation onto the scaffolds, as shown in FIGS. 7 and 8. Cell nuclei were stained with DAPI. FIGS. 7 and 8 show confocal images of nuclei attached to a 3 wt% 50/50 scaffold, wherein FIG. 7 is at lOx and FIG. 7 is at 20x; [0100] FIG. 9 shows test results of ultimate tensile strength for various scaffolds. FIGS. 10a- b shows work-to-failure results via stress versus strain charts for the six tested scaffolds. In FIGS. lOa-b, the charts use representative trials rather than an average.

[0101] SEM images demonstrate the effect of solution concentration on fiber diameter between scaffolds of same material composition. 3 wt% scaffolds (A, C, and E shown in FIG. 10a) have narrower average fiber diameter than 5 wt% scaffolds (B, D, and F shown in FIG. 10b) because there is less polymer dissolved in the initial polymer solution. Ultimate tensile stress was higher in the 3 wt% scaffolds (A, C, and E as shown in FIG. 10a) than their 5 wt% counterparts (B, D, and F as shown in FIG. 10b) because the fibers are bigger and there is more polymer in the solution. In these examples, wt% refers to the concentration of dissolved polymer in the solution that is then extruded through the needle tip. For example, 3 wt% PHB means 0% silk for these examples. This 3 wt% was used this to get a baseline of how the PHB material is compared to the mix of the materials. The wt% can be considered as w/v% for the dissolved polymer solution.

[0102] The stress versus strain curves of FIGS. lOa-b show how failure modes vary between different wt%. Samples A and B fail more abruptly, whereas E and F fail gradually. C and D resemble a cross of both A/B and E/F. Silk in C-F allows the sample to stretch farther.

[0103] As shown, more PHB generally correlates to higher ultimate strength. The tensile strength for the nanofibers can be at least 2 MPa, but may be approximately 5 MPa. Other tensile strength values are possible. Other wt% values are possible. The wt% can be from 0.5 to 10 wt% for PHB or silk fibroins.

[0104] Tensile tests indicated that aligned, 3 wt% fibers had the largest ultimate tensile strength, as shown in FIG. 11. There was high variation within samples for 3 wt% samples compared to 5 wt% samples, but the maximum strength was consistently higher.

EXAMPLE 2

[0105] This example provides a description of an embodiment of the nanofibrous scaffold described herein.

[0106] In this example, an electrospinner was used to fabricate homogenously mixed PHB and SF scaffolds. The scaffolds were composed of 3% PHB and SF wt/v dissolved in hexafluoroisopropanol (HFIP), at different ratios (100/0, 75/25, 50/50, 25/75, and 0/100) in the same, homogenous solution before being electrospun and imaged via scanning electron microscopy (SEM) (FIG. 12). Fiber diameters ranged from 300nm to 1 pm, which has been shown to be beneficial to fibroblast adhesion and proliferation. Scaffolds were created with varying amounts of fiber alignment on a spinning drum collector with the goal of promoting fibroblast growth and collagen deposition in line with the direction of the fibers.

[0107] Embodiments of the present disclosure are premised on the structural integrity and improved mechanical properties of the PHB material while exploiting increased cell attachment points of SF. Results have shown that samples with increased ratio of PHB have higher tensile properties, with and without the addition of cells. From this preliminary data, it can be anticipated that scaffolds with higher wt/v% PHB cores will similarly have increased mechanical properties. FIG. 13 shows that while there was no statistical difference in ultimate tensile strength (UTS) between the same scaffolds cultured with and without human vaginal fibroblasts (hVFs), there was a statistical difference between scaffold type (P < 0.05). Studies have shown uniaxial tensile properties of control and prolapsed vaginal tissue resulting in 0.79±0.05 and 0.60±0.02 MPa, respectively. Also, it was determined that the maximum force exerted on the mesh would be approximately 1.37N which both materials can withstand. Note that the 75/25 PHB/SF scaffolds have increased mechanical properties compared to native vaginal tissue. Vaginal tissue decreases in mechanical properties both due to prolapse and menopause which contributes to requiring pelvic floor reconstructive surgeries. A scaffold with increased mechanical support has the potential to improve this and reduce the need for additional surgery. Thus, creating a coaxial scaffold with a core of PHB will optimize the mechanical properties for urinary sling applications and the external SF will facilitate fibroblast adhesion.

[0108] Human vaginal fibroblast cellular attachment to different ratios of PHB/SF scaffolds was imaged using confocal microscopy after 2 weeks of culture, showing an increased number of cells with higher SF (FIG. 14). Scaffolds were stained with 4’,6-diamidino-2-phenylindole (DAPI) to visualize the number of cells as well as their morphology on the scaffold. The fibroblasts infiltrated and grew on the preliminary combined scaffolds, demonstrating that combined PHB and SF were not cytotoxic and promoted growth and proliferation. The scaffolds containing 3% wt/v 50/50 PHB/SF had a higher concentration of cells. While preliminary data showed an increased cell population on higher concentration of SF, further investigation of collagen deposition needed to be quantified. Utilization of these hVFs was useful for preliminary studies to develop an understanding of compatibility with the scaffold material. However, adipose-derived stem cells (ADSCs) and endometrial mesenchymal stem cells (MSCs) were both recognized as promising cell types moving towards clinical implantation. Further combination of these materials in a coaxial construct was imperative in the search to find an alternative to polypropylene surgical mesh. Embodiments of the present disclosure may be placed using the same surgical technique and approach as current state-of- the-art practices.

[0109] To create a product that can be used in delicate, soft tissue areas, the materials in question may need to be fully characterized and understood in cellular in vitro environments. SEM, mechanical testing, and confocal imaging was used to determine cell viability, structural integrity, and proliferation differences associated with each nanofibrous scaffold. To create appropriate electrospun fibers during fabrication, a cone shape must form at the tip of the needle, representing successful electrospinning. To form the cone shape, also known as a Taylor cone, the applied voltage and flow rate, as well as material concentration must be optimized. The addition of fibroblasts allows for preliminary biocompatibility testing and demonstrates the degree of cellular affinity to the scaffolds. Due to the prevalence of SUI in women, applications of the present disclosure may consist of hVF pre-seeded scaffolds and relevant animal models, such as ovine. [0110] Embodiments of the present disclosure include a tunable protocol to electrospin coaxial scaffolds with a PHB core and SF exterior. Embodiments center on maintenance of mechanical properties and contiguous material fabrication during scaffold manufacturing which enhanced mechanical and cellular interaction with the scaffold. The radially-organized, continuous material structure maximized the mechanical properties while allowing for maximized cell interaction. Embodiments of the present disclosure include a tunable protocol to fabricate coaxial scaffolds at different concentrations and ratios while maintaining consistent Taylor cones and effective electrospinning.

[OHl] In an embodiment, SF was extracted and concentrated from Bombyx Mori silkworm cocoons by removing the adhesive sericin proteins resulting in a concentration of approximately 8 wt%. Once lyophilized, SF can be stored indefinitely before being dissolved in hexafluoroisopropanol (HFIP) in the desired weight in volume (w/v) concentration. In an embodiment of the coaxial protocol, commercially purchased PHB and SF was dissolved separately in the HFIP solvent at 1-3% wt/v, as previously described. The coaxial setup required an additional syringe pump to extrude both solvents simultaneously. A coaxial needle tip was used to extrude both solutions at the same time. However, the extrusion rates ranged from 6-10 ml/hour for the two solutions to create an even solvent evaporation and Taylor cone formation (FIGS. 4a-b). Using the parameters from the preliminary fabrication of combined scaffolds, coaxial scaffolds will be made starting at 8-9 kV positive voltage, 1-3 negative voltage, 10-15 cm working distance, and drum rotation of 1000-2000 rpm. Utilizing low concentrations of 1-3% wt/v for both solutions will allow for the correct size of fiber diameter to be obtained. It has been demonstrated that optimal fibroblast attachment and proliferation occurs on an average fiber diameter of 500nm. Embodiments of the scaffolds fabricated in this example possessed an average fiber diameter ranging from 400-800nm, a bracket including the desired fiber size. However, due to the required coaxial extrusion of two materials, these concentrations needed to be decreased to 1, 2, and 3% wt/v in order to produce appropriately sized fibers that possess both a core and external shell. To determine whether the fabricated fibers are coaxial, a cross-sectional cut was made, and electron dispersive spectroscopy (EDS) and x-ray diffraction (XRD) verified the different PHB and SF rich regions. To quantify the fiber diameter, scaffolds were gold sputter coated and imaged using an SEM at 30kV accelerating voltage, similar to FIG. 12, and analyzed using ImageJ and MATLAB. In embodiments of the present disclosure, optimization of concentrations varied from 1-3% to ensure effective electrospinning.

[0112] Embodiments of the present disclosure include methods of fabricating coaxial scaffolds with mechanical properties greater than 0.8MPa (UTS) and 9.45MPa (Modulus) to demonstrate the strength appropriate to mechanically support the urethra. It was hypothesized that coaxial scaffold with PHB cores and an SF exterior have better mechanical properties than homogenously mixed scaffolds because of the congruency of PHB core and superior bulk properties relative to SF. Embodiments of the present disclosure increase mechanical properties of the scaffold to provide sufficient support to the urethra in vivo and alleviate the symptoms of SUI during the time of fibrous tissue regrowth.

[0113] Mechanical properties of the scaffold were analyzed using two modalities: uniaxial tensile tests and dynamic mechanical analysis (DMA). Uniaxial tensile tests are widely used mechanical tests to characterize the ultimate tensile strength (UTS) and develop stress-strain relationships. Samples with dimensions of 2x4cm were strained at a rate of lOmm/min. Outputted data collected were UTS, Young’s Modulus, and elongation at break, as well as force-displacement and stress-strain curves. Recognizing the viscoelastic nature of the scaffold materials and the dynamic loading of the pelvic floor, DMA was employed to identify the storage and loss modulus of the coaxial materials. Failure modes unique to dynamic testing were identified as appropriate. As the pelvic floor is dynamically loaded and not an easily defined stress state, dynamic mechanical analysis is a relevant testing modality.

[0114] In this example, 4x15mm samples were placed in a TA Instruments Q600 DMA in tensile mode. The sample was elongated at a constant rate of 0.8mm/s to 8mm length or to the equivalent of 40% strain. A sinewave was superimposed to the linear elongation with a frequency of 50 Hz and a 0.1mm peak-to-peak amplitude. The measurements were taken every second and the sample was incrementally increased in length. Stiffness was calculated over the average of three consecutive sinewaves and a force-stiffness relationship was obtained.

[0115] In this example changes in ECM deposition and fibroblast proliferation were quantified on the co-axially spun scaffolds versus previously characterized combination scaffolds. It was hypothesized that coaxial scaffolds would initiate ECM deposition from the fibroblasts, as well as increased proliferation compared to polypropylene mesh. In this example, the deposition of ECM proteins were quantified to validate that the material and fabrication method promoted healthy, collagen-rich fibrous tissue formation. Embodiments of the present disclosure may include patientspecific devices with harvested fibroblasts that promote scar tissue generation to provide support as the scaffold dissolves.

[0116] Embodiments of the present disclosure may allow for fibrotic promotion of ECM deposition that would establish in vivo production of healthy fibrotic tissue, providing the permanent support necessary to alleviate SUI symptoms. In an in vivo testing, all scaffolds may be sterilized in 70% ethanol for 30 min followed by 3 by 10 min of sterile phosphate buffered saline (PBS) washes. The seeding technique used in this example includes adding 50,000 cells/cm² to each scaffold with 250uL of media/cm². Once initial attachment to the scaffold has occurred, the scaffolds may be inverted and the same number of cells may be added to the other side using the same protocol. Human vaginal fibroblasts attachment may occur within the first 24-48 hours of being seeded onto the scaffolds. Fibroblast adherence and morphology may be analyzed using SEM at 2, 5, and 7 days as seen in FIG. 15. Once favoravke morphology is confirmed, the cells’ production of elastin, collagen I and III, and fibronectin may be examined through immunostaining, and confocal fluorescence microscopy at 5 different timepoints: 2, 5, 7, 14, and 21 days. These timepoints are relevant due to the proliferation of fibroblasts to confluency in a 75cc plate being about 7 days. To gain full saturation of the scaffold, it is important to measure the time it takes to gain confluence and to understand the difference between scaffolds based on their fiber size and material makeup. DAPI and collagen dye may be used to determine the amount of collagen production by cells calculated by the absorbance of stain per gram of scaffold (FIG. 16). Staining of the scaffolds with AlamarBlue may be used to determine metabolic activity of fibroblasts based on the absorbance of the cells and the values of colorimetric absorbance. To determine the level of cell-scaffold saturation and deposition of ECM, staining and imaging is critical. Confocal images may be analyzed through Fiji/ImageJ to determine concentration of each fluorophore.

[0117] In this example, each scaffold was made under identical environments resulting in the only variable being the concentration ratios of PHB/SF compared to established relevant tissue properties. The statistical analysis conducted was a two-way ANOVA tests to determine the different mechanical and cellular properties between weight percentages and ratio of compositions. Both variables are hypothesized to influence the fiber diameter and ECM deposition, as demonstrated in initial data collection. One type of cell line, hVFs, will be used for all scaffolds and in vitro testing to eliminate cellular variation. All study sample sizes were calculated prior to experiments based on a power analysis with a=0.05 and =0.20. Estimates of sample variance and effect size were based on power calculations and preliminary data at N=3, but to account for slight manufacturing and biologic variability N=6 will be used.45

[0118] In an embodiment of the present disclosure, the addition of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) on the scaffolds can enhance the proliferation and integration into the scaffolds. In vitro tests may include long term degradation, relevant biomechanical mechanical tests, and elution studies.

[0119] Although the present disclosure has been described with respect to one or more particular embodiments and/or examples, it will be understood that other embodiments and/or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A bioresorbable nanofibrous scaffold comprised of nanofibers of silk fibroin and polyhydroxybutyrate (PHB), wherein the silk fibroin and the PHB are arranged so that an area of the bioresorbable nanofibrous scaffold comprises concentrated amounts of the silk fibroin and another area of the bioresorbable nanofibrous scaffold comprises concentrated amounts of the PHB.

2. The bioresorbable nanofibrous scaffold of claim 1, wherein the silk fibroin and the PHB are coaxially arranged such that the PHB forms a center of the nanofibers and the silk fibroin surrounds the PHB concentrically.

3. The bioresorbable nanofibrous scaffold of claim 1, wherein the silk fibroin and the PHB are arranged such that the silk fibroin and the PHB are layered.

4. The bioresorbable nanofibrous scaffold of claim 1, wherein a diameter of the nanofibers is from 50 nm to 10 pm.

5. The bioresorbable nanofibrous scaffold of claim 1 , wherein a tensile strength of the nanofibers is from 2MPa to 5MPa.

6. The bioresorbable nanofibrous scaffold of claim 1, further comprising biological materials disposed on the nanofibers.

7. The bioresorbable nanofibrous scaffold of claim 6, wherein the biological materials comprise TGF-Beta, fibroblast growth factor (FGF), collagen, fibronectin, cognitive tissue growth factor (CTFG), integrin, proteoglycan, elastin, tenascin, and/or other small molecules.

8. The bioresorbable nanofibrous scaffold of claim 6, wherein the biological materials are coated onto the bioresorbable nanofibrous scaffold.

9. The bioresorbable nanofibrous scaffold of claim 1, further comprising fibroblasts harvested and isolated from a patient disposed on the nanofibers.

10. The bioresorbable nanofibrous scaffold of claim 9, wherein the fibroblasts are seeded onto the bioresorbable nanofibrous scaffold.

11. The bioresorbable nanofibrous scaffold of claim 1, further comprising focal adhesion points.

12. The bioresorbable nanofibrous scaffold of claim 1, wherein the bioresorbable nanofibrous scaffold is configured to be implanted in a human body to provide temporary support for medical applications.

13. The bioresorbable nanofibrous scaffold of claim 12, wherein the bioresorbable nanofibrous scaffold is configured to dissolve and promote autonomous fibrous growth to replace the temporary support of the bioresorbable nanofibrous scaffold.

14. The bioresorbable nanofibrous scaffold of claim 13, wherein the bioresorbable nanofibrous scaffold dissolves over a period from 6 months to 3 years.

15. The bioresorbable nanofibrous scaffold of claim 1, wherein the nanofibers are electrospun.

16. A bioresorbable nanofibrous scaffold comprised of nanofibers of silk fibroin and copolymers of polyhydroxybutyrate (PHB), wherein the silk fibroin and the co-polymers of PHB are arranged so that an area of the bioresorbable nanofibrous scaffold comprises concentrated amounts of the silk fibroin and another area of the bioresorbable nanofibrous scaffold comprises concentrated amounts of the co-polymers of PHB.

17. The bioresorbable nanofibrous scaffold of claim 1, wherein the silk fibroin and the copolymers of PHB are coaxially arranged such that the co-polymers of PHB forms a center of the nanofibers and the silk fibroin surrounds the co-polymers of PHB concentrically.

18. The bioresorbable nanofibrous scaffold of claim 1, wherein the silk fibroin and the copolymers of PHB are arranged such that the silk fibroin and the co-polymers of PHB are layered.

19. The bioresorbable nanofibrous scaffold of claim 16, wherein the co-polymers of PHB comprise poly 3-hydroxybutyrate (P3HB) or prolyl 4-hydroxylase (P4HB).

20. The bioresorbable nanofibrous scaffold of claim 16, wherein a diameter of the nanofibers is from 50 nm to 10 pm.