FIBER-BASED SCAFFOLDS FOR TENDON CELL MIGRATION AND REGENERATION

This application claims priority of U.S. Provisional Application Nos. 62/962,708, filed January 17, 2020, and 62/916,120, filed October 16, 2019, the contents of each of which are hereby incorporated by reference.

Throughout this application, various publications are referenced by author and publication date within parentheses. Full citations for these publications may be found at the end of the specification or at the end of each experimental section. The disclosures of these publications are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

This invention was made with government support under W81XWH-15-1- 0685 awarded by the Department of Defense Translational Research Award. The government has certain rights in the invention.

TECHNICAL FIELD

This disclosure relates to a mesh scaffold configured to support and promote tendon cell migration and regeneration and methods for producing the mesh scaffold configured to support and promote tendon cell migration and regeneration.

BACKGROUND

There is a significant unmet clinical demand for functional tendon grafts that can bridge severed tendon stumps and promote healing.This is due to several factors, but largely because tendon transections are one of the most common injuries that occur in military personals, both on-duty and off-duty, and in civilians, including a high incidence of hand lacerations and Achilles tendon ruptures. Additionally, tendon transections are a leading cause of medical evacuation (MEDEVAC) and battle attrition in combat operations. They occur in many trauma scenarios, including lacerations,blasts, gunshot wounds, crushes, falls, and in sports. Acute rupture of the Achilles tendon occurs in up to 40 out of 100,000 people per year and are typically sports-related (Lemme et al., 2018). Additionally, there were approximately 50,000 avocado related cutting injuries from 1998- 2017, 0.5% of which were reported as a tendon injury (Farley et al., 2020). Unfortunately, 97% of ruptured tendons show degenerative changes post-injury (Pankaj et al., 2005). Tendon transections are the most common orthopedic injury causing long-term disability (Durgam et al., 2017).

Tendon healing is generally slow (Sharma et al., 2006) and is of limited reparative capacity (Durgam et al., 2017).Adhesion formation prevents sheath gliding and fibrovascular scar formation can occur (Howell et al., 2017). Moreover, a lacerated tendon often presents a large gap between transected tendon ends post-injury (see, e.g., Figure 2), making it difficult to repair.

Even though not ideal, tendon repair is typically delayed by up to four days to over a week, rather than repaired on-site, and repair becomes more difficult with increased time post-injury (Tang et al. 1994). This delay is because of the challenges with current tendon repair,which uses sutures.Specifically, sutures are used to securely attach severed tendon ends together (see, e.g., Figure 3). However, this requires an operating surgeon's skill and instruments and is time consuming. A delay beyond four days is unadvisable due to the risk of tendons slipping out of the sheath and becoming enlarged due to fibrosis.Then it is difficult to reinsert the tendon into the sheath. Importantly, delayed repair prevents the recovery of full function. Specifically, persistent impaired function can occur (Howell et al., 2017) and lower biomechanical resistance can lead to recurrent injuries (Frauz et al., 2019). The lacerated tendon will recover significantly better if it is repaired within twelve hours of injury, even if the repair is just to bridge the large gap between transected tendon ends and hold the severed tendon ends together.

The following three topics related to tendon injuries and the current treatment for such injuries are discussed below: (i) Injuries of deployed military personnel; (ii) Previous methods for tendon laceration repair; and (iii) Biologically-derived grafts for tendon repair.

Injuries of deployed military personnel:

There are various types of injuries that can occur in a battle zone. According to Waterman et al., between April 2009 and December 2012 globally, approximately 51% of orthopedic injuries involved the upper extremity, including 197 hand injuries, followed by injuries involving the lower extremity (37%). Remarkably, 48% of all musculoskeletal injuries suffered by soldiers who were medically evacuated from the battle zone were noncombat injuries. Furthermore, MEDEVACs for upper extremity injuries were nearly two-fold greater than for lower extremity complaints, and hand injuries comprised 34% of all injury cases directed for evacuation for orthopaedic injuries. Prior reports have underscored the prevalence of traumatic, non-combat related hand injuries, accounting for between 4.4% and 17.5% of all injured patients.Notably, up to 23% of deployment hand injuries also required nerve or tendon repairs on the extensor digitorum (ED), flexor digitorum superficialis (FDS), or flexor digitorum profundus (FDP) tendons of zone II due to laceration.

Since military personnel on operations are required to handle personal weapons and to operate armored vehicles, often in close proximity to other soldiers and the local civilian population, injuries to the hand are of particular significance as they limit function.This emphasizes the need for forward treating providers to be well prepared for injuries and fractures of the hand, including clinical evaluation, management, and interdisciplinary coordination with ancillary services such as occupational therapy.

Previous methods for tendon laceration repair:

While sutures are the most widely used method for repairing lacerated tendons, they are inherently disadvantageous when used with soft tissue, such as tendons. Stress concentrations caused by sutures and errors made due to inexperience can lead to severe problems such as tearing of the tendon which can cause insufficient healing. Additionally, sutures are for long term repair and if the surgery is rushed or incorrectly performed in any way, the results lead to sub- optimal results.

Nitinol sleeves have also been used to reattach severed tendon stumps. These implants use laser cut tines to grip on to separate sides of the lacerated tendon and connect them without using sutures. However, these implants are expensive and time consuming to manufacture as they require high tolerance laser cutting and custom-made jig to heat set the tines in the correct orientation.

Other devices use a bed of spikes to connect the two tendon pieces together. However, such devices only use mechanical means to hold the severed tendon together.

Biologically-derived grafts for tendon repair:

Tendon regeneration remains a significant clinical challenge, given its inherently low self-healing potential, undesirable scar-dominated repair response, and poor graft-host integration. These challenges are exacerbated by the large number of tendon injuries reported in an aging, yet active, population in the armed services and in the general population.Biological matrices such as collagen-rich dermis and small intestinal submucosa have been marketed as tendon patches to reinforce surgical repair of tendons. While promising results were noted in animal models, the use of biologically-derived grafts for tendon repair is limited due to the gross mismatch in mechanical properties and rapid graft remodeling in the physiologically demanding joint. Thus, there is significant interest in the development of tissue engineered tendon grafts.

Nanofibers are an attractive platform for tendon repair and regeneration because their structural and mechanical properties can be readily tailored to match the native collagenous matrix.

Electrospinning is an established fiber fabrication method in which a polymer is dissolved in a solvent, and the resulting polymer solution is loaded into a syringe. Electrostatic forces are applied to the tip of the syringe needle, and charged polymeric fibers are ejected from the syringe at a constant rate. Electrospun fibers can be collected and used in a wide variety of applications, including but not limited to tissue engineering applications such as bone, cartilage, tendon, and ligament repair or regeneration. In such applications, a variety of growth factors, ceramic components, and other materials may be incorporated into the electrospinning solution so that resulting fibers contain advantageous material and biochemical properties.

Matrices may be fabricated via electrospinning, in which a polymer melt is ejected from an electrically charged syringe, resulting in porous, fibrous structures that can be functionalized for optimal tissue engineering outcomes.

However, no technology exists to our knowledge applying polymeric nanofibers to tendon cell migration and regeneration.

SUMMARY

This disclosure reflects the results from efforts to advance the state of the art for securely and rapidly re-attaching lacerated tendons in Prolonged Field Care in order to extend the window of primary repair for an injured tendon-muscular unit. Such result may be achieved by incorporating advances from the domains of biomaterial fabrication and tissue regeneration into the practice of reconstructive surgery. For example, a mesh scaffold, such as disclosed herein, may be used to secure severed tendon ends while mimicking the native tendon structure.

This disclosure describes various inventive mesh scaffolds that are biocompatible and formed of nanofibers suitable for supporting and promoting guided migration and regeneration of tendon cells and a method for producing such mesh scaffold. Various embodiments and examples are described. For example, the mesh scaffold preferably comprises a mesh of nanofibers formed of two or more polymers, rolled along a longitudinal axis of the scaffold to mimic native tendon structure. Further, the mesh is preferably a gelatin nanofiber-based mesh. Further, the two or more biomimetic polymers can preferably be polylactide-co-glycolide (PLGA) and polycaprolactone (PCL). Further, the rolled mesh preferably forms a layered structure, with one layer on another. Further, in a cross-sectional view of the rolled mesh, the rolled mesh preferably has a spiral arrangement.

Further, the mesh that is rolled is preferably formed of electrospun nanofibers, and the nanofibers preferably include aligned fibers aligned in an alignment direction parallel to the longitudinal axis of the scaffold.

Further, the mesh scaffold is preferably formed of a biomimetic material for repairing a gap between transected tendon ends due to an injury. Further, the rolled mesh may be sutured to the transected tendon ends. The mesh scaffold preferably further comprises a non fouling coating to prevent a foreign body response and/or an antibiotic coating. The mesh scaffold preferably further comprises inductive biomolecules and/or one or more growth factors, which promote faster healing of the tendon, applied to, or embedded in, the nanofibers or the mesh of nanofibers.

Further, the mesh scaffold preferably forms a 3-dimensional conduit configured for positioning between, and bridging together, transected tendon ends. Further, the mesh scaffold preferably forms a collar configured for surrounding an injured tendon, and/or for positioning between, and bridging together, transected tendon ends. Further, the mesh scaffold can form a graft collar formed of a biomimetic material that permits migration of a tendon cell population from the graft collar to the injured tendon. Further, the graft collar is preferably formed of a biomimetic material that permits depositing of physiologically relevant extracellular matrix.

The method for producing a mesh scaffold preferably includes dissolving gelatin and a polymeric blend of PLGA and PCL in a solvent to form a gelatin polymer solution and electrospinning the gelatin polymer solution onto a rotating collecting drum to form a mesh scaffold comprising aligned nanofibers.Further, the method preferably includes rolling the mesh scaffold around a rod, the nanofibers of the rolled mesh including fibers aligned in an alignment direction parallel to the longitudinal axis of the scaffold and form a multi layer scaffold structure mimicking native tendon structure.

Further, the solvent is preferably 2,2,2,-trifluoroethanol. Further, the method preferably comprises crosslinking each side of the mesh scaffold with glutaraldehyde.Further, the method preferably comprises soaking the mesh scaffold in media. Further, the concentration of the polymeric blend is preferably at least 32% weight by volume of the solvent. Further, the ratio of PLGA to PCL of the polymeric blend is preferably 5:1 weight by weight %.

Such inventive mesh scaffolds can be employed in meshes, implantable devices, grafts and other tissue engineering tools, etc.

Various other inventive aspects can be integrated or employed, as discussed infra.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a graphical representation of an apparatus employing electrospinning for generating a mesh of fibers, for tissue engineering.

Figure 2 shows a gap between transected tendon ends due to laceration of the tendon (Dashboard M., 2012).

Figure 3 shows a graphical representation of suture repair of a lacerated tendon (Jarrett P., 2016).

Figure 4 shows a graphical representation of hierarchical structure of a tendon (Spang C., 2015).

Figure 5 shows a graphical representation of a nanofiber-based mesh scaffold. Figure 6 shows an SEM (scanning electron microscopy) image of a nanofiber mesh that can be employed as a biomimetic scaffold for tendon regeneration.

Figure 7 shows an SEM image providing an end view of a rolled mesh that may operate as a Tendon-Scaffold Junction.

Figure 8 shows a notional representation of a spiral arrangement, such as may be similar to a cross-sectional view of a rolled mesh.

Figure 9 shows a transected patellar tendon displaying injury surface (S).

Figure 10 shows a schematic diagram of an experimental set-up for cell migration study.

Figure 11 shows a notional representation of tendon cell migration from an injured tendon to a nanofiber mesh.

Figure 12 shows SEM images of cell migration and morphology.

Figure 13A shows that patellar tendon stumps exhibited a zone of cell death at the injury surface (S) at 1-Day post-transection, while viable cells were seen within the bulk of the tissue (green = live; red = dead).

Figure 13B shows that cells migrated onto the mesh over time and viability was maintained over 14 days of culture.

Figure 14 shows graphs demonstrating that cell growth, collagen, and proteoglycan production increased with time on fiber scaffolds [where * indicates significance from previous time point (*p<0.05, n=5)].

Figure 15A shows a graph of tendon cell deposition on nanofibers compared to tissue culture plastic (n=5; ^L r<0.05 between groups;

*p<0.05 over time). Tendon cells migrated out and remained viable on nanofibers up to 28 days. Figure 15B shows graphs demonstrating collagen and proteoglycan production (iug) increase on nanofibers compared to tissue culture plastic (n=5; ^L r<0.05 between groups; *p<0.05 over time). Tendon cells migrated out and remained viable on nanofibers up to 28 days.

Figure 15C shows graphs demonstrating collagen and proteoglycan production (pg/cell) increase on nanofibers compared to tissue culture plastic (n=5; ^L r<0.05 between groups; *p<0.05 over time). Tendon cells migrated out and remained viable on nanofibers up to 28 days.

Figure 16 shows that tendon cells migrated out and remained viable on nanofibers up to 28 days similar to tissue culture plastic.

Figure 17A shows a notional representation of a mesh of fibers in which a non-fouling coating 171, and/or an antibiotic coating 172, has been applied to the mesh, to prevent a foreign body response.

Figure 17B shows a notional representation of a mesh to which inductive biomolecules 173 and/or one or more growth factors 174, have been applied, to promote faster healing of the tendon. The white dots representative of the inductive biomolecules 173 or one or more growth factors 174 are not to scale.

Figure 18 shows a flow chart for a method for producing a mesh scaffold, according to an embodiment.

DETAILED DESCRIPTION

The following embodiments and examples (including details thereof) are set forth to aid in an understanding of the subject matter of this disclosure but are not intended to, and should not be construed to, limit in any way the invention that is claimed.

Terms

In order to facilitate an understanding of the material which follows, except as otherwise expressly provided herein, each of the following terms, as used in this application, shall have the meaning set forth below.

As used herein, "active agent" shall mean a component incorporated into the fibrous, polymeric mesh scaffolds, which when released over time, supports alignment, proliferation and matrix deposition of a selected cell. Examples include but are in no way limited to growth factors such as transforming growth factor-beta 3(TGF-3), growth/differentiation factor-5 (gdf-5), bone morphogenetic protein (BMP) 1 through 14, fibroblast growth factor (FGF) and basic fibroblast growth factor (bGF).A single active agent or a combination of active agents may be incorporated into the fibrous, polymeric mesh scaffolds of this application. By "active agent" it is also meant to include an active pharmaceutical ingredient such as, but not limited to, an anti-inflammatory, an antibiotic or a pain medicament added to the fibrous, polymeric mesh scaffolds to enhance treatment and/or healing of the subject upon implantation.

As used herein, "aligned fibers" shall mean groups of fibers which are oriented generally in an alignment direction along the same directional axis. Examples of aligned fibers include, but are not limited to, groups of parallel fibers.

As used herein, "bioactive" shall include a quality of a material such that the material has an osteointegrative potential, or in other words the ability to bond with bone.Generally,materials that are bioactive develop an adherent interface with tissues that resist substantial mechanical forces.

As used herein, a "biocompatible" material is a synthetic or natural material used to replace part of a living system or to function in intimate contact with living tissue. Biocompatible materials are intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body. The biocompatible material can perform with an appropriate host response in a specific application and does not have toxic or injurious effects on biological systems.Nonlimiting examples of biocompatible materials include a biocompatible ceramic, a biocompatible polymer or a biocompatible hydrogel.

As used herein, "biocompatible matrices" shall mean three-dimensional structures fabricated from biocompatible material. The biocompatible material can be biologically-derived or synthetic.

As used herein, "biodegradable" means that the material, once implanted into a host, will begin to degrade.

As used herein, "biomimetic" shall mean a resemblance of a synthesized material to a substance that occurs naturally in a human body and which is not rejected by (e.g., does not cause an adverse reaction in) the human body.

As used herein, "biopolymer mesh" shall mean a mesh including any material derived from a biological source. Examples of a biopolymer mesh include, but are not limited to, collagen, chitosan, silk and alginate.

As used herein, "damaged soft tissue" shall mean damage of muscles, ligaments and tendons throughout the body. Damaged soft tissue can include injuries such as a sprain, strain, a one-off blow resulting in a contusion or overuse of a particular part of the body.

As used herein, "effective amount" and/or "sufficient concentration" shall mean a level, concentration, combination or ratio of one or more components added to the fibrous, polymeric mesh scaffolds which promotes differentiation of stem cells to a selected cell type and/or enhances proliferation of desired cells.

As used herein, "electrostatic forces" shall mean a force (attractive or repulsive) that exist between electrically charged particle or objects where the attractive or repulsive forces between particles that are caused by their electric charges. In electrospinning, the electric charge is applied to the needle attached to the syringe. As used herein, "fibroblast" shall mean a cell of connective tissue, mesodermally derived, that secretes proteins and molecular collagen including fibrillar procollagen, fibronectin and collagenase, from which an extracellular fibrillar matrix of connective tissue may be formed.

As used herein, "fully synthetic" matrices mean that the matrices are composed of man-made material, such as synthetic polymer, or a polymer-ceramic composite, but it does not preclude further treatment with material of biological or natural origin, such as seeding with appropriate cell types, (e.g., seeding with osteoblasts, osteoblast like cells, and/or stem cells), or treating with a medicament, (e.g., anti-infectives, antibiotics, bisphosphonate, hormones, analgesics, anti-inflammatory agents, growth factors, angiogenic factors, chemotherapeutic agents, anti-rejection agents, and RGD peptides).

As used herein, "functional" shall mean affecting physiological or psychological functions but not organic structure.

As used herein, "gelatin" shall mean a substance that is typically a glutinous mixture of peptides and proteins derived from collagen taken from animal parts, from seaweed extracts, from plant extracts, etc.

As used herein, "graft" shall mean the device to be implanted during medical grafting, which is a surgical procedure to transplant tissue without a blood supply, including but not limited to soft tissue graft, synthetic grafts, and the like. The graft can be an allograft or an autograft. An "allograft" is tissue taken from one person for transplantation into another. Allografts can include, most commonly, Achilles and tibialis, patellar and quadricep's tendons. An "autograft" or "autologous graft" is a graft comprising tissue taken from the same subject to receive the graft. Graft can also be allogeneic (e.g., derived from a material originating from the same species as that of the subject receiving the graft) or xenogenic (e.g., derived from a material originating from a species other than that of the subject receiving the graft). The graft can be a soft tissue graft, such as a tendon.The graft can be a graft for a ligament in a subject, including the ACL. The tendon graft can be a bone- patellar tendon-bone (BPTB) graft, a semitendinosus or a hamstring- tendon (HST) graft.

As used herein, "graft collar" shall mean a device embodying a graft and configured like a collar, that is, having a hollow cylindrical body in a longitudinal direction.A graft collar can be permeable, so the tissue can survive.

As used herein, "growth factors" shall mean proteins that regulate many aspects of cellular function, including survival, proliferation, migration and differentiation. Examples of growth factors can include cytokines, therapeutic peptides/proteins to aid in tendon cell repair or regeneration, hormones that bind to specific receptors on the surface of their target cells, PDGF, etc.

As used herein, "implantable" or "suitable for implantation" means surgically appropriate for insertion into the body of a host, (e.g., biocompatible), or having the design and physical properties set forth in more detail below.

As used herein, "matrix" shall mean a three-dimensional structure fabricated from biomaterials. The biomaterials can be biologically- derived or synthetic.

As used herein, "mesh" means a network of material. In one embodiment, the mesh may be woven synthetic fibers, non-woven synthetic fibers, microfibers and nanofibers suitable for implantation into a mammal, (e.g., a human). The woven and non-woven fibers may be made according to well-known techniques.The microfiber or nanofiber mesh may be made according to techniques known in the art and those disclosed in, e.g., PCT International Application No. PCT/US2008/001889, filed February 12, 2008 to Lu et al., which application is incorporated by reference as if recited in full herein. Fibers of the mesh may be aligned or unaligned. As used herein, "musculoskeletal cell" shall mean a chondrocyte, fibrochondrocyte, fibroblast or osteoblast.

As used herein, "nanofiber mesh" shall mean a flexible netting of nanofibers, oriented such that at least some of the nanofibers are not parallel to others of the nanofibers.

As used herein, "nanofiber scaffold" is constructed of "nanofibers."

As used herein, "nanofibers" shall mean fibers with diameters not more than about 1000 nanometers (and preferably less than 1000 nanometers). According to the invention, a "nanofiber" is a biodegradable polymer that is electrospun into a fibrous, polymeric mesh scaffold as described in more detail herein below. The nanofibers of the fibrous, polymeric mesh scaffolds are oriented in such a way (i.e., aligned or unaligned) so as to mimic the natural architecture of the soft tissue to be repaired. Moreover, the nanofibers and the subsequently formed fibrous,polymeric mesh scaffolds are controlled with respect to their physical properties, such as for example, fiber diameter, pore diameter, and porosity so that the mechanical properties of the nanofibers and fibrous, polymeric mesh scaffolds are similar to the native tissue to be repaired, augmented or replaced.Thus, in the case of tendon repair, the fibrous, polymeric mesh scaffolds are able to mimic the native tendon structure and bridge the gap between lacerated tendon ends. The fibrous, polymeric mesh scaffolds may be engineered to remain in place for as long as the treating physician deems necessary. Typically, the fibrous, polymeric mesh scaffolds will be engineered to have biodegraded between 6-18 months after implantation, such as for example 12 months.

As used herein, "PDGF" shall mean platelet-derived growth factor.

As used herein, "PGA" shall mean polyglycolide or poly(glycolic acid).

As used herein, "PLA" shall mean poly(lactic acid) or polylactic acid or polylactide. As used herein, "PLGA" shall mean poly(lactic-co-glycolic acid).

As used herein, "PCL" shall mean polycaprolactone.

As used herein, "polymer" means a chemical compound or mixture of compounds formed by polymerization and including repeating structural units. Polymers may be constructed in multiple forms and compositions or combinations of compositions. Polymers may be natural or synthetic. Examples of natural polymers include collagen, cellulose, silk fibroin, keratin, gelatin and polysaccharides such as chitosan and alginate. Examples of synthetic polymers include poly(lactic acid) (PLA), polycaprolactone (PCL), polyurethane (PU), poly(lactic-co- glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(ethylene-co-vinylacetate) (PEVA).

As used herein, "polymer solution" shall mean a solution in which a polymer has been dissolved.

As used herein, "polymeric blend" shall mean a blend of two or more polymers to form a material having physical properties different than each polymer alone.

As used herein, "polymeric fibers" shall mean a subset of man-made fibers, which are based on a polymer.

As used herein, "polymeric matrices" shall mean matrices produced from fibrous polymer.

As used herein, "soft tissue" includes, as the context may dictate, tendon and ligament, as well as the bone to which such structures may be attached. Preferably, "soft tissue" refers to tendon- or ligament- bone insertion sites requiring surgical repair, such as for example tendon-to-bone fixation.

As used herein, "soft tissue graft" shall mean a graft which is not fully synthetic, and can include autologous grafts, syngeneic grafts, allogeneic grafts, and xenogeneic graft. As used herein, "stem cell" means any unspecialized cell that has the potential to develop into many different cell types in the body, such as mesenchymal osteoprogenitor cells, osteoblasts, osteocytes, osteoclasts, chondrocytes, and chondrocyte progenitor cells. Preferably the cells are from a compatible human donor. More preferably, the cells are from the patient (i.e., autologous cells).

As used herein, "synthetic" shall mean that the material is not of a human or animal origin.

As used herein, all numerical ranges provided are intended to expressly include at least the endpoints and all numbers that fall between the endpoints of ranges.

Embodiments

The following embodiments and examples are set forth to aid in an understanding of the subject matter of this disclosure but are not intended to and should not be construed to, limit in any way the inventions applicant claims.

Figure 1 shows an electrospinning apparatus 1 including a syringe 3 configured with a vessel 11 and connected at one end of the vessel 11 to a needle 7 in the form of a cone from which a jet of solution 13 ejects toward the grounded plate 17. A syringe plunger 7 connects to the vessel 11 at the other end of the syringe 3, and applies pressure on the content of the cylindrical vessel, in particular the content in the cylindrical vessel is the polymer solution 5. The polymer solution 5 ejects towards the grounded plate 17 and forms a batch of polymeric fibers 15 on the grounded plate 17. The syringe 3 is configured with a volume of 5 ml, the needle 7 connected to cylindrical vessel of the syringe at one end is configured with a 26G needle. The syringe plunger that provides pressure on the content of the cylindrical vessel, ejects the content at a flow rate in the range of 0.4-1.0 ml/hr. The electrospinning apparatus is in an enclosing cabinet, which includes a humidifier that maintains the humidity in the enclosing cabinet in a range of 45% to 55%. Electrospinning, short for electrostatic spinning, involves the fabrication of fibers by applying a high electric potential to a polymer solution. The material to be electrospun, is loaded into a syringe or spoon, and a high potential is applied between the solution and a grounded substrate. As the potential increases, the electrostatic force applied to the polymer solution overcomes surface tension, distorting the solution droplet into a Taylor cone from which a jet of solution ejects toward the grounded plate or a cylindrical vessel. The jet splays into randomly oriented fibers. These fibers have diameters ranging from nanometer scale to greater than 1 pm and are deposited onto the grounded plate or onto objects inserted into the electric field forming a non-woven batch of polymeric fibers.

Further, a distance between the tip of the needle and the grounded plate during the electrospinning of the batch of polymeric fibers, is in a range of 10-15 cm. A voltage applied to the needle during the electrospinning of the batch of polymeric fibers is in the range of 8-15 kV.

MESH SCAFFOLD

In this disclosure, a mesh scaffold is described. According to an exemplary embodiment, a mesh scaffold includes a mesh of nanofibers formed of a polymeric blend of two or more polymers. In a further embodiment, the mesh of electrospun nanofibers is rolled along a longitudinal axis of the scaffold to mimic native tendon structure. In a further embodiment, the mesh is a gelatin nanofiber-based mesh. In a further embodiment, the polymeric blend includes polylactide-co- glycolide (PLGA) and polycaprolactone (PCL). In a further embodiment, the mesh scaffold formed of the rolled mesh of nanofibers is biomimetic and supports and promotes guided migration and regeneration of tendon cells. In a further embodiment, the rolled mesh forms a layered structure, with one layer on another layer. In another embodiment, in a cross-sectional view of the rolled mesh, the rolled mesh has a spiral arrangement. In a further embodiment, the mesh that is rolled is formed of electrospun nanofibers, and the nanofibers include aligned fibers aligned in an alignment direction parallel to the longitudinal axis of the scaffold.

In another embodiment, the rolled mesh of electrospun nanofibers forms a 3-dimensional conduit configured for positioning between, and bridging together, transected tendon ends. In a further embodiment, the rolled mesh is sutured to the transected tendon ends.

In another embodiment, the rolled mesh of electrospun nanofibers forms a collar configured for surrounding an injured tendon, and/or for positioning between, and bridging together, transected tendon ends. In a further embodiment, the collar is a graft collar formed of a biomimetic material that permits migration of a tendon cell population from the graft collar to the injured tendon. In a further embodiment, the collar is a graft collar formed of a biomimetic material that permits depositing of physiologically relevant extracellular matrix.

In yet another embodiment, the mesh scaffold is formed of a biomimetic material for repairing a gap between transected tendon ends due to an injury.

The mesh characteristics can be customized by altering green electrospinning parameters.For example, fiber diameter and morphology can be altered, including the formation of the fibers, by controlling applied voltage and polymer solution surface tension and viscosity. Also, fiber orientation can be controlled by rotating the grounded plate. This high degree of customizability and ability to use many different materials, such as biodegradable polymers and silks, grant this fabrication method a high potential in the development of materials for biomedical application. Management of fiber diameter allows surface area to be controlled, and polymers with different degradation rates can be combined in various ratios to control fiber degradation, both of which are significant in drug delivery applications. Also, controlling the orientation of fiber deposition grants a degree of control over cell attachment and migration. Moreover, the ability to electrospin fiber meshes onto non-metal objects placed in the electric field enables the fabrication of multiphasic scaffold systems.

The mesh scaffold can also include additional components.For example, the mesh can include an active agent that is released from the mesh over time, inductive biomolecules, or one or more growth factors to promote faster healing of the tendon. Further, the mesh scaffold can include selected musculoskeletal cells or stem cells which differentiate into the musculoskeletal cells, or with soft tissue graft to replace or repair damaged soft tissue. Examples of musculoskeletal cells which can be seeded onto the mesh scaffold include chondrocytes, fibro chondrocytes, fibroblasts and osteoblasts.

In an embodiment, the mesh scaffold comprises inductive biomolecules applied to, or embedded in, the nanofibers or the mesh of nanofibers. In another embodiment, the mesh scaffold comprises one or more growth factors, which promote faster healing of the tendon, applied to, or embedded in, the rolled mesh of nanofibers.

In another embodiment, the mesh scaffold comprises a non-fouling coating to prevent a foreign body response. In another embodiment, the mesh scaffold comprises an antibiotic coating.

The polymer used in the mesh scaffold can include polylactide-co- glycolide (PLGA), PLA or PGA. Another example of the polymer in the mesh scaffold can include polycaprolactone (PCL). The polymer in the mesh scaffold can also include a blend of polylactide-co-glycolide (PLGA), PLA and/or PGA and polycaprolactone (PCL). The polymer solution used for electrospinning the mesh can include a concentration of at least 32% (w/v) 5:1 blend of poly(lactide-co-glycolide) (PLGA) and poly(e-caprolactone) (PCL) in a solvent.

The mesh scaffold is preferably biodegradable, and can integrate with host bone tissue, and/or exhibits distinct regions with different chemical and mechanical properties which allow it to support the growth of multiple tissue types, and to be bioactive.

METHOD

In this disclosure, a method is described for producing a mesh scaffold. Such method, according to an exemplary embodiment, includes a) dissolving gelatin and a polymeric blend of PLGA and PCL in a solvent to form a gelatin polymer solution; b) electrospinning the gelatin polymer solution onto a rotating collecting drum to form a mesh scaffold comprising aligned nanofibers; and c) rolling the mesh scaffold around a rod, the nanofibers of the rolled mesh including fibers aligned in an alignment direction parallel to the longitudinal axis of the scaffold and form a multi-layer scaffold structure mimicking native tendon structure.

In one embodiment, the solvent is 2,2,2,-trifluoroethanol. In another embodiment, the method further comprises crosslinking each side of the mesh scaffold with glutaraldehyde. In another embodiment, the method further comprises soaking the mesh scaffold in media. In another embodiment, the concentration of the polymeric blend is at least 32% weight by volume of the solvent. In another embodiment, the ratio of PLGA to PCL of the polymeric blend is 5:1 weight by weight %.

Additional steps may optionally be added to the method to impart additional features or characteristics. For example, the method may include adding additional components to the mesh scaffold. The additional components may include active agents, one or more growth factors, hydroxyapatite or a calcium phosphate, calcium-deficient apatite (CDA).Such additional components may permit the mesh scaffold to provide a functional interface between multiple tissue types. Such components can further include an active pharmaceutical ingredient such as, but not limited to, an anti-inflammatory, an antibiotic or a pain medicament added to the mesh scaffold to enhance treatment and/or healing of the subject upon implantation of the mesh scaffold. The method may further include seeding the mesh scaffold with selected musculoskeletal cells or stem cells which differentiate into the musculoskeletal cells, or with soft tissue graft to replace or repair damaged soft tissue. Examples of musculoskeletal cells which can be seeded onto the mesh scaffold include chondrocytes, fibro chondrocytes, fibroblasts and osteoblasts.

In another embodiment, the method further includes applying to, or embedding in, the electrospun nanofibers or the mesh of electrospun nanofibers, an active agent. After implanting the mesh scaffold with the active agent into a subject, the active agent may release over time. In this embodiment, release of the active agent over time supports tendon cell migration and regeneration. Examples of the active agent include but are in no way limited to growth factors. A single active agent or a combination of active agents may be incorporated into the mesh scaffold. By active agent it is also meant to include an active pharmaceutical ingredient such as, but not limited to, an anti-inflammatory, an antibiotic or a pain medicament added to the mesh scaffold to enhance treatment and/or healing of the subject upon implantation.

In yet another embodiment, the method includes applying to, or embedding in, the electrospun nanofibers or the mesh of electrospun nanofibers, one or more inductive biomolecules.

In yet another embodiment, the method includes applying to, or embedding in, the rolled mesh of electrospun nanofibers, one or more growth factors, which may promote faster healing of the tendon. Such growth factors may release from the rolled mesh over time once in a subject. Examples of the growth factors include, but are in no way limited to, growth factors such as transforming growth factor-beta 3(TGF-3), growth/differentiation factor-5 (gdf-5), bone morphogenetic protein (BMP) 1 through 14, fibroblast growth factor (FGF) and basic fibroblast growth factor (bGF). Preferably, the growth factors in the mesh are in a range of up to 200 pg per electrospinning. The nanofiber-based mesh scaffolds of this application can be engineered to remain in place for as long as the treating physician deems necessary. Typically, these nanofiber-based mesh scaffolds are engineered to biodegrade between 6-18 months after implantation, such as for example 12 months. Examples of polymers which can be selected for the mesh scaffolds include, but are not limited to, biodegradable polymers selected from the group consisting of aliphatic polyesters, poly(amino acids), modified proteins, polydepsipeptides, copoly(ether-esters), polyurethanes, polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,poly(£-caprolactone)s,polyanhydrides,polyar ylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, modified polysaccharides, polycarbonates, polytyrosinecarbonates, polyorthocarbonates, poly(trimethylene carbonate), poly(phosphoester)s, polyglycolide, polylactides, polyhydroxybutyrates, polyhydroxyvalerates, polydioxanones, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(maleic anhydride), polyvinylalcohol, polyesteramides, polycyanoacrylates, polyfumarates, poly(ethylene glycol), polyoxaesters containing amine groups, poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic acid)s, poly(dioxanone)s, poly(alkylene alkylate)s, biopolymers, collagen, silk, chitosan, alginate, and a blend of two or more of the preceding polymers. In one embodiment, the polymer comprises at least one of polylactide-co- glycolide (PLGA), PLA or PGA. In another embodiment, the polymer can include at least one of polycaprolactone (PCL). In yet another embodiment, the polymer can include at least a blend of polylactide- co-glycolide (PLGA), PLA and/or PGA and polycaprolactone (PCL). In one embodiment, the polymer is a copolymer, such as for example a poly(D,L-lactide-co-glycolide (PLGA) and/or poly-caprolactone (PCL). In one embodiment, a concentration of the polymer is at least 32% (w/v) of 5:1 blend of poly(lactide-co-glycolide) (PLGA) and poly(s- caprolactone) (PCL) in a solvent.

Selection of a polymer or polymers used in the mesh scaffold is based upon the length of time needed to remain in place as well as the polymer's degradation characteristics which control release of the active agent or agents from the mesh scaffold. For example, a polymer such as PLGA is bulk-eroding while a polymer such as PCL is surface eroding.By using only a bulk-eroding polymer or only a surface eroding polymer or combining both of these types of polymers into a mesh scaffold, release of the active agent or agents from the mesh scaffold can be controlled and a temporal gradient of release of the active agent or agents supportive of tendon cell migration and regeneration can be created.

Further non-limiting details are described in the following Experimental Details section which is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the scope of this disclosure.

Experimental Details

A tendon cell migration study was performed to investigate the therapeutic potential of a PLGA:PCL nanofibrous mesh for tendon injury in an in-vitro tendon transection model using bovine patellar tendon and sheath explants. The study further assessed which tendon cell population the mesh targets for repair.

The inventors hypothesized that tendon cells would migrate from injury site onto the nanofiber-based mesh, and deposit tendon extracellular matrix (collagen and proteoglycans). The inventors further hypothesized that more cells would originate from the tendon proper compared to the tendon sheath.

The outcomes measured in the study were cell viability, migration, collagen and proteoglycan deposition.

Materials and Methods:

Injury model:

Patellar tendons were isolated from fetal bovine knees (aged 3-5 months) acquired from a local abattoir (Green Village, NJ) and transected (19 x 5 x 5 mm) into explants (See Figure 10). Fabrication of mesh scaffolds:

Aligned nanofibers were fabricated by electrospinning 5:1 w/w% PLGA:PCL (PLGA = 50:50,MW-123.6 kDa; Evonik ; PCL = MW-70,000-90,000; Sigma-Aldrich) polymer blends onto a rotating mandrel or rod (2400 RPM) to form a mesh (see, e.g., Figure 6). The aligned PLGA:PCL blend was 32% w/v of the solvent.

1 ml/hour of the gelatin polymer solution was electrospun at 15 kV with needle placement 15 cm from the drum.

Post-fabrication modifications include crosslinking each side of the scaffold with glutaraldehyde vapor for 15 minutes in a vacuum chamber. Crosslinked scaffolds will then be rolled around a rod (formed of, for example, PTFE—polytetrafluoroethylene) so that the electrospun fibers are aligned parallel to the longitudinal axis of the scaffold and sealed along the outside flap with glutaraldehyde.

The PLGA:PCL meshes were soaked in DMEM media overnight.

Experiment:

Injury surfaces (S) of the transected tendons were either placed atop aligned scaffolds (10 mm diameter; experimental group; n=5) or polystyrene tissue culture plates (control; n=5) throughout 14 days (see Figure 10). They were cultured at 37°C in 12-well plates with DMEM media.

Cell growth and matrix production:

Cell viability and migration, glycosaminoglycans (GAG), and collagen production (n=5) were assessed by Live/Dead staining (Invitrogen), DNA (Quant-iT Picogreen dsDNA assay, Invitrogen), 1,9- dimethylmethylene blue (DMMB) and hydroxyproline assays, respectively.

Statistical analysis:

Statistical analysis was performed using the Tukey-Kramer post-hoc test for all pair-wise comparisons (p<0.05, JMP-IN). Results and discussion:

A zone of cell death was observed at the cut surface (S), while cells remained viable through the bulk of the tendon (-300 mpi below the surface) at 24 hours post-transection.

Cells migrated onto nanofibers by Day 1, and cell number increased significantly overtime. The scaffolds supported tendon cell migration and proliferation.

Migrated cells produced both collagen and proteoglycans after 14 days of culture that reflected the typical composition of the native tendon matrix: 65-80% collagen and 1-5% proteoglycans.

Future studies aim to optimize cell migration, mechanical properties of the mesh scaffold, and production of a tendon-like matrix by the cells.

The inventive mesh scaffolds described herein support cell migration and viability, are mechanically competent, bridge large gaps between transected tendon ends in a lacerated tendon, guide tissue formation, and mimic native tendon architecture. These mesh scaffolds facilitate a tendon repair strategy that guides healing and aims to prevent scar and adhesion formation for an improved restoration of function post- injury.

The subject matter disclosed herein will bring a paradigm shift in two ways: first, in the treatment of transected tendons in Prolonged Field Care because it prolongs the window for primary care for a highly prevalent problem; and second, in broader orthopedic and reconstructive surgery since it paves the way for suture-free mesh scaffolds that can be used to attach lacerated tendon ends.

The aforementioned nanofiber-based mesh scaffolds are configured to minimize scarring and promote tendon regeneration.Such mesh scaffolds can be rolled along a longitudinal axis of the scaffold to mimic native tendon structure and provide a supporting structure through which tendon cells from either end of injury can migrate to join ends of the injury. It has not heretofore been suggested that such mesh scaffolds are so configured as to be suitable to bridge the large gap between transected tendon ends and therefore facilitate functional repair of tendon injuries with large gaps. The scaffold can be used along with sutures to bridge the gap between transected tendon ends. The mesh scaffolds guide cell growth and provide bulk mechanical support.

The mesh scaffolds disclosed herein are configured so that even an inexperienced field medic can easily and rapidly re-attach a lacerated tendon without using sutures.

This work is unique from work that develops biomaterials to statically attach tendon to bone, since the developed scaffolds will be attached to tendons that are intended to move relative to surrounding tissue. It is envisioned that every military kit will have these mesh scaffolds.

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