WO2011035017A2 - Artificial meniscal implants - Google Patents

Abstract

Provided are three dimensional shaped porous, fibrous polymer scaffolds capable of mimicking the functional properties of native tissue of a knee meniscus, and methods for manufacturing and using said shaped polymer scaffolds.

Description

ARTIFICIAL MENISCAL IMPLANTS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application

Serial No. 61/243,660 filed September 18, 2009, which is incorporated by reference in its entirety

TECHNICAL FIELD

[0002] The present invention relates to a shaped, three dimensional porous, fibrous polymer scaffolds capable of mimicking the functional properties of native tissue of a knee meniscus, and methods for manufacturing and using said shaped polymer scaffolds.

BACKGROUND.

[0003] Fibrous tissues are collagen-rich structures present throughout the musculoskeletal system that serve a variety of vital load bearing roles. The organization of these tissues is paramount to their mechanical function and is to great extent dictated by the mechanical

environments in which they operate. For example, those tissues that function under cyclic near- uniaxial tension (such as flexor tendons) are comprised of collagen fibers organized in a single predominant direction. Tissues that function in more complex loading environments, such as the knee meniscus reveal more complex hierarchical collagen organization. Despite the refined characteristics that function has imparted on the form of these tissues, traumatic injury and degeneration are common occurrences and can interrupt normal mechanical function. As a result, there remains unmet clinical needs for engineered replacements for damaged or diseased native fibrous tissues.

[0004] When engineering fibrous tissue replacements, the demanding physical environment in which they will perform must be taken into consideration. For these scaffolds to function in vivo, they must either recapitulate the full dynamic mechanical range of the native tissue upon

implantation, or must foster cell infiltration and matrix deposition so as to enable construct maturation to meet these criteria. Tissue engineered constructs should also, at minimum, recapitulate the subfailure stress-strain response of the tissue.

[0005] Commonly, the clinical solution for fibrous tissue damage remains the autologous or allogeneic transfer of tissue to the defect site. However, the low availability of suitable grafting tissues and high failure rates engender the need for tissue engineered solutions.

[0006] To specifically address the hierarchical and structural organization of fibrous tissues, many tissue engineering strategies have focused on collagen gel-based constructs. However, these collagen gel-based constructs remain very soft, even after long culture durations, in comparison to native fibrous tissue. One biologic scaffold using collagen, the "collagen meniscus implant" (CMI, produced from decellularized bovine Achilles tendon) was recently used in a randomized clinical trial in humans and showed some promise in patients with degenerative meniscus damage (e.g., Rodkey, et al., "Comparison of the collagen meniscus implant with partial meniscectomy. A prospective randomized trial," J. Bone Joint Surg. Am., 2008; 90(7): 1413-1426). However, this scaffold provides none of the functional properties characteristic of actual knee menisci.

[0007] Hierarchical structure has been imparted through the use of macro-scale polymer fibers (including biodegradable polyesters and silk) to engineer fiber-reinforced constructs. These scaffolds better recapitulate the native tissue mechanical response, and in some cases have moved toward clinical implementation.

[0008] An alternative strategy that has recently become more prevalent for engineering such tissues is based on the process of electrospinning. Compared to macrofibrous construction methods, electrospinning generates nano- and micron-scale fibers, thereby providing a ready means to recapitulate the organizational features and length-scales of many collagenous tissues. In its most basic form, electrospinning involves the application of a high voltage potential and resulting gradient to draw a polymer solution into thin fibers which can then be collected en masse.

[0009] More recently, several groups have begun to examine the role of fiber alignment in nanofibrous scaffolds, and its potential application for fiber-reinforced tissue engineering. Aligned fibers can be formed by simply focusing deposition onto a moving surface. These aligned arrays mimic the structure of numerous fiber-reinforced and anisotropic tissues. A significant limitation in these scaffolds is that surface-seeded cells have difficulty infiltrating through the small pore-sizes that result from the dense packing of aligned fibers. This slow infiltration rate results in inhomogeneous ECM deposition and incomplete cell colonization even in relatively thin scaffolds (~lmm thick) and over long culture durations (>10 weeks).

[0010] Attempts to overcome this problem using sacrificial fibers have also had problems.

While removal of a sufficient fraction of sacrificial fibers enhanced infiltration, it also resulted in an overall loss in scaffold structural integrity as cell-mediated traction forces compacted the construct.

[0011] Additionally, most electrospinning efforts to date have focused on single polymeric fibers for scaffold formation. This provides articles with simple mechanical properties. By contrast, the mechanical behavior of native tissues is complex, with most showing not only pronounced anisotropy but also non-linear stress-strain profiles. In knee menisci, both 'toe' and 'linear' properties are critical for normal tissue response; 'toe' region (low strain) properties are essential for flexibility, while 'linear' region (higher strain) properties resist extreme deformations at high loads. These non-linear properties are dependent on the complex interplay of numerous tissue constituents, including fibers (e.g., collagens), matrix (e.g., proteoglycans and water), and fiber-matrix

interactions (e.g., cross linking molecules). It is somewhat unreasonable to suppose that a single electrospun fiber population could recapitulate the intricate mechanical behavior of native tissues. This would therefore limit scaffold applications to non-load bearing situations, or rely on cell- mediated ECM deposition to provide tissue specific functionality.

[0012] So while aligned nanofibrous scaffolds hold tremendous potential for the engineering of dense connective tissues, much of this potential remains unrealized.

SUMMARY

[0013] In the face of these shortfalls in the existing art, the present invention describes combining multiple fiber populations, each with distinct mechanical and bio-erosion characteristics, into a single composite nanofibrous scaffold. The differing mechanical properties provide for development of a more complex scaffolding architecture, more representative of the native tissue. The inclusion of differentially eroding fiber populations serves to maintain scaffold integrity initially, and then gradually erode to augment pore size and porosity as the composite evolves.

Inclusion of these multiple fiber populations thus provide time-dependent characteristics in the composite scaffold, and can also be used to further refine the mechanical properties. [0014] This invention describes the multiple fiber populations of implantable knee scaffolds in terms of at least two fiber materials, each with specific mechanical properties and different biodegradation or bioerodible characteristics.

[0015] The scaffold also optionally provides for a porogen material, capable of quickly dissolving or biodegrading, such that in total, an implantable knee meniscus scaffold may comprise materials characterized as having slow, medium, and fast-eroding elements, each having different tensile properties.

[0016] More specifically, the invention provides various embodiments for an implantable knee meniscus scaffold exhibiting an overall circumferential modulus that is in the range of about 10 MPa to about 100 MPa, preferably at least about 20 MPa, more preferably at least about 40 MPa, still more preferably at least about 60 MPa, and most preferably at least about 80 MPa, at a strain region of about 10%, and / or an overall circumferential modulus in the range of about 5 to about 50 MPa, preferably at least 10 MPa, more preferably at least 20 MPa, and most preferably about 30-35 MPa, at a strain region of about 3%, and these properties are either retained or developed when the scaffold is subjected to physiological implant conditions for time sufficient to allow cell infiltration and meniscal healing, during and after which the components of the matrix are dissolved, bioeroded, or biodegraded into the patient. Preferably the modulus of the scaffold, after exposure to

physiological fluids under physiological conditions, retains at least about 60%> of its value after 7 days, and more preferably at least about 50% of its value after 60 days. In order to retain these modulus levels this invention also provides that the scaffolds have correspondingly, proportionately higher initial values.

[0017] The invention further provides for an implantable scaffold wherein the porogen fiber is substantially removed. It also provides for embodiments where the rates of biodegradation of the first and second fibers differ, where the second fiber biodegrades more quickly under physiological conditions than does the first, and where the relative rates of the first and second fibers and the porogen fibers can be characterized as slow, medium, and fast.

[0018] Such an approach, for the first time, allows for scaffolds characterized as having time dependent non-linear and anisotropic stress-strain properties. This invention further provides for scaffolds which allow for inclusion of at least one therapeutic agent, biofactor, population of cells, catalyst, or mixture or combination thereof, optionally attached to or within a biocompatible matrix or encapsulants.

[0019] Additionally, methods of making such scaffolds are described, as are methods of treating a patient therewith.

BRIEF DESCRIPTION OF THE DRAWINGS.

[0020] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments of the invention; however, the invention is not limited to the specific methods, compositions, and devices disclosed. In addition, the drawings are not necessarily drawn to scale. In the drawings:

[0021] FIGURE 1 schematically shows the (FIGURE 1A) radial and (FIGURE IB) axial

(longitudinal) dimensions of a solid body of rotation, such as comprises a knee meniscus, as discussed herein. FIGURE 1A also describes the term circumferential. FIGURE 1C illustrates the circumferential and radial orientations as applied to a schematic of a knee mensicus

[0022] FIGURE 2 schematically illustrates a method for providing composite nanofibrous scaffolds containing three distinct fiber populations were fabricated with a custom electrospinning device. FIGURE 2A provides a schematic representation of the formation of electrospun scaffolds containing fast, medium, and slow-degrading fiber populations. FIGURE 2B depicts a temporal evolution of porosity in composite scaffolds that lose fiber elements in a pre-programmed fashion via differing degradation profiles. FIGURE 2C illustrates the novel electrospinning device used in this work for forming single- and multi-polymer fibrous scaffolds by co-electrospinning from up to three jets onto a common rotating mandrel. FIGURE 2D shows an SEM image of fiber morphology in one of the composites in this work (scale bar: ΙΟμιη). FIGURE 2E illustrates an illustration of fluorescently labeled fiber populations show the presence and interspersion of each element (scale bar: ΙΟμιη).

[0023] FIGURE 3 illustrates the formation of anatomically shaped scaffolds. FIGURE 3A shows the process as a function of electrospinning time. FIGURE 3B illustrates the targeted shape for knee meniscus implants as well as the representation that additional particulate materials may be included in the scaffold at different radial distances, as required or desired. FIGURE 3C is a picture of one type of mandrel and form used to develop such shaped scaffolds.

[0024] FIGURE 4 provides an overview of the unique stress-strain profiles associated with electrospun PCL, PLGA, and PEO scaffolds as described in Study I. FIGURE 4A illustrates several stress-strain profiles of PCL, PEO, PLGA, and Composite scaffolds extended in the fiber direction. Modulus (FIGURE 4B) and yield strain (FIGURE 4C) for each scaffold (n=5/group). FIGURE 4D illustrates properties from Samples from Composite scaffolds removed along the length of the mandrel showed a range of behaviors, likely dependent upon the relative fractions of PLGA or PCL fibers at each location.

[0025] FIGURE 5 provides an overview of the scaffold tensile behaviors associated with single- and multiple-fiber populations as a function of composition and degradation time as described in Study II. FIGURE 5A illustrates the stress-strain curves of day 0 PCL, Blend, and

Composite scaffolds tested in the fiber direction. FIGURES 5B and FIGURE 5C show the yield strain modulus, respectively, of samples tested in the fiber direction over 63 days (n=5/group per time point). FIGURE 5D shows the modulus in the transverse direction as a function of time

(n=5/group per time point). FIGURE 5E shows the percent mass loss relative to dry, as-formed samples over the 63 day time course (n=5/group per time point). Note that Composite scaffolds on day 0 lose -22% of their starting mass due to removal of the PEO fiber population during hydration. *: p<0.01 versus day 0.

[0026] FIGURE 6 provides a characterization of single-fiber population (PCL and Blend) scaffold tensile behavior with a hyperelastic fiber-reinforced constitutive model. Curve fit results (lines) on day 0 are shown along with experimental data (circles) for the transverse (FIGURE 6A) and fiber (FIGURE 6D) directions. From transverse direction testing, matrix parameters μ

(FIGURE 6B) and v (FIGURE 6C) were determined at each time point. These values, coupled with fits to fiber direction data at each time point were used to determine fiber parameters, γ (FIGURE 6E) and ξ (FIGURE 6F).

[0027] FIGURE 7 - Material parameters from single-component scaffolds successfully predict Composite scaffold behavior. (FIGURE 7A) Stress-strain curves for Composites tested in the fiber direction. Stress-strain profiles diminished as degradation occurred over 63 days, due to decreasing properties of the Blend fiber population. Model predictions of Composite stress response when tested in the fiber direction on days 0 (FIGURE 7B), 7 (FIGURE 7C), 21 (FIGURE 7D), 42 (FIGURE 7E), and 63 (FIGURE 7F) showed good agreement with experimental measures. Each plot contains all five experimental curves (dotted lines) and the corresponding five model-generated predictions (solid lines).

[0028] FIGURE 8 - Simulation of Composite scaffolds of any formulation. (FIGURE 8A)

Behavior of Composites covering the full range of possible PCL/Blend combinations described herein (as indicated by the initial mass fraction of Blend fibers, 0^B) was simulated for as-formed samples and with degradation over time. Modulus for each theoretical composite is denoted both by height as well as color (or gradations of gray in black and white depictions). (FIGURE 8B) Stress- strain behavior of Composites of varying formulations on day 0. Note that the color (or corresponding shade of gray) of each line serves only to illustrate the resulting modulus of the curve.

[0029] FIGURE 9 shows the results of testing of the stress-strain response of the circumferential samples taken from 10 sheep menisci, benchmarked against various polymer scaffolds, as described in Example 11.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS.

[0030] The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying Figures and Examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of any claimed invention. Similarly, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the invention herein is not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement. Throughout this text, it is recognized that the descriptions refer both to the method of preparing such devices and to the resulting, corresponding physical devices themselves, as well as the referenced and readily apparent applications for such devices.

[0031] In the present disclosure the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to "a material" is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth.

[0032] When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. In general, use of the term "about" indicates approximations that can vary depending on the desired properties sought to be obtained by the disclosed subject matter and is to be interpreted in the specific context in which it is used, based on its function, and the person skilled in the art will be able to interpret it as such.

Where present, all ranges are inclusive and combinable.

[0033] It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further, references to values stated in ranges include each and every value within that range.

[0034] Generally terms are to be given their plain and ordinary meaning such as understood by those skilled in the art, in the context in which they arise. To avoid any ambiguity, however, several terms are described herein.

[0035] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.

[0036] The present invention discloses an implantable knee meniscus scaffold, comprising a fiber matrix, electrospun in the presence of a porogen fiber, and oriented substantially circumferentially to an axis of rotation. The tensile characteristics of the scaffold replicate those of a mammalian knee menisus. Since the materials of construction in some cases exhibit non-linear stress responses to strain and / or are biodegrade or bioerode when subjected to physiological fluids under physiological conditions, and the scaffold must continue to provide tensile support during this period of biodegradation or bioerosion over a range of strain conditions, it is necessary to characterize the scaffold in terms of these parameters. That is, in various embodiments, the scaffold of the present invention exhibits an overall circumferential modulus that is in the range of about 10

MPa to about 200 MPa, preferably at least about 20 MPa, more preferably at least about 40 MPa, still more preferably at least about 60 MPa, and most preferably at least about 80 MPa, at a strain region of about 10%, and / or an overall circumferential modulus in the range of about 5 to about 60

MPa, preferably at least 10 MPa, more preferably at least 20 MPa, and most preferably about 30-35 MPa, at a strain region of about 3%, and these properties are either retained or developed when the scaffold is subjected to physiological implant conditions for time sufficient to allow cell infiltration and meniscal healing, during and after which the components of the matrix are dissolved, bioeroded, or biodegraded into the patient. Preferably the modulus of the scaffold, after exposure to

physiological fluids under physiological conditions, retains at least about 60% of its value after 7 days, and more preferably at least about 50% of its value after 60 days. In order to retain these modulus levels this invention also provides that the scaffolds have correspondingly, proportionately higher initial values. Unless otherwise stated herein, any reference to a specific target modulus is intended to reflect an initial value (i.e., before biodegradation or bioerosion and the changes in mechanical properties that develop as cells infiltrate and deposit new, load-bearing extracellular matrix within the scaffold substance).

[0037] The present invention also discloses a method to achieve this implantable knee meniscus scaffold, and its method of making and use, comprising a first and a second biocompatible fiber, electrospun together with a porogen fiber so as to be oriented substantially circumferentially to an axis of rotation. This invention also teaches methodologies for selecting materials to make and use these scaffolds, and gives examples of modeling the properties of the scaffolds made from these materials.

[0038] One purpose of this invention is to match the timing of the biodegradation of the scaffold with that of the ingress of cells and tissue regeneration. One embodiment provides for the selection of materials for the fibers of sufficiently high modulus such that as one of the fibers degrades, the scaffold retains the required modulus, for example at least 20 MPa, preferably at least 40 MPa, more preferably at least 60 MPa, and more preferably at least about 80 MPa, at a strain region of about 10%, and / or an overall circumferential modulus in the range of about 5 to about 60 MPa, preferably at least 10 MPa, more preferably at least 20 MPa, and most preferably at least about 30-35 MPa, at a strain region of about 3%, as defined herein, for sufficient time, for example over 10-20 weeks, under physiological conditions.

[0039] In another embodiment, the first fiber comprises a material characterized as having a yield strain at least about 1%, preferably at least about 4%, more preferably at least about 8% and most preferably at least 10%. This fiber must be biocompatible, but may or may not be

biodegradable, though it is preferably so. Absolute tensile properties of this material are less important than are those of the second fiber, since it is the combination of the moduli of the first and second fibers, especially as a function of time of exposure to physiological conditions, that are important, but to accomplish this, the modulus of the first fiber material should be at least about 20 MPa at lower (3%) strain levels. Higher values are preferred, for example, preferably at least about 100 MPa, and most preferably at least about 200 MPa, especially at higher (10%) strain levels. In one embodiment, the first fiber material comprises poly(caprolactone). In other embodiments, this first fiber comprises a poly(P-amino ester) or an acrylate terminated poly(P-amino ester). Such materials are described, for example, in Anderson, et al., "A Combinatorial Library of

Photocrosslinkable and Degradable Materials," Adv. Materials, vol. 18 (19), 2006, this reference being incorporated by reference in its entirety.

[0040] The second fiber comprises a biodegradable material characterized as having a modulus in the range from about 10 MPa to about 500 MPa, when subjected to a strain in the range of from about 1% to about 10% and measured in the direction of the fiber alignment; in another embodiment, this modulus is in the range of about 20 MPa to about 500 MPa; in another, this modulus is in the range of about 300 MPa to about 500 MPa, especially at higher (10%) strain levels. In another embodiment, the second fiber comprises poly(glycolic acid). In yet another embodiment, the second fiber comprises a blend of poly(caprolactone) and poly(glycolic acid). In other embodiments, this second fiber also comprises a poly(P-amino ester) or an acrylate terminated poly(P-amino ester). Yield stress for this fiber material should be at least 1%, preferably at least about 4%, and most preferably at least about 8%.

[0041] One embodiment of the invention provides that the first and second fibers are be biodegradable, and that the rates of biodegradability of the two fibers are different, when subjected to similar or the same physiological conditions. In one embodiment, the second fiber biodegrades more quickly than the first. In this embodiment, when taken together, the relative rates of biodegradability (or biosorption or dissolution) of the first fiber, the second fiber, and the porogen fiber can be considered slow, medium, and fast. It is preferred that the relative lifetime of the second fiber in vivo is sufficiently long so as to provide a sustained basis for tissue regeneration - generally on the order of weeks under physiological conditions. The relative lifetime of the second fiber in vivo can be determined or approximated by measuring tensile properties or weight loss of the scaffold under simulated physiological conditions.

[0042] In one embodiment, the scaffolding contains a porogen fiber, co-spun with the first and the second fibers, comprising an amount in the of about 10 to about 80 weight percent based on the total weight of electrospun fibers, preferably in the range of about 20 to about 60 weight percent, more preferably in the range of about 30 to about 60 weight percent, and most preferably in about 40-55 weight percent, all with respect to the total weight of electrospun fibers. As used herein, the term "porogen" refers to sacrificial materials added during the production of a scaffold (for example, during electrospinning) and subsequently removed, whose purpose is to occupy space during the construction process, such that their subsequent removal results in what amounts to engineered porosity. In tissue engineering, materials such as inorganic salt like sodium chloride, crystals of saccharose, gelatin spheres or paraffin spheres are used to introduce particulate porosity. In the present invention, the use of porogen fibers provides porosity aligned with the remaining fibers.

[0043] In another embodiment, this porogen fiber has been removed, such that the resulting scaffold contains spacings defined by the absence of this porogen material. This removal can be accomplished in several ways, though the most usual way of doing so is by selective dissolution. Preferably, this porogen fiber is capable of selectively and substantially dissolving in physiological fluids, such as water, saline, meniscal fluid, simulated body fluid, or synovial fluid, such dissolution occurring within one hour, preferably within 30 minutes, and most preferably within 10 minutes of contact with the physiological fluid at ambient or physiological conditions. In such circumstances, one non-limiting example, the porogen fiber comprises poly(ethylene oxide). However, the means of removal is not limited to use of physiological fluids. For example, depending on the porogen, hydrocarbons or other organic solvents may also be used {ex vivo). Also the removal can be accomplished inside or outside the patient. One of the purposes of applying and then removing this porogen fiber material is to provide spaces within the matrix to expedite cellular ingress into the scaffold matrix, whose fiber densities otherwise inhibit this incursion. For this reason, one skilled in the art would appreciate that removing the porogen material from the matrix before seeding with, for example, cell populations, and implanting into the patient can be a desirable scenario.

[0044] Other embodiments describe the relative proportion of the first and second fiber. In one such embodiment, the first fiber comprises an amount in the range of about 20% by weight to about 80% by weight, relative to the combined weight of the first and second fiber. Other embodiments define the relative amount of the first fiber to be in the range of about 40% by weight to about 60%) by weight, or about 50%> by weight, each relative to the combined weight of the first and second fiber. The specific ratio of the two fibers will depend on the particular choice of fibers, and one skilled in the art would be able to understand the most appropriate ratio for a given set of fiber materials based on the teaching herein.

[0045] The invention teaches that the electrospun fibers may individually comprise the individual polymers or copolymers, or blends of polymers or copolymers or both. Within the scaffold and / or within the individual fibers, the first fiber material may be present in the range of about 1 to about 80 weight percent, and the second fiber may be present in the range of about 80 to about 1 weight percent, each with respect to the total weight of electrospun fibers.

[0046] Together, the first and second fibers form a scaffold whose circumferential modulus in the range of about 10 MPa to about 100 MPa, preferably in the range of about 60 MPa to about 90 MPa, more preferably in the range of about 70 MPa to about 85 MPa, most preferably about 80 MPa. This is accomplished by combining the fiber materials, applied either as individual fibers or co-spun as blended materials, such that the weighted average of the materials according to their individual moduli provide the target scaffold circumferential modulus. One skilled in the art would be able to measure and / or calculate the combined modulus as a function of such a composite. In the simplest case, this relationship can be characterized according to the Rule of Mixtures equation:

[0047]

[0048] where Φ_χ represents the weight fraction of the x^th component (strictly speaking, the rule of mixtures deals with volume fractions, but to a good approximation, and assuming polymers of comparable densities are used, use of weight fractions provides an equivalent means of characterization) .

[0049] Tensile modulus is a property which is often defined in terms relative to the total cross-sectional area of the fiber or fiber bundle, or in this case, to the circumferential alignment of fibers. So as to maintain internal consistency, as described herein, whether the scaffold contains or has had removed the porogen fiber, the moduli are calculated and described so as to consider the cross-sectional area of the porogen fiber, but not to consider the tensile properties of that porogen fiber. For example, in but one non-limiting example, a mixture comprising 25% by weight (of the total polymer weight) of a first polymer, having a modulus of ca. 20 MPa, and 25% by weight a second fiber, having a modulus of 300 MPa, and 50%> by weight of a porogen fiber, having a modulus of 100 MPa is described herein as having a composite modulus of 80 MPa for the composite (i.e., (25% x 20 MPa) + (25% x 300 MPa) + (50% x 0 MPa) = (5 + 75 + 0) = 80 MPa), and not 130 MPa (as would result if the modulus of the scaffold retained the contribution of the porogen; i.e., (25% x 20 MPa) + (25% x 300 MPa) + (50% x 100 MPa) = (5 + 75 +50) = 130 MPa) or 160 MPa (as would result if the cross-sectional area of the porogen were ignored; i.e., (50%> x 20 MPa) + 50% x 300 MPa) = (10 + 150) = 160 MPa. It should be appreciated that this definition provides a more rigorous requirement for tensile modulus for the scaffold than if the tensile contribution of the porogen material had been considered or if the cross sectional area of the porogen material had been ignored.

[0050] Other embodiments of this invention lift the constraint that the first fiber have a particular yield stress value, and allowing the second fiber to have a modulus lower in value than described above, replacing these requirements with one that the combination of first and second fibers maintain a mean circumferential scaffold modulus of at least about 40 MPa, preferably about 60 MPA, and most preferably at least about 80 MPa, at higher (10%>) strain levels, when subjected to physiological fluids under physiological conditions for times sufficient to allow for cell ingress and proliferation, typically on the order of weeks. As described earlier, it is highly desirable that the scaffold maintain a minimum modulus during the time of this cell ingress and proliferation, corresponding to healing.

[0051] It is also understood that electrospinning provides fibrous solid bodies which contain a degree of porosity which can be affected by the materials of construction - both fibers and other incorporated materials - and the method of making. Accordingly, certain embodiments of this invention describe porous solids whose void volumes are on the order of about 5 to 99 volume percent; other embodiments describe porous solids with void volumes at the lower end of this range, e.g., in the range from about 5 volume percent to about 25 volume percent; still other embodiments describe porous solids with void volumes in the middle of this range, e.g., in the range from about 25 volume percent to about 75 volume percent; and still other embodiments describe solids with void volumes at the high end of this range, e.g., in the range from about 50 volume percent to about 95 volume percent. Other exemplary sub-ranges contemplated by the present invention include the range of about 80 to about 99 volume percent, the range of about 85 to about 95 volume percent, and the range of about 90 to about 95 volume percent.

[0052] Moreover, the scaffolds of the present inventions, are described in terms of radial distance. As used herein, the term "radial" refers to the distance defined by the length between the mandrel and the outer surface of the body, or when used to connote direction, from the mandrel- defined surface to the outer surface of the body (see FIGURES 1A). Again, depending on the form and shape of the top and bottom surfaces, this can range from the nanometer to the millimeter scale. Depending on the application, this radial distance can be further defined as being in the range of about 50 microns to about 1000 microns, in the range of about 1 micron to about 1 millimeter, or in the range of about 1 millimeter to about 10 millimeters.

[0053] Similarly, the solids are characterized by their thickness, or axial / longitudinal distance (when referenced to the mandrel on which it is formed) which are generally defined by the particular application of interest. As used herein, the terms "axial" or "longitudinal" refer to the thickness (see FIGURE 2B), or mean distance between the top and bottom surfaces. In the present invention, this distance is practically limited only by the length of the mandrels on which the body can be formed. For mensical implants, this thickness can be in the range of about 10 microns to about 10 millimeters, in the range of about 100 microns to about 10 millimeters, or in the range of about 100 microns to about 1 millimeter.

[0054] The term "fiber" refers to a plurality of fibers comprising the indicated material, and not necessarily to a single strand of fiber. When an electrospun solid is made of a single fiber (e.g. nanofiber), the fiber is folded thereupon, hence can be viewed as a plurality of connected fibers. It is to be understood that a more detailed reference to a plurality of fibers is not intended to limit the scope of the present invention to such particular case. Thus, unless otherwise defined, any reference herein to a "plurality of fibers" applies also to a single fiber and vice versa. The polymeric nanofibers can have a general random orientation, or a preferred orientation, as desired e.g., when the nanofibers are collected on a cylindrical collector such as a drum, the polymeric nanofibers can be aligned predominantly radially or predominantly circumferentially. Different layers of the electrospun solid can have different orientation characteristics. For example, without limiting the scope of the present invention to any specific ordering or number of layers, the nanofibers of a first layer can have a first predominant orientation, the nanofibers of a second layer can have a second predominant orientation, and the nanofibers of third layer can have general random orientation. Similarly, by changing the composition, cross-sectional shapes, or dimensions of the fibers throughout the spinning process, it is possible to provide solids wherein the different layers are comprised of different compositions, cross-sectional shapes, or thicknesses. Several means to achieve this alignment are described in Baker and Mauck, "The effect of nanofiber alignment on the maturation of engineered meniscus constructs," Biomaterials, 28 (2007) 1967-1977, which is incorporated in its entirety by reference for this purpose.

[0055] The invention is not constrained by the thickness or shape of the electrospun fibers generated and used. Accordingly, the cross-sections of the fiber or fibers may be circular, oval, rectangular, square, or any shape which can be defined by the spinneret. Similarly, the fibers can have thickness dimensions in the range of about 1 nm to about 10 microns, in the range of about 20 nm to about 1000 nm, in the range of about 100 nm to about 1000 nm, or in the range of about 1 micron to about 10 microns. As used herein, the phrase "nanofibers" refers to polymer fibers having diameters typically between 10 nm and 1000 nm. Exemplary sub-ranges contemplated by the present invention include between 100 and 1000 nm between 100 and 800 nm, between 100 and 600 nm, and between 100 and 400 nm. Other exemplary ranges include 10-100 nm, 10-200 nm and 10- 500 nm. As mentioned, the nanofibers of the solids of the present invention are preferably generated by an electrospinning processes.

[0056] The size and shape of the fibers, as well as the relationship between the rotational speed of the mandrel, the distance between the spinneret and the mandrel, and the strength of the potential field between the spinneret and the mandrel define the density and the degree of porosity, and the degree to which fiber-fiber welding occurs in the final porous body.

[0057] As described herein, the various fibers may comprise materials which are natural, synthetic, biocompatible, biodegradable, non-biodegradable, and / or biosorbable, and unless specifically restricted to one or more of these categories, the fibers may comprise materials from any one of these categories, provided that the material satisfies the required physical properties (e.g., tensile properties).

[0058] The phrase "synthetic polymer" refers to polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. Examples include, but are not limited to, aliphatic polyesters, poly(amino acids), copoly(ether- esters), polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, polysiloxanes, and combinations thereof.

[0059] Suitable synthetic polymers for use according to the teachings of the present invention can also include biosynthetic polymers based on sequences found in collagen, elastin, thrombin, fibronectin, starches, poly(amino acid), poly(propylene fumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin, hyaluronic acid, polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene), polycarbonate, polypropylene and poly(vinyl alcohol), ribonucleic acids, deoxyribonucleic acids, polypeptides, proteins, polysaccharides, polynucleotides and combinations thereof.

[0060] The phrase "natural polymer" refers to polymers that are naturally occurring. Non- limiting examples of such polymers include, silk, collagen-based materials, chitosan, hyaluronic acid and alginate.

[0061] The phrase "biocompatible polymer" refers to any polymer (synthetic or natural) which when in contact with cells, tissues or body or physiological fluid of an organism does not induce adverse effects such as immunological reactions and/or rejections and the like. It will be appreciated that a biocompatible polymer can also be a biodegradable polymer.

[0062] The phrase "biodegradable polymer" refers to a synthetic or natural polymer which can be degraded (i.e., broken down) in the physiological environment such as by enzymes, microbes, or proteins. Biodegradability depends on the availability of degradation substrates (i.e., biological materials or portion thereof which are part of the polymer), the presence of biodegrading materials (e.g., microorganisms, enzymes, proteins) and the availability of oxygen (for aerobic organisms, microorganisms or portions thereof), carbon dioxide (for anaerobic organisms, microorganisms or portions thereof) and/or other nutrients. Aliphatic polyesters, poly(amino acids), polyalkylene oxalates, polyamides, polyamido esters, poly(anhydrides), poly(beta-amino esters), polycarbonates, polyethers, polyorthoesters, polyphosphazenes, and combinations thereof are considered biodegradable. More specific examples of biodegradable polymers include, but are not limited to, collagen (e.g., Collagen I or IV), fibrin, hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(Lactide-co-Glycolide) (PLGA),

polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG), Collagen, PEG- DMA, alginate or alginic acid, chitosan polymers, or copolymers or mixtures thereof.

[0063] The phrase "non-biodegradable polymer" refers to a synthetic or natural polymer which is not degraded (i.e., broken down) in the physiological environment. Examples of nonbiodegradable polymers include, but are not limited to, carbon, nylon, silicon, silk, polyurethanes, polycarbonates, polyacrylonitriles, polyanilines, polyvinyl carbazoles, polyvinyl chlorides, polyvinyl fluorides, polyvinyl imidazoles, polyvinyl alcohols, polystyrenes and poly( vinyl phenols), aliphatic polyesters, polyacrylates, polymethacrylates, acyl-sutostituted cellulose acetates, nonbiodegradable polyurethanes, polystyrenes, chlorosulphonated polyolefms, polyethylene oxides, polytetrafluoroethylenes, polydialkylsiloxanes, and shape-memory materials such as poly (styrene- block-butadiene), copolymers or mixtures thereof.

[0064] The phrase "biosorbable" refers to those polymers which are absorbed within the host body, either through a biodegradation process, or by simple dissolution in aqueous or other body fluids. Water soluble polymers, such as poly(ethylene oxide) are included in this class of polymers.

[0065] It will be appreciated that more than one polymer may be used to fabricate the scaffolds of the present invention. For example, the scaffold may be fabricated from a co-polymer. The term "co-polymer" as used herein, refers to a polymer of at least two chemically distinct monomers. Non-limiting examples of co-polymers which may be used to fabricate the scaffolds of the present invention include, PLA-PEG, PEGT-PBT, PLA-PGA, PEG-PCL and PCL-PLA. The use of copolymers or mixtures of polymers / copolymers provides a flexible means of providing the required blend of properties. In but one non-limiting example, functionalized poly(P-amino esters), which may be formed by the conjugate addition of primary or secondary amines with diacrylates, can provide a range of materials exhibiting a wide array of advantageous properties for this purpose. Such materials are described, for example, in Anderson, et al., "A Combinatorial Library of Photocrosslinkable and Degradable Materials," Adv. Materials, vol. 18 (19), 2006, this reference being incorporated by reference in its entirety.

[0066] Additionally, individual polymers or co-polymers may be physically mixed and co- spun through the same spinneret.

[0067] Similarly, according to this invention, the solid may be comprised of a mixture of simultaneously or sequentially delivered polymers and / or copolymers. This includes mixtures of at least two natural, synthetic, biocompatible, biodegradable, non-biodegradable, and / or biosorbable polymers and co-polymers.

[0068] Other embodiments of this invention provide that, where the solid comprises two or more fibers, that each may have a different biodegradation and / or biosorption profile in a physiological fluid, said fluids including water, saline, simulated body fluid, or synovial fluid.

[0069] Still other embodiments provide that the polymers, co-polymers, or blends thereof may be photolytically active, such that once electrospun, they may be made to crosslink on exposure to light, thereby improving the tensile characteristics of the scaffold, and increasing the diversity and range of properties available. See for example, Tan, et al, J. Biomed Matl. Res., vol. 87 (4), 2008, pp. 1034-1043, which is incorporated by reference in its entirety.

[0070] Certain embodiments provide that the electrospun fibers are oriented substantially circumferential. Moreover, but adjusting the process parameters as described below, the degree of orientation can vary according to at least one gradient within the composition, and that this at least one gradient can either across the radial and longitudinal distances (i.e., across the distance between the top and bottom surfaces of the solid), or both. These gradients can be continuous or step-wise, again, as determined by the processing parameters.

[0071] Another distinguishing feature of this invention is that the fibrous solid can be substantially anatomically shaped, for example, in the substantial shape of a knee meniscus, and that the solid can be formed in such a substantial shape. The invention further provides that these substantially anatomically shaped solids can be used as scaffold implants, to replace or repair their corresponding anatomical part - e.g., a knee meniscus. Formation of such shaped scaffolds can be accomplished through the use of shaped forms in tandem with the electrospinning mandrels, for example as shown in FIGURE 3. While FIGURE 3 shows a particular shape, it should be recognized that FIGURE 3 is not intended to be limiting; upper and lower forms can be individually flat, concaved, convex, or multiply contoured, where the resulting changes in thickness may be continuous or discontinuous, resulting in scaffolds which can provide customized shapes for varying circumstances.

[0072] In addition to the fibers, the fibrous solid can also comprise a variety of additional materials, added during or after the formation of the solid body (for example, as shown in FIGURE 3B). These may comprise biofactors, therapeutic agents, particles, or cells. The materials agents can be applied to at least a portion of the solid, using techniques well known in the art, by coating or impregnating or at least a portion of the polymer fibers prior to or during the process of

electrospinning, by co-applying them with the fibers, or by impregnating the electrospun scaffold by soaking the scaffold after spinning. Such attachments can be performed using e.g., cross-linking (chemical or light mediated) of the agent with the polymer solution or the electrospun fiber formed therefrom (e.g., PLC and the agent). Additionally or alternatively, the agent can be embedded in electrospun nanofibers having the core-shell structure essentially as described in Sun et al. (e.g., see Sun et al., "Compound Core/Shell Polymer Nanofibers by Co-Electrospinning", Advanced

Materials, 15, 22: 1929-1936, 2003), or adhered to the fibers either directly using biocompatible adhesives or through the use of biocompatible carriers, including microsphere encapsulants.

[0073] Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes. Methods of preparing microcapsules are known in the electrospinning art.

[0074] The invention further provides that these biofactors, therapeutic agents, particles, or other materials incorporated within the solid, as required or desired, may be biodegraded, dissolved, and/or released according to a predetermined time profile. In other embodiments, these changes occur so as to complement the entry and incorporation of cells and/or tissues within the fibrous solid.

[0075] The invention is flexible in allowing these biofactors, therapeutic agents, particles, or cells also to be present independently in the solid as at least one gradient within the composition, and this at least one gradient can either across the radial and longitudinal distances (i.e., across the distance between the top and bottom surfaces of the solid), or both. These gradients can be continuous or step-wise, again, as determined by the processing parameters. [0076] In one set of embodiments, these additional materials comprise at least one therapeutic compound or agent, capable of modifying cellular activity. Similarly, agents that act to increase cell attachment, cell spreading, cell proliferation, cell differentiation and/or cell migration in the scaffold may also be incorporated into the scaffold. Such agents can be biological agents such as an amino acid, peptides, polypeptides, proteins, DNA, RNA, lipids and/or proteoglycans.

[0077] These agents may also include growth factors [e.g., a epidermal growth factor, a transforming growth factor-a, a basic fibroblast growth factor, a fibroblast growth factor-acidic, a bone morphogenic protein, a fibroblast growth factor-basic, erythropoietin, thrombopoietin, hepatocyte growth factor, insulin-like growth factor-I, insulin-like growth factor-II, Interferon-β, platelet- derived growth factor, a nerve growth factor, a transforming growth factor, a tumor necrosis factor, Vascular Endothelial Growth Factor, an angiopeptin, or a homolog or combination thereof], cytokines [e.g., M-CSF, IL-lbeta, IL-8, beta-thromboglobulin, EMAP-II, G-CSF and IL- IO, or a homolog or combination thereof], proteases [pepsin, low specificity chymotrypsin, high specificity chymotrypsin, trypsin, carboxypeptidases, aminopeptidases, proline-endopeptidase, Staphylococcus aureus V8 protease, Proteinase K (PK), aspartic protease, serine proteases, metalloproteases, ADAMTS 17, tryptase-gamma, and matriptase-2, or a homolog or combination thereof] and protease substrates.

[0078] Suitable proteins which can be used along with the present invention include, but are not limited to, extracellular matrix proteins [e.g., fibrinogen, collagen, fibronectin, vimentin, microtubule-associated protein ID, Neurite outgrowth factor (NOF), bacterial cellulose (BC), laminin and gelatin], cell adhesion proteins [e.g., integrin, proteoglycan, glycosaminoglycan, laminin, intercellular adhesion molecule (ICAM) 1 , N-CAM, cadherin, tenascin, gicerin, RGD peptide and nerve injury induced protein 2 (ninjurin2)].

[0079] Additionally and/or alternatively, the solids of the present invention may comprise an antiproliferative agent (e.g., rapamycin, paclitaxel, tranilast, Atorvastatin and trapidil), an immunosuppressant drug (e.g., sirolimus, tacrolimus and Cyclosporine) and/or a non-thrombogenic or anti-adhesive substance (e.g., tissue plasminogen activator, reteplase, TNK-tPA, glycoprotein Ilb/IIIa inhibitors, clopidogrel, aspirin, heparin and low molecular weight heparins such as enoxiparin and dalteparin). [0080] Examples of immunosuppressive agents which can be used to minimize immunosuppression include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D- penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF-a, blockers, a biological agent that targets an inflammatory cytokine, IL-1 receptor antagonists, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, difiunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

[0081] Cytokines useful in the present invention include, but are not limited to,

cardiotrophin, stromal cell derived factor, macrophage derived chemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophage inflammatory proteins 1 alpha (MOP-1 alpha, 2, 3 alpha, 3 beta, 4, and 5, IL-, 11-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL- 13, TNF-a, and TNF-β. Immunoglobulins useful in the present invention include but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGFs (nerve growth factors), PFGF-AA, PDGF-BB, PDGF- AB, FGFb, FGFa, and BGF.

[0082] Additionally, the solids of the present invention can include organic and/or inorganic particles as well as the electrospun polymers. It will be appreciated that, the solids of the present invention may comprise a single type of particles or alternatively may comprise two or more types of particles. As used herein, the term "particles" refers to any finely divided solid non- cellular matter, including powders, filings, crystals, beads and the like, which are capable of being integrated into a scaffold, but without interfering with the scaffolds capability to support cells.

[0083] According to one aspect of the present invention, the particles are dispensed concomitantly with the dispensing of the electrospun polymers, although from a separate dispenser e.g. by air pressure from a pneumatic activator. It will be appreciated that the concomitant dispensing of the particles from a separate dispenser can result in particles being situated between the polymeric fibers and not necessarily embedded within the fibers. [0084] Another embodiment provides that the fibrous solid further comprises at least one population of cells. These populations of cells can exist within the solid as homogeneous or heterogeneous mixtures, and as at least one gradient either across the radial and axial/longitudinal distances, or both. These gradients can be continuous or step-wise, as with the other components, as determined by the processing parameters.

[0085] Techniques for seeding cells onto or into a scaffold are well known in the art, and include, without being limited to, static seeding, filtration seeding and centrifugation seeding. See, e.g., Baker and Mauck, "The effect of nanofiber alignment on the maturation of engineered meniscus constructs," Biomaterials, 28 (2007) 1967-1977, which is incorporated in its entirety by reference for this purpose. Static seeding includes incubation of a cell-medium suspension in the presence of the scaffold under static conditions and results in non- uniformity cell distribution (depending on the volume of the cell suspension); filtration seeding results in a more uniform cell distribution; and centrifugation seeding is an efficient and brief seeding method (see for example EP19980203774).

[0086] The cells may be seeded directly onto the scaffold, or alternatively, the cells may be mixed with a gel which is then absorbed onto the interior and exterior surfaces of the scaffold and which may fill some of the pores of the scaffold. Capillary forces will retain the gel on the scaffold before hardening, or the gel may be allowed to harden on the scaffold to become more self- supporting. Alternatively, the cells may be combined with a cell support substrate in the form of a gel optionally including extracellular matrix components.

[0087] The cells which can be used according to the teachings of the present invention may comprise non-autologous cells or non-autologous cells (e.g. allogeneic cells or xenogeneic cells), such as from human cadavers, human donors or xenogeneic (e.g. porcine) donors.

[0088] The cells may comprise a heterogeneous population of cells or alternatively the cells may comprise a homogeneous population of cells. Such cells can be for example, stem cells (such as adipose derived stem cells, embryonic stem cells, bone marrow stem cells, cord blood cells, mesenchymal stem cells, adult tissue stem cells, induced pluripotential stem cells,), progenitor cells (e.g. progenitor bone cells), or differentiated cells such as chondrocytes, meniscal

fibrochondrocytes, osteoblasts, osteoclasts, osteocytes, connective tissue cells (e.g., fibrocytes, fibroblasts, tenocytes, and adipose cells), endothelial and epithelial cells, or mixtures thereof. [0089] As used herein, the phrase "stem cell" refers to cells which are capable of differentiating into other cell types having a particular, specialized function (i.e., "fully

differentiated" cells) or remaining in an undifferentiated state hereinafter "pluripotent stem cells".

[0090] Furthermore, such cells may be of autologous origin or non-autologous origin, such as postpartum-derived cells (as described in U.S. Application Nos. 10/887,012 and 10/887,446). Typically the cells are selected according to the tissue being generated.

[0091] It will be apparent that the invention has applications beyond medical tissue engineering. For example, certain embodiments comprise industrial applications, including dimensionally shaped filters, textile, composites, catalyst scaffolds, electrolytic cell diaphragms, battery separators, and fuel cell components.

[0092] Certain other embodiments provide that the fibrous solid also comprises a catalyst, including biological catalysts, for example, but not limited to, enzymes, anti-microbials, or antifungals. The means for incorporating these catalysts onto or within electrospun solids are well known in the art as being analogous to those used to incorporate particles as described above. The means for functionalizing, immobilizing and/or attaching catalysts onto polymer solids are equally known to those skilled in the art.

[0093] In addition to the fibrous solid bodies themselves, this invention teaches several methods for making and using them. In one embodiment, a fibrous solid of rotation can be made using the steps comprising: (a) electrospinning a first and a second biocompatible fiber, with a porogen fiber onto a rotating mandrel, said first fiber comprising a material characterized as having a yield strain in the range of about 6% to about 12%; and said second fiber comprising a biodegradable material characterized as having a modulus in the range from about 10 MPa to about 500 MPa when subjected to a strain in the range of from about 1% to about 8%, and said porogen fiber present in the range of about 20 to about 60 weight percent based on the total weight of electrospun fibers and comprising a material capable of being selectively and substantially removed from the first and the second fibers; such that the resulting scaffold is characterized as having a mean circumferential modulus in the range of about 10 MPa to about 100 MPa, when measured in the direction of the fibers and neglecting the tensile contribution of the porogen fiber.

[0094] This invention also includes those solids produced by the methods provided, to the extent that the methods provide solids which are different than those otherwise characterized herein. [0095] Similarly, it will be apparent that the solids and their method of making are intimately related to the machinery used for this purpose, and such equipment to do so is considered within the scope of this invention. That equipment is also considered within the scope of this invention which is necessary or convenient to produce the solids or use the methods described herein.

[0096] The invention further provides for embodiments describing the use of these fibrous solids in patients.

[0097] For medical applications, the anatomically shaped fibrous solids can be implanted to treat diseases characterized by connective tissue or meniscal damage or loss. As used herein, the phrase "connective tissue" refers to tissues which surround, protect, bind and support all of the structures in the body. Examples of connective tissues include, but are not limited to, cartilage (including, elastic, hyaline, and fibrocartilage), collagen, adipose tissue, reticular connective tissue, embryonic connective tissues (including mesenchymal connective tissue and mucous connective tissue), tendons, ligaments, and bone.

[0098] Patients for which such implants may be considered include mammals, said mammals including humans. It should be appreciated that while not necessarily required in all applications, it is at least highly preferred that the materials of construction appropriate regulatory approval, at least for use in human patients; e.g., in the United States, approval by the U.S. Food and Drug Administration. Other countries have similar approval requirements.

[0099] As used herein, the term "treating" refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition in an individual suffering from, or diagnosed with, the disease, disorder or condition, or repairing breaks, rips, or tears in the tissue, such as a complete or partial

meniscectomy. Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.

[00100] Those skilled in the art are capable of determining when and how to implant the solid as a scaffold to thereby induce tissue regeneration and treat the pathology. The site of implantation is dependent on the disease to be treated. For example, if the pathology to be treated is a torn meniscus the scaffold is seeded with chondrocytes or stem cells and following the required days in culture the scaffold is preferably implanted in the damaged knee. The solids of the present invention are suitable for ex vivo tissue formation to be utilized in 1 surgical procedures. According to another embodiment, tissue formation is effected in vivo - in this case the solid scaffold supported cells are typically implanted into the subject immediately following seeding.

[00101] Embodiments in which the substance comprises cells include cells that can be cultured in vitro, derived from a natural source, genetically engineered, or produced by any other means. Any natural source of prokaryotic or eukaryotic cells may be used. Embodiments in which the matrix is implanted in an organism can use cells from the recipient, cells from a conspecific donor or a donor from a different species, or bacteria or microbial cells. Cells harvested from a source and cultured prior to use are included.

[00102] As those skilled in the art will appreciate, numerous modifications and variations of the present invention are possible in light of these teachings, and all such are contemplated hereby.

EXAMPLES

[00103] Example 1. General Methods

[00104] Electrospinning was performed using a custom device designed to focus three electrospun polymer jets from separate spinnerets towards a common, centralized rotating mandrel (FIGURE 2). Each solution was driven through the spinnerets via syringe pump (KDS100, KD Scientific, Holliston, MA) at lml/h (Study I) or 2.5ml/h (Study II) from an 18G needle that was translated (with custom fanning devices) over a 5 cm distance along the vertical mandrel (which rotated at a linear surface velocity of ~10m/s). All three spinnerets were charged to +13kV and placed 15 cm from the mandrel surface, which was itself charged to -2kV to enhance fiber collection. Additionally, aluminum plates were positioned at the vertices of this setup and charged to either +9kV (Example 2) or +8kV (Example 3) to focus the electrospun cloud so as to further aid in efficient fiber capture. In both studies, each polymer solution was electrospun individually to generate a mesh containing a single fiber population, then, all three polymers were co-electrospun onto the same mandrel simultaneously to generate multi-fiber composites. That is, this apparatus was used to generate both single fiber population and multiple fiber population composite aligned nanofibrous scaffolds.

[00105] Example 2. Scaffold Fabrication - Study I

[00106] Solutions of PCL, PLGA, and PEO were electrospun either individually or simultaneously to generate scaffolds with a single fiber population or with multiple fiber populations (Composite), respectively. All polymers were dissolved by stirring over 18 hours at 40°C. PEO (10% w/v, 200kDa, Polysciences, Warrington, PA) was prepared in 90%> ethanol while PLGA (50:50 lactic acid:glycolic acid, 22.2% w/v, lOOkDa, Durect, Pelham, AL) and PCL (14.3% w/v, 80kDa, Sigma, St. Louis, MO) were each dissolved in equal parts dimethylformamide and tetrahydrofuran (DMF:THF, Sigma).

[00107] Example 3. Scaffold Fabrication - Study II

[00108] In Study II, the PLGA solution of Study I (Example 2) was replaced with a mixture of PCL and PLGA (termed Blend). In Study II, PCL and PEO solutions were prepared as described in Example 2, and PCL (7.2% w/v) and PLGA (11.1% w/v) were dissolved together in DMF:THF to produce the blended polymer solution (TABLE 1).

[00109] Table 1 - Scaffold Fabrication. Polymer formulations used to fabricate composite nanofibrous constructs in Study I and Study II.

[00110]

[00111] Example 4. Imaging

[00112] In additional studies, polymer solutions were fluorescently doped and electrospun to confirm the presence and interspersion of the three distinct fiber populations. Cell Tracker Red, 7- dimethylaminocoumarin-4-acetic acid (Invitrogen, Carlsbad, CA), and fluorescein (Sigma) were added at 0.2%> w/v to solutions of PCL, PEO, and blended PCL/PLGA, respectively. Fluorescently labeled solutions were then co-electrospun for 15s onto glass cover slips affixed to the rotating mandrel. Fibers were imaged at 20^x magnification using a Nikon T30 inverted fluorescent microscope equipped with a CCD camera and the NIS Elements software (Nikon Instruments, Inc., Melville, NY). Additionally, PCL, Blend, and Composite scaffolds were examined by scanning electron microscopy at each time point. Samples were Au-Pd sputter coated and imaged with a JEOL 6400 scanning electron microscope operating at an accelerating voltage of lOkV (Penn School of Medicine Microscopy Core Center).

[00113] In each case, analysis of the fluorescently labeled fibers demonstrated successful interspersion of discrete fibers throughout the composite scaffold. One skilled in the art would appreciate that by adjusting the parameters of the electrospinning device, it is possible to adjust the relative amounts of each fiber, relative to one another, as a function of radial and longitudinal/axial positioning within the scaffold.

[00114] Example 5. Methods Used in Mechanical Testing

[00115] From each mat, 30mm by 5mm strips were excised in the prevailing fiber direction

(Fiber) or perpendicular to this direction (Transverse). For Study I (Example 2), all testing was performed on as-formed, dry samples. In Study II (Example 3), acellular scaffolds were rehydrated and maintained in a standard culture environment (37°C, 5% C0₂) for nine weeks in order to measure degradation-dependent behavior. After determining the initial dry mass, samples were UV sterilized, rehydrated in diminishing fractions of ethanol (100, 70, 50, 30%), and incubated in DMEM containing lx PSF at 37°C until testing. For samples from both studies, four measurements of the cross-sectional area were acquired with a custom LVDT/laser system. The strips were airbrushed with black enamel to generate texture before mounting in an Instron 5848 Microtester (Instron, Canton, MA). After a nominal preload of 0. IN, the samples were allowed 1 min to equilibrate. Following this, the strips were extended beyond their yield point at a rate of 0.1% of the gauge length per second. Images of the central third of each specimen were captured at 0.5Hz for subsequent texture-correlation analysis via Vic2D to determine two-dimensional Lagrangian strain (E) (Correlation Solutions, Columbia, SC). Modulus was determined from the linear region of the stress-strain plot. Linear regressions were performed iteratively over ranges of 0.6% strain and the

2 2 yield strain was demarcated when the R transitioned below 0.996. This strain range and R threshold was chosen as a conservative and reproducible method for defining the end of the linear portion of the stress-strain curve, and was maintained for analysis throughout the entire study. After tensile testing, samples were dried and reweighed to determine mass loss at each time point.

[00116] Example 6. Determination of Fiber Fractions [00117] Day 0 samples from Study II were enzymatically digested to estimate starting fiber fractions of Composite scaffolds. PCL, Blend, and Composite scaffolds were digested with lOU/ml of Pseudomonas sp. lipase (Type XIII, Sigma) in phosphate buffered saline and incubated at 37°C for 48h with agitation. After lipase-mediated digestion of the PCL component of each scaffold, samples were washed twice in distilled water before being dried and reweighed.

[00118] Example 7. Constitutive Modeling of Composites

[00119] A hyperelastic fiber-reinforced constitutive model was employed to describe the tensile behavior of hydrated composite nanofibrous scaffolds in Study II. The constitutive laws for scaffolds composed of single fiber populations of Blend or PCL were first determined. These were then applied, using a constrained mixture approach, to predict the time-varying behavior of the two- component composite scaffold (after removal of PEO). Upon validation, the model was used to simulate time and composition dependent mechanics.

[00120] The composite and single component scaffolds were modeled using the constitutive theory of highly anisotropic solids, in which the strain energy density function is decomposed into the sum of 'fiber' and 'matrix' functions.

[00121] Single Component Constitutive Laws

[00122] For each Blend and PCL scaffold, the matrix phase was described as a compressible

Neo-Hookean material, while the fibers were described with an exponential law. As detailed in Nerurkar et. al. ⁽^'Integrating theoretical and experimental methods for functional tissue engineering of the annulus fibrosus," Spine 2008) the resulting constitutive law is given by:

[00123]

i

Where the superscript i = B, C for Blend and PCL, respectively; T is the Cauchy stress tensor, and are the two matrix parameters that characterize matrix modulus and material

compressibility;

are fiber parameters representing the fiber stiffness and the degree of their stress-strain nonlinearity; I₃ = det(C) is the third invariant of the Right Cauchy Green Tensor

C = F^TF, where F is the deformation gradient tensor; the scalar I₄ is an invariant defined as I₄ = a · Ca , where a is a unit vector along the fiber direction. Fiber contributions were restricted to tensile stresses only (I₄ = 1 for I₄ < 1).

[00124] Therefore, each single fiber population scaffold, Blend or PCL, was described by four scalar material parameters: μ' and v¹ to describe the matrix phase, and ¹ and ζ to represent the fiber phase. The implementation of this model for single-component electrospun scaffolds was performed as described previously. In brief, a least squares curve fit of the model to the transverse stress-strain data was performed at each time point to yield matrix material parameters, μ and V. The average of the resulting values was used to fit the fiber direction stress-strain curves in order to determine the time-matched values of fiber parameters, ¹ and ξ'. Accordingly, the four material parameter values were obtained for each component (Blend and PCL) at each time point (days 0, 7, 21, 42, 63).

[00125] Composite Constitutive Law

[00126] To model the composite scaffold mechanics, it was assumed that the composite properties were determined entirely by the constituent material parameters and the relative amounts of each constituent present, or:

where Τ is the total stress of the composite and

is the mass fraction of each component. Specifically, a constrained-mixture approach was used, whereby each constituent deforms identically, according to the overall deformation of the composite, or in other words F = F^* for i = Blend, PCL. Additionally, the composite stress is the sum of constituent stresses scaled to their current mass fraction:

[00127] Conventionally, it is required that the sum of mass fractions always be unity.

Although this constraint was enforced at Day 0, it was relieved at subsequent time points in order to account for the loss of intact, load-bearing fibers with degradation; mass fractions were computed at each time point as the current constituent mass normalized to the initial total mass. At Day 0, it was assumed that

_? based upon experiments using lipase to remove the PCL component from both Blend and Composite scaffolds. From Days 7 to 63, mass fractions were approximated by assuming all mass loss of the composite (measured experimentally) was due to loss of the Blend component, while

_Was maintained for all time points. The contention that PCL mass does not degrade significantly over 63 days was supported by the experimental results. The model was then validated by predicting the fiber direction stress-strain behavior of the composite scaffold at each time point using time-matched material parameters and mass fractions. For each sample, the experimentally measured 2-D deformations were input into the model and the resulting model- computed stresses were compared with experimentally measured stress. Agreement of the model predicted stress with the corresponding experimental stress was assessed to indicate the suitability of the proposed model as a full quantitative description of the nanofibrous composite scaffold.

[00128] Model Simulation

[00129] Upon validation, the model was used to simulate the time-varying mechanical behavior of composite nanofibrous scaffolds for a range of initial compositions. In order to approximate the time dependence of the material parameters, each parameter was assumed to vary linearly with time and linear regression were utilized. This assumption proved reasonably accurate for all time-varying parameters (see Results). The moduli of simulated composites were determined by fitting a tenth order polynomial to the model-generated stress-strain curve, and evaluating the derivative of the polynomial at E=0.05.

[00130] Statistical Analyses

[00131] For experimental data, ANOVA with Bonferroni post-hoc tests was used to make comparisons between groups. Data is presented as the mean ± standard deviation from a minimum of 5 samples for each condition and time point. The quality of model fits are reported by R values as well as the Bland-Altman limits of agreement (B-A, bias ± standard deviation), reported in MPa

[00132] Example 8. Analysis of the Tensile Characteristics of the Scaffolds of Study I

[00133] PCL, PLGA, and PEO scaffolds formed as in Example 2 were individually tested in uniaxial tension in the fiber direction. Stress-strain profiles for each single fiber population scaffold were distinct (FIGURE 4A). PEO and PCL scaffolds had comparable tensile moduli while the modulus of PLGA scaffolds was -20 times higher (FIGURE 4B). While PCL scaffolds exhibited a significant toe region and yielded at a higher strain (E=0.100), both PEO and PLGA scaffolds lacked toe regions and were significantly less extensible, yielding at E=0.012 and E=0.014, respectively (FIGURE 4C). When all three polymers were co-electrospun into a composite scaffold, the resulting mesh displayed characteristics of its constituents (FIGURE 4A). Composite scaffolds had a modulus intermediate to PCL and PLGA (FIGURE 4B) and a toe region was observed, though composite scaffolds still yielded at low strains (E=0.026) relative to pure PCL scaffolds (FIGURE 4C). Moreover, whereas the fiber to transverse modulus ratio for PCL was found to be 10: 1, for the Blend it was only 3: 1. Of note, these scaffolds were all taken from the same location on the rotating mandrel. However, when composite strips were excised from different locations along the length of the mandrel we observed a gradient in the deposition of PCL and PLGA fibers (FIGURE 4D). Where PLGA fibers were dominant, stress-strain profiles revealed a linear behavior with a higher modulus. Conversely, increases in the PCL fiber fraction resulted in a more pronounced toe region but a lower modulus. This suggests that care must be taken in ensuring full interspersion of composite fibers, particularly when one component has markedly higher mechanical properties than the others. It was also noted that, upon hydration, both PLGA and Composite scaffolds contracted in length by -70% and -60%, respectively (data not shown). Due to this behavior, as well as the low yield strains of PLGA fibers, the PLGA solution was replaced with a blend of PCL and PLGA in Study II.

[00134] Single fiber population PLGA scaffolds were very stiff (~20-fold higher than PCL), however, two significant limitations were observed. First, pure PLGA scaffolds could only be deformed by -1% before succumbing to permanent deformation and/or failure. Thus when PLGA was electrospun into the Composite scaffold, the resulting yield strain was reduced by a factor of four, to E=0.025. As most fibrous tissues experience deformations on the order of 5-10%, we considered this attribute to be a serious deterrent to using pure PLGA fibers. Additionally, and through a mechanism not yet fully understood, PLGA scaffolds underwent severe contraction and thickening immediately upon hydration, precluding a longitudinal study of mechanical properties with degradation.

[00135] Example 9. Analysis of the Tensile Characteristics of the Scaffolds of Study II

[00136] Scaffolds comprised of a single fiber population of Blend PCL/PLGA fibers could be hydrated without noticeable decreases in scaffold length or fiber morphology in SEM images (not shown). This allowed for evaluation of scaffold mechanical behavior over a time course of degradation, without the influence of complicating factors such as gross scaffold distortion. For this study, composite scaffolds were hydrated before mechanical analysis. As expected, upon hydration, PEO scaffolds dissolved completely, and so are excluded from further analysis of mechanics or mass loss. On day 0 (immediately after hydration), PCL and Blend scaffolds tested in the fiber direction demonstrated distinct stress-strain profiles (FIGURE 5A). As in Study I, PCL revealed a significant toe region with yield strains of E=0.115 (FIGURE 5B). Blend scaffolds were linear and lacked a toe region, but were extensible to higher strains (E=0.080) before yield (compared to yield at ~1% for pure PLGA scaffolds evaluated in Study I). Similar to Study I, composites comprised of PCL and Blend fibers showed characteristics of the two remaining constituents (recall that PEO is removed upon hydration). Initially, composite scaffolds had moduli intermediate to PCL and Blend (FIGURE 5C), but lower strains at yield than both of its constituents (FIGURE 5B). Testing performed on transverse samples revealed the high degree of anisotropy in all three types of scaffolds as evidenced by the significantly lower moduli in the transverse direction (FIGURE 4D).

[00137] After the initial day 0 hydration and testing, scaffolds were incubated in DMEM+PSF at 37°C and fiber and transverse properties as well as changes in mass were assessed on days 7, 21, 42, and 63. As observed in previous studies, the mass of pure PCL scaffolds did not diminish up to 63 days (FIGURE 5E, p=1.0). Paralleling the lack of mass loss, the mechanical behavior of PCL scaffolds did not change appreciably, with moduli in both the fiber and transverse directions remaining constant for the entire course of the study (FIGURES 5C, 5D, p=1.0). Interestingly, the yield strain of PCL increased with time, and was significantly higher than starting values by day 42 (FIGURE 5B). Contrasting the immutability of PCL scaffolds, Blend scaffolds began degrading from the outset, as indicated by the significant mass loss by day 7. Additional decreases in mass were observed at all remaining time points, cumulating to a -40% mass loss by day 63 (FIGURE 5E). Degradation of these Blend fibers resulted in decreases in both fiber and transverse moduli in the scaffolds (FIGURES 5C, 5D, p<0.001). Additionally, yield strains of Blend scaffolds extended in the fiber direction increased with degradation time (FIGURES 5B, p<0.001).

[00138] The mechanism by which Blend scaffolds (or fibers) degrade was not assessed, nor does this invention depend on such a mechanism. While the idealized mechanism shown in

FIGURE 2B suggests degradation resulting in complete fiber removal, and without being bound to any single theory, Blend fibers may undergo scission and complete removal or may experience internal changes that leave the fiber present but in a weakened state. SEM imaging over the time course did not reveal obvious structural changes, although this may be an inherent limitation of the 2D imaging modality. [00139] Composite scaffolds showed time dependent characteristics as well. These scaffolds lost -22% of their original mass upon hydration as a result of the immediate dissolution of the PEO fiber population (FIGURES 5E). Lipase digestion after PEO removal showed that Blend fibers made up 54.3% of the remaining Composite scaffold, while pure PCL fibers made up 45.7%. Due to the presence of Blend fibers, Composites continually lost mass for the duration of the study resulting in an additional 18% decrease in mass by day 63. As observed in Blend scaffolds, Composite scaffolds showed temporal changes in mechanical properties, with both fiber and transverse moduli decreasing in a time-dependent manner (FIGURES 5C, 5D, p<0.001).

Contrasting Blend behavior, the fiber-direction modulus of Composites decreased dramatically by day 7 to levels well below PCL and Blend values (p<0.001), then appeared to stabilize towards later time points. As with PCL and Blend scaffolds, Composite yield strains increased with time

(p<0.001) approaching E=0.122 by day 63.

[00140] Example 10. Simulation Results

[00141] The constitutive model was successfully fit to transverse PCL and Blend constructs at all time points to yield matrix constants μ and V (FIGURES 6B, 6C and TABLE 2), with average Pv²=0.986 and B-A -0.002±0.004 MPa. As an example, model fits to experimental data of day 0 samples tested in the transverse direction are shown in FIGURE 6A. The Blend matrix parameter μ^Β decreased linearly with time by up to 2.5-fold on day 63. No significant changes were observed in μ , v^c or V^s with time. The constitutive model also successfully fit Blend and PCL samples along the fiber direction to yield fiber parameters y¹ and ξ' with average R²=0.988 and B-A -0.018±0.055 MPa (FIGURES 6E, 6F). Model fits to fiber direction experimental data of day 0 samples are shown in FIGURE 6D. The fiber modulus parameter y¹ decreased approximately 2-fold with time for Blend samples, but did not change with time for PCL. ξ increased slightly by Day 63, and ξ was zero at all time points.

[00143] Having determined the time-varying material parameters for each single fiber population scaffold, this information was used to predict the behavior of the composite scaffold. Accurate prediction of the composite scaffold mechanics would indicate that the model is valid for this application. The model was successfully validated at each time point, using only the deformation, the relative amount of Blend to PCL fibers remaining, and the material parameters from each time point to predict the Composite scaffold stress under uniaxial extension in the fiber direction (FIGURE 7, average R²=0.997 and B-A -0.013±0.053 MPa, TABLE 2). Although at Day 0 the model under-predicted stresses, it provided strikingly close predictions of experimental stress- strain curves at each subsequent time point (TABLE 2).

[00144] Using the validated model, the modulus and curvature of theoretical scaffolds consisting of any combination of PCL and Blend at any time point were predicted. The model simulation demonstrated that the composite scaffold modulus can be tuned between 20 and 45 MPa, simply by varying the initial content of Blend or PCL (FIGURE 7). Additionally, the composite mechanics are increasingly sensitive to degradation with time as the Blend content is increased. Finally, by varying the balance between Blend and PCL fiber populations, the model demonstrated that the magnitude and the nonlinearity of the composite stress-strain behavior can both be modulated.

[00145] With the validated model, the complete range of possible Blend and PCL

combinations was simulated. This simulation demonstrated that the magnitude and time- dependence of Composite scaffold mechanics can be modulated in a predictable fashion by altering the initial composition. Simulations also showed that it is possible to tune not only linear region modulus (FIGURE 8A), but also the profile of the stress-strain curve. For instance, introducing an increasing fraction of PCL fibers to the Composite reduces the modulus, while conferring nonlinearity to the overall material behavior. This model result is consistent with experimental observations of Study I and II (FIGURE 4D). Because many fiber reinforced soft tissues are subject to physiologic deformations below the transition strain, it is important to implement a method for scaffold design that incorporates full material nonlinearity, and not only linear region metrics such as a single modulus value.

[00146] Example 11. Measured Benchmarks Compared with Experimental and Modeling

Results

[00147] Circumferential samples were taken from 10 sheep menisci, and the stress-strain response of these samples were measured. These results were fit to a common non-linear formulation [σ=Α(ε^Βε-1)], as well as to the hyperelastic model (described above). FIGURE 9 shows the average stress-strain response derived from these fits, with the upper and lower bounds defined by data points from the strongest and weakest samples tested. This grey region represents a

'meniscus domain', and includes the full set of points that define a successful tissue engineered construct. There are however several regions to this domain, constituting both 'toe' (low modulus) and linear (high modulus) regions. Indeed, the moduli computed from these models was 32±14 MPa (range: 15-50) in the 'toe' (3% strain) region and 82±33 MPa (range: 46-120) in the 'linear' (10% strain) region.

Claims

What is Claimed:

An implantable knee meniscus scaffold, comprising a first and a second biocompatible fiber, electrospun together with a porogen fiber to be oriented substantially circumferentially to an axis of rotation, said first fiber comprising a material characterized as having a yield strain in the range of about 1% to about 20%; said second fiber comprising a biodegradable material characterized as having a modulus in the range from about 10 MPa to about 500 MPa, when subjected to a strain in the range of from about 1% to about 10%; said porogen fiber comprising an amount in the range of about 20 to about 60 weight percent based on the total weight of fibers; and said scaffold having a mean circumferential modulus, when measured in the direction of the fibers and including the cross-sectional area of the porogen fiber, in the range of about 20 MPa to about 200 MPa, when subjected to a strain of about 10%.

An implantable knee meniscus scaffold, comprising a first and a second biocompatible fiber, electrospun together with a porogen fiber to be oriented substantially circumferentially to an axis of rotation, said first fiber comprising a material characterized as having a yield strain in the range of about 1% to about 20%; said second fiber comprising a biodegradable material characterized as having a modulus in the range from about 10 MPa to about 500 MPa, when subjected to a strain in the range of from about 1% to about 10%>; said porogen fiber comprising an amount in the range of about 20 to about 60 weight percent based on the total weight of fibers; and said scaffold having a mean circumferential modulus, when measured in the direction of the fibers and including the cross-sectional area of the porogen fiber, in the range of about 10 MPa to about 60 MPa, when subjected to a strain of about 3%.

The implantable scaffold of claim 1 wherein the porogen fiber is substantially removed.

The implantable scaffold of claim 2 wherein the porogen fiber is substantially removed.

The scaffold of any of claims 1 to 4 wherein the yield strain of the scaffold is in the range of about 4% to about 20%.

The scaffold of any of claims 1 to 4 wherein the yield strain of the scaffold is at least 6%. The scaffold of claim 1 or claim 3 characterized as having an initial mean circumferential modulus of at least 40 MPa.

The scaffold of claim 1 or claim 3 characterized as having an initial mean circumferential modulus of at least 60 MPa.

The scaffold of claim 1 or claim 2 characterized as having an initial mean circumferential modulus of about 80 MPa.

The scaffold of claim 3 or claim 4 wherein the mean circumferential modulus of the scaffold after 7 days exposure to physiological fluid at a temperature in the range of 35°C to 40°C is at least 60 % of the initial mean circumferential modulus of the scaffold.

The scaffold of claim 3 or claim 4 wherein the mean circumferential modulus of the scaffold after 60 days exposure to physiological fluid at a temperature in the range of 35°C to 40°C is at least 50 % of the initial mean circumferential modulus of the scaffold.

The scaffold of any of claims 1 to 4 wherein the porogen fiber is capable of being selectively and substantially removed from the first and second fibers by dissolving or biodegrading in a physiological fluid.

The scaffold of any of claims 1 to 4 wherein the physiological fluid is water, saline, meniscal fluid, simulated body fluid, or synovial fluid.

The scaffold of any of claims 1 to 4 wherein the porogen electrospun fiber comprises a poly(ethylene oxide).

The scaffold of any of claims 1 to 4 wherein the first fiber and the second fiber each comprise an organic polymer.

The scaffold of any of claims 1 to 4 wherein the first fiber or the second fiber or both comprise an aliphatic polyester, poly(amino acid), polyalkylene oxalate, polyamide, polyamido ester, poly(anhydride), poly(beta-amino ester), polycarbonate, polyether, polyorthoester, polyphosphazene, polyurethane, polycarbonate, polyacrylonitrile, polyaniline, polyvinyl carbazole, polyvinyl fluoride, polyvinyl imidazole, polyvinyl alcohol, polystyrene, poly( vinyl phenol), polyacrylate, polymethacrylate, acyl-substitituted cellulose acetate, polyethylene oxides, polyfluorinated ethylene, polydialkylsiloxane, a copolymer or a mixture thereof.

The scaffold of any of claims 1 to 4 wherein the first fiber comprises poly(caprolactone) or a poly(P-aminoester).

The scaffold of any of claims 1 to 4 wherein the second fiber comprises poly(glycolic acid) or a poly(P-aminoester).

The scaffold of any of claims 1 to 4 wherein at least one of the first fiber material or the second fiber comprises a co-spun blend of polymers.

The scaffold of any of claims 1 to 4 wherein the second fiber comprises a co-spun blend of poly(caprolactone) and poly(L-glycolic acid).

The scaffold of any of claims 1 to 4 comprising a first fiber in the range of about 1 to about 80 weight percent and the second fiber in the range of about 80 to about 1 weight percent, each with respect to the total weight of electrospun fibers.

The scaffold of any of claims 1 to 4 wherein both the first and the second fibers biodegrade, biosorb, or both biodegrade and biosorb in a physiological fluid.

The scaffold of claim 22 wherein the physiological fluid is water, saline, meniscal fluid, simulated body fluid, or synovial fluid.

The scaffold of any of claims 1 to 4 wherein the first and the second fibers each biodegrade, biosorb, or both biodegrade and biosorb at different rates. The scaffold of claim 24 wherein the second fiber biodegrades, biosorbs, or both biodegradi and biosorbs at a slower rate than the first fiber.

The scaffold of any of claims 1 to 4 wherein at least one of the electrospun fibers has a diameter dimension in the range of about 1 nm to about 10 microns.

The scaffold of any of claims 1 to 4 wherein at least one of the electrospun fibers has a diameter dimension in the range of about 20 nm to about 1000 nm.

The scaffold of any of claims 1 to 4 wherein at least one of the electrospun fibers has a diameter dimension in the range of about 100 nm to about 1000 nm.

The scaffold of any of claims 1 to 4 wherein at least one of the electrospun fibers has a diameter dimension in the range of about 1 micron to about 10 microns.

The scaffold of any of claims 1 to 4 wherein the degree of degree of orientation of the electrospun fibers varies across the radial distance of the scaffold.

The scaffold of any of claims 1 to 4 wherein the degree of degree of orientation of the electrospun fibers varies of at least one gradient varies continuously.

The scaffold of any of claims 1 to 4 wherein the composition of the fibers varies across the radial distance of the scaffold.

The scaffold of any of claims 1 to 4 wherein the composition of the fibers varies across the distance between the top and bottom surfaces of the scaffold.

The scaffold of any of claims 1 to 4 wherein the scaffold is substantially anatomically shaped.

The scaffold of claim 34 wherein the shape of the scaffold is designed to be used as a medical implant scaffold in a mammal. The scaffold of claim 35 wherein the mammal is a human.

The scaffold of any of claims 1 to 4 further comprising at least one therapeutic agent.

The scaffold of claim 37 wherein the at least one therapeutic agent comprises an amino acid, an antibiotic, an anti-inflammatory, an antiproliferative agent, a DNA or R A, an

immunosuppressant, a peptide or polypeptide, a prosteoglycan, a protease or protease substrate, a protein, a vitamin, or a mixture thereof.

The scaffold of claim 37 wherein the at least one therapeutic agent is attached to or contained within a biocompatible material.

The scaffold of claim 39 wherein the biocompatable material provides for a timed release of the at least one therapeutic agent.

The scaffold of claim 37 wherein the at least one therapeutic agent varies across the radial distance of the scaffold.

The scaffold of claim 37 wherein the at least one therapeutic agent varies across the distance between the top and bottom surfaces of the scaffold.

The scaffold of any of claims 1 to 4 further comprising at least one biofactor.

The scaffold of claim 43 wherein the at least one biofactor is a growth factor

The scaffold of claim 43 wherein the at least one growth factor comprises a basic fibroblast growth factor, an insulin-like growth factor, a nerve growth factor, an epidermal growth factor, a platelet derived growth factor, a transforming growth factor, a tumor necrosis factor, a vascular endothelial growth factor, or a homolog or combination thereof.

The scaffold of claim 43 wherein the at least one growth factor is attached to or contained within a biocompatible material. The scaffold of claim 43 wherein the biocompatible material provides for a timed release of the at least one growth factor.

The scaffold of claim 43 wherein the concentration of the at least one growth factor varies across the radial distance of the scaffold.

The scaffold of claim 43 wherein the at least one growth factor varies across the distance between the top and bottom surfaces of the scaffold.

The scaffold of any of claims 1 to 4 further comprising at least one population of cells.

The scaffold of claim 50 wherein the at least one population of cells comprises adipose derived stem cells, chondrocytes, connective tissue cells, embryonic stem cells, induced pluripotential stem cells, meniscal fibrochondrocytes, mesenchymal stem cells, osteoblasts, or mixtures thereof.

The scaffold of claim 50 wherein the concentration of cells varies across the radial distance of the scaffold.

The scaffold of claim 50 wherein the at least one population of cells varies across the distance between the top and bottom surfaces of the scaffold.

The scaffold of any of claims 1 to 4 infiltrated with growing tissue.

The scaffold of any of claims 1 to 4 further comprising a catalyst.

The scaffold of claim 55 wherein the catalyst comprises an enzyme. A method of making an implantable knee meniscus scaffold comprising: a) electrospinning a first and a second biocompatible fiber, with a porogen fiber onto a

rotating mandrel, said first fiber comprising a material characterized as having a yield strain in the range of about 1% to about 20%; said second fiber comprising a

biodegradable material characterized as having a modulus in the range from about 10 MPa to about 500 MPa, when subjected to a strain in the range of from about 1% to about 10%; said porogen fiber comprising an amount in the range of about 20 to about 60 weight percent based on the total weight of fibers; and said scaffold having a mean circumferential modulus, when measured in the direction of the fibers and including the cross-sectional area of the porogen fiber, in the range of about 20 MPa to about 200 MPa, when subjected to a strain of about 10%>.

A method of making an implantable knee meniscus scaffold comprising: a) electrospinning a first and a second biocompatible fiber, with a porogen fiber onto a

rotating mandrel, said first fiber comprising a material characterized as having a yield strain in the range of about 1% to about 20%>; said second fiber comprising a

biodegradable material characterized as having a modulus in the range from about 10 MPa to about 500 MPa, when subjected to a strain in the range of from about 1% to about 10%; said porogen fiber comprising an amount in the range of about 20 to about 60 weight percent based on the total weight of fibers; and said scaffold having a mean circumferential modulus, when measured in the direction of the fibers and including the cross-sectional area of the porogen fiber, in the range of about 10 MPa to about 60 MPa, when subjected to a strain of about 3%.

The method of claim 57 or claim 58 wherein the porogen material is capable of dissolving or biodegrading in physiological fluid within one hour of exposure to said fluid at ambient or physiological temperatures.

The method of claim 59 wherein the physiological fluid is water, saline, meniscal fluid, simulated body fluid, or synovial fluid. The method of claim 57 or claim 58 wherein the fibers comprise organic polymers.

The method of claim 57 or claim 58 wherein the first fiber or the second fiber first or both comprise co-spun blends of organic polymers.

The method of claim 57 or claim 58 wherein the porogen fiber is removed.

The method of claim 63 wherein the porogen fiber is removed by dissolution in physiological fluid.

The method of claim claim 57 or claim 58 further comprising providing at least one therapeutic agent, biofactor, population of cells, catalyst, growing tissue, or mixture or combination thereof.

The method of claim 65 wherein the at least one therapeutic agent, biofactor, population of cells, catalyst, or mixture or combination thereof is provided attached to or within a biocompatible matrix or encapsulants.

A method of treating a mammalian patient comprising implanting the scaffold of any of claims 1 to 4.

The method of claim 67 wherein the patient is human.