WO2008100534A2 - Biomimetic nanofiber scaffold for soft tissue and soft tissue-to-bone repair, augmentation and replacement - Google Patents

Abstract

An implantable device is provided for soft-tissue or soft tissue-to-bone repair, fixation, augmentation, or replacement that includes a biomimetic and biodegradable nanofiber scaffold. More particularly, there is provided an implantable device for repair, fixation, augmentation, or replacement of a rotator cuff or a tendon- to-bone interface thereof. In one aspect, this device includes a biphasic, biomimetic, and biodegradable nanofiber scaffold having a first phase that includes nanofibers whose anisotropy mimics that of a tendon and non-mineralized fibrocartilage, which nanofibers are made from a biodegradable polymer and a second phase coupled to the first phase, which second phase includes nanofibers whose anisotropy mimics that of mineralized fibrocartilage and bone, which nanofibers are made from a biodegradable polymer and a biocompatible ceramic, wherein the first and second phases are continuous. Methods of using such devices are also provided

Description

BIOMIMETIC NANOFIBER SCAFFOLD FOR SOFT TISSUE AND SOFT TISSUE- TO-BONE REPAIR. AUGMENTATION AND REPLACEMENT

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0001] The invention was made with government support under NSF

Graduate Fellowship (GK-12 0338329) awarded by the National Science Foundation. The government may have certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims benefit to U.S. provisional patent application nos. 60/901 ,047 and 60/905,649 filed on February 12, 2007 and March 7, 2007, respectively, each of which is incorporated by reference in its entirety as if recited in full herein.

FIELD OF THE INVENTION

[0003] The present invention provides a biomimetic nanofiber scaffold for repair, augmentation, fixation and/or replacement of damaged soft tissue and/or soft tissue-to-bone interfaces, such as for example, a rotator cuff tendon. Methods of making and using such nanofiber scaffolds are also provided.

BACKGROUND OF THE INVENTION

[0004] The rotator cuff consists of a group of four muscles and tendons, including the supraspinatus, infraspinatus, teres minor, and subscapularis, which function in synchrony to stabilize the glenohumeral joint as well as to actively control shoulder kinematics. The supraspinatus tendon inserts into the humeral head via a direct insertion exhibiting region-dependent matrix heterogeneity and mineral content.

[0005] Four distinct yet continuous tissue regions are observed at the tendon- bone junction (Figure 9): tendon proper, non-mineralized fibrocartilage, mineralized fibrocartilage and bone (86, 87, 97). The tendon proper consists of fibroblasts found between aligned collagen fibers in a matrix rich in collagen I, with small amounts of collagen III and proteoglycans (88). The non-mineralized fibrocartilage region is composed of fibrochondrocytes in a matrix of collagen I₁ II, and III with fibers oriented perpendicular to the calcified interface region (99). The mineralized fibrocartilage region consists of hypertrophic fibrochondrocytes within a matrix of collagen I and Il (99) as well as collagen X (70). The last region of the insertion site is bone which consists of osteoblasts, osteoclasts, and osteocytes in a mineralized matrix rich in type I collagen.

[0006] This controlled matrix heterogeneity exhibited by the tendon-bone interface serves to minimize stress concentrations and to mediate load transfer between two distinct tissue types (70, 97). Due to its functional significance, interface regeneration is a pre-requisite for biological fixation.

[0007] Rotator cuff tears are among the most common injuries afflicting the shoulder, with greater than 75,000 repair procedures performed annually in the United States alone (1). Clinical intervention is required because injuries to the rotator cuff do not heal, largely due to the complex anatomy noted above and the extended range of motion of the shoulder joint, as well as the relative weakening and hypovascularization of the cuff tendons (2-4). Moreover, chronic degeneration increases both the frequency and size of cuff tears with age (5) and is considered the main contributing factor in the pathogenesis of rotator cuff tendon tears (4,6). Early primary anatomic repair followed by carefully controlled rehabilitation is currently the treatment of choice for symptomatic rotator cuff tears (4). [0008] Rotator cuff repair has evolved from traditional open repair to "mini- open" to primarily arthroscopic (7-11). This transition has occurred due to advances in surgical techniques and fixation methods, with the current technique being a double row "suture-bridge" technique which simulates the compression afforded by transosseous sutures previously used in open and mini-open repairs (12). These methods have been shown to improve mechanical strength and graft stability (13). The focus in the field now centers on how to address the challenge of achieving functional rotator cuff healing and/or augmentation, which is essential for long term clinical success.

[0009] Currently, a significant demand exists for a functional tendon grafting system which can augment and promote rotator cuff healing post surgical repair, due to the relatively high failure rates associated with current repair procedures as well as the clinical need to treat large tears and chronic degeneration of the rotator cuff tendons. For example, failure rates as high as 90% have been reported after primary repair of chronic rotator cuff injuries (8), generally attributed to factors such as osteoporotic bone, degenerative and poorly vascularized tendons, severe tendon weakening, muscle atrophy, and size of the original defect (14-18). Moreover, the primary repair of chronic degenerative cuff injuries often results in excessive tension on the cuff tissues and at the repair site (4, 19, 20). To improve healing, synthetic grafts (21 , 22) have been designed to reconstruct large rotator cuff defects. See also, e.g., U.S. Pat. No. 7,112, 417. However, these devices are suboptimal due to concerns of biocompatibility as well as their inability to meet the functional demand of the native tendon.

[0010] Recently, biological matrices such as acellularized allogeneic and xenogeneic extracellular matrix scaffolds have emerged as promising grafts for rotator cuff repair and augmentation (4, 23). Both collagen-rich dermis and small intestinal submucosa (SIS) (24) have been marketed commercially as graft patches for reinforcing soft tissue repair following rotator cuff surgery (25, 26). See also, e.g., U.S. Pat. Nos. 6,638,312 and 7,160,333. SIS is particularly attractive as it exhibits a biomimetic, collagen nanofiber-based architecture and alignment, thus it can be readily remodeled by host cells while encouraging angiogenesis and neo-collagen production (24).

[0011] Highly promising results have been reported for SIS in several animal models (4, 23), but unfortunately suboptimal outcomes were observed in human trials (26, 27), in which augmentation with SIS did not improve the rate of tendon healing or clinical outcome scores. Similar outcomes have been reported for other biological grafts used in rotator cuff repair (27, 28).

[0012] The suboptimal results of biologically-derived grafts may be attributed to mismatch in mechanical properties and the rapid matrix remodeling experienced in the physiologically demanding and often diseased shoulder joint. Utilizing a canine model, Derwin et al. (25) performed a systematic comparison of the biomechanical properties of commercially available extracellular matrices for rotator cuff augmentation. A mismatch in mechanical properties with the canine infraspinatus tendon was observed for all types of extracellular matrix tested. [0013] Moreover, it has been reported that the mechanical properties of SIS decreased as resorption occurred prematurely (25). Thus the mismatch in the kinetics of graft remodeling and neo-collagen formation compromised the clinical outcome. Therefore, the debilitating effect of rotator cuff tears coupled with the high incidence of failure associated with existing graft choices emphasize the clinical need for functional rotator graft augmentation solutions.

SUMMARY OF INVENTION

[0014] While the mechanism for interface regeneration is not known, knowledge of the structure-function relationship at the tendon-bone insertion (70, 71) provides invaluable clues in biomimetic nanofiber scaffold design for interface regeneration. Combining biomechanical testing with the quasi-linear viscoelastic model (QLV) (91), Thomopoulos et al. (70) determined the mechanical properties of the rat supraspinatus tendon insertion sites and later related it to collagen orientation using a finite element model (71). It was found that controlled collagen organization plays an important role in reducing stress concentration at the tendon-bone insertion (71). Specifically, the average collagen fiber angle varied from 83-98° in the non- mineralized and 86-103° in the mineralized fibrocartilage region, indicating that the interface fiber architecture deviated minimally from the tendon proper. [0015] In addition to collagen alignment, another intrinsic parameter of the interface is the region-dependent mineral distribution across the insertion site (87, 97). Calcium phosphate is a prime modulator of both the biochemical milieu and the nature of mechanical stimuli presented to cells. Moreover, the spatial variation in mineral content at the interface is mechanically relevant, as increased mineral content has been associated with higher mechanical properties (89, 90, 93, 94). For example, Ferguson et al. found a positive correlation between indentation modulus and hardness with mineralization in calcified human articular cartilage. Moffat et al. reported that increases in compressive modulus of the mineralized fibrocartilage region at the ligament-bone insertion corresponded to the presence of minerals (93). These observations collectively suggest that both collagen alignment and mineral content are critical design parameters for interface tissue engineering. [0016] Accordingly, one embodiment of the present invention is an implantable device for soft-tissue or soft tissue-to-bone fixation, repair, augmentation, or replacement comprising a biomimetic and biodegradable nanofiber scaffold, which scaffold comprises one or more continuous phases. [0017] Another embodiment of the invention is an implantable biphasic biomimetic and biodegradable nanofiber device for soft tissue or soft tissue-to-bone interface fixation, repair, augmentation, or replacement. This device comprises a first phase comprising nanofibers made from a biodegradable polymer and a second phase coupled to the first phase, which second phase comprises nanofibers made from a biodegradable polymer and a biocompatible ceramic, wherein the first and second phases are continuous.

[0018] A further embodiment of the invention is an implantable device for fixation, repair, augmentation, or replacement of a rotator cuff or a tendon-to-bone interface thereof. This devices comprises a biphasic, biomimetic, and biodegradable nanofiber scaffold having a first phase comprising nanofibers whose anisotropy mimics that of a tendon and non-mineralized fibrocartilage, which nanofibers are made from a biodegradable polymer and a second phase coupled to the first phase, which second phase comprises nanofibers whose anisotropy mimics that of mineralized fibrocartilage and bone, which nanofibers are made from a biodegradable polymer and a biocompatible ceramic, wherein the first and second phases are continuous. [0019] Another embodiment of the present invention is a method for fixation of, repairing, augmenting, or replacing a damaged soft tissue or soft tissue-to-bone interface in a patient. This method comprises affixing a biomimetic, biodegradable, continuous multi-phasic nanofiber scaffold to a surgically relevant site in order to fixate, repair, augment, or replace the damaged soft tissue or soft tissue-to-bone interface.

[0020] Yet another embodiment of the present invention is a method for fixating, repairing, augmenting, or replacing a damaged rotator cuff in a patient. This method comprises affixing a biomimetic and biodegradable continuous multiphase nanofiber scaffold to a surgically relevant site in order to repair, augment, or replace the damaged rotator cuff.

BRIEF DESCRIPTION OF FIGURES

[0021] Figure 1 shows the structural properties of the aligned and unaligned nanofiber scaffolds. Scanning electron micrographs of A) aligned and B) unaligned nanofiber scaffolds are shown (200Ox, bar=20 μm).

[0022] Figure 2 shows mechanical properties of aligned and unaligned nanofiber scaffolds. A representative stress-strain curve for aligned and unaligned nanofiber scaffolds tested in uniaxial tension is shown.

[0023] Figure 3 shows the effects of nanofiber organization on cell morphology. The top panel shows confocal microscopy of human rotator cuff fibroblasts at day 1 and day 14 (2Ox, bar=100 μm). The bottom panel shows scanning electron micrographs of cells grown on aligned and unaligned nanofiber scaffolds at day 1 and day 14 (1000x, bar=50 μm). The fibroblasts remained viable and grew on both types of substrate over time, with the rotator cuff cells exhibiting phenotypic elongated morphology on the aligned nanofiber scaffolds. [0024] Figure 4 shows gene expression on aligned and unaligned nanofiber scaffolds over time, lntegrin expression differed between the aligned and unaligned groups while types I and III collagen expression were maintained on both nanofiber scaffold types. The α2 integrin was consistently expressed on the aligned nanofiber scaffolds, while no α2 expression was detected on the unaligned nanofiber scaffolds at all time points evaluated.

[0025] Figure 5 shows quantitative analysis of fibroblast response on aligned and unaligned nanofiber scaffolds at day 1 and day 14 post-plating. Nanofiber organization guided cell attachment and was analyzed based on analysis of A) Mean Vector Angle (O=horizontally aligned) and B) Mean Vector Length (0=random, 1=aligned, *p<0.05). The inserted pictures in Figure 5A show confocal images of cell on aligned (top) and unaligned (bottom) nanofibers (6Ox, bar=50 μm). [0026] Figure 6 shows cell proliferation and matrix elaboration on aligned and unaligned nanofiber scaffolds. Figure 6A shows that cells grew on both types of nanofiber scaffolds independent of fiber alignment. Figure 6B shows immunohistochemical staining for types I and III collagen (Day 7, 2Ox, bar=100 μm). Figure 6C demonstrates that matrix production on the nanofiber scaffolds was also guided by nanofiber organization, with an aligned collagen I matrix found on the aligned nanofiber scaffolds (Day 7, mean angle analysis).

[0027] Figure 7 shows the effects of in vitro culture on nanofiber scaffold mechanical properties. Mechanical properties decreased due to polymer degradation, and aligned nanofiber scaffolds were significantly stronger than unaligned nanofiber scaffolds, Figure 7A shows ultimate tensile strength; ^*:p<0.05; Figure 7B shows elastic modulus, *:p<0.05 vs. unaligned, and #:p<0.05 vs. unaligned cellular scaffolds; Figure 7C shows yield strength, ^*:p<0.05 for aligned vs. as-fabricated aligned nanofiber scaffolds, and #:p<0.05 day 1 vs. day 14.

[0028] Figure 8 shows an arthroscopic image of a torn supraspinatus tendon in the right shoulder, posterior view.

[0029] Figure 9 shows a supraspinatus tendon-to-bone insertion site (100),

CT: Tendon, UF: Uncalcified Fibrocartilage, CF: Calcified Fibrocartilage, B: Bone.

[0030] Figures 10A-E shows one embodiment of a clinical application of a bi- phasic nanofiber scaffold according to the present invention.

[0031] Figure 11A-C shows that calcium and phosphorous peaks acquired through EDAX analysis confirmed incorporation of hydroxyapatite (HA) into the

PLGA nanofibers. Fiber roughness was found to increase with increasing hydroxyapatite content (1% (A), 5% (B), and 15% (C) HA) as shown by scanning electron microscopy.

[0032] Figure 12 shows a bi-phasic nanofiber scaffold according to the present invention at different magnifications (A and B) under scanning electron microscopy. The nanofiber scaffold was fabricated with Phase A consisting of PLGA with 0% HA and Phase B containing 5% HA. The phases (A and B) of Figure 12 are continuous. When tested in tension (C), the elastic modulus of the bi-phasic constructs (n=3) was found to be significantly greater than either Phase A or Phase

B alone (n=5).

[0033] Figure 13 shows that increasing HA content in PLGA nanofiber meshes had no significant effect on alkaline phosphatase gene expression at days 3 and 21 as indicated by semiquantitative analysis of PCR band intensities (n=2).

[0034] Figure 14 shows that (A) matrix and (B) cellular morphology corresponded to fiber alignment on PLGA-HA (5%) nanofiber meshes at day 21 as shown by SEM and confocal fluorescence microscopy, respectively. (C) Extracellular matrix consisted of both type I and type III collagen as shown by immunohistochemistry.

[0035] Figure 15 shows co-culture of fibroblasts (FB) and osteoblasts (OB) on a continuous bi-phasic nanofiber scaffold according to the present invention. Distinct cellular regions were obtained on the nanofiber scaffold with fibroblasts (green, DiO) and osteoblasts (red, DiI) attached only on Phase A and Phase B, respectively, as indicated by fluorescence confocal microscopy (2Ox).

[0036] Figure 16A and B show top-side views of two embodiments of the present invention.

[0037] Figure 17 shows top-side views of multi-phasic embodiments of the invention that are layered - biphasic (A) and tri-phasic (B). In these embodiments, the phases are layered along a vertical axis (y).

[0038] Figure 18 shows top-side views of multi-phasic embodiments of the invention that are aligned along a horizontal axis (x) - biphasic (A) and tri-phasic (B).

Figure 18 C shows an expanded view of an embodiment where one of the phases is comprised of multiple layers.

[0039] Figure 19 is a schematic depicting the electrospinning process according to the present invention.

[0040] Figure 20 is a series of micrographs showing how nanofiber orientation and alignment change with the drum surface velocity during electrospinning.

DETAILED DESCRIPTION

[0041] The ideal nanofiber scaffold for rotator cuff tendon repair must be able to meet the functional demand of the native tendon by matching its mechanical properties as well as promoting host cell-mediated healing by mimicking the ultrastructure organization of the native tendon. In addition to being biomimetic and able to promote cell attachment and growth, the nanofiber scaffold must be biodegradable so it can be gradually replaced by new tissue without compromising graft mechanical properties. To this end, the present invention is directed to a nanofiber scaffold for, inter alia, rotator cuff repair, augmentation, or replacement, including fixation of tendon-to-bone. These nanofiber scaffolds are highly advantageous for orthopedic tissue engineering due to their superior biomimetic potential and physiological relevance because they exhibit high aspect ratio, surface area, porosity and closely mimic the native extracellular matrix (29-33). [0042] Nanofiber scaffolds have been investigated for bone (34-37), meniscus

(38), intervertebral disk (39), cartilage (40, 41), ligament (42, 43) as well as tendon tissue engineering (44), and they are likely to be a promising solution for the functional augmentation of rotator cuff repairs. Moreover, nanofiber organization and alignment can be readily modulated during fabrication (33, 45-47). See also, e.g., U.S. Pat. No. 6,689,166. Thus, nanofiber scaffold systems exhibit significant versatility in their ability to tailor structural and material properties to meet the functional demands of, e.g., the rotator cuff.

[0043] Accordingly, one embodiment of the present invention is an implantable device for soft-tissue or soft tissue-to-bone fixation, repair, augmentation, or replacement. The device comprises a biomimetic and biodegradable nanofiber scaffold, which scaffold comprises one or more continuous phases. The present invention is well suited for soft-tissue repairs in mammals, particularly humans. As used herein, "soft tissue" refers to tendon and ligament, as well as the bone to which such structures may be attached. Preferably, "soft tissue" refers to tendon- or ligament-bone insertion sites requiring surgical repair, such as for example tendon-to-bone fixation.

[0044] An "implantable device" according to the present invention is a surgically appropriate, e.g., biocompatible, apparatus having the design and physical properties set forth in more detail below. Preferably, the implantable device is designed and dimensioned to function in the surgical repair, augmentation, or replacement of damaged soft tissue, such as, e.g., a rotator cuff, including fixation of tendon-to-bone.

[0045] More particularly, the implantable device comprises a "nanofiber scaffold". As used herein, the "nanofiber scaffold" is constructed of "nanofibers." In the present invention, a "nanofiber" is a biodegradable polymer that is electrospun into a fiber as described in more detail herein below. The nanofibers of the scaffold are oriented in such a way (i.e., aligned or unaligned) so as to mimic the natural architecture of the soft tissue to be repaired. Moreover, the nanofibers and the subsequently formed nanofiber scaffold are controlled with respect to their physical properties, such as for example, fiber diameter, pore diameter, and porosity so that the mechanical properties of the nanofibers and nanofiber scaffold are similar to the native tissue to be repaired, augmented or replaced.

[0046] Thus, in the case of a rotator cuff repair, the nanofiber scaffold is able to regenerate the native insertion of tendon-to-bone through interface tissue engineering and promote tendon-to-bone integration and biological fixation. In the present invention, such a nanofiber scaffold may be multiphasic, such as e.g., bi- phasic. One aspect of such multiphasic nanofiber scaffolds is that each phase is "continuous" with the phase adjacent to it. Thus, in the present nanofiber scaffolds, the interface between one phase and the next is designed, e.g., by electrospinning and other means described in more detail below, to mimic the natural anatomical transition between, e.g., tendon and bone at a tendon-to-bone interface. By designing the nanofiber scaffolds of the present invention so that the phases are continuous, improved fixation and function is achieved by minimizing stress concentrations and mediating load transfer between tendon and bone compared to prior systems.

[0047] The implantable devices of the present invention may be used in open surgical procedures, so-called "mini-open" procedures, and arthroscopic procedures as may be required and determined by a surgeon. Preferably, the implantable devices of the present invention are used in arthroscopic procedures. [0048] The nanofiber scaffold of the implantable device is biomimetic and biodegradable. In the present invention, "biodegradable" means that the nanofiber scaffold, once implanted into mammalian body, will begin to degrade. The rate of biodegradation may be engineered into the nanofiber scaffold based on the polymers used, the ratio of copolymers used, and other parameters well known to those of skill in the art. Moreover, in certain embodiments of the present invention, when the nanofiber scaffold is a multi-phasic nanofiber scaffold, the rate of biodegradation of each phase may be separately engineered according to the needs of the particular surgery to be performed.

[0049] In the present invention, the nanofiber scaffold may be engineered to remain in place for as long as the treating physician deems necessary. Typically, the nanofiber scaffold will be engineered to have biodegraded between 6-18 months after implantation, such as for example 12 months. As used herein, all numerical ranges provided are intended to expressly include at least the endpoints and all numbers that fall between the endpoints of ranges. [0050] As used herein, "biomimetic" when used in connection with the

"nanofiber scaffold" means that the nanofiber scaffold is biologically inert (i.e., will not cause an immune response/rejection) and is designed to resemble a structure (e.g., soft tissue anatomy) that occurs naturally in a mammalian, e.g., human, body and that promotes healing when implanted into the body.

[0051] Thus, in this embodiment of the present invention, the nanofiber scaffold comprises a plurality of nanofibers that are made from a biodegradable polymer. In the present invention, the biodegradable polymer may be selected from biodegradable polymer is selected from the group consisting of aliphatic polyesters, poly(amino acids), modified proteins, polydepsipeptides, copoly(ether-esters), polyurethanes, polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, poly(ε-caprolactone)s, polyanhydrides, polyarylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, modified polysaccharides, polycarbonates, polytyrosinecarbonates, polyorthocarbonates, poly(trimethylene carbonate), poly(phosphoester)s, polyglycolide, polylactides, polyhydroxybutyrates, polyhydroxyvalerates, polydioxanones, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(maleic anhydride), polyvinylalcohol, polyesteramides, polycyanoacrylates, polyfumarates, poly(ethylene glycol), polyoxaesters containing amine groups, poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic acid)s, poly(dioxanone)s, poly(alkylene alkylate)s, biopolymers, collagen, silk, chitosan, alginate, and a blend of two or more of the preceding polymers.

[0052] Preferably, the polymer comprises at least one of poly(lactide-co- glycolide), poly(lactide), and poly(glycolide). More preferably, the polymer is a copolymer, such as for example a poly(D,L-lactide-co-glycolide (PLGA). [0053] As noted above, the ratio of polymers may be varied in the biocompatible polymer of the nanofibers in order to achieve certain desired physical properties, including e.g., strength, ease of fabrication, degradability, and biocompatibility. Preferably, the ratio of polymers in the biocompatible polymer, e.g., the PLGA copolymer, is between about 25:75 to about 95:5. More preferably, the ratio of polymers in the biocompatible polymer, e.g., the PLGA copolymer, is between about 85:15. Generally, a ratio of about 25:75 in the PLGA copolymer will equate to a degradation time of about six months, a ratio of about 50:50 in the PLGA copolymer will equate to a degradation time of about twelve months, and a ratio of about 85:15 in the PLGA copolymer will equate to a degradation time of about eighteen months.

[0054] As noted above, the anisotropy of the nanofibers in the nanofiber scaffold may be controlled. In the present invention, the anisotropy of the nanofibers in the nanofiber scaffold may be varied between substantially aligned to substantially unaligned, depending on the anatomical structure of the soft tissue or soft tissue-to- bone interface to be repaired, augmented, fixated, or replaced. For example, the nanofiber alignment and orientation may be designed with reference to the alignment and orientation of various extracellular matrix components, such as collagen, which as noted above, has an average fiber angle of 83-98° (non-mineralized) and 86-103° (mineralized and fibrocartilage) depending on the region measured (71). In one aspect of the invention, it is preferred that the nanofibers are aligned. In another aspect, the nanofibers are unaligned. In a further aspect, the nanofiber scaffold may contain regions where the orientation of the nanofibers varies from substantially aligned to substantially unaligned. Thus, in one embodiment, the nanofiber scaffold comprises both aligned and unaligned nanofibers. [0055] As noted above, the nanofiber alignment and orientation mimics the anatomy of the soft-tissue or soft tissue-to-bone interface to be repaired, augmented, fixated, or replaced. Preferably, the soft tissue to be repaired, augmented, or replaced is a ligament or tendon. More preferably, the soft tissue is a rotator cuff. For example, the nanofiber alignment and orientation mimics the anatomy of a tendon-to-bone interface, such as, e.g., a rotator cuff tendon-to-bone interface. Thus, in one aspect, the nanofiber scaffold is designed to mimic soft tissue and comprises a preformed interface region Other soft tissue and soft tissue-to-bone interfaces in a mammal, particularly a human, are well known to those of skill in the art and are contemplated herein.

[0056] In the present invention, the nanofiber scaffold may have one or more phases, depending on the anatomical architecture of the soft tissue or soft tissue-to- bone interface to be repaired, fixated, augmented, or replaced. An exemplary number of phases is from about 1 to about 10, such as for example, from about 2 to about 4. In one aspect of the invention, phases may be adjacent to each other in a single sheet (Figure 18 A (biphasic, with phases 70 and 80) and B (triphasic, with phases 90, 100, and 110). In this embodiment, each phase is aligned along a horizontal axis (x). In another aspect, the phases may be layered, one over another (Figure 17 A (two layers, 20, 30) and B (three layers (40, 50, and 60)). In this embodiment, the phases are aligned/layered along a vertical axis (y). In another embodiment, at least two phases are layered along a vertical axis and at least two phases are aligned along a horizontal axis. In another aspect, at least one of the phases comprises more than one layer, e.g., from about 2 to about 20 layers (see, e.g., Figure 18 C insert showing a phase (80) comprised of an exemplary three layers (120, 130, and 140)), and each layer may be composed of the same or different nanofiber polymer and/or biocompatible ceramic, nanofiber alignment and orientation, and coating. For example, in one biphasic embodiment, a first phase may contain two layers: a first layer having nanofibers aligned in a parallel arrangement and a second layer having the nanofibers arranged in an unaligned manner. Similarly, in another biphasic embodiment, a first phase may contain two layers: a first layer having nanofibers aligned in a perpendicular arrangement and a second layer having the nanofibers arranged in an unaligned manner. Thus, depending on the physical properties desired in the overall scaffold, one or more layers of nanofibers may be arranged, wherein each layer has the same or different alignment (e.g., parallel, perpendicular, unaligned (or any variation therebetween)). Thus, different properties, particularly viscoelastic responses that mimic the natural architecture of a native soft tissue or soft tissue-to-bone interface, may be engineered into the scaffold by, e.g., varying the number and alignment of each layer within a particular phase.

[0057] Preferably, the nanofiber scaffold is multi-phasic, such as for example biphasic. As noted above, in such multiphasic embodiments, each phase of the scaffold is continuous from phase-to-phase.

[0058] Referring now to Figure 17, in one embodiment of the biphasic design, the implantable device (15) includes a first phase (20) comprising nanofibers made from a biodegradable polymer. The implantable device includes a second phase (30), which is coupled to the first phase. In the present invention, the phases are coupled to each other using standard techniques, such as those disclosed in more detail in the examples. The second phase comprises nanofibers made from a biodegradable polymer and a biocompatible ceramic. In this embodiment, the first phase is continuous with the second phase [0059] In this embodiment, the biocompatible ceramic may be incorporated into the biodegradable polymer by any conventional means. For example, the biocompatible ceramic may be incorporated into the biodegradable polymer to form a composite nanofiber by solution immersion (50, 54), liposome delivery, or electrospinning (84, 85) as described in more details in the Examples. Preferably, the biocompatible ceramic is incorporated into the nanofibers of the second phase by electrospinning.

[0060] The biocompatible ceramic may be selected from any ceramic material that is biologically inert (or substantially inert), is incorporatable into the nanofiber scaffold, and will enhance the nanofiber scaffold's mimicry of mineralized and non- mineralized anatomy in a soft tissue or soft tissue-to-bone interface to be repaired, fixated, augmented, or replaced. For example, the biocompatible ceramic may be selected from silicon nitride-based ceramics, Pseudowollastonite ceramics (β- CaSiOs), bredigite (Ca₇MgSi₄θi₆) ceramics, mono-phase ceramics of monticellite (CaMgSiO(4)), akermanite ceramics (Ca₂MgSi₂O₇), tricalcium silicate (Ca(3)SiO(5)), hydroxyapatite, bio-active glass, calcium phosphate, dense calcium sulfate (DCaS), porous silicated calcium phosphate (Si-CaP), tricalcium phosphate (TCP), calcium pyrophosphate (CPP), and combinations thereof. Preferably, the biocompatible ceramic is hydroxyapatite or bio-active glass, such as, e.g., 45S5® bioglass (Novabone, Alachua, Florida).

[0061] The biocompatible ceramic may be incorporated into the nanofibers of the scaffold at any convenient concentration based on the method of incorporation used and the desired physical properties of the scaffold. By way of example, in a composite nanofiber scaffold design according to the present invention, nanofibers that range from 0 to about 25% biocompatible ceramic may be electrospun. For example, nanofibers containing about 1%, about 5%, about 15%, and about 25% hydroxyapatite may be electrospun.

[0062] In another aspect of the invention, a bioactive agent may be incorporated into the nanofiber scaffold of the implantable device. In the present invention, the "bioactive agent" may be any pharmaceutically acceptable entity that does not deleteriously effect the structure or function of the nanofiber scaffold and which may provide an added benefit to the patient.

[0063] The bioactive agent may be incorporated into a portion or the entirety of one or more phases (or layers) of the nanofiber scaffolds of the present invention. One or more bio-active agents may be distributed throughout a nanofiber scaffold. In another aspect of the invention, one or more bioactive agents may be incorporated into a first phase of a multi-phasic nanofiber scaffold and one or more other bioactive agents (the same or different from those incorporated into the first phase) may be incorporated into another phase of the multi-phasic nanofiber scaffold. For example, in the case of a biphasic nanofiber scaffold for a rotator cuff repair, a growth factor that promotes growth of tendon fibroblasts may be incorporated into the first phase, which is attached to the tendon and a growth factor that promotes the growth of osteoblasts may be incorporated into the second phase, which is attached to bone.

[0064] The bioactive agents may be incorporated into the nanofiber scaffold using procedures well known in the art, including, e.g., immersion, impregnation, vacuum suction, spraying, and the like.

[0065] Non-limiting representative examples of suitable bioactive agents according to the present invention include an anti-infective, an extracellular matrix component, an antibiotic, bisphosphonate, a hormone, an analgesic, an anti- inflammatory agent, a growth factor, an angiogenic factor, a chemotherapeutic agent, an anti-rejection agent, an RGD peptide, and combinations thereof. [0066] Non-limiting representative examples of suitable growth factors according to the present invention include a member of the Transforming Growth Factor (TGF) super family, a vascular endothelial growth factor (VEGF), a platelet- derived growth factor (PDGF)₁ an insulin-derived growth factor (IGF), a modulator of a growth factor, and combinations thereof. In one aspect of this embodiment, a member of the TGF super family is selected from TGF-β, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), Activin A and Activin B, lnhibin A, lnhibin B, anti-mullerian hormone, Nodal, and combinations thereof. [0067] In another aspect of this embodiment, the TGF-β is selected from TGF- β1 , TGF-β2, TGF-β3, and combinations thereof. In a further aspect of this embodiment, the BMP is selected from the group consisting of BMP1-20 and combinations thereof. In yet another aspect of this embodiment, the GDFs are selected from GDF1-15 and combinations thereof. In a further aspect of this embodiment, the IGF is selected from IGF1 , IGF2, insulin growth factor binding proteins 1-6 (IGFBP1-6), and combinations thereof. In a further aspect of this embodiment, a modulator of a growth factor is a SMAD (small mothers against decapentaplegic) selected from SMAD1-9 and combinations thereof. [0068] The nanofiber scaffold may be treated with other materials to enhance or provide other additional biological benefits as desired. For example, the nanofiber scaffold may further contain a hydrogel disposed on all or a portion of the scaffold. In a multi-phasic nanofiber scaffold, such as for example, the bi-phasic nanofiber scaffold, the hydrogel may be disposed on at least a portion of one or both of the phases (or one or more layers of a phase). [0069] The hydrogel may be disposed/incorporated into the nanofiber scaffold using procedures well known in the art, including immersion, impregnation, vacuum suction, spraying, and the like.

[0070] As used herein, a "hydrogel" is a pharmaceutically compatible network of polymer chains that are water-insoluble, super absorbent, made of natural or synthetic polymers, and possess a degree of flexibility very similar to natural tissue, due to their significant water content.

[0071] Non-limiting representative examples of suitable hydrogels according to the present invention are composed of a material selected from agarose, carageenan, polyethylene oxide, polyethylene glycol, tetraethylene glycol, triethylene glycol, trimethylolpropane ethoxylate, pentaerythritol ethoxylate, hyaluronic acid, thiosulfonate polymer derivatives, polyvinylpyrrolidone-polyethylene glycol-agar, collagen, dextran, heparin, hydroxyalkyl cellulose, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, dextran sulfate, pentosan polysulfate, chitosan, alginates, pectins, agars, glucomannans, galactomannans, maltodextrin, amylose, polyalditol, alginate-based gels cross-linked with calcium, polymeric chains of methoxypoly(ethylene glycol) monomethacrylate, chitin, poly(hydroxyalkyl methacrylate), poly(electrolyte complexes), poly(vinylacetate) cross-linked with hydrolysable bonds, water-swellable N-vinyl lactams, carbomer resins, starch graft copolymers, acrylate polymers, polyacrylamides, polyacrylic acid, ester cross-linked polyglucans, and derivatives and combinations thereof.

[0072] In the present invention, hydrogels may contain mammalian cells, such as, e.g., human cells, in order to promote tissue repair. Non-limiting representative examples of suitable cells that may be incorporated into the hydrogel and subsequently the nanofiber scaffold include fibroblasts, chondrocytes, osteoblasts, osteoblast-like cells, stem cells, and combinations thereof. Preferably the cells are from a compatible human donor. More preferably, the cells are from the patient (i.e., autologous cells).

[0073] The cells may be incorporated into a portion or the entirety of one or more phases (or one or more layers of a phase) of the nanofiber scaffolds of the present invention, with or without use of a hydrogel. Moreover, one or more cell types may be distributed throughout a nanofiber scaffold. In another aspect of the invention, one or more cell types may be incorporated into a first phase of a multiphasic nanofiber scaffold and one or more other cell types (the same or different from those incorporated into the first phase) may be incorporated into another phase of the multi-phasic nanofiber scaffold. For example, in the case of a biphasic nanofiber scaffold for a rotator cuff repair, tendon fibroblasts may be incorporated into the first phase, which is attached to the tendon and osteoblasts may be incorporated into the second phase, which is attached to bone. [0074] In another aspect of the invention, fibroblasts, stem cells, chondrocytes, or combinations thereof are disposed on at least a portion of the first phase of a biphasic nanofiber scaffold. In a further aspect, chondrocytes, osteoblasts, osteoblast-like cells, stem cells, or combinations thereof are disposed on at least a portion of the second phase of a biphasic nanofiber scaffold. In yet another aspect of this embodiment, fibroblasts stem cells, and chondrocytes are disposed on at least a portion of the first phase of a nanofiber scaffold and chondrocytes, osteoblasts, osteoblast-like cells, stem cells, or combinations thereof are disposed on at least a portion of the second phase of the nanofiber scaffold. [0075] In one aspect of these embodiments, the stem cells are undifferentiated prior to disposition on the implantable device. In another aspect, the stem cells are pre-differentiated prior to disposition on the implantable device. The pre-differentiated stem cells may be selected for lineages that are specific for the type of repair to be carried out. For example, stem cells that will differentiate into osteoblasts and/or osteoclasts lineages in the case of a tendon-to-bone interface may be incorporated into the phase of the implantable device that will be fixated to bone. Whereas, stem cells that will differentiate into, e.g., fibroblasts, chondrocytes, and the like may be disposed on the phase of the implantable device that will be attached to the tendon.

[0076] As disclosed above, the nanofibers and the nanofiber scaffold of the implantable device are designed to mimic the anatomic architecture of the soft tissue or soft tissue-to-bone interface to be repaired, fixated, augmented, or replaced. Thus, the physical and mechanical properties of the nanofibers and nanofiber scaffold must approximate those of the soft tissue or soft tissue-to-bone interface to be repaired, fixated, augmented, or replaced. In the case of rotator cuff repair, implantable devices of the present invention that are biphasic and biomimetic have been designed and made. As discussed in more detail below, these nanofiber scaffolds have physical properties that are the same as or substantially the same as the in vivo architecture.

[0077] For example, a single layer of a scaffold composed of aligned nanofibers according to the present invention may have a yield strength of about 9.8±1.1 MPa, an elastic modulus of about 341 ±30 MPa, and an ultimate stress of about 12.0±1.5 MPa. In the case of a scaffold composed of non-aligned nanofibers, a single layer of such a nanofiber scaffold may have a yield strength of about 2.5±0.4 MPa, an elastic modulus of about 107±23 MPa, and an ultimate stress of about 3.7±0.2 MPa. [0078] Moreover, in one implantable device according to the present invention, the nanofiber scaffold is composed of nanofibers with a fiber diameter of between about 568 to about 615 nm, a pore diameter of about 4.2 to about 4.9 μm, a porosity of about 80.7 to about 81.8%, and a permeability of about 5.7 to about 7.9 X 10^"12 m⁴/N s. As will be understood by one skilled in the art, the nanofiber scaffolds may be designed having the physical properties of the soft tissue or soft tissue-to- bone interface to be repaired. Thus, the parameters of each physical characteristic (e.g., yield strength, elastic modulus, ultimate stress, fiber diameter, pore diameter, and permeability) will be designed according to the repair to be carried out. The specific values for these characteristics may be determined from the literature and/or are readily measured using conventional techniques.

[0079] As discussed in more detail below, each of these physical properties can be modified, as desired, to approximate the natural architecture of the soft tissue to be repaired, augmented, or replaced by making the appropriate selection of polymer and/or polymer ratio, by modifying the electrospinning process, and by the selection of biocompatible ceramic materials and/or hydrogel for incorporation into the nanofiber scaffold.

[0080] For example, the orientation and alignment of the nanofibers may be modified based on whether the nanofibers are spun onto a static surface, which produces fibers of decreased orientation and alignment or whether the nanofibers are spun onto a rotating drum. As shown in Figure 20 and described in more detail in Example 1 , increasing drum surface velocity increases the degree of fiber alignment and orientation.

[0081] In another embodiment of the present invention, there is provided an implantable biphasic biomimetic and biodegradable nanofiber device for soft-tissue or soft tissue-to-bone interface fixation, repair, augmentation, or replacement. This device comprises a first phase comprising nanofibers made from a biodegradable polymer and a second phase coupled to the first phase, which second phase comprises nanofibers made from a biodegradable polymer and a biocompatible ceramic, wherein the first and second phases are continuous. [0082] In a further embodiment of the present invention, there is provided an implantable device for fixation, repair, augmentation, or replacement of a rotator cuff or a tendon-to-bone interface thereof. This device comprises a biphasic, biomimetic, and biodegradable nanofiber scaffold that mimics a tendon-to-bone interface. This device has a first phase comprising nanofibers whose anisotropy mimics that of a tendon and non-mineralized fibrocartilage, which nanofibers are made from a biodegradable polymer and a second phase coupled to the first phase, which second phase comprises nanofibers whose anisotropy mimics that of mineralized fibrocartilage and bone, which nanofibers are made from a biodegradable polymer and a biocompatible ceramic, wherein the first and second phases are continuous. [0083] In these last two embodiments, the biodegradable polymer is selected from the group consisting of aliphatic polyesters, poly(amino acids), modified proteins, polydepsipeptides, copoly(ether-esters), polyurethanes, polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, poly(ε-caprolactone)s, polyanhydrides, polyarylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, modified polysaccharides, polycarbonates, polytyrosinecarbonates, polyorthocarbonates, poly(trimethylene carbonate), poly(phosphoester)s, polyglycolide, polylactides, polyhydroxybutyrates, polyhydroxy valerates, polydioxanones, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(maleic anhydride), polyvinylalcohol, polyesteramides, polycyanoacrylates, polyfumarates, poly(ethylene glycol), polyoxaesters containing amine groups, poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic acid)s, poly(dioxanone)s, poly(alkylene alkylate)s, biopolymers, collagen, silk, chitosan, alginate, and a blend of two or more of the preceding polymers.

[0084] In these last two embodiments, the biocompatible ceramic is selected from silicon nitride-based ceramics, Pseudowollastonite ceramics (β-CaSiθ₃), bredigite (Ca₇MgSi₄OiS) ceramics, mono-phase ceramics of monticellite (CaMgSiO(4)), akermanite ceramics (Ca₂MgSi₂O₇), tricalcium silicate (Ca(3)SiO(5)), hydroxyapatite, bio-active glass, calcium phosphate, dense calcium sulfate (DCaS), porous silicated calcium phosphate (Si-CaP), tricalcium phosphate (TCP), calcium pyrophosphate (CPP), and combinations thereof.

[0085] In another aspect of these embodiments, at least one of the phases further comprises a bioactive agent selected from an anti-infective, an extracellular matrix component, an antibiotic, bisphosphonate, a hormone, an analgesic, an antiinflammatory agent, a growth factor, an angiogenic factor, a chemotherapeutic agent, an anti-rejection agent, an RGD peptide, and combinations thereof. [0086] In these last two embodiments, non-limiting representative examples of suitable growth factors according to the present invention include a member of the Transforming Growth Factor (TGF) super family, a vascular endothelial growth factor (VEGF), a platelet-derived growth factor (PDGF), an insulin-derived growth factor (IGF), a modulator of a growth factor, and combinations thereof. In one aspect of these embodiments, a member of the TGF super family is selected from TGF-β, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), Activin A and Activin B, lnhibin A, lnhibin B, anti-mullerian hormone, Nodal, and combinations thereof.

[0087] In another aspect of these embodiments, the TGF-β is selected from

TGF-β1 , TGF-β2, TGF-β3, and combinations thereof. In a further aspect of this embodiment, the BMP is selected from the group consisting of BMP1-20 and combinations thereof. In yet another aspect of these embodiments, the GDFs are selected from GDF1-15 and combinations thereof. In a further aspect of these embodiments, the IGF is selected from IGF1 , IGF2, insulin growth factor binding proteins 1-6 (IGFBP1-6), and combinations thereof. In a further aspect of these embodiments, a modulator of a growth factor is a SMAD (small mothers against decapentaplegic) selected from SMAD1-9 and combinations thereof. [0088] In these last two embodiments, the implantable device may further comprise a hydrogel disposed on at least a portion of one or both of the phases (or one or more layers of a phase). In this aspect, the hydrogel is composed of a material selected from agarose, carageenan, polyethylene oxide, polyethylene glycol, tetraethylene glycol, triethylene glycol, trimethylolpropane ethoxylate, pentaerythritol ethoxylate, hyaluronic acid, thiosulfonate polymer derivatives, polyvinylpyrrolidone-polyethylene glycol-agar, collagen, dextran, heparin, hydroxyalkyl cellulose, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, dextran sulfate, pentosan polysulfate, chitosan, alginates, pectins, agars, glucomannans, galactomannans, maltodextrin, amylose, polyalditol, alginate- based gels cross-linked with calcium, polymeric chains of methoxypoly(ethylene glycol) monomethacrylate, chitin, poly(hydroxyalkyl methacrylate), poly(electrolyte complexes), poly(vinylacetate) cross-linked with hydrolysable bonds, water-swellable N-vinyl lactams, carbomer resins, starch graft copolymers, acrylate polymers, polyacrylamides, polyacrylic acid, ester cross-linked polyglucans, and derivatives and combinations thereof.

[0089] In these embodiments, the implantable device may further comprise fibroblasts, chondrocytes, osteoblasts, osteoblast-like cells, stem cells, or combinations thereof. In this aspect, fibroblasts, stem cells, chondrocytes, or combinations thereof are disposed on at least a portion of the first phase. In another aspect, chondrocytes, osteoblasts, osteoblast-like cells, stem cells, or combinations thereof are disposed on at least a portion of the second phase. In a further aspect, fibroblasts, stem cells, and chondrocytes are disposed on at least a portion of the first phase and chondrocytes, osteoblasts, osteoblast-like cells, stem cells, or combinations thereof are disposed on the second phase.

[0090] In one aspect of these embodiments, the stem cells are undifferentiated prior to disposition on the implantable device. In another aspect, the stem cells are pre-differentiated prior to disposition on the implantable device. The pre-differentiated stem cells may be selected for lineages that are specific for the type of repair to be carried out. For example, stem cells that will differentiate into osteoblasts and/or osteoclasts lineages in the case of a tendon-to-bone interface may be incorporated into the phase of the implantable device that will be fixated to bone. Whereas, stem cells that will differentiate into, e.g., fibroblasts, chondrocytes, and the like may be disposed on the phase of the implantable device that will be attached to the tendon.

[0091] In the present invention, the nanofiber scaffolds of the implantable device may be manufactured in manner that is convenient for surgical delivery. Preferably, the nanofiber scaffolds are manufactured in a manner that closely mimics the architectural anatomy to be repaired, fixated, augmented, or replaced. Thus, to a certain degree, the soft tissue or soft tissue-to-bone interface to be repaired will drive the dimensions of the nanofiber scaffolds.

[0092] Referring now to Figure 16 A, and by way of example, for a rotator cuff, the implantable device (1) will be about 0.2 to about 2.0 mm thick (D). The shape of the implantable device is not critical, but should be informed by surgeon preference. Typically, the implantable device (1) may be about 5.0 cm long (L) and about 5.0 cm wide (W). Alternatively, referring to Figure 16 B, the implantable device (10) may be about 10.0 cm in diameter (D). Preferably, the implantable device will be dimensioned so that it is larger than required for the repair, fixation, augmentation, or replacement, so that the surgeon may adjust the dimensions to fit the particular anatomy of the patient.

[0093] Another embodiment of the invention is a method for fixation of, repairing, augmenting, or replacing a damaged soft tissue or soft tissue-to-bone interface in a patient. This method comprises affixing a biomimetic, biodegradable continuous multi-phasic nanofiber scaffold according to the present invention to a surgically relevant site in order to repair, fixate, augment, or replace the damaged soft tissue or soft tissue-to-bone interface.

[0094] A further embodiment of the present invention is a method for fixating, repairing, augmenting, or replacing a damaged rotator cuff in a patient. This method comprises affixing a biomimetic and biodegradable continuous multiphase nanofiber scaffold according to the present invention to a surgically relevant site in order to repair, augment, or replace the damaged rotator cuff.

[0095] In sum, the present invention relates to a biodegradable polymer-based nanofiber scaffold designed for repair, fixation, augmentation, or replacement of tendon-to-bone insertion site damage, such as, for example, rotator cuff repair. Data disclosed herein include human rotator cuff fibroblast response on the degradable nanofiber scaffolds as well as the effects of nanofiber organization (aligned vs. unaligned fibers) on cell attachment and matrix deposition. The novel nanofiber scaffold of the present invention has been designed to match the structural and mechanical properties of the rotator cuff tendon. Although not wishing to be bound by a particular theory, it is believed that fibroblast attachment, morphology and matrix elaboration will be guided by the underlying organization of nanofiber scaffolds.

Characterization of the Aligned and Unaligned Nanofiber Scaffolds [0096] Both aligned and unaligned PLGA nanofiber scaffolds were successfully fabricated and characterized. Structural properties of aligned and unaligned nanofiber scaffolds are summarized in Table 1.

Table 1. Structural Properties of Unaligned and Aligned Nanofiber Scaffolds

Scaffold Fiber Pore Porosity Permeability

Thickness Diameter Diameter (%) (m4/N s)

(mm) (nm) (μm)

Aligned 0.22 ± 0.02 615 ± 152 4 228 ± 1.056 80.745 ± 2.966 (7.87 ± 2.47JxIO^'1'

(n=5)

Unaligned 0.19 ± 0.02 568 ± 147 4.914 ± 0.777 81.760 ± 3.929 (5.72 ± 0.63)x10^"12 (n=5)

[0097] The average nanofiber diameter for the aligned nanofiber scaffolds was

615 ± 152 nm, while fiber diameter of the unaligned group measured 568 ± 147 nm. No significant difference was found between the two groups. Similarly, nanofiber scaffold porosity, pore diameter and permeability were also found to be comparable between the aligned and unaligned nanofiber scaffolds (Table 1). [0098] In contrast, the mechanical properties of the as-fabricated aligned and unaligned nanofiber scaffolds differed significantly (p<0.05). As shown in Figure 2, the aligned nanofiber scaffold exhibited a markedly different stress-strain profile when compared to the unaligned nanofiber scaffold, with a significantly higher tensile modulus, yield stress as well as ultimate tensile stress. Table 2 summarizes the mechanical properties of aligned and unaligned nanofiber scaffolds, with significantly higher mechanical properties found in the aligned nanofiber scaffolds (*: p<0.05).

Table 2. Mechanical Properties of Aligned and Unaligned Scaffolds (*: p<0.05)

Elastic Modulus (MPa) Yield Strength (MPa) Ultimate Stress (MPa)

Aligned 341 ± 30* 9. 8 ± 1. 1* 12.0 ± 1 .5*

(n=5)

Unaligned 107 ± 23 2 .5 ± 0 .4 3.7 ± 0 .2

(n=5)

[0099] Specifically, the aligned nanofiber scaffold exhibited a three-fold higher elastic modulus, and nearly four-fold higher yield strength and ultimate tensile strength when compared to the unaligned nanofiber scaffolds. It is emphasized here that the nanofiber scaffold structural properties (porosity, permeability) are similar to values reported for soft tissue (52, 59-65) and the mechanical properties of the aligned nanofiber scaffolds are within range of those reported for human supraspinatus tendon (66).

Effects of Nanofiber Organization on Fibroblast Attachment and Alignment Cell Attachment Morphology

[0100] The attachment morphology and growth of human rotator cuff fibroblasts on aligned and unaligned nanofiber scaffolds were visualized via both electron and confocal microscopy. As shown in Figure 3, the fibroblasts attached to the nanofiber scaffold but assumed distinct morphologies on the two types of nanofiber scaffolds. Specifically, the cells grown on the aligned fibers adopted a phenotypic elongated morphology and oriented in the direction of the long axis of the fiber. In contrast, fibroblasts seeded on the unaligned mesh exhibited a polygonal

morphology without preferential orientation. Moreover, while the human fibroblasts proliferated on both types of substrates over time, these morphological differences were maintained over the two-week culturing period (Figure 3).

Gene Expression

[0101] Fibroblast gene expression was compared over time on aligned and unaligned nanofiber scaffolds (Figure 4). All groups expressed the housekeeping gene GAPDH at all time points. It was observed that integrin expression differed between the aligned and unaligned nanofiber scaffolds while the cells expressed both types I and III collagen on the nanofiber scaffolds. The expression of α2 integrin was observed on the aligned nanofiber scaffolds at days 1 , 3 and 14, while no α2 expression was seen on the unaligned nanofiber scaffolds. All genes evaluated were expressed at all time points on the monolayer control (data not shown). Quantitative Analysis of Cell Attachment and Alignment

[0102] The attachment response of human rotator cuff fibroblasts to the inherent organization of the nanofiber scaffolds (aligned vs. unaligned) was further analyzed using quantitative circular statistical analysis (58, 67). Specifically, cell alignment and orientation over time were compared to those of the underlying nanofiber substrate, focusing on mean vector angle (Figure 5A) and angular deviation as well as mean vector length (Figure 5B). Table 3 summarizes the

alignment analysis results.

Table 3. Summary of Cell and Fiber Orientation

Day 1 (n=3) Mean Angle ± Angular Deviation (°)

Aligned Cells 4.24 ± 19.73 Aligned Fibers 4.32 ± 17.00 Unaligned Cells -63.46 ± 37.70

Unaligned Fibers -55.69 ± 35.97

Day 14 (n=3) Mean Angle ± Angular Deviation (°)

Aligned Cells 6.19 ± 18.74 Aligned Fibers 4.62 ± 17.78

Unaligned Cells -19.45 ± 35.93 Unaligned Fibers -20.84 ± 38.14

[0103] At day 1 , fibroblast attachment on the aligned nanofiber scaffolds was significantly more aligned than on unaligned nanofiber scaffolds. As shown in Figure 5A, analysis of cell and fiber orientation and alignment at day 1 revealed that cells grown on the aligned nanofiber scaffolds are oriented horizontally, exhibiting a similar mean angle distribution and narrow angular deviation as that of the aligned nanofibers on which the cells are seeded. Interestingly, fibroblasts on unaligned nanofiber scaffolds also conformed to matrix organization at day 1 , demonstrating a similar random orientation with a wide angular deviation approximating those of the unaligned nanofiber scaffold matrix (Figure 5A, Table 3).

[0104] At day 14, fibroblast growth and morphology continued to be dictated by the underlying nanofiber organization, with significantly higher alignment measured for fibroblasts cultured on the aligned nanofiber scaffolds compared to those found on the unaligned nanofiber scaffolds (p<0.05). However, within each nanofiber scaffold type (aligned or unaligned), circular statistical analysis of fibroblast growth revealed no significant change in alignment parameters when compared to day 1 results. Specifically, the mean angle (Figure 5A) and angular deviation (Table 3), as well as the mean vector length (Figure 5B) values of both cell and nanofiber scaffold samples at day 14 did not differ significantly from those found at days 1. Effects of Nanofiber Organization on Fibroblast Growth and Matrix Deposition [0105] The human tendon fibroblasts proliferated on the nanofiber scaffolds over the two week period, with no significant difference in cell proliferation found between the aligned and unaligned groups (Figure 6A). Matrix deposition by human fibroblasts on nanofiber scaffolds was also evaluated over time, and the cells produced a collagen-rich matrix containing both type I and type III collagen (Figure 6B). Additionally, circular statistical analysis of the immunohistochemistry images revealed that collagen matrix deposition was also guided by nanofiber organization, with an aligned type I collagen matrix found only when the fibroblasts were cultured on the aligned nanofiber scaffold (Figure 6C).

Effects of In Vitro Culture on Nanofiber Scaffold Mechanical Properties [0106] The mechanical properties of fibroblast-seeded PLGA nanofiber scaffolds were also determined over time and compared as a function of in vitro culture (cellular vs. acellular nanofiber scaffolds) as well as nanofiber organization (aligned vs. unaligned). As expected for PLGA, in vitro culture decreased nanofiber scaffold mechanical properties over time, with a significantly lower ultimate tensile stress and yield strength found over time for both aligned and unaligned nanofiber scaffolds when compared to the as-fabricated nanofiber scaffolds (Figure 7A,C; p<0.05). In contrast, in vitro culture has no significant effect on nanofiber scaffold elastic modulus (Figure 7B). When compared to the acellular controls, the ultimate tensile stress, elastic modulus and the yield strength of the fibroblast-seeded nanofiber scaffolds did not vary significantly over time (Figure 7). [0107] At all examined time points and culture conditions, the elastic modulus, ultimate tensile stress, and yield strength of the aligned nanofiber scaffolds were significantly greater than those of the unaligned nanofiber scaffolds (p<0.05), and this trend was consistently observed in both cellular and acellular groups. Interestingly, a significant decrease in ultimate tensile stress was detected at day 1 for the unaligned nanofiber scaffold, while such a decrease was not found until a week later for the aligned group (Figure 7A, day 7). For yield strength, a significant decrease was measured for aligned nanofiber scaffolds at day 1 compared to the as- fabricated group (Figure 7C, p<0.05), and the yield strength of the day 14 aligned nanofiber scaffolds was significantly lower compared to day 1 (p<0.05). In contrast, no significant decrease was observed for the aligned nanofiber scaffolds at any time point.

[0108] By controlling nanofiber organization (aligned vs. unaligned), nanofiber scaffolds with controlled matrix anisotropy mimicking those of any muscle- to-bone insertion site, e.g., a rotator cuff, may be engineered. In the present invention, the effects of nanofiber organization on the resultant nanofiber scaffold structural and mechanical properties, as well as the response of primary cells derived from the human rotator cuff tendons were systematically investigated. Additionally, the effects of fibroblast culture and duration on the ability of the nanofiber scaffold to maintain stable mechanical properties were also evaluated. It was found that nanofiber organization controls nanofiber scaffold mechanical properties and is the primary factor guiding the attachment morphology, alignment, gene expression as well as matrix deposition by human rotator cuff fibroblasts. Moreover, while in vitro culture resulted in an expected decrease in nanofiber scaffold mechanical properties with polymer degradation, these changes were modulated by nanofiber organization. Based on these observations, it is clear that nanofiber organization exerts significant control over cell response as well as nanofiber scaffold properties, and it is a critical parameter in nanoftber scaffold design for functional muscle-to-bone repair, e.g., rotator cuff repair. [0109] In addition to possessing a fiber diameter approximating that of collagen fibers present in the tendon extracellular matrix, the nanofiber scaffolds of the present invention exhibit structural properties that are optimal for soft tissue repair, including its over 80% porosity and high permeability which can facilitate nutrient transport as well as promote cell viability and tissue growth in vitro and in vivo. Additionally, the permeability of the nanofiber scaffold is within range of those reported for musculoskeletal tissues (52, 59-65). For the unaligned and aligned nanofiber scaffolds of the present invention, tensile (elastic) modulus varied from 107 MPa to 341 MPa and the ultimate tensile stress from 3.7 MPa to 12.0 MPa, respectively. While the elastic modulus of the nanofiber scaffold was unaffected by in vitro culture, both yield strength and ultimate tensile stress decreased significantly over time. However, the magnitude of nanofiber scaffold mechanical properties remained within the range of those reported for human rotator cuff tendons (66). Moreover, the mechanical properties of the nanofiber scaffolds of the present invention can be further tailored by varying, e.g., either the average molecular weight or co-polymer ratio of the degradable polymers (54).

[0110] The observed differences in mechanical properties between the aligned and unaligned nanofiber scaffolds are similar to those seen in other types of nanofiber scaffolds (32, 38, 42, 68, 69). These reports collectively highlight another distinct advantage of the nanofiber scaffold in that by varying nanofiber organization and alignment, matrix anisotropy can be pre-engineered into nanofiber scaffold design. This is especially desirable for tendon repair or regeneration, as nanofiber scaffolds with controlled matrix anisotropy can be fabricated to recapitulate the inherent structure-function relationship of, e.g., rotator cuff tendons. [0111] When ltoi et al. evaluated the tensile mechanical properties of the human supraspinatus tendon at the anterior, middle and posterior regions of the tendon; it was found that both the elastic modulus and ultimate tensile stress varied as a function of tendon region. Specifically, the elastic modulus values ranged from 50 MPa to 170 MPa when progressing from the posterior to the superficial region, while the ultimate tensile stress spanned from 4.1 MPa to 16.5 MPa from the posterior to the superficial tendon region. Moreover, Thomopoulos et al. (70) reported that collagen organization and alignment play an important role in reducing stress concentration at the supraspinatus tendon-to-bone insertion (71). By controlling nanofiber organization (alignment or layering) and/or other nanofiber scaffold parameters such as polymer composition or molecular weight, nanofiber scaffolds with biomimetic collagen alignment and region-dependent mechanical properties can be readily engineered and utilized for, e.g., rotator cuff repair, augmentation and regeneration.

[0112] Nanofiber organization also exerted profound effects on cellular response. The nanofiber scaffolds of the present invention were pre-designed with similar structural properties (nanofiber diameter, porosity, pore size, permeability). Thus, any observed differences in cell response can be primarily attributed to differences in nanofiber organization (aligned vs. unaligned). [0113] Human rotator cuff fibroblast attachment and matrix production were guided by the organization of nanofibers of the aligned or unaligned nanofiber scaffolds. Both qualitative and quantitative analyses revealed that the cells exhibited phenotypic morphology and attached preferentially along the fiber axis of the aligned nanofiber scaffolds, while random cell attachment was found on the unaligned nanofiber scaffold. These differences were maintained over time and more importantly, subsequent cell-mediated matrix production also conformed to scaffold nanofiber organization. This contact guidance phenomena, in which surface topography of the biomaterial substrate regulates the spatial distribution of focal contacts and the direction of cell spreading (72-77), is similar to those reported for connective tissue cells cultured on synthetic or natural polymer-based nanofiber scaffolds (32, 38, 40, 42, 43, 53, 69, 78). As discussed above, these observed differences in cell response on aligned or unaligned nanofiber scaffolds and the resultant matrix properties can be readily exploited for, e.g., functional repair of rotator cuff injuries as well as the formation of complex tissues. [0114] Interestingly, the data presented herein suggest that the human rotator cuff fibroblasts may detect differences in nanofiber alignment during initial attachment as well as post-adhesion matrix synthesis. It was identified here that while the expression of integrins such as α5 and αV were consistently observed on both aligned and unaligned nanofiber scaffolds, α2 expression was only detectable on the aligned nanofiber scaffolds (Figure 4). Li et al. (40) compared fetal bovine chondrocytes response in monolayer culture to those seeded on an unaligned poly(ε-caprolactone) nanofiber scaffold, and found that α2 expression was suppressed when compared to monolayer controls. It has been reported that α2 is a key integrin that mediates cell attachment to collagenous matrix (79-83). Thus, expression of α2 integrin by rotator cuff fibroblasts on the aligned nanofiber scaffold suggests that matrix fiber alignment may also regulate integrin expression. Moreover, compared to the unaligned nanofibers, the aligned nanofiber scaffold may better mimic the native extracellular matrix produced by the rotator cuff fibroblasts. [0115] Fibroblasts proliferated on both aligned and unaligned nanofiber scaffolds of the present invention over time, with no significant difference observed between groups at all time points. These results are in agreement with previous studies which also reported minimal effect on cell proliferation due to nanofiber organization (38, 42). Additionally, no apparent differences in types I and III collagen deposition were observed between the aligned and unaligned groups in immunohistochemical staining. While collagen production was not quantified in this study, Baker and Mauck (38) reported that fiber architecture has little effect on normalized collagen content for both bovine mesenchymal stem cells and meniscal fibrochondrocytes cultured on aligned and unaligned poly(ε-caprolactone) nanofiber scaffolds. Interestingly, Lee et al. (42) reported significantly higher total collagen synthesis by human ligament fibroblasts grown on aligned polyurethane nanofiber scaffolds subjected to mechanical loading. These results collectively indicate that mechanical stimulation and fiber organization may be coupled to promote overall collagen production over time, and in turn improve cuff healing and long term clinical response.

[0116] The intended application of the novel nanofiber scaffold system presented here is to improve, e.g., the repair and/or augmentation of tears in muscle- to-bone insertion sites, such as, e.g., rotator cuff tendons, specifically by providing a biomimetic substrate with physiologically relevant mechanical properties that will enable functional and stable repair or augmentation of the damaged area, e.g., rotator cuff. Therefore, it is important to characterize whether nanofiber scaffold mechanical properties will be maintained during in vitro culture and if cell-mediated matrix production will compensate for changes in mechanical properties due to nanofiber degradation.

[0117] As expected, nanofiber scaffold mechanical properties decreased due to hydrolytic degradation of the PLGA nanofibers (54). Interestingly, the aligned PLGA nanofiber scaffolds maintained the as-fabricated tensile mechanical properties longer than the unaligned group. The ultimate tensile stress of the unaligned nanofiber scaffold decreased significantly by day 1 , while no such change was detected for the aligned nanofiber scaffolds until day 7. These observations also suggest that degradation kinetics of the nanofiber scaffolds and associated changes in mechanical properties are dependent on fiber organization. No significant increase in nanofiber scaffold mechanical properties due to fibroblast culture was observed, which is likely due to the relatively short culturing time evaluated, as reported significant increases in mechanical properties in cell-seeded nanofiber scaffolds are not found until day 70 of in vitro culture (38). Effects of Co-Culture on Osteoblast and Ligament Fibroblast Phenotypes [0118] We propose that heterotypic cellular interactions are important in the maintenance and repair of the tendon-to-bone interface. When damage to the interface region during rotator cuff injury or subsequent repair results in non- physiologic exposure of normally segregated tissue types (e.g., bone and tendon), heterotypic cellular interactions (osteoblast-fibroblast) may initiate repair and direct the regeneration of a neo-interface between these two tissues. [0119] It is well documented that in post anterior cruciate ligament reconstruction using soft tissue-based grafts, tendon-bone healing within the bone tunnel results in the formation of a fibrocartilage-like interface (95). Using a rabbit model, Koike et al. (92) evaluated tendon-bone healing and found that after resection of the enthesis, the reattachment of the supraspinatus tendon to the greater tuberosity of the humerus led to the regeneration of a new fibrocartilage-like interface. Interestingly, chondrocytes were not observed until two weeks post reattachment and their number increased to the level of the positive control by week six. In vitro co-culture of ligament fibroblast and osteoblast have shown that their interaction regulates cell phenotype (96) and results in the expression of fibrocartilage-like markers such as aggrecan, cartilage oligomeric matrix protein and collagen II. These observations suggest that the fibrocartilage interface can be formed when the tendon is juxtaposed with bone, and fibroblast-osteoblast interactions may be critical in initiating this interface regeneration process. Biphasic Nanofiber Scaffold for Tendon-to-Bone Interface Tissue Engineering [0120] Nanofiber scaffolds represent promising matrices for interface tissue engineering due to their superior biomimetic potential and physiological relevance because they exhibit high aspect ratio, surface area, porosity and closely mimic the extracellular matrix (29, 30, 31 , 32, 33). These nanofiber scaffolds have been investigated for bone (35, 36, 37, 98), meniscus (38), intervertebral disc (39), cartilage (40, 41), ligament (42, 43) as well as tendon tissue engineering (44). [0121] Inspired by the structure and organization of the tendon-bone insertion site, and focusing on mimicking collagen alignment and exercising spatial control in mineral content, we have developed a nanofiber-based biphasic nanofiber scaffold. In this biphasic design, Phase A consists of nanofibers of biodegradable polymer, such as, e.g., polylactide-co-glycolide (PLGA)₁ while Phase B is of composite nanofibers of a biocompatible ceramic, such as, e.g., hydroxyapatite (HA) nanoparticles and a biodegradable polymer, such as, e.g., PLGA (Figs. 10 and 17). It is expected that this novel nanofiber scaffold will provide the necessary structural and mechanical properties as well as mineral distribution to guide the regeneration of the complex heterogeneous tendon-to-bone interface. Additionally, by implementing nanofibers that mimic the alignment of collagen fibers at the insertion site, the biphasic nanofiber scaffold provides the foundation for guided matrix deposition and tendon-bone healing. Moreover, the biphasic nanofiber scaffold exhibits tensile mechanical properties similar to those reported for human supraspinatus tendon (66), and supports, e.g., fibroblast, osteoblast culture in preliminary studies. The nanofiber scaffolds of the present invention may be fabricated into a multi-phasic, e.g., a biphasic patch for, e.g., rotator cuff repair (Figure 10). The nanofiber scaffold can be sutured onto the tendon during cuff repair and will integrate with bone through the PLGA-HA phase.

Fibroblast Response on PLGA-HA Nanofiber Scaffolds

[0122] A preliminary evaluation was carried out of the mineralization potential of human rotator cuff tendon fibroblasts as a function of HA content (0, 1 , 5, 15 wt%) in Phase B. Briefly, fibroblasts derived from explant cultures of human tissue (male, aged 49-79 yrs) were seeded on a PLGA-HA nanofiber scaffold according to the present invention (3.14x10⁴ cells/cm²). It was found that fibroblasts remained viable (Figure 14) and proliferated on all substrates. The cells were elongated and aligned along the long axis of the fibers. Gene expression for alkaline phosphatase (ALP) was similar among all groups at both days 3 and 21, and only basal ALP activity levels were measured in these cultures (Figure 13). Collagen I & III deposition was maintained on the PLGA-HA nanofiber scaffolds, with no observable differences found between groups (Figure 14). These results suggest that increased HA content had no adverse effect on fibroblast phenotype and does not appear to induce ectopic mineralization. Co-Culture on the Biphasic Nanofiber Scaffold (Phases A & B) [0123] To demonstrate the feasibility of co-culture on the biphasic nanofiber scaffold, bovine osteoblasts and fibroblasts derived from explant culture were labeled with Vybrant dyes (green:D\O = fibroblasts, red:D\\ = osteoblasts). The fibroblasts were then seeded on Phase A (PLGA) and osteoblasts on Phase B (PLGA-HA, 5%), and allowed to attach for 15 minutes before adding media. Cell distribution on each nanofiber scaffold phase, as well as the cross-section, were imaged at day 1 using fluorescence confocal microscopy. As seen in Figure 15, with the biphasic design, fibroblasts remained in Phase A while osteoblasts were localized to Phase B. These results demonstrate the feasibility of co-culture and suggest that the phase-specific cell distribution may lead to multi-tissue formation. [0124] Disclosed herein is the design and systematic in vitro evaluation of a novel biomimetic, biodegradable nanofiber scaffold for soft tissue repair, augmentation, or replacement, such as, e.g., for functional rotator cuff repair. The present invention discloses that nanofiber organization has a significant effect on human rotator cuff fibroblast response, with the structural anisotropy of the aligned and isotropy of the unaligned nanofiber scaffold directly guiding cell attachment and matrix deposition. Controlled cell response resulted in a more physiologically relevant matrix for, e.g., rotator cuff repair on the biomimetic nanofiber scaffold. Moreover, physiologically relevant nanofiber scaffold mechanical properties were maintained in vitro. Our results demonstrate that the novel nanofiber scaffold has significant potential for enabling tendon regeneration and offers a functional tissue engineering solution for soft tissue repair, augmentation, or replacement, such as, e.g., rotator cuff repair. DEFINITIONS

[0125] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et a/., Dictionary of Microbiology and Molecular Biology (2nd Ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them.

[0126] As used herein, "aligned fibers" shall mean groups of fibers which are oriented along the same directional axis. Examples of aligned fibers include, but are not limited to, groups of parallel fibers.

[0127] As used herein, "fibroblast" shall mean a cell of connective tissue, mesodermally derived, that secretes proteins and molecular collagen including fibrillar procollagen, fibronectin and collagenase, from which an extracellular fibrillar matrix of connective tissue may be formed.

[0128] As used herein, "polymer" shall mean a chemical compound or mixture of compounds formed by polymerization and including repeating structural units. Polymers may be constructed in multiple forms and compositions or combinations of compositions.

[0129] As used herein, "porosity" shall mean the ratio of the volume of interstices of a material to a volume of a mass of the material. [0130] The following examples are provided to further illustrate the devices and methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1

Nanofiber Scaffold Fabrication

[0131] PoIy(D, L-lactide-co-glycolide) co-polymer (85:15 PLGA, Mw » 123.6 kDa; Lakeshore Biomaterials, Birmingham, AL) nanofiber scaffolds were produced via electrospinning (45, 48, 49). Briefly, a 35% (v/v) solution of PLGA was prepared in an organic solvent mixture consisting of 55% N.N-dimethylformamide (Sigma- Aldrich, St. Louis, MO) and 10% ethyl alcohol. The polymer solution was loaded in a 5 mL syringe with a 18.5-G stainless steel blunt tip needle and electrospun at 8-1OkV using a custom designed electrospinning device. Both aligned and unaligned nanofiber scaffolds were fabricated. For unaligned nanofiber scaffolds, the collecting surface consisted of a stationary plate, while a rotating mandrel having a diameter of 2 inches and a length of 20 inches, which mandrel rotated at 20 m/s was utilized to produce aligned nanofiber scaffolds. The polymer solution was deposited using a syringe pump (Harvard Apparatus, Holliston, MA; 1 mL/hour) with the distance between the needle and the collecting target distance (air gap distance) set at 10.5 cm. See, Figs. 19 and 20. As shown in Figure 20, the drum surface velocity may be varied by changing the gear in the pump, which provides control over fiber orientation and alignment.

Example 2: Nanofiber Scaffold Characterization

[0132] The structural and material properties of the nanofiber scaffolds were characterized post fabrication. Specifically, nanofiber morphology and diameter were imaged by Scanning Electron Microscopy (SEM, 5 kV, FEI Quanta 600, FEI Co. Hillsboro, OR). The nanofiber scaffolds were sputter coated with palladium prior to SEM analysis in order to reduce charging effects. Fiber diameter was quantified by image analysis of SEM micrographs (n=3, 200Ox) using NIH ImageJ (version 1.34s, Bethesda, MD). In addition, nanofiber scaffold porosity and pore diameter (n=5) were determined by mercury porosimetry (Micromeritics Autopore III, Norcross, GA) following published protocols (50). In this method, the construct porosity was determined by measuring the volume of mercury infused into the structure during analysis. Nanofiber scaffold permeability (n=5) was directly determined using a custom designed device (51 , 52), by first measuring the pressure difference and then calculating permeability via Darcy's Law:

AΔP where k is nanofiber scaffold permeability (m⁴/N s), Q is the fluid flow rate through nanofiber scaffold (300 mL/hr), ΔP is the pressure difference (N/m), h is the thickness of nanofiber scaffold (m) and A is the nanofiber scaffold surface area (m²). [0133] The mechanical properties of the as-fabricated aligned and unaligned nanofiber scaffolds were evaluated under uniaxial tensile testing (53). Briefly, the nanofiber scaffolds (6 cm x 1 cm) were secured with custom clamps and mounted on an lnstron (Model 8841 , Norwood, MA) with an average sample gauge length of 3 cm. The samples were tested to failure at a strain rate of 5 mm/min, and the aligned nanofiber scaffolds were tested along the long axis of the aligned fibers. Both the yield stress and ultimate tensile stress were determined, and nanofiber scaffold elastic modulus was calculated from the linear region of the stress-strain curve.

Example 3

In Vitro Culture of Human Tendon Fibroblasts on Nanofiber Scaffolds Cells and Cell Culture

[0134] Human rotator cuff fibroblast-like cells were obtained from explant cultures of tissue samples obtained as surgical waste following rotator cuff repair surgery (exempted from IRB approval). For this study, the cells were derived exclusively from female patients (n=3, aged 65 to 70 years). The tissue samples were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% penicillin/streptomycin and 1% amphotericin B. Only cells obtained from the second and third migration were used in order to ensure a relatively homogeneous cell population (54). All media and supplements were purchased from Mediatech (Herndon, VA). Cell Seeding on Nanofiber Scaffolds

[0135] Prior to cell seeding and to prevent nanofiber scaffold contraction

(55), the ends of aligned and unaligned nanofiber scaffolds were secured to nanofiber scaffold holders using a sterile adhesive (Fisher Scientific, Pittsburgh, PA). The nanofiber scaffolds were sterilized by UV irradiation (30 minutes/side) and to promote cell adhesion, the nanofiber scaffolds were pre-incubated in fully supplemented media at 37°C and 5% CO₂ for 16 hours. Human rotator cuff fibroblasts were seeded on the nanofiber scaffolds (1 cm x 1 cm cell seeding area) at a density of 3 x 10⁴ cells/cm². The cells were allowed to attach on the nanofiber scaffolds for 15 minutes, after which fully supplemented media was added to each culture well. Cells were cultured on the aligned and unaligned nanofiber scaffolds for two weeks, and the effects of nanofiber organization on cell morphology, attachment, proliferation and matrix production were determined at days 1 , 7 and 14. In addition, the effects of in vitro culture on nanofiber scaffold mechanical properties were also determined over the two week period. Both monolayer culture of the human tendon fibroblasts and acellular nanofiber scaffolds (aligned as well as unaligned) served as controls.

Cell Viability and Attachment Morphology

[0136] Cell attachment morphology on the nanofiber scaffolds (n=3/group) were evaluated by SEM (FEI Quanta 600, FEI Co. Hillsboro, OR) at days 1 , 7 and 14. The samples were first rinsed with 0.1 M sodium cacodylate buffer (Sigma- Aldrich) and fixed in Karnovsky's fixative (56, 57) for 24 hours at 4°C, and subsequently dehydrated with an ethanol series. The nanofiber scaffolds were coated with palladium prior to SEM analysis to reduce charging effects. Cell viability as well as attachment morphology were evaluated by Live/Dead staining (Molecular Probes, Eugene, OR) imaged using confocal microscopy. Specifically, the samples (n=3/group) were rinsed twice with PBS and stained following the manufacturers suggested protocol. The samples were then imaged with a laser scanning confocal microscope (Olympus Fluoview IX70, Center Valley, PA) at wavelengths of 488 nm and 568 nm. Gene Expression

[0137] Gene expression was measured by reverse transcriptase polymerase chain reaction (RT-PCR) at days 1 , 3 and 14. The nanofiber scaffolds were first rinsed with PBS and total RNA was isolated using the Trizol extraction method (Invitrogen, Carlsbad, CA). The isolated RNA was reverse-transcribed into complementary DNA (cDNA) using the Superscript First-Strand Synthesis System (Invitrogen), and the cDNA product was then amplified using recombinant Taq DNA polymerase (Invitrogen). Expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (GAPDH sense, δ'-GGCGATGCTGGCGCTGAGTA-S' (SEQ ID NO:1); antisense, δ'-ATCCACAGTCTTCTGGGTGG-S' (SEQ ID NO:2)), integrin α2 (sense, 5'-CAGAATTTGGAACGGGACTT-S' (SEQ ID NO:3); antisense, δ'-CAGGTAGGTCTGCTGGTTCA-S' (SEQ ID NO:4)), integrin α5 (sense, 5'- GTGGCCTTCGGTTTACAGTC-3' (SEQ ID NO:5); antisense, 5'- AATAGCACTGCCTCAGGCTT-3' (SEQ ID NO:6)), integrin αV (sense, 5'- GATGGACCAATGAACTGCAC-3' (SEQ ID NO:7); antisense, 5'- TTGGCAGACAATCTTCAAGC-S" (SEQ ID NO:8)), collagen I (sense, 5'- TGCTGGCCAACTATGCCTCT-3' (SEQ ID NO:9); antisense, 5' TTGCACAATGCTCTGATC-3' (SEQ ID NO:10)) and collagen III (sense, 5'- CCAAACTCTATCTGAAATCC-3' (SEQ ID NO: 11); antisense, 5'- GGACTCATAGAATACAATCT-3' (SEQ ID NO: 12)) were determined. All genes were amplified for 30 cycles in a thermocycler (Eppendorf Mastercycler gradient, Brinkmann, Westbury, NY).

Quantitative Analysis of Cell Attachment on Nanofiber Scaffolds [0138] The effects of nanofiber organization (aligned vs. unaligned) on fibroblast attachment and alignment on the nanofiber scaffolds over time were quantified following the methods of Costa et a/. (58). Specifically, confocal microscopy images (1024 x 1024 pixel resolution, n=3) of both fibroblasts seeded on the nanofiber scaffolds and acellular nanofiber scaffolds at days 1 , 7 and 14 were analyzed using circular statistics software customized for measurement of fiber alignment (Fiber3). The circular statistics parameters determined include mean vector angle (-90° ≤ θ ≤ 90°; 0° indicates horizontal orientation) which represents the average fiber alignment in the matrix, mean vector length (0 ≤ r < 1) which ranges from zero for a randomly distributed sample to unity for a perfectly aligned sample, and angular deviation (0-40.5°) which characterizes the dispersion of the non- Gaussian angle distribution of the nanofibers. Specifically, angular deviation of 0° is, in general, found in a perfectly aligned sample, while 40.5° is indicative of random distribution. Cell Proliferation

[0139] Cell proliferation (n=5) was determined at days 1 , 3, 7 and 14 by measuring total DNA content using the PicoGreen double-stranded DNA assay (Molecular Probes) following the manufacturers suggested protocol. At designated time points, each nanofiber scaffold was rinsed twice with PBS, then treated with 0.1% Triton X solution (Sigma-Aldrich) and homogenized by sonication (Kontes, Vineland, NJ) in order to remove adhered cells from the nanofiber scaffold. Sample fluorescence was measured with a microplate reader (Tecan, Research Triangle Park, NC), at the excitation and emission wavelengths of 485 nm and 535 nm, respectively. The total number of cells in the sample was determined by converting the amount of DNA per sample to cell number using the conversion factor of 8 pg DNA/cell (53). Cell Matrix Production

[0140] The elaboration of types I and III collagen (n=3/group) by fibroblasts seeded on the aligned and unaligned nanofiber scaffolds were evaluated by immunohistochemistry at days 7 and 14. Briefly, the samples were rinsed twice with PBS₁ fixed with 10% neutral buffered formalin for 24 hours at room temperature. Monoclonal antibodies for type I collagen (1 :20 dilution) and type III collagen (1 :100) were purchased from EMD Chemicals (Calbiochem, San Diego, CA) and Sigma- Aid rich, respectively. Before staining for type III collagen, the samples were treated with 1% hyaluronidase for 30 minutes at 37°C and incubated with primary antibody overnight. Following a PBS wash, biotinylated secondary antibody and Streptavidin conjugate (LSAB2 System-HRP, DakoCytomation, Carpinteria, CA) were added. Positive staining with the colorimetric substrate (AEC Substrate Chromogen, DakoCytomation) was indicated by the formation of brown precipitates and visualized with an inverted light microscope (Ziess Axiovert 25, Zeiss, Germany). At day 7, alignment of the collagen type I produced by the human fibroblasts was also evaluated using the circular statistics software (Fiber3) described above.

Example 4

Mechanical Properties of the Fibroblast-Seeded Nanofiber Scaffolds [0141] The effects of in vitro fibroblast culture on the mechanical properties of the aligned and unaligned nanofiber scaffolds were determined at days 1 , 7 and 14. The human rotator cuff fibroblasts were grown on both types of nanofiber scaffolds (6 cm x 1 cm) at a density of 3x10⁴ cells/cm² in fully supplemented media at 37°C and 5% CO2. For the control groups, the aligned and unaligned nanofiber scaffolds without cells (acellular) were incubated in fully supplemented DMEM and analyzed at days 1 , 7 and 14. At each designated time point, the samples were rinsed twice with PBS, and then tested to failure under uniaxial tension following the protocol described for the as-fabricated nanofiber scaffolds. The elastic modulus, yield stress and ultimate tensile stress of the samples (n=5) were determined.

Example 5 Statistical Analysis

[0142] Results are presented in the form of mean ± standard deviation, with n equal to the number of samples analyzed. One-way analysis of variance (ANOVA) was performed to determine the effects of fiber organization of the as-fabricated nanofiber scaffolds on material and mechanical properties. Two-way ANOVA was used to determine nanofiber scaffold fiber organization and temporal effects on cellular alignment and cell proliferation. Additionally, two-way ANOVA was performed to determine the effects of cellularity and culture time on nanofiber scaffold tensile mechanical properties. The Tukey-Kramer post-hoc test was utilized for all pair-wise comparisons and statistical significance was attained at p<0.05. All statistical analyses were performed using JMP statistical software (SAS Institute, Can/, NC). [0143] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

CITED DOCUMENTS

[0144] The following documents, as well as those cited within this specification, are specifically incorporated by reference to the extent that they provide or teach exemplary methodology, techniques and/or compositions supplemental to those employed herein.

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Claims

WHAT IS CLAIMED IS:

1. An implantable device for soft-tissue or soft tissue-to-bone fixation, repair, augmentation, or replacement comprising a biomimetic and biodegradable nanofiber scaffold, which scaffold comprises one or more continuous phases.

2. The implantable device according to claim 1 , wherein the nanofiber scaffold comprises a plurality of nanofibers made from a biodegradable polymer.

3. The implantable device according to claim 2, wherein the biodegradable polymer is selected from the group consisting of aliphatic polyesters, poly(amino acids), modified proteins, polydepsipeptides, copoly(ether-esters), polyurethanes, polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, poly(ε-caprolactone)s, polyanhydrides, polyarylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, modified polysaccharides, polycarbonates, polytyrosinecarbonates, polyorthocarbonates, poly(trimethylene carbonate), poly(phosphoester)s, polyglycolide, polylactides, polyhydroxybutyrates, polyhydroxyvalerates, polydioxanones, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(maleic anhydride), polyvinylalcohol, polyesteramides, polycyanoacrylates, polyfumarates, poly(ethylene glycol), polyoxaesters containing amine groups, poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic acid)s, poly(dioxanone)s, poly(alkylene alkylate)s, biopolymers, collagen, silk, chitosan, alginate, and a blend of two or more of the preceding polymers.

4. The implantable device according to claim 3, wherein the polymer comprises at least one of poly(lactide-co-glycolide), poly(lactide), and poly(glycolide).

5. The implantable device according to claim 3, wherein the polymer is a copolymer.

6. The implantable device according to claim 5, wherein the copolymer is poly(D,L-lactide-co-glycolide (PLGA).

7. The implantable device according to claim 6, wherein the ratio of polymers in the PLGA copolymer is between about 25:75 to about 95:5.

8. The implantable device according to claim 7, wherein the ratio of polymers in the PLGA copolymer is between about 85:15.

9. The implantable device according to claim 2, wherein the nanofibers are aligned.

10. The implantable device according to claim 2, wherein the nanofibers are unaligned.

11. The implantable device according to claim 2, wherein the nanofiber scaffold comprises both aligned and unaligned nanofibers.

12. The implantable device according to claim 2, wherein the nanofiber alignment and orientation mimics the anatomy of a soft-tissue or a soft tissue-to-bone interface to be repaired, augmented, fixated, or replaced.

13. The implantable device according to claim 12, wherein the soft tissue is a ligament or tendon.

14. The implantable device according to claim 2, wherein the nanofiber alignment and orientation mimics the anatomy of a tendon-to-bone interface.

15. The implantable device according to claim 13, wherein the soft tissue is a rotator cuff.

16. The implantable device according to claim 1, wherein the scaffold is designed to mimic soft tissue and comprises a preformed interface region.

17. The implantable device according to claim 1, wherein the nanofiber scaffold is multi-phasic and the scaffold is continuous from phase-to-phase.

18. The implantable device according to claim 17, wherein the phases are layered along a vertical axis.

19. The implantable device according to claim 17, wherein each phase is aligned along a horizontal axis.

20. The implantable device according to claim 17, wherein at least two phases are layered along a vertical axis and at least two phases are aligned along a horizontal axis.

21. The implantable device according to claim 17, wherein the nanofiber scaffold is biphasic.

22. The implantable device according to claim 21 , wherein at least one of the phases comprises more than one layer and each layer is composed of the same or different nanofiber polymer and/or biocompatible ceramic, nanofiber orientation, and coating.

23. The implantable device according to claim 21 , which comprises a first phase comprising nanofibers made from a biodegradable polymer and a second phase coupled to the first phase, which second phase comprises nanofibers made from a biodegradable polymer and a biocompatible ceramic, and the first phase is continuous with the second phase.

24. The implantable device according to claim 23, wherein the biocompatible ceramic is selected from the group consisting of silicon nitride-based ceramics, Pseudowollastonite ceramics (β-CaSiθ₃), bredigite (Ca₇MgSi₄Oi₆) ceramics, monophase ceramics of monticellite (CaMgSiO(4)), akermanite ceramics (Ca₂MgSi₂O₇), tricalcium silicate (Ca(3)SiO(5)), hydroxyapatite, bio-active glass, calcium phosphate, dense calcium sulfate (DCaS), porous silicated calcium phosphate (Si-CaP), tricalcium phosphate (TCP), calcium pyrophosphate (CPP), and combinations thereof.

25. The implantable device according to claim 23, wherein at least one of the phases further comprises a bioactive agent selected from the group consisting of an anti-infective, an extracellular matrix component, an antibiotic, bisphosphonate, a hormone, an analgesic, an anti-inflammatory agent, a growth factor, an angiogenic factor, a chemotherapeutic agent, an anti-rejection agent, an RGD peptide, and combinations thereof.

26. The implantable device according to claim 25, wherein the growth factor is selected from the group consisting of a member of the Transforming Growth Factor (TGF) super family, a vascular endothelial growth factor (VEGF), a platelet-derived growth factor (PDGF), an insulin-derived growth factor (IGF), a modulator of a growth factor, and combinations thereof.

27. The implantable device according to claim 26, wherein a member of the TGF super family is selected from the group consisting of TGF-β, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), Activin A and Activin B, lnhibin A, lnhibin B, anti-mullerian hormone, Nodal, and combinations thereof.

28. The implantable device according to claim 27, wherein the TGF-β is selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, and combinations thereof.

29. The implantable device according to claim 27, wherein the BMP is selected from the group consisting of BMP 1-20 and combinations thereof.

30. The implantable device according to claim 27, wherein the GDFs are selected from the group consisting of GDF1-15 and combinations thereof.

31. The implantable device according to claim 26, wherein the IGF is selected from the group consisting of IGF1 , IGF2, insulin growth factor binding proteins 1-6 (IGFBP1-6), and combinations thereof.

32. The implantable device according to claim 26, wherein a modulator of a growth factor is a SMAD (small mothers against decapentaplegic) selected from the group consisting of SMAD1-9 and combinations thereof.

33. The implantable device according to claim 23, further comprising a hydrogel disposed on at least a portion of one or both of the phases.

34. The implantable device according to claim 33, wherein the hydrogel is composed of a material selected from the group consisting of agarose, carageenan, polyethylene oxide, polyethylene glycol, tetraethylene glycol, triethylene glycol, trimethylolpropane ethoxylate, pentaerythritol ethoxylate, hyaluronic acid, thiosulfonate polymer derivatives, polyvinylpyrrolidone-polyethylene glycol-agar, collagen, dextran, heparin, hydroxyalkyl cellulose, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, dextran sulfate, pentosan polysulfate, chitosan, alginates, pectins, agars, glucomannans, galactomannans, maltodextrin, amylose, polyalditol, alginate-based gels cross-linked with calcium, polymeric chains of methoxypoly(ethylene glycol) monomethacrylate, chitin, poly(hydroxyalkyl methacrylate), poly(electrolyte complexes), poly(vinylacetate) cross-linked with hydrolysable bonds, water-swellable N-vinyl lactams, carbomer resins, starch graft copolymers, acrylate polymers, polyacrylamides, polyacrylic acid, ester cross-linked polyglucans, and derivatives and combinations thereof.

35. The implantable device according to claim 33 further comprising fibroblasts, chondrocytes, osteoblasts, osteoblast-like cells, stem cells, and combinations thereof.

36. The implantable device according to claim 33, wherein fibroblasts, chondrocytes, stem cells, and combinations thereof are disposed on at least a portion of the first phase.

37. The implantable device according to claim 33, wherein chondrocytes, osteoblasts, osteoblast-like cells, stem cells, and combinations thereof are disposed on at least a portion of the second phase.

38. The implantable device according to claim 33, wherein fibroblasts, stem cells, and chondrocytes are disposed on at least a portion of the first phase and chondrocytes, osteoblasts, osteoblast-like cells,. stem cells, and combinations thereof are disposed on at least a portion of the second phase.

39. The implantable device to any one of claims 35, 36, 37, or 38, wherein the stem cells are undifferentiated.

40. The method according to any one of claims 35, 36, 37, or 38, wherein the stem cells are pre-differentiated prior to disposition on the implantable device.

41. The implantable device of claim 1 , wherein the nanofiber scaffold has a fiber diameter of between about 568 to about 615 nm, a pore diameter of about 4.2 to about 4.9 μm, a porosity of about 80.7 to about 81.8%, and a permeability of about 5.7 to about 7.9 X IO^{^12} m⁴/N s.

42. An implantable biphasic biomimetic and biodegradable nanofiber device for soft-tissue or soft tissue-to-bone interface fixation, repair, augmentation, or replacement comprising a first phase comprising nanofibers made from a biodegradable polymer and a second phase coupled to the first phase, which second phase comprises nanofibers made from a biodegradable polymer and a biocompatible ceramic, wherein the first and second phases are continuous.

43. An implantable device for fixation, repair, augmentation, or replacement of a rotator cuff or a tendon-to-bone interface thereof comprising a biphasic, biomimetic, and biodegradable nanofiber scaffold having a first phase comprising nanofibers whose anisotropy mimics that of a tendon and non-mineralized fibrocartilage, which nanofibers are made from a biodegradable polymer and a second phase coupled to the first phase, which second phase comprises nanofibers whose anisotropy mimics that of mineralized fibrocartilage and bone, which nanofibers are made from a biodegradable polymer and a biocompatible ceramic, wherein the first and second phases are continuous.

44. The implantable device according to any one of claims 42 or 43, wherein the biodegradable polymer is selected from the group consisting of aliphatic polyesters, poly(amino acids), modified proteins, polydepsipeptides, copoly(ether-esters), polyurethanes, polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, poly(ε-caprolactone)s, polyanhydrides, polyarylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, modified polysaccharides, polycarbonates, polytyrosinecarbonates, polyorthocarbonates, poly(trimethylene carbonate), poly(phosphoester)s, polyglycolide, polylactides, polyhydroxybutyrates, polyhydroxyvalerates, polydioxanones, polyalkylene Oxalates, polyalkylene succinates, poly(malic acid), poly(maleic anhydride), polyvinylalcohol, polyesteramides, polycyanoacrylates, polyfumarates, poly(ethylene glycol), polyoxaesters containing amine groups, poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic acid)s, poly(dioxanone)s, poly(alkylene alkylate)s, biopolymers, collagen, silk, chitosan, alginate, and a blend of two or more of the preceding polymers.

45. The implantable device according to any one of claims 42 or 43, wherein the biocompatible ceramic is selected from the group consisting of silicon nitride-based ceramics, Pseudowollastonite ceramics (β-CaSiθ₃), bredigite (CayMgSi-iOie) ceramics, mono-phase ceramics of monticellite (CaMgSiO(4)), akermanite ceramics (Ca₂MgS-^O_?), tricalcium silicate (Ca(3)SiO(5)), hydroxyapatite, bio-active glass, calcium phosphate, dense calcium sulfate (DCaS), porous silicated calcium phosphate (Si-CaP), tricalcium phosphate (TCP), calcium pyrophosphate (CPP), and combinations thereof.

46. The implantable device according to any one of claims 42 or 43, wherein at least one of the phases further comprises a bioactive agent selected from the group consisting of an anti-infective, an extracellular matrix component, an antibiotic, bisphosphonate, a hormone, an analgesic, an anti-inflammatory agent, a growth factor, an angiogenic factor, a chemotherapeutic agent, an anti-rejection agent, an RGD peptide, and combinations thereof.

47. The implantable device according to claim 46, wherein the growth factor is selected from the group consisting of a member of the Transforming Growth Factor (TGF) super family, a vascular endothelial growth factor (VEGF), a platelet-derived growth factor (PDGF), an insulin-derived growth factor (IGF), a modulator of a growth factor, and combinations thereof.

48. The implantable device according to claim 47, wherein a member of the TGF super family is selected from the group consisting of TGF-β, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), Activin A and Activin B, lnhibin A, lnhibin B, anti-mullerian hormone, Nodal, and combinations thereof.

49. The implantable device according to claim 48, wherein the TGF-β is selected from the group consisting of TGF-β1 , TGF-β2, TGF-β3, and combinations thereof.

50. The implantable device according to claim 48, wherein the BMP is selected from the group consisting of BMP1-20 and combinations thereof.

51. The implantable device according to claim 48, wherein the GDFs are selected from the group consisting of GDF1-15 and combinations thereof.

52. The implantable device according to claim 47, wherein the IGF is selected from the group consisting of IGF1 , IGF2, insulin growth factor binding proteins 1-6 (IGFBP1-6), and combinations thereof.

53. The implantable device according to claim 47, wherein a modulator of a growth factor is a SMAD (small mothers against decapentaplegic) selected from the group consisting of SMAD1-9 and combinations thereof.

54. The implantable device according any one of claims 42 or 43, further comprising a hydrogel disposed on at least a portion of one or both of the phases.

55. The implantable device according to claim 54, wherein the hydrogel is composed of a material selected from the group consisting of agarose, carageenan, polyethylene oxide, polyethylene glycol, tetraethylene glycol, triethylene glycol, trimethylolpropane ethoxylate, pentaerythritol ethoxylate, hyaluronic acid, thiosulfonate polymer derivatives, polyvinylpyrrolidone-polyethylene glycol-agar, collagen, dextran, heparin, hydroxyalkyl cellulose, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, dextran sulfate, pentosan polysulfate, chitosan, alginates, pectins, agars, glucomannans, galactomannans, maltodextrin, amylose, polyalditol, alginate-based gels cross-linked with calcium, polymeric chains of methoxypoly(ethylene glycol) monomethacrylate, chitin, poly(hydroxyalkyl methacrylate), poly(electrolyte complexes), poly(vinylacetate) cross-linked with hydrolysable bonds, water-swellable N-vinyl lactams, carbomer resins, starch graft copolymers, acrylate polymers, polyacrylamides, polyacrylic acid, ester cross-linked polyglucans, and derivatives and combinations thereof.

56. The implantable device according to claim 55 further comprising fibroblasts, chondrocytes, osteoblasts, osteoblast-like cells, stem cells, and combinations thereof.

57. The implantable device according to claim 56, wherein fibroblasts, stem cells, chondrocytes, and combinations thereof are disposed on at least a portion of the first phase.

58. The implantable device according to claim 56, wherein chondrocytes, osteoblasts, osteoblast-like cells, stem cells, and combinations thereof are disposed on at least a portion of the second phase.

59. The implantable device according to claim 56, wherein fibroblasts, stem cells, and chondrocytes are disposed on at least a portion of the first phase and chondrocytes, osteoblasts, osteoblast-like cells, stem cells, and combinations thereof are disposed on at least a portion of the second phase.

60. The implantable device according to any one of claims 56, 57, 58, or 59 wherein the stem cells are undifferentiated.

61. The method according to any one of claims 56, 57, 58, or 59 wherein the stem cells are pre-differentiated prior to disposition on the implantable device.

62. The implantable device according any one of claims 42 or 43, which is about 0.2 to about 2.0 mm thick.

63. The implantable device according to claim 62, which is about 5.0 cm in length and about 5.0 cm in width.

64. The implantable device according to claim 62, which has a diameter of about 10 cm.

65. A method for fixating, repairing, augmenting, or replacing a damaged soft tissue or soft tissue-to-bone interface in a patient comprising affixing a biomimetic, biodegradable, continuous multi-phasic nanofiber scaffold to a surgically relevant site in order to repair, augment, or replace the damaged soft tissue or soft tissue-to-bone interface.

66. A method for fixating, repairing, augmenting, or replacing a damaged rotator cuff in a patient comprising affixing a biomimetic and biodegradable continuous multiphase nanofiber scaffold to a surgically relevant site in order to repair, augment, or replace the damaged rotator cuff.