Biomimetic soft composite orthopedic implants having radial fibers

Abstract

Provided herein are biomimetic composite(s) tissue analogue / implant having radial fibers, and method(s) of fabricating the same, for human tissue substitution / replacement of a knee meniscus or intervertebral disc (IVD).

Description

[0001] BIOMIMETIC SOFT COMPOSITE ORTHOPEDIC IMPLANTS HAVING RADIAL FIBERS

[0002] FIELD OF INVENTION

[0003] The present disclosure generally pertains to the field of orthopedic implants, more specifically the disclosure relates to biomimetic composite implants having radial fibers.

[0004] BACKGROUND OF INVENTION

[0005] Over millions of years of evolution, biological soft fibrous tissues have developed intricate structural mechanisms that enable exceptional mechanical performance, especially in enduring large deformations. These tissues are composite materials of repeating building blocks with diverse structural motifs, such as fiber fraction, orientation, hierarchy, crimping, and weak interfaces.

[0006] The knee is the largest and most complex joint in the human body. It comprises hard (bones: femur, tibia, and patella) and soft tissues (ligaments, meniscus, cartilage, tendons, and muscles). The meniscus is an essential structure for the normal function of the knee, responsible for load bearing, load distribution, and joint stabilization. These functions are strongly dependent on its complex structure.

[0007] Internal knee injuries have become increasingly common in today's active lifestyles, with over 6 million incidents occurring annually in the US alone. This number continues to rise among younger individuals as extreme sports gain popularity and health awareness increases. Approximately 32% of these injuries involve the meniscus tissue, making it the most common intra-articular knee injury. Meniscus tears may occur from an acute traumatic injury or as part of a degenerative process with age. Clinical symptoms of pain, swelling, locking, catching, and losing knee motion may occur due to a meniscus injury. These injuries alter the load distribution in the joint and may lead to degenerative processes that ultimately result in an early onset of osteoarthritis (OA) and knee degeneration.

[0008] Meniscus repair is accomplished by closing the tear with sutures or anchors. Unrepairable meniscal tears usually require partial or complete removal of the meniscus. Total or near-total meniscectomies require the use of allograft and synthetic implants.

[0009] These implants do not have biological ingrowth properties, and their long-term stability is insufficient. Furthermore, these implants are homogenous porous scaffolds that do not mimic the complex internal structure and the anisotropic biomechanics of the native meniscus tissue and fail to prevent further degeneration.

[0010] The degeneration of the intervertebral disc (IVD) and the resulting lower back pain (LBP) present a significant worldwide healthcare challenge, causing substantial economic implications through increased morbidity, hospitalization, and prolonged recovery periods. This issue affects an estimated 632 million individuals globally. LBP is the primary contributor to disability, resulting in expenses surpassing $100 billion and causing 149 million days of work to be forgone annually in the United States. Over 70% of the world’s population experiences LBP throughout their lifetime.

[0011] Most current treatments involve conservative methods or surgical interventions. The latter fails to restore the mechanical function of the IVD; in some cases, it actually accelerates degeneration. Numerous strategies have been proposed to mimic the annulus fibrosus (AF), nucleus pulposus core (NP), or disc. However, most of these studies have failed to replicate the complex IVD structure and mechanics, which are imperative for the proper functioning of the native tissue. Some IVD implants are available but the complexity of the IVD makes it a long- range goal for successful development.

[0012] There is therefore a need for a durable biomimetic composite orthopedic implant which mimics the complex internal structure and the anisotropic biomechanics of native tissue.

[0013] SUMMARY OF INVENTION

[0014] According to some aspects, the present disclosure provides a biomimetic composite(s) tissue analogue / implant with radial fibers, and method(s) of fabricating the same, for human tissue substitution / replacement of a knee meniscus or intervertebral disc (IVD).

[0015] Advantageously, fibers’ orientation in the biocomposite simulates the main directions of the human tissue, including the transverse / radial fibers that provides mechanical shielding / structural integrity to the composite(s) / implant(s), as also evident in a computational biomimetic composite model of the tissue.

[0016] The addition of radial fibers to the structure furnishes the biocomposite(s) with advantageous and surprising mechanical tolerance or resistant behavior and properties more similar to the human tissue than composite(s) lacking radial fibers or improved with respect to composite(s) lacking radial fibers, in some embodiments, this maintenance of mechanical performance relative to the human tissue included maintenance of strength [ultimate tensile strength (UTS) (MPa)], stiffness [stress (MPa) vs. strain (mm / mm)], and toughness (MJ / m3) and favorable change in strain [ultimate strain (mm / mm)], while in other embodiments, by distribution of stress in the tissue.

[0017] In some embodiments, the biocomposite(s) with radial fibers is characterized by increased shear force (N) between the circumferential AF lamellae rings and the NP core under compression; in some other preferred embodiments, the biocomposite(s) with radial fibers is characterized by reduction in width change (%) and / or preservation of transverse deformation [ultimate strain (mm / mm)] under stretching relative to laminate without radial fibers.

[0018] According to additional embodiments, the biocomposite of the present invention is characterized by an advantageous quantitatively controlled amount of fibers that surprisingly furnishes the composite / implant with mechanical durability / tolerance or resistance, and include in some specific embodiments a total volume fraction (VF) of fibers of at least about 10% (v / v) or at some specific embodiment between about 10% and about 85%, or between about 15% and about 75%; or between about 20% and about 65%.

[0019] According to an aspect there is provided a cross-plied (CP) biocomposite laminates / layered tissue analogue / implant comprising hydrogel matrix reinforced with longitudinal and transverse / radial fibers for human tissue substitution / replacement of a medial knee meniscus and / or lateral knee meniscus, wherein an orientation of the fibers in the biocomposite simulates main directions of the meniscus fibers, and wherein the transverse / radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability / tolerance and / or resistance.

[0020] According to another aspect there is provided an annulus fibrosus - nucleus pulposus (AF-NP) disc-shaped biocomposite construct tissue analogue / implant comprising a plurality of circumferential rings and an NP core made of hydrogel matrix reinforced with radial fibers for human tissue substitution / replacement of an intervertebral disc (IVD), wherein each circumferential ring comprises AF lamellae made of hydrogel matrix reinforced with angle- plied (AP) fibers, and wherein the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reinforcing the disc-shaped biocomposite; and wherein an orientation of the fibers in the biocomposite simulates main directions of the IVD fibers, and wherein the radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability / tolerance or resistance.

[0021] According to some embodiments, the radial fibers create a refined complex network of fibers across the full IVD tissue, that can work together with the other fibers; therefore, these fibers contribute to the overall connectivity and thus provide better robustness to graft and tissue.

[0022] According to some embodiments, the biocomposite comprises a quantitatively controlled amount / volume of fibers.

[0023] According to related embodiments, the quantitatively controlled amount / volume of fibers comprises a total volume fraction (total VF) consisting of radial fibers volume and longitudinal fibers volume of at least about 10% of the laminate hydrogel matrix (v / v), or a total volume fraction (total VF) consisting of radial fibers and circumferential AP fibers of at least about 10% of the AF lamellae hydrogel matrix (v / v). Each possibility is a separate embodiment.

[0024] According to specific embodiments, the quantitatively controlled amount / volume of fibers comprises a total volume fraction (VF) consisting of radial fibers volume and longitudinal fibers volume of between about 10% and about 85% of the laminate hydrogel matrix (v / v), or a total volume fraction (VF) consisting of radial fibers and circumferential AP fibers of between about 10% and about 85% of the AF lamellae hydrogel matrix (v / v). Each possibility is a separate embodiment.

[0025] In some embodiments, the mechanical durability of the biocomposite is more similar to human or bovine meniscus compared to biocomposite lacking radial fibers, and / or the mechanical durability of the biocomposite is compared to biocomposite lacking radial fibers. Each possibility is a separate embodiment.

[0026] In additional embodiments, the mechanical durability comprises tolerance or resistance of a biomimetic model composite using structure-function relationships of the human tissue, wherein the biomimetic of the structure-function relationships comprises the main direction(s) of the fibers of the human tissue, corresponding to the radial and / or longitudinal fibers of the cross-plied (CP) biocomposite, or corresponding to the radial and / or circumferential fibers of the AF-NP composite construct. Each possibility is a separate embodiment.

[0027] In related embodiments, the structure-function relationships model of the human tissue comprises the meniscus in a knee joint model, or the whole IVD as a functional spinal unit. Each possibility is a separate embodiment.

[0028] According to further related embodiments, the mechanical durability comprises tolerance or resistance towards mechanically applied or computational simulated stretching, compression, and / or physiological moments. Each possibility is a separate embodiment.

[0029] According to some embodiments, the mechanical durability comprises at least one mechanical behavior / property selected from the group consisting of: structural width change (%), stress (MPa) vs. strain (mm / mm), Poisson’s ratio, tensile modulus (MPa), ultimate strains (mm / mm), UTS (MPa), toughness (MJ / m3), shear force (N), and rotation (degrees), or any combination thereof. Each possibility is a separate embodiment.

[0030] In some embodiments, the hydrogel matrix includes one or more materials selected from a protein(s), a polysaccharide(s), a polypeptide(s), a peptide, a lipid, a polylipid(s), a synthetic polymer (s), a natural polymer(s), or any combination thereof. Each possibility is a separate embodiment.

[0031] In other embodiments, the hydrogel includes one or more of alginate hydrogel, Hyaluronic acid hydrogel, PEG hydrogel, cellulose hydrogel, Agar hydrogel, and Interpenetrating network (IPN) hydrogel, or any combination thereof. Each possibility is a separate embodiment.

[0032] In additional embodiments, the fibers comprise one or more type(s) of fiber(s) selected from woven fibers, twisted fibers, braided fibers, knitted fibers, tie fibers, and sutured fibers, or any combination thereof. Each possibility is a separate embodiment.

[0033] In related embodiments, the fibers material comprises one or more material selected from a protein(s), a polysaccharide(s), a saccharide, a polypeptide(s), a peptide, a lipid, a polylipid(s), a synthetic polymer(s), natural polymer(s), or any combination thereof. Each possibility is a separate embodiment. In further embodiments, fibers comprise one or more fiber(s) selected from silk, silk- made fibroin fibers, collagen fibers, cellulose fibers, synthetic fibers, or any combination thereof. Each possibility is a separate embodiment.

[0034] In specific embodiments, the fibers comprise silk and / or collagen fibers. Each possibility is a separate embodiment.

[0035] In specific embodiments, the silk is derived from spider and / or silkworm. Each possibility is a separate embodiment. In further specific embodiments, the silk-made fibroin fibers are derived from Bombyx mori.

[0036] According to some embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers, wherein said fiber orientation comprises: a. the transverse / radial fibers of the implant projecting in a direction from about a center towards a perimeter of a c-shaped meniscus; and / or b. the longitudinal fibers of the implant projecting in a circumferential direction along the perimeter of the c-shaped meniscus.

[0037] According to specific embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers, wherein said fiber orientation comprises: the transverse / radial fibers of the implant (about 90°) oriented essentially on a horizontal axis about perpendicular to a vertical femur-tibia axis, projecting in a direction from about a center towards a perimeter of a c-shaped meniscus (about 90°).

[0038] According to specific embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers, wherein said fiber orientation comprises: the longitudinal fibers (0°) of the implant oriented essentially on a same horizontal axis, about perpendicular (0°) to the radial fibers, projecting in a circumferential direction along the perimeter of the c-shaped meniscus.

[0039] According to some embodiments, the mechanical durability comprises reduction in width change and / or preservation of transverse deformation, under stretching relative to laminate without radial fibers. Each possibility is a separate embodiment.

[0040] According to some embodiments, the CP composite comprising quantitatively controlled amount / volume of fibers; wherein said quantitatively controlled amount / volume of fibers comprises a total volume fraction (VF) consisting of radial fibers volume and longitudinal fibers volume of between about 20-70% (for example, about 30-60%, such as, 42% and about 55%) of the laminate hydrogel matrix (v / v), providing the biocomposite with similar mechanical durability / behavior to the mechanical durability / behavior of human meniscus under uniaxial tension / stretching. Each possibility is a separate embodiment.

[0041] According to some embodiments, the CP composite comprising a quantitatively controlled amount / volume of fibers, wherein the quantitatively controlled amount / volume of fibers comprises a range of ratios of transverse / radial fibers volume to longitudinal fibers volume of between about 1 :3 (25%:75% transverse / radial: longitudinal vf(0°)) and about 4:1 (80%: 20% transverse / radial: longitudinal vf(0°)), thereby providing mechanical resistance / shielding, at least under stretching. Each possibility is a separate embodiment.

[0042] According to some embodiments, the CP composite comprising a quantitatively controlled amount / volume of fibers, wherein the quantitatively controlled amount / volume of fibers comprises a range of ratios of transverse / radial fibers volume to longitudinal fibers volume of between about 4:6 (40%:60% transverse / radial: longitudinal vf(0°)) and about 9:1 (90%: 10% transverse / radial: longitudinal vf(0°)), thereby providing mechanical resistance / shielding, at least under stretching. Each possibility is a separate embodiment.

[0043] According to some embodiments, the structural shielding comprises an increased ratio of transverse / radial: longitudinal fibers volume thereby reducing the change in average behavior of stress (MPa) vs. strain (mm / mm) under stretching.

[0044] In some embodiments, the structural shielding is characterized by a non-linear increase in tensile modulus (MPa) under stretching, compared to a linear increase of laminates lacking radial fibers, thereby providing mechanical durability / resistance under uniaxial tension / stretching. Each possibility is a separate embodiment.

[0045] In some embodiments, the CP biocomposite is characterized by decreased ultimate strain under stretching, compared to laminates lacking radial fibers.

[0046] In some embodiments, the CP biocomposite is characterized by an increased UTS (MPa) under stretching, compared to laminates lacking radial fibers. In some embodiments, the CP biocomposite is characterized by similar or increased toughness (MJ / m3) under stretching, compared to laminates lacking radial fibers, thereby providing mechanical durability.

[0047] In some embodiments, the mechanical durability is compared to laminates lacking radial fibers, and / or wherein a fiber volume of longitudinal fibers vf(0°) is within the range of about 10%-40% (for example, 12-36%) for a total fiber volume fraction (VF) of about 50%±5.0%. Each possibility is a separate embodiment.

[0048] In some embodiments, the CP biocomposite is characterized by reduced change in stress (MPa) vs. strain (mm / mm) under stretching compared with laminate lacking radial / transverse fibers, thereby providing mechanical durability / resistance.

[0049] In some embodiments, the CP biocomposite is characterized by reduction of Poisson’s ratio under stretching.

[0050] In some embodiments, the transverse / radial fibers reduce pressure on an articular cartilage under compression.

[0051] In some embodiments, the transverse / radial fibers distribute stress within the implant / tissue.

[0052] According to specific embodiments, the orientation of the fibers in the AF-NP biocomposite simulates main directions of the IVD fibers, wherein said fiber orientation comprises: a. the radial fibers of the implant projecting in a direction from about a center of the NP core towards a perimeter of the disc / AF rings; and / or b. the angle-plied (AP) fibers projecting in a circumferential direction relative to the horizontal axis.

[0053] According to specific embodiments, the orientation of the fibers in the AF-NP biocomposite simulates main directions of the IVD fibers, wherein said fiber orientation comprises: the radial fibers (about 90°) of the implant oriented essentially along a horizontal axis about perpendicular 90° to a vertical spinal axis, projecting in a direction from about a center of the NP core towards a perimeter of the disc / AF rings. According to specific embodiments, the orientation of the fibers in the AF-NP biocomposite simulates main directions of the IVD fibers, wherein said fiber orientation comprises: the angle-plied (AP) (about ±30°) fibers arranged essentially parallel to / along the vertical spine axis within the circumferential AF lamellae rings, about perpendicular to the radial fibers, projecting in a circumferential direction of about ±30° relative to the horizontal axis.

[0054] In some embodiments, the mechanical durability comprises increased stress (MPa) vs. strain (mm / mm) under compression.

[0055] In some embodiments, the mechanical durability comprises increased shear force (N) between the circumferential AF lamellae rings and the NP core under compression providing structural integrity / shielding.

[0056] According to additional embodiments, the structural shielding comprises reduced delamination between the AF lamellae, compared with biomimetic model lacking radial fibers, thereby providing mechanical durability.

[0057] According to yet additional embodiments, the mechanical durability comprises maintaining rotation (degrees) under at least one of flexion, extension, bending, and / or torsion, with respect to biomimetic without radial fibers. Each possibility is a separate embodiment.

[0058] According to another aspect there is provided a method for fabricating cross-plied (CP) biocomposite laminates / layered meniscal tissue analogue / implant, the method comprises: (a) wrapping fibers around each of a plurality of 3D frame in longitudinal (0°) and / or transverse / radial (about 90°) orientation; (b) stacking the plurality of 3D frames containing the longitudinally and / or radially oriented fibers in CP orientation consisting of alternating 0° and about 90° directions; (c) placing the stack in a mold and filling the mold with hydrogel solution; and (d) cross-linking the hydrogel; thereby, obtaining a cross-plied (CP) biocomposite laminates comprising longitudinal (0°), and transverse / radial (about 90°) fibers at orientation that simulates main directions of the meniscus tissue fibers.

[0059] In some embodiments, the CP biocomposite comprises a wedge shape.

[0060] According to an additional aspect there is provided a method for fabricating an annulus fibrosus - nucleus pulposus (AF-NP) biocomposite construct intervertebral disc (IVD) tissue analogue / implant, the method comprising: (a) wrapping fibers around a frame in angle-plied (AP) (about ±30°) orientation, inserting them to a 3D mold with hydrogel solution and crosslink it, thereby obtaining a plurality lamella made of hydrogel matrix reinforced with angle-plied (AP) fibers; (b) locating the plurality of AP AF laminates comprising alternating (±30°) orientations in the circumference of a 3D cylinder frame; (c) suture the AF laminates using a fiber in a radial direction of the cylinder connecting the circumferential AP AF lamella rings and NP core of following step (d), while keeping the laminates hydrated; and (d) adding / injecting hydrogel into the AF rings construct center to obtain an NP core embedded with the radial fibers and surrounded by the AF rings; thereby, obtaining a disc-shaped AF-NP construct / biocomposite comprising / reinforced with angle-plied fibers (about ±30° relative to horizontal axis), and radial fibers (about 90° relative to vertical spinal axis) at orientation that simulates main directions of the IVD tissue fibers.

[0061] In some embodiments, the AF lamellae and the NP core are made of same or different hydrogel matrix. Each possibility is a separate embodiment.

[0062] According to some embodiments, the cross-linking of the hydrogel with the fibers comprises chemical, biochemical, and / or physical-based cross-linking. Each possibility is a separate embodiment.

[0063] In some embodiments, the hydrogel matrix material comprises one or more material selected from a protein(s), a polysaccharide(s), a saccharide, a peptide, a polypeptide(s), a lipid, a polylipid(s), a synthetic polymer, a natural polymer(s), or any combination thereof. Each possibility is a separate embodiment.

[0064] In related embodiments, the hydrogel is selected from one or more of alginate hydrogel, Hyaluronic acid hydrogel, PEG hydrogel, cellulose hydrogel, Agar hydrogel, and Interpenetrating network (IPN) hydrogel, or any combination thereof. Each possibility is a separate embodiment.

[0065] In specific embodiments, the hydrogel is alginate hydrogel.

[0066] In additional embodiments, the fibers comprise one or more type(s) of fiber(s) selected from woven fibers, twisted fibers, braided fibers, knitted fibers, tie fibers, and sutured fibers, or any combination thereof. Each possibility is a separate embodiment. According to one embodiment, the fibers comprise tie fibers.

[0067] In some embodiments, the fibers material comprises one or more material selected from a protein(s), a polysaccharide(s), a polypeptide(s), a polylipid(s), a synthetic polymer(s), or any combination thereof. Each possibility is a separate embodiment.

[0068] In related embodiments, the fibers comprise one or more fiber(s) selected from silk, silk-made fibroin fibers, collagen fibers, and cellulose fibers, or any combination thereof. Each possibility is a separate embodiment.

[0069] In specific embodiments, the fibers comprise silk and / or collagen fibers. Each possibility is a separate embodiment.

[0070] In specific embodiments, the fibers comprise silk or silk-made fibroin fibers. Each possibility is a separate embodiment. In further specific embodiments, the silk is derived from spider and / or silkworm. Each possibility is a separate embodiment. In further specific embodiments, the silk-made fibroin fibers are derived from Bombyx mori.

[0071] Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art from the figures, descriptions and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some or none of the enumerated advantages.

[0072] BRIEF DESCRIPTION OF THE FIGURES

[0073] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0074] FIGs. 1A-1F present the CP biocomposite laminate and constructs with radial and longitudinal fiber, biomimicking the knee meniscus fibers orientation.

[0075] FIG. 1A shows a schematic representation of the circumferential and radial / transverse fibers in the meniscus corresponding to the longitudinal and radial / transverse fibers orientations in the cross-plied (CP) biocomposite laminates. The biomimetic fiber orientations in the longitudinal biocomposite laminates (control lacking radial fibers) mimics the orientation of the meniscus circumferential fibers, and the biomimetic fiber orientations in the CP biocomposite laminates mimic the orientation of both the circumferential and radial fibers in the meniscus. Shown are the Lateral meniscus and the Medial meniscus. The fibers are oriented on a horizontal axis perpendicular to a vertical femur-tibia axis.

[0076] FIGs. 1B-1C shows a schematic representation of the knee joint, meniscus, and the biomimetic meniscus implant.

[0077] FIG. IB shows a knee joint and the 3D layered structure of the meniscal tissue, including. (1) The superficial layer, (2) the lamellar layer, and (3) the circumferential central main layer containing circumferential and radial / trans verse fibers.

[0078] FIG. 1C shows a wedge-shaped biomimetic meniscus implant “transplanted” into a C-shaped meniscus.

[0079] FIG. ID schematically illustrates the fabrication process of a wedge-shaped meniscal implant, (i) The fibroin fibers wrapped around the 3D printed frames and (ii) arranged in a stack, (iii) The stack was inserted into a mold which was filled with alginate solution and then (iv) inserted into the dialysis membrane and immersed in a CaCh solution for hydration and cross -linking, (v) The meniscal construct / implant was obtained with layers of alternately arranged fibers (red arrowheads). Each marked layer contains fibers in both orientations (longitudinal 0° / radial 90°).

[0080] FIG. IE shows a histogram presenting the distribution of the silk fibroin fibers in the crossplied (CP) biocomposite laminates in longitudinal (0°) and Cross-plied (0-90°) orientations. Shown are (i) longitudinal laminates exhibiting distribution of fibers peaking around (0°) with deviations of ± 10°, and (ii) CP laminates exhibiting distribution of longitudinal fibers peaking around (0°) with deviations of ± 20° and of transverse / radial fibers peaking around (90°) with deviations of ± 20°.

[0081] FIG. IF shows micrograph images of scanning electron microscopy (SEM) presenting (i) longitudinal biomimetic silk laminate, and (ii) human meniscus.

[0082] FIGs. 2A-2E show average mechanical behavior and properties of the biomimetic biocomposite laminates. FIG. 2A shows a line graph presenting the average mechanical behavior, measured as stress (MPa) vs. strain (mm / mm), of the longitudinal and CP laminates and the human meniscus.

[0083] FIG. 2B-2E show dot graphs presenting the average mechanical properties, measured as tensile modulus (MPa)(FIG. 2B), ultimate strains (mm / mm) (FIG. 2C), UTS (MPa) (FIG. 2D), and toughness (MJ / m3) (FIG. 2E), of the longitudinal and CP laminates versus the fiber volume fraction in the tensile direction vf (0°) (i.e., the longitudinal fibers).

[0084] FIGs. 3A-3E shows structural width change of the biocomposite laminates under tension, comparing mechanical behavior of longitudinal (control without radial fibers) and CP laminates.

[0085] FIG. 3A shows imaging of the longitudinal and CP laminates at the beginning of stretching (left; beginning) and under tensile at the UTS point (right; end).

[0086] FIG. 3B schematic representations of the longitudinal and CP laminates under tension. A significant narrowing in the center of the sample was obtained during stretching for the longitudinal laminates compared to the CP laminates.

[0087] FIG. 3C shows a graph presenting the axial vs. transverse strains of the laminates. The CP laminates exhibited smaller transverse strains during stretching than the longitudinal laminates.

[0088] FIGs. 3D-3E show graphs presenting the average mechanical behavior and Poisson’s ratios of the longitudinal (FIG. 3D) and CP laminates (FIG. 3E) up to failure.

[0089] FIG. 4A-4C presents the 3D Finite Elements (FE) model of the knee joint, and the meniscal composite models that were generated, according to some embodiments.

[0090] FIG. 4A shows illustration presenting 3D Finite Elements (FE) model of the knee joint, including the femur and tibia bones, femoral and tibial cartilage, ligaments (ACL, MCL, LCL, PCL), and the medial and lateral menisci (posterior view). The reference point on which the axial displacement was applied (Z-direction), and the fixation of the bottom of the tibia are presented. (ACL, anterior cruciate ligament; PCL, posterior cruciate ligament; MCL, anterior cruciate ligament; LCL, anterior cruciate ligament.) FIG. 4B shows illustration presenting a refined composite structure of the meniscus based on the main body of the medial meniscus (Matrix), including circumferential fibers (red), radial fibers (blue) and both circumferential fibers and radial fibers combined (green).

[0091] FIG. 4C shows a line graph presenting a validation of the FE knee model against experimental results. The graph shows similar total contact area on the tibial cartilage for five meniscal composite models: Circ, Rad and Circ-Rad, and two control composites Iso and Coll.

[0092] FIG. 5 shows stress maps presenting max principal stresses of the meniscal composite models. The columns from left to right show the max principal stresses in the components of the Circ composite, Rad composite, and Circ-Rad composite models. In the Circ composite model, there is stress concentration in the middle of the central-posterior portion of the circumferential medial and lateral fibers (black arrows).

[0093] FIG. 6 shows stress distribution maps presenting the contact pressure / stress distribution on the tibial cartilage with the Circ composite, Rad composite, and Circ-Rad composite meniscus models under similar axial compression of (i) 478-524 N, (ii) 990-995 N, and (iii) under equal axial displacement of 0.91 mm. The addition of radial fibers reduces the stress concentration on the tibial articular cartilage in comparison to Circ composite model.

[0094] FIGs. 7A-7C show bar graphs presenting the average width changes in the meniscal composite models including only circumferential fiber model composite (Circ), mainly radial fiber model composite (Rad), and both circumferential and radial fiber model composite (Circ-Rad, representing a CP orientation).

[0095] FIG. 7A presents the average width change of Medial and Lateral meniscus models. Showing the effect of the radial fibers on the meniscus expansion.

[0096] FIG. 7B presents the relative width change of the medial meniscal matrix at the anterior, central, and posterior regions.

[0097] FIG. 7C presents the relative width change of the lateral meniscal matrix at the anterior, central, and posterior regions.

[0098] FIG. 8 Anatomical dissection of the medial meniscus tissue and sample preparation for SEM and histology analysis, (i) Anterior view of the knee following patella removal. The tibial plateau superior view, with the medial meniscus (MM), lateral meniscus (LM), and the insertion points of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL). (ii) The medial meniscus and the radial section, (iii) Schematic representation of the central and posterior regions in the meniscus from which samples were taken for SEM and histology analyses, respectively. Representation of a radial sample obtained from the radial sections and the inner and outer regions.

[0099] FIGs. 9A-9B shows micrograph images presenting meniscal tissue sub-structures of a left knee joint of bovine (femur and tibia bones) using scanning electron microscopy (SEM)

[0100] FIG. 9A: SEM micrographs of the radial section of the central part of the medial meniscus, including control (undigested) (i-iv) and partially digested meniscal samples (v-viii). (i) A circumferential fascicle close to the surface from the femoral side (dash yellow circle). Non- homogeneous fiber bundles with non-circular cross-sections (dash purple circles). Twisted or wavy tie-fibers surround the fascicle edges and the fiber bundles. Twisted or wavy tiefibers surround the fascicle edges and the fiber bundles, (ii) Dense bundles with a smooth cross-section containing pores within and between them. Twisted and tangled tie-fiber between bundles (yellow arrow), (iii, iv) Many thin fibrils assemble the circumferential fibers, (v) The internal region has tie-fibers, which arborize and penetrate the main body of the tissue from the lamellar layer of the tibial side (white arrowheads), (vi) Gaps formed between the circumferential fiber bundles (white arrowheads) and tie-fiber with a twisted shape close to them (yellow arrow), (vii) The bundle is divided into individual fibers composed of many fibrils, some of which may collapse or stretch between gaps (yellow arrows), (viii) Thin, delicate fibrils formed cross-bridges-like structures inside the gaps between two adjacent fibers.

[0101] FIG. 9B - Meniscal tissue sub-structures using scanning electron microscopy, (i) the different layers in radial section including the radial fibers, circumferential fibers, and bundle, (ii) Fibrous structure of meniscal collagen.

[0102] FIG. 10: Schematic illustration of meniscus longitudinal and radial tear patterns.

[0103] FIGs. 11A-11D: present illustration of the tested annulus fibrosus - nucleus pulposus (AF-NP) construct with orientations of the fibers with respect to the IVD and additional, the process of fabrication of AF-NP construct with its structural characteristic features including radial fibers, and Bovine IVD sample preparation.

[0104] FIG.11A shows an illustration of the tested AF-NP disc-shaped biocomposite construct including a plurality of circumferential rings and an NP core made of hydrogel matrix reinforced with radial fibers. Each illustrated circumferential ring comprises AF lamellae made of hydrogel matrix reinforced with angle-plied (AP) fibers, and the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reinforcing the disc-shaped biocomposite; the orientation of the fibers in the biocomposite simulates main directions of the IVD fibers.

[0105] FIG.11B presents from left to right: suture of two biocomposite laminates for lap test, tensile test, and lap test.

[0106] FIG.11C presents from left to right: biomimetic AF laminates, creation of radial fibers and the final fabricated AF-NP construct with silk radial fibers, and illustration of the shear test apparatus.

[0107] FIG.11D Bovine IVD sample cleaning and mechanical testing (compression).

[0108] FIG. 12 shows a line graph presenting the tensile behavior of angle-plied (AP) suture AF-only biocomposites (+30° and -30°)(VF 0.46, n=5) vs. silk biocomposite (±30°)(VF 0.42). Also shown are: an angle-plied collagen biocomposite (FVF 0.35), circumferential bovine tail AF, and circumferential ovine thoracic AF specimens. The Angle-plied (AP) suture - is equivalent to suturing two laminates together using out-of-plain silk fiber sutures (radial fibers only in the AF). The silk biocomposite is a single laminate consisting of ±30° fibers.

[0109] FIG.13A shows a line graph presenting the mechanical behavior (stress (MPa) vs. strain (mm / mm)) of the AF-NP biomimetic construct with radial fibers (triangles) under compression (upper graph) compared with bovine native IVD (black solid line) (upper graph). The addition of the radial fibers demonstrated stiffer behavior and more similar to the bovine native IVD. The upper graph also shows biomimetic NP under confined (blue) or unconfined compression (green), and a biomimetic AF-NP construct without radial fibers under semi-confined (i.e., confined by AF rings, red) compression. These biomimetic NPs (blue and green lines) and the AF-NP construct without radial fibers are also seen in the lower graph.

[0110] FIG.13B shows an illustration of a self-designed apparatus for shear test, and a bar graph presenting the measured shear forces (N) between the AF and NP in AF-NP biomimetic construct with and without radial fibers. The addition of the radial fibers significantly increases the shear forces, therefore strengthening the binding between the AF and NP and contributing to its structural integrity / durability. FIG.14 shows an illustration of the IVD computational model, including from Left-to right: FE model constructs L3-L4 FSU-FE Model with facets and ligaments, IVD model, IVD model with AF structural fiber representation, AF structural fiber Network, AF-NP fibers structural Network.

[0111] FIGs.l5A-15E compression and moment-rotation of biomimetic IVD computational model.

[0112] FIG.15A shows a line graph presenting a load-displacement curve of the biomimetic with radial fibers model compared with the IVD validated model and in vitro measurements.

[0113] FIG.15B shows a bar graph presenting Intra-discal pressure (IDP) of the biomimetic with radial fibers compared with the validated model and in vitro measurements.

[0114] FIGs.l5C-15E shows a line graph presenting physiological loading modes of the biomimetic IVD with radial fibers vs. validated model results and in vitro measurements, for Flexion and extension (FIG.15C), Eateral bending (FIG.15D), and Torsion (FIG.15E).

[0115] FIGs.l6A-16B show stress maps presenting internal and local influence of the inter- structural radial fibers on the different parts of the IVD.

[0116] FIG. 16A presents the inner effect of adding AF-NP radial fibers on the IVD matrix under left bending and compression

[0117] FIG. 16B presents the inner effect of adding AF-NP radial fibers on the circumferential fibers under right torsion and right bending.

[0118] DETAILED DESCRIPTION

[0119] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0120] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, governs.

[0121] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise, “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the stated object, unless the context clearly dictates otherwise. By way of example, “a fiber” means one or more fibers.

[0122] The term “may” refer to an optional / possible approach / possibility, but not a requirement. The term “can” refer to a permissible / plausible approach / possibility, but not a requirement.

[0123] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0124] As used herein, the term "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass deviations / variations of ±20% or in some embodiments ±10%, or in some embodiments ±5%, or in some embodiments ±1%, or in some embodiments ±0.1% from the specified value, as such deviations are appropriate to perform the disclosed methods.

[0125] As used herein, the terms “essentially” and “substantially” are synonymous and when referring to a stated material such as a composition, a substance, and the like, is meant to encompass variations of in some embodiments, ±0.1%, or in some embodiments, ±1%, or in some embodiments, ±2%, or in some embodiments, ±5% from a stated amount, as such variations / deviations are appropriate to perform the disclosed methods. According to some embodiments, the term “essentially devoid of’ may refer to a stated material as either entirely absent or present in a residual amount, such as less than 5%, or less than 2%, or less than 1%, or less than 0.1% are present. Each possibility is a separate embodiment. According to some embodiments, the term “substantially made of’ may refer to a stated material as either entirely present or absent in a neglectable amount, such as more than 95%, or more than 98%, or more than 99%, or more than 99.9% are present. Each possibility is a separate embodiment. As used herein, the term “comprising” is synonymous with the terms "including," "containing," or "characterized by," and is inclusive or open-ended i.e. does not exclude additional, unrecited elements. According to some embodiments, the term comprising may be replaced with the term with the term “consisting of’ which excludes any element, step, or ingredient not specified in the claim. According to some embodiments, the term comprising may be replaced with the term “consisting essentially of’ which limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention.

[0126] As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, “alleviate”, and “inhibit” are used interchangeably.

[0127] As used herein, the terms “enhanced”, “increased”, “elevated” are used interchangeably.

[0128] As used herein, the term “plurality” may refer to at least two (two or more). According to some embodiments, the plurality includes at least two, or at least three, or at least five, at least about ten, or about twenty, or more. Each possibility is a separate embodiment.

[0129] As used herein, the terms "subject", "patient" or "individual" may be used interchangeably and generally refer to a human suffering from, or at risk of developing, orthopedic conditions related to the knee meniscus or invertebrate disk (IVD), including, for example, but not limited to injuries, inflammation, and degeneration.

[0130] As used herein, the term "treating" refers to an approach for obtaining beneficial or desired results, including clinical results in treating orthopedic conditions related to the knee meniscus or invertebrate disk (IVD). Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of disease, stabilization of the state of disease, prevention of deterioration of the disease or condition, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total).

[0131] The terms “disease”, “condition” and “disorder” may be used interchangeably.

[0132] The term “treatment” as used herein refers to both therapeutic treatment and prophylactic or preventative measures, including for example medical intervention in the form of pharmaceuticals or surgery, including for example tissue substitution or implant transplantation. In some embodiments, those in need of treatment include those already having a disorder as well as those in which the disorder is to be prevented.

[0133] As used herein, the term “administering” includes routes of administration which allow the compositions of the invention to perform their intended function including, for example, but not necessarily limited to, orthopedic surgery.

[0134] According to some embodiments, provided herein are biomimetic composite(s) tissue analogue / implant with radial fibers, and method(s) of fabricating the same, for human tissue substitution / replacement of a knee meniscus or intervertebral disc (IVD).

[0135] In some embodiments, the fibers’ orientation in the biocomposite are configured to simulate the main directions of the human tissue, including the transverse / radial fibers that provide mechanical shielding / structural integrity to the composite(s) / implant(s), as also evident, in some embodiments, by computational results of a biomimetic composite model that uses a structure-function relationships of the human tissue (i.e., the knee meniscus or the intervertebral disc (IVD)).

[0136] As used herein, the term “biomimetic” relate to a biocomposite or a computational model of a composite, and either way refers to a composite including structural features that simulate / mimic corresponding features of the target tissue intended for sub stituti on / repl acement .

[0137] As used herein, the term “biocomposite” or “construct” may be interchangeable while referring to a composite suitable for functioning as an implant, or part of it, for tissue substitution / replacement at least with respect of having favorable properties that make it physiologically compatible, such properties may include, for example, but are not limited to: having low immunogenic properties, and proper biostability under physiological conditions.

[0138] According to one aspect, there is provided a wedge-shaped cross-plied (CP) biocomposite laminates / layered tissue analogue / implant comprising hydrogel matrix reinforced with longitudinal and transverse / radial fibers for human tissue substitution / replacement of a medial knee meniscus and / or lateral knee meniscus. In some embodiments, the transverse / radial fibers provide the CP biocomposite laminates implant with structural shielding / integrity characterized by mechanical durability / (tolerance or resistance).

[0139] According to another aspect, there is provided an annulus fibrosus - nucleus pulposus (AF-NP) disc-shaped biocomposite construct tissue analogue / implant comprising a plurality of circumferential rings and an NP core made of hydrogel matrix reinforced with radial fibers for human tissue substitution / replacement of an intervertebral disc (IVD), wherein each circumferential ring comprises AF lamellae made of hydrogel matrix reinforced with angle- plied (AP) fibers, and wherein the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reinforcing the disc-shaped biocomposite.

[0140] In some embodiments, the transverse / radial fibers provide the AF-NP biocomposite construct / laminates implant with structural shielding / integrity characterized by mechanical durability / (tolerance or resistance).

[0141] According to additional embodiments, the CP biocomposite or the AF-NP biocomposite, comprises a quantitatively controlled amount / volume of fibers. Each possibility is a separate embodiment.

[0142] In some embodiments, the structural shielding comprises introducing to the biocomposite of the invention a quantitatively controlled amount / volume of fibers.

[0143] In some specific embodiments, the quantitatively controlled amount / volume of fibers comprises a total volume fraction (total VF) consisting of radial fibers volume and longitudinal fibers volume of at least about 20% of the laminate hydrogel matrix (v / v).

[0144] According to some related embodiments, the quantitatively controlled amount / volume of fibers comprises a total volume fraction (VF) consisting of radial fibers volume and longitudinal fibers volume of between about 10% and about 80% (for example, between 25% and about 65%, or between about 35% and about 60%), of the laminate hydrogel matrix (v / v). Each possibility is a separate embodiment. In some specific embodiments, the quantitatively controlled amount / volume of fibers comprises a total volume fraction (total VF) consisting of radial fibers and circumferential AP fibers of at least about 20% of the AF lamellae hydrogel matrix (v / v).

[0145] According to some related embodiments, the quantitatively controlled amount / volume of fibers comprises a total volume fraction (VF) consisting of radial fibers and AP circumferential fibers of between about 10-80%, 25% and about 65%, or between about 35% and about 60%, of the AF lamellae hydrogel matrix (v / v). Each possibility is a separate embodiment.

[0146] In some embodiments, the mechanical durability of the biocomposite is more similar to human meniscus or bovine meniscus, compared to biocomposite lacking radial fibers. Each possibility is a separate embodiment.

[0147] In some embodiments, the mechanical durability of the biocomposite is improved compared to biocomposite lacking radial fibers.

[0148] In some embodiments, the mechanical durability comprises tolerance or resistance to physically applied stress or to computationally simulated stress. Each possibility is a separate embodiment.

[0149] In some embodiments, the mechanical durability comprises tolerance or resistance to mechanical stress applied on the biocomposite. Each possibility is a separate embodiment.

[0150] In some embodiments, the mechanical durability comprises tolerance or resistance to simulated stress on a biomimetic model composite. Each possibility is a separate embodiment.

[0151] In some related embodiments, the biomimetic composite modeling comprises a structure-function relationships of the human tissue, wherein the modeling comprises the main direction(s) of the fibers of the human tissue, corresponding to the radial and / or longitudinal fibers of the cross-plied (CP) biocomposite. Each possibility is a separate embodiment.

[0152] In some related embodiments, the biomimetic composite modeling comprises a structure-function relationships of the human tissue, wherein the modeling comprises the main direction(s) of the fibers of the human tissue, corresponding to the radial and / or AP circumferential fibers of the AF-NP composite construct. Each possibility is a separate embodiment. In some specific embodiments, the structure-function relationships model of the human tissue comprises the meniscus in a knee joint model.

[0153] In some specific embodiments, the structure-function relationships model of the human tissue comprises the whole IVD as a functional spinal unit.

[0154] According to further specific embodiments, the mechanical durability comprises tolerance or resistance towards mechanically applied or computational simulated stretching, compression, and / or physiological moments. Each possibility is a separate embodiment.

[0155] According to some embodiments, the structural shielding characterized by mechanical durability comprises at least one mechanical behavior / property selected from the group consisting of structural width change (%), stress (MPa) vs. strain (mm / mm), Poisson’s ratio, tensile modulus (MPa), ultimate strains (mm / mm), UTS (MPa), toughness (MJ / m3), shear force (N), and rotation (degrees), or any combination thereof. Each possibility is a separate embodiment.

[0156] In some embodiments, the hydrogel comprises, or essentially consists of, one or more materials, including, a protein(s), a polysaccharide(s), a polypeptide(s), a polylipid(s), a synthetic polymer(s), or any combination thereof. Each possibility is a separate embodiment.

[0157] In some embodiments, the hydrogel comprises, or essentially consist of, one or more type(s) of hydrogel selected from alginate hydrogel, Hyaluronic acid hydrogel, PEG hydrogel, cellulose hydrogel, Agar hydrogel, and Interpenetrating network (IPN) hydrogel, or any combination thereof. Each possibility is a separate embodiment.

[0158] In some embodiments, the hydrogel comprises, or essentially consists of, alginate hydrogel.

[0159] In some embodiments, the fibers comprise, or essentially consist of, one or more type(s) of fiber(s) including, woven fibers, twisted fibers, braided fibers, knitted fibers, tie fibers, and sutured fibers, or any combination thereof. Each possibility is a separate embodiment.

[0160] In some embodiments, the fibers comprise, or essentially consist of, tie fibers. In some embodiments, the fibers comprise, or essentially consist of, one or more material selected from a protein(s), a polysaccharide(s), a polypeptide(s), a polylipid(s), a synthetic polymer(s), or any combination thereof. Each possibility is a separate embodiment.

[0161] In some embodiments, the fibers comprise, or essentially consist of, one or more type(s) of fibers selected from silk, silk-made fibroin fibers, collagen fibers, and cellulose fibers, or any combination thereof. Each possibility is a separate embodiment.

[0162] In some embodiments, the fibers comprise, or essentially consist of, silk or silk-fibroin. Each possibility is a separate embodiment.

[0163] In some embodiments, the silk is derived from spider and / or silkworm. Each possibility is a separate embodiment. In some embodiments, the silk-made fibroin fibers are derived from Bombyx mori.

[0164] I. soft orthopedic biomimetic composite implants with radial fibers: cross-plied

[0165] (CP) biocomposite laminate with radial fibers

[0166] The present invention provides cross-plied (CP) biomimetic composite including radial fibers, structural and functional characterization thereof, and methods of fabricating such CP biocomposite laminates.

[0167] According to an aspect of the disclosure, there is provided a cross-plied (CP) biocomposite laminates / layered tissue analogue / implant comprising hydrogel matrix reinforced with longitudinal and transverse / radial fibers for human tissue substitution / replacement of a medial knee meniscus and / or lateral knee meniscus, wherein an orientation of the fibers in the biocomposite simulates main directions of the meniscus fibers, and wherein the transverse / radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability / (tolerance or resistance).

[0168] According to some embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers, wherein said fiber orientation comprises: a. the transverse / radial fibers of the implant projecting in a direction from about a center towards a perimeter of a c-shaped meniscus; and / or b. the longitudinal fibers of the implant projecting in a circumferential direction along the perimeter of the c-shaped meniscus. In some embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers; In some embodiments, the longitudinal fibers of the CP biocomposite are at about 0°; In some embodiments, the transverse / radial fibers of the CP biocomposite are at about 90°.

[0169] According to an aspect of the disclosure, there is provided a cross-plied (CP) biocomposite laminates / layered tissue analogue / implant comprising hydrogel matrix reinforced with longitudinal (0°) and transverse / radial (about 90°) fibers for human tissue substitution / replacement of a medial knee meniscus and / or lateral knee meniscus, wherein an orientation of the fibers in the biocomposite simulates main directions of the meniscus fibers, and wherein the transverse / radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability / (tolerance or resistance).

[0170] According to specific embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers, wherein said fiber orientation comprises: the transverse / radial fibers of the implant (about 90°) oriented essentially on a horizontal axis about perpendicular to a vertical femur-tibia axis, projecting in a direction from about a center towards a perimeter of a c-shaped meniscus (about 90°).

[0171] According to specific embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers, wherein said fiber orientation comprises: the longitudinal fibers (0°) of the implant oriented essentially on a same horizontal axis, about perpendicular (0°) to the radial fibers, projecting in a circumferential direction along the perimeter of the c-shaped meniscus.

[0172] In some embodiments, the orientation of the fibers in the CP biocomposite simulates main directions of the meniscus fibers, wherein said fiber orientation comprises: a. the transverse / radial fibers (about 90°) of the implant oriented essentially on a horizontal axis about perpendicular to a vertical femur-tibia axis, projecting in a direction from about a center towards a perimeter of a c-shaped meniscus (about 90°); and / or b. the longitudinal fibers (0°) of the implant oriented essentially on a same horizontal axis, about perpendicular (0°) to the radial fibers, projecting in a circumferential direction along the perimeter of the c-shaped meniscus. Each possibility is a separate embodiment. Each possibility is a separate embodiment. Reference is made to FIG. 1A showing a schematic representation of the circumferential and radial / transverse fibers of the meniscus, corresponding to the longitudinal and radial / transverse fibers orientations in the cross-plied (CP) biocomposite laminate of the invention. FIGs. 1B-1C show a schematic representation of the knee joint, meniscus, and a “transplanted” biomimetic meniscus implant. FIGs. 1D-1E present a wedge-shaped meniscus construct, fabrication thereof, and the orientation of its fibers.

[0173] In some embodiments, the CP biocomposite laminate comprises a quantitatively controlled amount / volume of fibers; wherein said quantitatively controlled amount / volume of fibers comprises a total volume fraction (VF) consisting of radial fibers volume and longitudinal fibers volume of between about 42% and about 55% of the laminate hydrogel matrix (v / v), providing the biocomposite with similar mechanical durability / behavior to the mechanical durability / behavior of human meniscus at least under uniaxial tension / stretching.

[0174] In some embodiments, the CP biocomposite laminate comprises a quantitatively controlled amount / volume of fibers, wherein the quantitatively controlled amount / volume of fibers comprises a range of ratios of transverse / radial fibers volume to longitudinal fibers volume of between about 1 :3 (25%: 75% transverse / radial: longitudinal vf(0°)) and about 4: 1 (80%: 20% transverse / radial: longitudinal vf(0°)), thereby providing mechanical resistance / shielding, at least under stretching. In some embodiments, the ratio may be about 1 : 1.5 to about 10: 1 (transverse / radial : longitudinal vf).

[0175] In some embodiments, the CP biocomposite laminate comprises a quantitatively controlled amount / volume of fibers, wherein the quantitatively controlled amount / volume of fibers comprises a range of ratios of transverse / radial fibers volume to longitudinal fibers volume of between about 4:6 (40%: 60% transverse / radial: longitudinal vf(0°)) and about 9: 1 (90%: 10% transverse / radial: longitudinal vf(0°)), thereby providing mechanical resistance / shielding, at least under stretching. In some embodiments, the ratio may be about 1 : 1.5 to about 15: 1 (transverse / radial: longitudinal vf(0°)).

[0176] In some embodiments, the CP biocomposite laminate comprises a quantitatively controlled amount / volume of fibers, wherein the quantitatively controlled amount / volume of fibers comprises a range of ratios of transverse / radial fibers volume to longitudinal fibers volume of between about 1 :3 (25%: 75% transverse / radial: longitudinal vf(0°)) and about 9: 1 (90%: 10% transverse / radial: longitudinal vf(0°)), thereby providing mechanical resistance / shielding, at least under stretching.

[0177] Reference is made to FIG. 2 A showing average mechanical behavior and properties of the biomimetic biocomposite laminates. FIG. 2A shows a line graph presenting the average mechanical behavior, measured as stress (MPa) vs. strain (mm / mm), of the longitudinal and CP laminates and the human meniscus. The figure shows a range of behaviors of longitudinal and cross-plied biocomposites laminates in comparison to native human meniscal tissue.

[0178] In some embodiments, the fiber volume of longitudinal fibers vf(0°) is within the range of about 7-50% (such as, 10-40%, 12%-36%) for a total fiber volume fraction (VF) of about 50%±5.0%.

[0179] In some embodiments, the structural shielding comprises an increased ratio of transverse / radial: longitudinal fibers volume thereby reducing the change in average behavior of stress (MPa) vs. strain (mm / mm) under stretching.

[0180] In some embodiments, the structural shielding is characterized by a non-linear increase in tensile modulus (MPa) under stretching, compared to a linear increase of laminates lacking radial fibers, thereby providing mechanical durability / resistance under uniaxial tension / stretching.

[0181] In some embodiments, the biocomposite is characterized by decreased ultimate strain under stretching, compared to laminates lacking radial fibers.

[0182] In some embodiments, the biocomposite is characterized by an increased UTS (MPa) under stretching, compared to laminates lacking radial fibers.

[0183] In some embodiments, the biocomposite is characterized by similar or increased toughness (MJ / m3) under stretching, compared to laminates lacking radial fibers, thereby providing mechanical durability.

[0184] In some embodiments, the mechanical durability is compared to laminates lacking radial fibers.

[0185] Reference is now made to FIGs. 2B-2E. FIG. 2B shows dot graph presenting the average mechanical properties, measured as tensile modulus (MPa) as a function of the fiber fraction in the longitudinal direction. FIGS. 2C-E show line graphs of ultimate strains (mm / mm) (FIG. 2C), UTS (MPa) (FIG. 2D), and toughness (MJ / m3) (FIG. 2E), of the longitudinal and CP laminates versus the fiber volume fraction in the tensile direction vf (0°) (i.e., the longitudinal fibers). The figures exemplify the effect of the addition of radial fibers on biocomposite laminates (structural shielding) for different fractions of fibers in the longitudinal direction.

[0186] In some embodiments, the mechanical durability comprises reduction in width change under stretching relative to laminate without radial fibers.

[0187] In some embodiments, the mechanical durability comprises preservation of transverse deformation, under stretching relative to laminate without radial fibers.

[0188] Reference is now made to FIGs. 3A-3C presenting mechanical width change and FIGs 7A-7C presenting computational width change.

[0189] In some embodiments, the CP biocomposite is characterized by reduced change in stress (MPa) vs. strain (mm / mm) under stretching compared with laminate lacking radial transverse / fibers, thereby providing mechanical durability / resi stance.

[0190] In some embodiments, the CP biocomposite is characterized by reduction of Poisson’s ratio under stretching.

[0191] Reference is now made to FIGs. 3D-3E, showing graphs of the average mechanical behavior and Poisson’s ratios of the longitudinal (FIG. 3D) and CP laminates (FIG. 3E) up to failure.

[0192] In some embodiments, the transverse / radial fibers reduce pressure on an articular cartilage under compression.

[0193] In some embodiments, the transverse / radial fibers distribute stress within the implant / tissue.

[0194] Reference is now made to FIGs. 4A-C, FIG. 5 and FIG. 6, showing the 3D Finite Elements (FE) model of the knee joint, and the meniscal composite models that were generated, stress maps presenting max principal stresses of the meniscal composite models, and stress distribution maps presenting the contact pressure / stress distribution on the tibial cartilage, respectively. FIG. 4A shows illustration presenting 3D Finite Elements (FE) model of the knee joint, including the femur and tibia bones, femoral and tibial cartilage, ligaments (ACL, MCL, LCL, PCL), and the medial and lateral menisci (posterior view). The reference point on which the axial displacement was applied (Z-direction), and the fixation of the bottom of the tibia are presented. (ACL, anterior cruciate ligament; PCL, posterior cruciate ligament; MCL, anterior cruciate ligament; LCL, anterior cruciate ligament. FIG. 4B shows illustration presenting a refined composite structure of the meniscus based on the main body of the medial meniscus (Matrix), including circumferential fibers (red), radial fibers (blue) and both circumferential fibers and radial fibers combined (green) and FIG. 4C shows a line graph presenting a validation of the FE knee model against experimental results. The graph shows similar total contact area on the tibial cartilage for five meniscal composite models: Circ, Rad and Circ-Rad, and two control composites Iso and Coll. FIG. 5 shows the max principal stresses in the components of the Circ composite, Rad composite, and Circ-Rad composite models. In the Circ composite model, there is stress concentration in the middle of the centralposterior portion of the circumferential medial and lateral fibers (black arrows). FIG. 6 shows stress distribution maps presenting the contact pressure / stress distribution on the tibial cartilage with the Circ composite, Rad composite, and Circ-Rad composite meniscus models under similar axial compression of (i) 478-524 N, (ii) 990-995 N, and (iii) under equal axial displacement of 0.91 mm. The addition of radial fibers reduces the stress concentration on the tibial articular cartilage in comparison to Circ composite model.

[0195] According to some embodiments, provided herein is a method for fabricating crossplied (CP) biocomposite laminates / layered meniscal tissue analogue / implant, the method comprises: a. wrapping fibers around each of a plurality of 3D frame in longitudinal (0°) and / or transverse / radial (about 90°) orientation; b. stacking the plurality of 3D frames containing the longitudinally and / or radially oriented fibers in CP orientation consisting of alternating 0° and about 90° directions; c. placing the stack in a mold and filling the mold with hydrogel solution; d. cross-linking the hydrogel; thereby, obtaining a cross-plied (CP) biocomposite laminates comprising longitudinal (0°), and transverse / radial (about 90°) fibers at orientation that simulates main directions of the meniscus tissue fibers.

[0196] In some embodiments, the CP biocomposite comprises a wedge shape; In some embodiments, the CP biocomposite comprises a wedge triangular shape.

[0197] In some embodiments, the cross-linking of the hydrogel with the fibers comprises chemical, biochemical, and / or physical -based cross-linking.

[0198] CP biocomposite

[0199] The herein disclosed biocomposite laminates demonstrated mechanical behavior in the same range as the human meniscus tissue. In some embodiments, adding a transverse reinforcement allows the reduction of the fiber volume fraction in the stretching direction and obtains a similar mechanical behavior (FIG. 2A). In some embodiments, the mechanical properties of the laminates are affected by the presence of fibers, and the transverse fibers contribute to the material stiffness and strength without affecting the toughness but reduce strains. Further, in some embodiments, adding fibers about perpendicular to the tensile direction leads to smaller transverse strains and, consequently, smaller Poisson ratios.

[0200] The biocomposite laminates of the present invention demonstrated Poisson’s ratios in the same range as the meniscus tissue. The reported Poisson’s ratio values for the meniscus range from 0.52 to 2.13 for circumferential samples and 0.28 to 1.50 for radial samples. Apart from the significant changes in the Poisson’s values during the stretching, the presence and amount of radial fibers in a specific meniscal specimen are possibly different, leading to a wide range of Poisson’s ratios.

[0201] The meniscus can transmit and distribute loads across the joint, even with large radial tears that disrupt the circumferential fibers. That is, even when many circumferential fibers are tom, the meniscus can function to a certain extent. The Rad Composite meniscal model only had a 0.05 volume fraction of circumferential fibers. However, it showed similar contact results, was resistant to width changes, and produced minimal contact pressure on the tibial cartilage. Similarly, as exemplified herein below, CP laminates demonstrated an advantageous ability to maintain their mechanical performance in the loading direction, even with only a third of the fiber VF in this direction (FIGs. 2B-2E) thereby providing “structural shielding” characterized by the maintenance of strength, stiffness, and toughness and a strain decrease of the laminates. Accordingly, provided herein are biomimetic biocomposite laminates and fabrication method thereof, and meniscus structural models, including radially oriented fibers for novel meniscus substitutes. Provided herein is a biocomposite including fibroin fiber-reinforced alginate hydrogel with fibers structurally mimicking materials and FE models of the meniscus. A composite model containing circumferential and radial fibers was found to be an adequate structural representation of the meniscus tissue in the knee joint. As exemplified herein, the advantageous radially oriented fibers, have a role in preventing excessive transverse deformations, which is important for protecting the articular cartilage from considerable contact pressures, and for stress distribution within the tissue.

[0202] Moreover, as exemplified herein, the biocomposite of the present invention simulates the meniscal mechanical behavior by including an advantageous quantitatively controlled amount / volume of fibers and fiber directions / orientations and or distribution, that simulate the meniscal fiber orientation. The biocomposite’s internal structure thus affects the deformations of the meniscus tissue.

[0203] Finite elements (FE) model

[0204] An FE model of the knee was generated containing biomimetic representations of the meniscus based on the internal meniscal structure. To this aim, structural models of the meniscus as composite material reinforced with fibers were created and tested under axial compression in the knee joint, as detailed in Example 1 herein below.

[0205] The contact properties of the meniscal fibroin composite models were within the range known in the art, including finite element analyses (FEA). Furthermore, in some embodiments, the composite models demonstrated better results in all cases than an isotropic meniscal model ( FIG. 7C).

[0206] The max principal stresses were distributed between the components of the composite meniscal models. Thus, the composite meniscal model allows some of the compressive forces to be transformed into hoop tensile forces, similar to the mechanical function of the native meniscus. In regions where the circumferential fibers were not stretched, there is an increase in the stresses in the radial fibers (FIG. 5). Where both the circumferential and radial fibers carry minor stresses, there is an increase in the stresses in the matrix. The matrix had to carry the most significant stresses without the radial fibers, as obtained in the Circ Composite model. These results indicate that all the meniscal components are essential for proper function. Testing the stresses in the components of the meniscal composite allows the examination of regions that are more prone to failure and may lead to meniscal injury. FIG. 10 shows a schematic illustration of two types of common meniscal tears, longitudinal and radial. The majority of meniscal tears are in the second third of the meniscus along the entire circumference. In this region, greater stresses were found on the circumferential fibers in the Circ Composite meniscus model (FIG. 5). One of the most common tear patterns in young populations ( < 40 years) and athletes is the longitudinal tear, which can propagate and become a bucket handle tear. The structural factor that resists the propagation is the radial fibers. Without the radially oriented fibers, the max principal stresses of the Circ Composite model were concentrated at the central-posterior circumferential fibers, and the matrix had to carry greater stresses.

[0207] The max principal stresses in the circumferential fibers of the lateral meniscus were distributed less uniformly than in the medial meniscus and were smaller in the inner circumference (Circ-Rad composite) (FIG. 5). Since the radial fibers adjacent to the inner boundary do not carry significant loads, the matrix has to withstand more considerable stresses. The current disclosure shows that in some embodiments the inner circumference may be more prone to failure, especially in the lateral meniscus. In some embodiments, radial tears are the most common tear pattern in the lateral meniscus. Thus, according to some embodiments, the current disclosure demonstrates the importance of the radial fibers in distributing the stresses in the meniscus tissue and / or in the biocomposite. Furthermore, adding radial fibers may be crucial to knee modeling and the meniscal substitute design.

[0208] According to some embodiment, the three fibroin-based composite models, Circ, Rad, and Circ-Rad, presented similar total mean contact pressure on the tibial cartilage (Table 5), the pressures were dispersed differently (FIG. 6). The PCP and the area in which it operates were larger using the Circ Composite model than the Rad and Circ-Rad models, and the value was the smallest for the Rad Composite. The total contact area was similar, but case subjects with incident symptomatic knee OA risk had maximum articular contact stress of 0.54 ± 0.77 MPa (mean ± SD) higher and more area in contact at higher stress levels than for control subjects. Advantageously, these results indicate that the radial fibers are essential for distribution and reducing the contact pressures on the tibial cartilage.

[0209] The Circ Composite model demonstrated a significant width increase compared to the other composite models, a difference related to the meniscal Poisson’s ratio. In some embodiments, collagen fibers architecture of biological materials contributes significantly to their Poisson’s ratio. Thus, the differences in the internal structure of the composite meniscal models lead to different width changes.

[0210] In some embodiments, vertical deformations occur during the stretching of the herein disclosed biocomposite laminates. The width differences of the meniscal models is inversely proportional to those observed in the fibroin biocomposite laminates tensile tests. The transverse strain or width decreased during the stretching of longitudinal laminates, corresponding to the width increase in compression of the Circ Composite meniscal model. In contrast, the CP laminates demonstrated minor negative transverse strain, representing a smaller width change, as obtained for the Rad and Circ-Rad Composite meniscal models. This exemplifies the different width changes of the composite models and indicates that the radial fibers prevent excessive deformations under compression in the knee joint.

[0211] Meniscus structure

[0212] The knee meniscus tissue is subjected to both tension and compression and, thus, contains relatively large amounts of collagen and proteoglycans. The current disclosure demonstrated that the radial section consists of many collagen fiber bundles oriented circumferentially, having an inhomogeneous cross-section (FIG. 9A (i), (v)). In some regions, the bundles form a larger fiber bundle (fascicle), which appears to have a more circular crosssection (480pm) ( FIG. 9A (i)). bovine meniscus includes collagen fascicles with a similar diameter range of 80-420pm. Tie fibers with twisted or wavy shapes were observed at the boundary of the fascicle and fiber bundles cross-section. These fibers penetrate from the lamellar layer, tying and weaving the circumferential bundles ( FIG. 9A (v)). In some embodiments, the considerable amount of tie fibers indicates that they have a mechanical role in meniscus functionality. In some related embodiments, interspersing random fibers within an aligned fiber composite promotes strain transfer to nearby disconnected fibers and suggests that the radial tie fibers encourage tear tolerance in the meniscus.

[0213] As exemplified herein below (Example 2), gaps exist between bundles of fibers and pores structures inside the fibers in the radial section of the control sample ( FIG. 9A (ii)). These structures also exist in the human meniscus. On the other hand, the porous structure was not visible in the partially digested sample, suggesting that the extracellular matrix was partially removed in the digestion process. Instead, the fiber bundles were separated by larger gaps ( FIG. 9A (vii)). Additionally, thin and delicate fibrils resembling cross-bridges-like structures were observed inside the gaps between two adjacent fibers. Histological examination of the architecture of radial tie fibers in the meniscus revealed they vary by location within the tissue, and their number and size increase from the anterior to the posterior region.

[0214] II. soft orthopedic biomimetic composite implants with radial fibers: AF-NP biomimetic composite construct with radial fibers

[0215] According to some embodiments, there are provided AF-NP intervertebral disc (IVD) biomimetic composite including radial fibers, structural and functional characterization thereof, and methods of fabricating such IVD biocomposite construct.

[0216] According to an aspect of the disclosure, there is provided an annulus fibrosus - nucleus pulposus (AF-NP) disc-shaped biocomposite construct tissue analogue / implant comprising a plurality of circumferential rings and an NP core made of hydrogel matrix reinforced with transverse / radial fibers for human tissue substitution / replacement of an intervertebral disc (IVD), wherein each circumferential ring comprises AF lamellae made of hydrogel matrix reinforced with angle-plied (AP) fibers, and wherein the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reinforcing the disc-shaped biocomposite; and wherein an orientation of the fibers in the biocomposite simulates main directions of the IVD fibers, and wherein the radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability / ( tolerance or resistance.

[0217] According to specific embodiments, the orientation of the fibers in the AF-NP biocomposite simulates the main directions of the IVD fibers, wherein said fiber orientation comprises: a. the radial fibers of the implant projecting in a direction from about a center of the NP core towards a perimeter of the disc / AF rings; and / or b. the angle-plied (AP) fibers projecting in a circumferential direction relative to the horizontal axis.

[0218] In some embodiments, the orientation of the fibers in the AF-NP biocomposite construct simulates main directions of the IVD fibers; In some embodiments, the angle-plied (AP) circumferential fibers of the AF-NP biocomposite construct are at about ±30°; In some embodiments, the transverse / radial fibers of the AF-NP biocomposite construct are at about 90°. According to an aspect of the disclosure, there is provided an annulus fibrosus - nucleus pulposus (AF-NP) disc-shaped biocomposite construct tissue analogue / implant comprising a plurality of circumferential rings and an NP core made of hydrogel matrix reinforced with transverse / radial (about 90°) fibers for human tissue substitution / replacement of an intervertebral disc (IVD), wherein each circumferential ring comprises AF lamellae made of hydrogel matrix reinforced with angle -plied (AP) fibers (about ±30°), and wherein the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reinforcing the disc- shaped biocomposite; and wherein an orientation of the fibers in the biocomposite simulates main directions of the IVD fibers, and wherein the radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability / (tolerance or resistance.

[0219] According to specific embodiments, the orientation of the fibers in the AF-NP biocomposite simulates main directions of the IVD fibers, wherein said fiber orientation comprises: the radial fibers (about 90°) of the implant oriented essentially along a horizontal axis about perpendicular 90° to a vertical spinal axis, projecting in a direction from about a center of the NP core towards a perimeter of the disc / AF rings.

[0220] According to specific embodiments, the orientation of the fibers in the AF-NP biocomposite simulates main directions of the IVD fibers, wherein said fiber orientation comprises: the angle-plied (AP) (about ±30°) fibers arranged essentially parallel to / along the vertical spine axis within the circumferential AF lamellae rings, about perpendicular to the radial fibers, projecting in a circumferential direction of about ±30° relative to the horizontal axis.

[0221] In some embodiments, the orientation of the fibers in the AF-NP biocomposite construct simulates main directions of the IVD fibers, said fibers orientation comprises: a. the radial fibers of the implant projecting in a direction from about a center of the NP core towards a perimeter of the disc / AF rings; and / or b. the angle-plied (AP) (about ±30°) fibers arranged essentially parallel to / along the vertical spine axis within the circumferential AF lamellae rings, about perpendicular to the radial fibers, projecting in a circumferential direction of about ±30° relative to the horizontal axis. Each possibility is a separate embodiment. Reference is now made to FIG. 11A showing a schematic representation of the orientation of circumferential and radial fibers of the human IVD, corresponding to fibers orientations in the AF-NP biocomposite construct of the invention, and to FIGs. 11B-11C presenting the fabrication process of AF-NP construct with radial fibers from fabricated AF laminate with sutures, testing of the construct, and of a bovine IVD.

[0222] In some embodiments, wherein the mechanical durability comprises increased stress (MPa) vs. strain (mm / mm) under compression.

[0223] In some embodiments, the mechanical durability comprises increased shear force (N) between the circumferential AF lamellae rings and the NP core under compression providing structural integrity / shielding.

[0224] In some embodiments, the mechanical durability comprises increased shear force (N) between the circumferential AF lamellae rings and the NP core under compression providing structural integrity / shielding.

[0225] In some embodiments, the structural shielding comprises reduced delamination between the AF lamellae, compared with biomimetic model lacking radial fibers, thereby providing mechanical durability (AF-NP biocomposite)

[0226] In some embodiments, the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reinforcing structural integrity of the disc-shaped biocomposite.

[0227] In some embodiments, the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reducing delamination between the AF lamellae, compared with biomemetic model lacking radial fibers, and providing mechanical durability.

[0228] FIG. 12 shows a line graph presenting the tensile behavior of angle-plied (AP) suture AF-only biocomposites (+30° and -30°)(VF 0.46, n=5) vs. silk biocomposite (±30°)(VF 0.42). Also shown are: an angle -plied collagen biocomposite (FVF 0.35), circumferential bovine tail AF, and circumferential ovine thoracic AF specimens. The Angle-plied (AP) suture - is equivalent to suturing two laminates together using out-of-plain silk fiber sutures (radial fibers only in the AF). The silk biocomposite is a single laminate consisting of ±30° fibers. FIG.13A shows a line graph presenting the mechanical behavior (stress (MPa) vs. strain (mm / mm)) of the AF-NP biomimetic construct with radial fibers (triangles) under compression (upper graph) compared with bovine native IVD (black solid line) (upper graph). The addition of the radial fibers demonstrated stiffer behavior and more similar to the bovine native IVD. The upper graph also shows biomimetic NP under confined (blue) or unconfined compression (green), and a biomimetic AF-NP construct without radial fibers under semi-confined (i.e., Confined by AF rings, red) compression. These biomimetic NPs (blue and green lines) and the AF-NP construct without radial fibers are also seen in the lower graph. FIG.13B shows an illustration of a self-designed apparatus for shear test, and a bar graph presenting the measured shear forces (N) between the AF and NP in AF-NP biomimetic construct with and without radial fibers. The addition of the radial fibers significantly increases the shear forces, therefore strengthening the binding between the AF and NP and contributing to its structural integrity / durability .

[0229] FIGs.l6A-16B show stress maps presenting internal and local influence of the inter- structural radial fibers on the different parts of the IVD. FIG. 16A presents the inner effect of adding AF-NP radial fibers on the IVD matrix under left bending and compression and FIG. 16B presents the inner effect of adding AF-NP radial fibers on the circumferential fibers under right torsion and right bending.

[0230] In some embodiments, the mechanical durability comprises maintaining rotation (degrees) under at least one of: flexion, extension, bending, and / or torsion, with respect to biomimetic without radial fibers.

[0231] Reference is now made to FIGs. 15A-15E demonstrating compression and moment-rotation of biomimetic IVD computational model. FIG.15A shows a line graph presenting a loaddisplacement curve of the biomimetic with radial fibers model compared with the IVD validated model and in vitro measurements. FIG.15B shows a bar graph presenting Intra-discal pressure (IDP) of the biomimetic with radial fibers compared with the validated model and in vitro measurements. FIGs.l5C-15E shows a line graph presenting physiological loading modes of the biomimetic IVD with radial fibers vs. validated model results and in vitro measurements, for Flexion and extension (FIG.15C), Lateral bending (FIG.15D), and Torsion (FIG.15E). According to another aspect, the disclosure provides a method for fabricating an annulus fibrosus - nucleus pulposus (AF-NP) biocomposite construct intervertebral disc (IVD) tissue analogue / implant, the method comprising: a. wrapping fibers around a frame in angle-plied (AP) (about ±30°) orientation, inserting them to a 3D mold with hydrogel solution and crosslink it, thereby obtaining a plurality lamella made of hydrogel matrix reinforced with angle- plied (AP) fibers; b. locating the plurality of AP AF laminates comprising alternating (±30°) orientations in the circumference of a 3D cylinder frame; c. suture the AF laminates using a fiber in a radial direction of the cylinder connecting the circumferential AP AF lamella rings and NP core of following step (d), while keeping the laminates hydrated; d. adding / inj ecting hydrogel into the AF rings construct center to obtain an NP core embedded with the radial fibers and surrounded by the AF rings; thereby, obtaining a disc-shaped AF-NP construct / biocomposite comprising / reinforced with angle-plied fibers (about ±30° relative to horizontal axis), and radial fibers (about 90° relative to vertical spinal axis) at orientation that simulates main directions of the IVD tissue fibers.

[0232] In some embodiments, the AF lamellae and the NP core are made of same or different hydrogel matrix.

[0233] In some embodiments, the cross-linking of the hydrogel with the fibers comprises chemical, biochemical, and / or physical -based cross-linking.

[0234] Reference is made to FIG. 11B, which demonstrates suture of two biocomposite laminates for lap test and tensile test and biomimetic AF laminates. FIG 11C demonstrates the creation of radial fibers and the final fabricated AF-NP construct with silk radial fibers and shear test of the construct. Further shown is an illustration of the shear test apparatus.

[0235] The AF-NP construct includes AF laminates made of silk fiber-reinforced alginate hydrogel that were added with suture as radial fibers and tested. The IVD construct includes biomimetic AF and biomimetic NP, analog hydrogels composed of sulfonate-containing precursor monomers, which were combined into novel biomimetic AF-NP construct with added radial fibers was tested under semi-confined compression, as in the native IVD, and its performance under physiological loading modes was further validated using an FE model.

[0236] The current disclosure provides new insights into the structure-function relationships of radial fibers in the IVD.

[0237] AF laminates cross-bridges / sutures

[0238] Several tests were performed to determine if the silk suture connection would be strong enough to hold the two or more of the biocomposite laminate together (out of plain radial fibers) without changing the biocomposite's mechanical behavior.

[0239] In some embodiments, the failure stress of the suture under lap test withhold high stress compared to other AF-repaired materials.

[0240] The capacity of the suture to withstand stress that are equivalent to or greater than native IVD proves that biomimetic AF laminates can be connected to native IVD as a patch. The mechanical behavior of the biomimetic AF laminates did not change much with the suture. Although the modulus of 19.62+8.5 MPa and 20.26+0.9 MPa, without and with sutures, respectively didn’t change much in its mean value the standard deviation (STDV) reduced from 43% without sutures to 4% with them. In some embodiments, the radial fibers prevented delamination between the AF lamella. In some other embodiments, the suture prevents the delamination between the two laminates, they force the laminates to function as one and they also prevent the energy dissipation. In some other embodiments, the addition of the inter- structural fibers by suture made the strain more uniform without sliding. Hence, it is mechanically compatible with native IVD and may allow connecting of multiple lamellae. Due to the quad and cross suture's increased connectivity and stress distribution, the possible applied load was more significant than the line suture. When creating a full biomimetic IVD with translamellar radial fibers, more sutures are done in quad or cross style and can prevent the delamination of the biomimetic AF under pressure. The cross suture failure strain is much higher it can hold even in unnaturel movment and might be the better way to connect the tissue back, if there is enough tissue to connect that way.

[0241] AF-NP construct with radial fibers In some embodiments, the addition of the radial fibers improved the connection between the biomimetic AF and NP. In some embodiments, the mechanical behavior of the AF-NP construct with the inter-structural radial fibers was neither confined nor unconfined but somewhere between them and much closer to the bovine IVD. The biomimetic laminates were sewed together, and inter- structural radial fibers from one side of the outer AF to another were created, thus forming radial structural bridges across the IVD from the center of the biomimetic NP core to the external biomimetic AF lamella. In some embodiments, these inter- structural fibers are shown to hold the AF-NP structure intact under compression. After the compression, no damage was detected across the biomimetic AF, and the radial fibers were intact. It shows that radial fibers mechanically connect the AF and NP and are crucial for mimicking the IVD.

[0242] The shear test demonstrated how, in some embodiments, the inter-structural radial fibers assist in keeping the NP in its place underpressure. Mechanically, in some embodiments, the radial fiber’s ability to resist shear loads maintains the structural integrity of the IVD. In some embodiments, it can prevent delamination and constrain the IVD movement under cyclic load.

[0243] The commercial silk sewing thread was used to create the inter- structural radial fibers. In some embodiments, the biomimetic AF laminates were sewed with more sutures than the mount of sewing across the NP region.

[0244] FE computational models

[0245] FE models provide an important tool in structure-mechanics relationship characterization. In the present disclosure, the FSU FE model included a radial fiber network from the NP‘s center (assuming a round shape to NP, i.e., 3D core) to the external AF. The effect of inter- structural radial fibers was tested under different physiological loading modes, as exemplified in Example 4 below herein.

[0246] One model also assumes that inter-structural radial fibers have mechanical properties similar to collagen or silk, which may overestimate radial fiber stiffness. However, it is likely that, while nonlinear, the actual properties are less stiff than those in the current model. The primary justification for the mechanical properties’ assumption is that, according to some embodiments, the overall volume fraction of the radial fibers is 0.1 percent of the total AF; thus, the effective mechanical behavior of the interlamellar space in the radial direction is primarily affected by matrix behavior (from a gross assumption using the rule of mixtures). The radial fibers have a local effect. Several assumptions and simplifications were used in the FE model of the IVD, such as uniform cross-section and the radial fibers distribution, as well as the omission of elastic fibers, which are also found in that exact locations. The fundamental reason for these simplifications is that the model is intended to comprehend the overall trends caused by radial fibers rather than provide absolute conclusions. Furthermore, although elastic fibers form a dense network in the same regions, strength and stiffness are derived from a collagenous component, and nonlinear behavior in soft tissues is caused by the combination of elastin and collagen fibers, with elastin working in the toe region and collagen providing stiffening.

[0247] The NP radial fibers addition had no significant influence at the macro level. In some embodiments, under compression, the silk radial fibers increased the maximum load of the model by 200 N without changing the mechanical behavior. The IDP increased under 1000 N by 12% for silk compared to the biomimetic model without the radial fibers. It shows that the radial fibers while having no significant impact on the overall behavior of the FSU, have an important internal effect.

[0248] The inter-structural radial fibers decrease the stress building in the AF-NP junction, thus showing the inter- structural radial fibers' impact on maintaining the IVD stability. It is worth noting that most stresses moved to the outer AF with the radial fibers, which are known to be the stiffer part. The radial expansion of the IVD decreased; thus, according to some embodiments, the radial fibers prevent delamination.

[0249] In some embodiments, under torsion, the most noticeable change was the prevention of delamination between the AF lamellae. The stiffer the radial fibers, the more prevention of delamination (seen by radial expansion) was prominent. In some embodiments, the NP radial fibers activated the radial fibers in the opposite region of the AF, hence, the applied load, and increased the stress on the AF radial fibers, thus distributing the load more efficiently. As shown in different physiological loading modes, in some embodiments, the radial fibers decreased the stress distribution on the matrix. While under torsion, that trend is less significant than in the other loading modes.

[0250] In some embodiments, under bending, flexion, and extension, the addition of inter- structural radial fibers assists with stress distribution. Under bending, the radial fibers created pressure mainly in the NP on the compressed compared to the more region under pressure without the inter- structural radial fibers. Therefore, the radial fibers that work in that direction are tensioned. Substantial loads were transferred between the lamellae through the interlamellar bonds. Stiffer radial fibers reduce the strains of the IVD but, in some embodiments, can increase stresses, as shown under flexion. In some embodiments, the radial fibers reduced the stress, but by doing so, they increased the local stress in the AF-NP junction. The AF radial fibers in the ventrolateral region were under noticeable stress. In some embodiments, stiffer radial fibers significantly increased the NP stresses under flexion.

[0251] To conclude, the present disclosure examplified that the inter-structural radial fibers network provides the maintenance and durability of the IVD. From mechanical consideration, the AF and NP, have to be connected to keep the IVD structure. In some embodiments, the presence of radial fibers lowers matrix sideways expansion, bringing the lamellae closer together and maintaining / shielding the IVD structure. Radial fiber deterioration is likely to result in annular tears. Moreover, high stresses in tissues are the lead cause of progressive damage and limited durability therefore, the AF-NP biocomposite structural mechanisms that distribute the load and reduce local stresses is beneficial for full IVD replacements.

[0252] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0253] EXAMPLES

[0254] Example 1 - Radial fibers provide the CP biocomposite laminates with structural shielding characterized by mechanical durability / tolerance or resistance

[0255] Materials and methods - Mechanical testing of CP Biocomposite laminates:

[0256] CP Biocomposite laminates fabrication - The Cross-Plied (CP) biocomposite laminates were constructed from an alginate matrix reinforced with fully degummed silk fibers (fibroin fibers). Briefly, natural silk fibers (Bombyx mori) were degummed twice, first in double-distilled water (DDW) and second in 0.02 M Na2COs solution at 100 °C for 30 minutes. Then, the fibers were rinsed with fresh DDW three times and dried with a warm air stream. The fibroin fibers were wrapped around thin custom three-dimensional (3D) printed frames to produce unidirectional-longitudinal (0°) and cross-plied (CP, 0° and 90° with the vertical axis) biocomposite laminates. These fiber orientations allow mimicking of the main directions of the collagen fibers in the meniscus, corresponding to circumferential (0°), and circumferential and radial (0° and 90° with respect to the vertical femur-tibia axis). The thickness of the fibers on each frame was modified and measured using a digital micrometer. The frames were inserted into cellulose dialysis membranes (MWCO 14000, Sigma- Aldrich, Israel) with a 6% (w / v) sodium alginate solution (Protanal® LF 10-60, FMC biopolymer, USA). Subsequently, the dialysis membranes were sealed, flattened, and soaked in a 0.1 M CaCh solution for at least 48 hours at room temperature to cross-link the alginate and form a hydrogel matrix.

[0257] Reference is now made to FIG. ID illustrating the fabrication process of the cross-plied (CP) biocomposite laminates, to FIG. IE demonstrating the orientation and distribution of fibers within the longitudinal and CP (0-90°) silk fibroin laminates, and to FIG. IF that shows micrograph images of scanning electron microscopy (SEM) presenting biomimetic silk laminate including micro-nano silk-based structures, and human meniscus.

[0258] Fiber volume fraction quantification - the total fiber volume fraction (total VF) was defined as the thickness ratio, calculated by dividing the fiber thickness by the laminate thickness. For CP laminates, the thicknesses of the fiber layers were modified to produce laminates with different percentages of fibers arranged in the stretching direction (longitudinal fibers (%), 0°), while the total VF was kept relatively constant. The percentage of longitudinal fibers was calculated by dividing their thickness by the total thickness of the fibers layer. In order to compare the two groups of laminates (longitudinal and CP), fiber volume fraction in the tensile direction, vf (0°), was also defined. The vf (0°) was calculated as the thickness of the fibers with 0° orientation divided by the final laminate thickness. The total VF equals the vf (0°) for the longitudinal laminates.

[0259] Mechanical testing of CP biocomposite laminates - The mechanical behavior of the biocomposite laminates was studied using unidirectional tensile test measurements. Tensile testing is performed using a tensile machine with a 222 N load cell (Psylotech® pTS system, IL, USA). The laminates were extracted from the frames using a surgical knife, and width and thickness were measured along the sample using a digital caliper and a micrometer. The average dimensions were used to calculate the cross-sectional area, fiber volume fractions (VF and vf (0°)), and longitudinal fiber percentage (and the complement percentage of radial fibers). The laminate samples were mounted to the tensile machine using custom-3D printed clamps, and the gage length of each sample was measured as the distance between the clamps. All samples were maintained in the CaCh solution until the tensile test.

[0260] The longitudinal and CP samples were stretched at a 3 mm / min rate parallel to the loading direction (0°). The preconditioning phase contains manual stretch to approximately 1 N and then five precondition cycles to 5% strain of the original length of the laminate samples. Subsequently, the samples were stretched to failure. Engineering stresses and strains were calculated to analyze the mechanical properties of the biocomposite laminates. The stresses were calculated as the ratio of the force to the initial cross-sectional area, and the axial strains as the change in the sample length divided by the original length. The tensile modulus was calculated in the initial linear section of the stress-strain curves, between 2.5 and 5.5% strain for all samples. The ultimate tensile strength (UTS) was defined as the maximum stress before failure, and the maximum strain was defined as the strain at UTS (ultimate strain). Toughness was defined as the area under the obtained stress-strain curve and was calculated up to a 10% decrease of the UTS value received by the trapezoidal rule using Excel software.

[0261] Poisson’s Ratio - The Poisson's ratios of the longitudinal and CP biocomposite laminates were measured using a camera video-extensometer. Measurements and data analyses were employed using a 12-bit CCD digital camera (MER-503-20GM-P, Daheng, China) with a macro lens (100 mm F2.8, Nikon) and PASCO capstone software (version 2.6.1). The 12-bit CCD camera consisted of a trigger that was connected to the Psylotch load frame for load data synchronization between the tensile machine and the camera. Test data were recorded using a camera rate of 20 Hz and were synchronized with the loading frame measurements. The sample preparation included marking tiny dots inside the laminate using Indian Ink injection with a 29G syringe to record two transverse widths (distance between dots) and measure the Poisson’s ratio in the plane parallel to the laminate surface. The transverse width measurements were taken from the center of the sample in order to reduce edge effects. The transverse strain is calculated as the average change in the sample width divided by the original average width between the dots. The Poisson’s ratio (v) is defined as:

[0262] .. . Transverse S train

[0263] (1) V = - Axial Strain

[0264] Experimental Results - Mechanical testing of CP Biocomposite laminates: Biomimetic biocomposite laminates of fibroin fiber-reinforced alginate matrix were fabricated and examined using unidirectional tensile tests parallel to the longitudinal direction of the fibers. The fiber orientation in the laminates simulates the main directions of the meniscus fibers, where laminates with longitudinal fibers mimic the circumferential fibers, and cross-plied (CP) laminates mimic the circumferential and radial fibers (FIG. 1A). The geometrical and mechanical properties of the longitudinal and CP biocomposite laminates are shown in Table 1, Table 2 and Table 3 below, respectively.

[0265] Table 1: Geometrical and mechanical properties of the longitudinal biocomposite laminates. n - number of samples; VF - total fiber volume fraction

[0266] Table 2: Geometrical and mechanical properties of the cross-plied (CP) biocomposite laminates. n - number of samples; VF - total fiber volume fraction; vf (0°) - fiber volume fraction in tensile direction.

[0267] Table 3: Geometrical and mechanical properties of the biocomposite laminates for Poisson’s ratios measurements. n - number of samples; VF - total fiber volume fraction; vf (0°) - fiber volume fraction in tensile direction.

[0268] As can be seen in FIG. 2A, the general average mechanical behavior of the biomimetic laminates was hyperelastic with large deformations. A toe region followed by a linear region that started at approximately 0.015 strain was observed in the resulting stress-strain curves. The longitudinal and CP laminates demonstrated increased mechanical behavior as the fiber volume fraction in the longitudinal direction increased (i.e., as the ratio of transverse / radial fibers volume to longitudinal (vf (0°)) fibers volume decreases). Although having different fiber volume fractions in the tensile direction (vf (0°)), longitudinal and CP laminates demonstrated similar mechanical behaviors. The longitudinal (VF = vf (0°); 0.24) and CP 21.9% (vf (0°); 0.12, VF: 0.53) laminates demonstrate the same behavior, as well as the same behavior of the longitudinal (VF = vf (0°); 0.42) and CP 74.9% (vf (0°); 0.36, VF: 0.47) laminates. Compared to the mechanical tensile behavior of the human meniscus in the circumferential direction, the laminates demonstrated behavior in the same range.

[0269] For CP, as the percentage of fibers in the radial direction increased, i.e., the percentage of longitudinal fibers decreased, the mechanical behavior decreased. However, the mechanical behavior change was smaller compared to the change in the total fiber volume fraction (VF) for the longitudinal laminates. The differences are reflected in the mechanical properties obtained (FIG. 2B-2E).

[0270] The mechanical properties of the two groups of laminates with a different fiber volume fraction in the tensile direction, vf (0°), are shown in FIGs. 2B-2E. Longitudinal and CP laminates demonstrated an increase in tensile modulus as the vf (0°) increased ( FIG. 2B). However, for a similar vf (0°), the values for the CP laminates were larger compared to the longitudinal laminates. For vf (0°) values range from 0 to 0.3, the ultimate strains of the CP laminates were smaller, and the UTS were larger than the longitudinal laminates (FIG. 2C and FIG.2D, respectively). The modulus and UTS values obtained for the CP laminates were similar to those of the longitudinal laminates with larger vf (0°). The differences between the toughness of the two groups of laminates were minor, and similar values were obtained for a similar vf (0°) (FIG. 2E).

[0271] During stretching, differences were visually observed in the width changes of the laminates. In this section, longitudinal laminates had a total fiber volume fraction, VF (equal to the fiber volume fraction in the tensile direction, vf(0°)) of 0.47, and the CP laminates had a VF of 0.49 and a vf(0°) of 0.23. The width of longitudinal samples decreased significantly during stretching compared to the width of the CP laminate samples (FIG. 3A and FIG. 3B). The transverse strains obtained in the center of the longitudinal and CP samples are consistent with these observations, with smaller negative transverse strain values demonstrated by the longitudinal laminates (FIG. 3C).

[0272] Examining the Poisson's ratio of the laminates showed the differences between the two groups during stretching (FIG. 3D and FIG. 3E). The CP laminates demonstrated a smaller Poisson ratio than the longitudinal laminates, resulting from smaller lateral contractions. The Poisson value decreased significantly until about the UTS point and then increased. In a strain range of 0.03 to 0.3, Poisson's ratio was reduced by 50%, from 1.22 to 0.65 and 0.52 to 0.25, for the longitudinal and CP laminates, respectively. The average Poisson’s ratio, in a strain range of 0.02 to 0.08, was 1.14 + 0.13 and 0.51 + 0.25 for the longitudinal and CP laminates, respectively.

[0273] Example 2 - Finite Elements (FE) 3D model of human meniscus and meniscal biomimetic composite models

[0274] Materials and Methods - FE analysis and biomimetic composite model of human meniscus: FE model geometry - An FE model of the knee joint is created based on the 3D human knee model from the Open Knee project (OpenKnee, SimTK). The geometry was extracted from Magnetic Resonance Images (MRI) of a female cadaveric right knee specimen (70 years old). This model includes the structures pertinent to the tibiofemoral joint: the femur and tibia bones, femoral cartilage, tibial cartilage, medial and lateral menisci, and ligaments (anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), and lateral collateral ligament (LCL)), as shown in FIG. 4A. The menisci were modeled with three types of structural models: linear elastic isotropic, composite with circumferential fibers, and composite with circumferential and radial fibers. The matrix and fiber geometries are shown in FIG. 4B.

[0275] The knee FE model was modified to allow investigation of contact mechanics. The number and type of elements used for each part in the model are shown in Table 4.

[0276] Table 4: element characteristics and material properties of the knee model substructures.

[0277] The meniscal horn attachments are defined using linear springs similar to the original model.

[0278] Each node on the meniscal horn surface is attached to a node on the tibia located approximately perpendicular to the horn face, resulting in 88 springs for each horn. The total spring stiffness is in the range of 1840-3529 NmnT1. These values reflect an error of up to 11.3% between the experimental contact and model variables.

[0279] Meniscal biomimetic models - A composite representation of the menisci is generated to simulate the native meniscal structure. Five knee models with various menisci representations were created, of which the first two meniscal models were used to validate the Finite elements (FE) knee model. (1) control Linear elastic isotropic model (Iso), (2) control composite model of collagen circumferential and radial fibers (Coll), (3) Composite model of fibroin circumferential fibers (Circ), (4) Composite model of mainly radial fibers of fibroin (Rad), and (5) Composite model of fibroin circumferential and radial fibers (Circ- Rad). The validation of the meniscal composite models Iso, Coll, Circ, Rad and Circ-Rad can be seen in FIG. 4C, and is further detailed in the experimental results section hereinbelow.

[0280] The upper and lower solid elements layers of the menisci were defined as was for the Iso model to mimic the superficial layers of the native meniscus. Truss elements representing the meniscal fibers are attached between the nodes of the solid central layers of elements (truss type T3D2H elements and solid C3D8H elements), creating a total of 6 truss element layers. Thus, each truss element shares its two nodes with the matched solid element to allow for translation-only kinematic constraint and the associated load transfer between the matrix and the fibers constituents. Hybrid solid elements are used due to the incompressible nature of biological materials. The Circ composite model contains an array of 6 layers of truss elements oriented circumferentially. For the other composites, the truss elements were arranged alternately in layers of circumferential and radial.

[0281] The fiber volume fraction (VF) of the truss elements representing the meniscal fibers is calculated from the volume of the central five layers of solid elements. The VF is chosen according to the VFs of our fabricated biocomposite laminates (~0.5) and the biochemical composition of the meniscus. More information, and details about the number of truss elements, VFs, and cross-sectional areas of the fibers for the composite models are presented in Table 5 below.

[0282] Table 5: The characteristics and material properties of the menisci composite models.

[0283]

[0284] Vcirc- Volume fraction of the circumferential fibers; VM- Volume fraction of the radial fibers; M- Medial meniscus; L- Lateral meniscus.

[0285] Material properties - The material properties of the knee substructures are described in Table 4 above. The femur and the tibia are modeled as rigid bodies. The ligaments are defined as homogenous, isotropic, and hyperelastic, and their mechanical properties are described using the Neo-Hookean material law as in the original model. Femoral and tibial articular cartilages are defined as linear elastic, isotropic, and homogenous materials, as described in various studies.

[0286] For the Coll model, the fibers are defined as homogenous, isotropic, and hyperelastic, and their mechanical properties are described using Marlow material law. The material parameters are computed by curve-fitting data considered for the annulus fibrosus collagen fibers in FE analysis of the intervertebral disc. The meniscal matrix is defined as a linear elastic, isotropic, homogenous material with an elastic modulus (E) of 8 MPa and Poisson’s ratio (v) 0.45.

[0287] For all other composite models, the mechanical properties of the fibers are described using the second-order Ogden material law. Material parameters were computed using curvefitting data from the tensile behavior of silk fibers, which are described using the Zener model. This model is developed for silk fibers that are fully degummed from the sericin layer. Thus, they are named fibroin for consistency. The meniscal matrix is defined as hyperelastic, isotropic, and homogenous material, and the mechanical properties are described using the Marlow material law. The material properties were computed using curve-fitting data from alginate tensile tests.

[0288] Boundary Conditions - The passive response of the knee joint is studied at full extension under axial displacement. A reference point in the central region between lateral and medial femoral epicondyles is coupled to the upper femoral surface using the MPC constraint method (FIG. 4A). The femur was fixed at full extension, whereas the bottom of the tibia was fixed in all translational and rotational DOF. An axial displacement of up to 1 mm is applied to the femoral condyle reference point.

[0289] The cartilage is connected to the femoral and tibial bones using the surface-to-surface tie constraint. The meniscal fibers were connected to the matrix by a tie constraint that ties the fiber nodes to the matrix nodes. Contact was modeled between the surfaces of femoral and tibial cartilage, femoral cartilage and menisci, and tibial cartilage and menisci for both the lateral and medial compartments, resulting in six contact- surface pairs. A tangential surface-to-surface contact pattern was defined between all articulating surfaces using the penalty method for contact constraint enforcement with a friction coefficient of 0.1. This value is within the wide range of the friction coefficients of the native cartilage (0.015-0.28).

[0290] Seven contact-related variables were determined: reaction axial compressive force, the tibial cartilage contact area, mean contact pressure, peak (maximum) contact pressure, max principal stresses of the composite components, and the width and height distance changes. The mean contact pressure was calculated as performed in experimental studies in vitro on cadaver human knees (axial force divided by contact area) to compare the results. The width difference was measured on the middle solid elements layer between nodes of the externalinternal circumference, and the height difference was measured between nodes on the upper and lower layers of the external circumference. At least 20 nodes were used for calculations. The differences were examined in the anterior, central, and posterior regions of the main body of the medial and lateral meniscus.

[0291] Computational Results -modeling of meniscus biomimetic composite:

[0292] The FE knee joint model with all five meniscal representations Iso, Coll, Circ, Rad and Circ-Rad was compared and validated FIG. 4C. The contact properties of the meniscal Circ, Rad, and Circ-Rad Composites models at various loads were found within the data range known in the art. Fibroin-based meniscal models demonstrated results closer to those known in the art, with a 13-22% larger total contact area (CA), 12-18% smaller mean contact pressure (MCP), and 5-20% smaller peak contact pressure (PCP) than the control (Iso and Coll) models, the contact properties of the meniscal models is shown in Table 6 below.

[0293] Table 6: contact properties based on the knee joint model. CA contact area; CAM contact area medial compartment; CAL contact area lateral compartment; MCP mean contact pressure; PCP peak contact pressure on medial tibial cartilage.

[0294] The max principal stresses in the components of the meniscal models under similar compression of 990-995 N are shown in FIG. 5. Stress concentration was observed in the middle of the central -posterior portion of the circumferential fibers in the Circ Composite menisci model (black arrows). In the Rad Composite model, the circumferential fibers carried more considerable stresses due to their small volume fraction. Compared to these models, it seems that the stress distribution was better in the circumferential fibers of the Circ -Rad Composite model. In regions where the circumferential fibers were not stretched, like in the anterior of the lateral meniscus, it can be seen that there is an increase in the stresses in the radial fibers.

[0295] The radially oriented fibers carried greater and widely distributed stresses in the Circ- Rad Composite than in the Rad Composite model. The matrices of the models showed consistent results. Regions with stress concentration in the matrix of the Circ Composite have almost completely disappeared in the other Composite models. The matrix of the Rad Composite had more area than the Circ-Rad Composite model for the same stress level. Significant stresses were obtained in the matrix for the Circ-Rad Composite in the posterior portion of the medial meniscus and the anterior portion of the lateral meniscus. In these regions, the circumferential and radial fibers carried minor stresses, suggesting that the meniscus components share mechanical loads.

[0296] The meniscal models obtained similar total CA and the MCP on the tibial plateau (Table 5), yet differences were observed in the pressure maps. The resulting contact pressures on the tibial cartilage under similar compression are shown in FIG. 6 (i-iii). With the Rad Composite meniscal model, the PCP on the tibial cartilage was the smallest (3.62 MPa), followed by the Circ-Rad Composite (3.96 MPa) and the Circ Composite models (4.82 MPa). These values were obtained in the region below the inner circumference of the medial meniscus for all the models. However, it can be seen that the area on which the peak pressures act is smaller for the Rad and Circ-Rad Composites than the Circ model. The considerable contact pressure obtained between the contacting cartilaginous surfaces of the lateral side for the Circ and Rad Composites significantly decreased in area using the Circ-Rad Composite. Furthermore, the pressure distribution improved beneath the meniscal anterior and posterior horns and the outer circumference using models with radial fibers. In other words, the addition of radial fibers reduces the stress concentration on the articular cartilage in comparison to circ and rad models.

[0297] The average width changes of the meniscal matrix under similar axial compression (990-995 N) are presented in FIGs. 7A-7C. The Circ Composite model demonstrated the most significant width increase. The largest change between the menisci was in the medial meniscus (2.46%, FIG. 7A), and the most varied regions were the central portion of the medial meniscus (3.26%, FIG. 7B) and the anterior portion of the lateral meniscus (4.57%, FIG. 7C). In contrast, the width differences for the Rad and Circ-Rad Composite models were minor, not greater than 0.10%. The width increased mainly at the central portion of the medial and lateral meniscus matrix (FIG. 7B and FIG. 7C ), while a width decrease was obtained in the anterior and posterior parts of the medial meniscus (FIG. 7B).

[0298] Example 3 - Bovine Meniscus Structure

[0299] Materials and methods - Meniscus Structure:

[0300] Meniscus sample preparation - A left knee joint of bovine (femur and tibia bones) was obtained from a local abattoir and is stored at -20°C until used for sample preparation. The patella, connective tissues, ligaments, and fat are removed to expose the menisci on the tibial plateau (FIG. 8 (i)). The menisci are dissected from their skeletal attachments, and samples are taken from the central and posterior regions of the medial meniscus (FIG. 8 (ii) and (iii)).

[0301] For scanning electron microscopy (SEM), the radial section was mounted with an optimal cutting temperature compound (O.C.T, Tissue-Tek®) and cut into 300pm slices at -20°C using SLEE MEV Semi-Automatic Cryostat (SLEE medical GmbH, Germany). Some radial samples were partially digested in 1 M NaOH solution for 3 min at room temperature, then rinsed twice with DDW. Subsequently, the samples are dried in a series of graded ethanol (50 - 100%, step = 10%) for 5 min each and placed in a vacuum desiccator until the scanning electron microscopy (SEM) investigation (5 days). The ImageJ® (NIH) software was used to measure the fascicle and fibril diameters. A radial section from the posterior part of the medial meniscus was analyzed.

[0302] Scanning Electron Microscopy (SEM) - The structural organization of the circumferential and radial collagen fibers in the bovine meniscus was investigated with ultra- high-resolution SEM (Maia 3 FE-SEM, Tescan). Samples are mounted on aluminum stubs using double adhesive tape and sputter-coated with a 10 nm thickness gold for conductivity. Some images were colored using software to distinguish between the different architectures of the fibers within the meniscus tissue.

[0303] Results - Meniscus structure:

[0304] The SEM micrographs of the control (undigested) and partially digested radial samples from the central part of the medial meniscus are shown in FIG. 9A. A cross-section of a circumferential fascicle was observed close to the femoral surface in the outer-middle region of the control sample with approximately 480 pm diameter, consisting of smaller bundles with non-circular cross-sections ( FIG. 9A (i)). The fascicle edges were surrounded by twisted or wavy tie fibers, which split, penetrated, and wrapped smaller bundles. A twisted tie fiber was observed in the gap between fiber bundles ( FIG. 9A (ii)). The surface appears smooth, and pores were observed between and within the fibers. The circumferential fibers appeared composed of many thin fibrils with relatively circular cross-sections with diameters of 154 ± 22 nm (mean ± SD, n=17) ( FIG. 9A (iii) and FIG. 9A (iv)).

[0305] In the inner region of the partially digested sample, tie fibers penetrating the main body of the tissue from the lamellar layer of the tibial surface were observed ( FIG. 9A (v)). The tie fibers arborize throughout the tissue and appear to wrap around circumferentially oriented fiber bundles. Whereas the surface was smooth in the undigested sample, it was rough in the digested sample ( FIG. 9A (vi)). The circumferential bundles separated, and spaces formed between them, which appeared as crevices. Tie fibers with a twisted shape were also found near these spaces. Individual circumferential bundles were divided into fibers with inhomogeneous cross-sections composed of many fibrils ( FIG. 9A (vii)). It appears that some of the fibrils have collapsed from the cross-sectional surface of the intact fiber and stretched between the gaps. However, inside the gaps between two adjacent fibers, there were thin, delicate fibrils that resemble cross-bridges-like structures ( FIG. 9A (viii)).

[0306] Additional digitally colored images of Meniscal tissue sub-structures are presented in FIG. 9B, exemplifies (i) the different layers in radial section including the radial fibers, circumferential fibers, and bundle, as well as (ii) Fibrous structure of meniscal collagen.

[0307] Example 4 - Radial fibers provide the AF-NP biomimetic construct composite with structural shielding characterized by mechanical durability / tolerance or resistance Materials and Methods - Mechanical testing of AF laminates and AF-NP construct Biocomposite, and Bovine IVD sample preparation:

[0308] Suture fabrication of AF laminates - Two laminates were fabricated on hexagonal frames, put into a custom-made stand in a ±30°, and sewn together (FIG. 11B). Three forms of sutures were chosen, a single-line suture, one continuous line in the middle, a quad suture, four lines in a square shape, and a cross suture, one horizontal line and one vartical line in a cross shape. A silk braided nonabsorbable suture with 19 mm 3 / 8 reverse cutting was used to create the sutures. The sutured biocomposite was extracted using a surgical knife. Sample geometry (width and thickness) was measured using a digital micrometer, and the gage length was measured on the tensile machine as the distance between the clamps.

[0309] Tensile tests for two sutured biomimetic AF lamellae - Tensile tests were performed using a tensile machine (Psylotech® pTS system, IL, USA) with 222N±0.05% load cell using displacement control mode at a static strain rate of Imm / min. The samples were gripped using custom-made clamps. The sample was stretched manually until IN load, then proceeded by five preconditioning cycles at a rate of 1 mm / min of 8% strain of the original sample length, and stretched to failure. The mechanical properties of the AF laminates were calculated as the engineering stresses and strains to allow the comparison with the data reported in the literature. The stresses and strains were calculated as the ratio of the force to the initial sample's cross- sectional area and as the change in the sample length divided by the original length, respectively.

[0310] Lap test for two sutured biomimetic AF lamellae - A lap test's mechanics, which typically consist of two plates sandwiched by a "matrix" material that binds them together, can be pretty complex. The plates, in this case, correspond to the biocomposite. The sutured laminates were grabbed on two separate, unconnected sides (FIG. 11B). The laminates were preconditioned with three cycles of 0-10% strain at 1% / s strain rate and then to failure at strain rate of 2% / s. Three different suture styles were chosen, a single suture, a small suture in a straight line, a quad suture, four continuous lines in a square shape, and a cross suture, one horizontal line and one vartical line in a cross shape. Load-displacement and stress-strain graphs were generated, and the failure stress and strain was taken.

[0311] Fabrication of AF-NP biomimetic construct with radial fibers - AF laminates (±30°) were fabricated as described above (suture fabrication) using a 3D printed parallelogram frame (L-150 mm). 2-3 laminates were wrapped around a cylinder with a diameter of 19 mm same as the mold for the confined test. The layers were designed with an increasing height from 11mm up to 18 mm to mimic the native IVD structure where the inner part is lower than the outer part. A silk braided nonabsorbable suture for radial fibers with a reverse cutting of 19 mm 3 / 8 was used. The biomimetic structure was secured using a suture mold (FIG. 11C). Calcium Chloride solution (0.1M) was poured on the laminates every few seconds during suturing to keep them from drying out. Then, the biomimetic NP was injected into the AF structure.

[0312] Reference is now made to FIG. 11B presenting the fabrication process of the tested AF laminates, and to FIG. 11C presenting the fabrication process of the AF-NP biocomposite construct with radial fibers.

[0313] Compression test for the AF-NP biomimetic construct - Compression tests were performed using a custom-made cylinder (d=19 mm, L=15 mm) that fully closed the sample inside the mold. The compression rate was 1 mm / min. The results and graphs shown in the current disclosure were calculated by engineering stress and strain to compare with the data reported in the literature.

[0314] Shear test for the AF-NP biomimetic construct - The shear test was performed using the custom-made cylinder (d=19 mm, L=15 mm) and custom-made holder (illustrated in FIG. 11C and 12B). The compression rate was 1 mm / min. The biomimetic IVD was placed in a mold with a hole in the bottom (d=19.1 mm) to allow the biomimetic NP to be pushed out of the biomimetic AF using a 19.05 mm cylinder. The biomimetic AF was held in place by the holder. The holder allows the biomimetic NP to be pushed out of the biomimetic AF. The diameter hole of the holder is 19.05 mm to prevent friction between the holder and the biomimetic NP (biomimetic NP diameter is 19 mm).

[0315] Bovine IVD sample preparation and mechanical testing - The bovine spine was obtained from a local abattoir and frozen (-20 °C) within 48 h of slaughter. While frozen, IVDs were dissected from the top vertebra and used the bottom vertebra to hold the sample. The IVD was cleaned in DPBS with 0.9 mM calcium chloride (human calcium concentration). Samples were kept in DPBS before mechanical testing. NP diameter was measured, and a custom-made cylinder was 3D printed with that same diameter (FIG. 11D). In mechanical testing the compression rate was 1 mm / min. A custom-made cylinder was printed according to the sample NP diameter. The NP was assumed to be round.

[0316] Experimental results - Mechanical testing of AF-NP construct Biocomposite: Biomimetic AF testing - Lap tests were performed on three forms of sutures, a single line suture (n=6, four unidirectional and two angle-plied), a quad suture (n=6, four unidirectional and two angle-plied) and a cross suture (n=6, four unidirectional and two angle- plied) (Fig 11B). The procedure was performed with silk fibers to check if the connection would be strong enough to hold the biocomposite lamella together. Both angle-plied and unidirectional laminates were tested, and no significant difference in failure values (strain and stress) was found. Failure was defined when the biocomposite matrix demonstrated raptures that reduced peak load.

[0317] Two distinct biocomposite laminates were sutured together, one in +30° and the other in -30° (quad / cross suture) with out-of-plain silk suture as radial fibers to create an angle-plied suture (+30° and -30°)biocomposite (n=5, VF=0.46±0.07) with an average thickness of 1.2+0.3 mm. Tensile tests were performed to ensure that the suture would not influence the mechanical behavior of the angle-plied laminate. As can be seen in FIG. 12 the behavior of the laminates that were sutured together with out-of-plain suture as radial fibers (angle -plied suture (VF 0.46)) is similar to the angle -plied single laminate (±30°) (silk biocomposite (VF 0.42)) and ovine and bovine specimens in the circumferential direction.

[0318] Biomimetic AF-NP Construct mechanical testing - The biomimetic IVD's binding between the AF and NP based on the NP-swell internal pressure was tested under compression.

[0319] The resultened stress-strain curves for IVD bovine samples (n=3) and the biomimetic construct with inter- structural radial fibers (n=5) under compression is presented in FIG. 13A, demonstrating that the inter-structural radial fibers result in a stress-strain curve that is between the confined and unconfined behavior, for both the IVD bovine and the biomimetic construct, where the presence of the inter- structural radial fibers stiffens the biomimetic construct, making it more similar to the bovine IVD.

[0320] The addition of the radial fibers created a better connection between the AF laminates and the biomimetic NP. Therefore, a shear test was produced to assess the effect of the addition of radial fibers to connect the biomimetic AF and biomimetic NP. The biomimetic IVD was placed in a mold with a hole in the bottom (d=19.1 mm) to allow the biomimetic NP to be pushed out of the biomimetic AF using a 19.05 mm cylinder ( FIG. 13B). Three samples of biomimetic IVD without radial fibers were tested, and the average shear load was 5.1+0.2 N, while the biomimetic with radial fibers (n=3) exhibited an average shear load of 10.7+0.2 N (FIG. 13B). Advanteagously,the addition of radial fibers doubled the possible shear load, demonstrating the improved durability / rolerance or resistance resulting from the connectivity between the biomimetic AF and the biomimetic NP using the radial fibers.

[0321] Example 5 - Finite Elements (FE) 3D model of invertebrate disc (IVD) and In Silico Physiological Loading of the Biomimetics AF-NP Construct

[0322] Materials and Methods - FE analysis of AF-NP radial network connectivity:

[0323] L3-L4 FSU computational model - A nonlinear, heterogeneous three-dimensional FE model of the L3- L4 FSU (functional spinal unit), or invertebrate disc (IVD), was generatedbased on data from computed tomography (CT) scan of a healthy subject with no spine-related pathology (FIG. 14).

[0324] Table 7 and Error! Reference source not found.8 below summarize the mechanical properties, type, and the number of elements used in every part of the model. The model included a motion segment - two vertebrae separated by the intervertebral disc. Each vertebra was divided into the cortical bone, trabecular bone, cartilaginous endplates, and facet cartilage. The model was expanded to include radial fibers that connect the NP and AF in the transition zone. Specifically, radial fibers were added from the center of the NP with a cross-section set at 0.01 mm2,and circumferential distribution was every 20°. The NP radial fibers were tied with the existing radial fibers in the AF and enchored to the center of the NP.

[0325] The current FSU model includes a refined network of the collagen fibers in the AF and the NP. The AF fibers were arranged in 20 lamellae with an alternate orientation of ±30° with the IVD base (i.e., about ±30° with respect to horizontal axis). The tension-only truss elements connecting the proximal and distal surfaces of the IVD served as the representation for the fibers. Every lamella consisted of 76 circumferential fibers with 4-16 nodes. The fiber angles in every lamella were assumed to be constant. The AF matrix and fibers were connected by constraint equations that tied the fiber nodes to the nearest AF matrix nodes. The AF radial fibers were added every 10 degrees and the NP radial fibers every 20 degrees to create a gradient change between the zones by having fewer fibers in the NP. Table 7 - The type and number of elements in the FE model and tested parameters of the radial models:

[0326] Table 8- Mechanical properties of the FSU model materials:

[0327]

[0328] (*) The bone behaves as transverse isotropic material, where the axial direction (a) represents the superior-inferior direction, and the transverse (t) direction represents the anterior-posterior and rightleft lateral directions. The mechanical behavior of the ligaments is bilinear, where the value between the two stiffnesses represents the transition strain for every ligament.

[0329] The distal surface of the L4 vertebra was fixed (in all 6 degrees of freedom), and a 1.6 mm vertical displacement was applied on the L3 proximal surface. This range was chosen to compare the results with the in vitro measurements. The intradiscal pressure (IDP) was compared at least with the native disc. The facets and ligaments were removed from the model to compare with the compression model's experimental measurements. A 10 N-m moment was applied to the model in flexion, extension, lateral bending, and torsion to test the biomimetic AF-NP with radial fibers under physiological loading modes. A range suitble for comparison of the results with vitro measurements of human IVD. Stress and strain measurements were done in the Abaqus post-processor. Stress and strains for the matrix were set as max principal. Fibers' stress and strain were set as maximal principal values. There was no difference between the max principal and S11 / LE11 because the fibers were defined as truss elements.

[0330] Computational results - In Silico Physiological Loading of the Biomimetics AF-NP Construct:

[0331] Next, the Biomimetics AF-NP Construct was tested under physiological loading modes, including flexion, extension, lateral bending, torsion, and compression (FIGs. ISA- ISE).

[0332] In compression, the silk radial fibers strengthened the load by 200N (at 1.6 mm) with no significant change in the mechanical behavior (FIG. 15A).

[0333] The IDP of the silk radial fibers was very similar to a validated model, Biomimetic model, and human IVD measurements in 300 and 1000 N (FIG. 15B). Under 300 N, the IDP of the silk radial fibers model was 0.34 MPa, the same as the in vitro human values. In addition, under 1000 N, the radial fibers model showed an increase in the IDP values of 12% compared to the biomimetic model without the radial fibers.

[0334] The moment-rotation results of the radial fibers model were compared to validated, biomimetic, and native human lumbar L3-L4 IVD models and in vitro measurements under flexion, extension, lateral bending, and torsion (FIG. 15C-15E). The models were similar in all movements, with minor changes compared to the biomimetic without the radial fibers. The addition of silk radial fibers restrict the IVD movement a bit.

[0335] Furthermore, fibers strains of the validated and biomimetic model with and without radial fibers was examined under extension and flexion (FIGs. 16A-16B).

[0336] The inner distribution of stresses changed due to the presence of the radial fibers. FIG. 16A displays the effect of the inter-structural radial fibers on the matrix compared to a model without these fibers under physiological loading modes. Under compression and left bending, the radial fibers reduced the stress in the AF-NP transition zone (TZ) (FIG. 16A). For all physiological loading modes, the stress distribution in the matrix decreased due to the radial fibers' ability to distribute the stress. The NP radial fibers contribute to the load decrease from the matrix by transfer it to the AF radial fibers. Furthermore, the NP radial fibers also activated the circumferential fibers and transferred the stress to the AF radial fibers (FIG. 16B). The circumferential fibers’ stress is reduced due to the stress distribution over more circumferential fibers and not just a narrow area. The radial fibers move and assist with stress decrease in the matrix and with constraining the matrix. The stress was reduced in the circumferential fibers of the AF inner part (FIG. 16B). Therefore, adding inter- structural radial fibers can reduce the delamination between the AF lamellae (presented by the circumferential fibers).

[0337] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

Claims

CLAIMS:What we claim is:

1. A cross-plied (CP) biocomposite laminate layered tissue analogue comprising hydrogel matrix reinforced with longitudinal and transverse / radial fibers for human tissue substitution / replacement of a medial knee meniscus and / or lateral knee meniscus, wherein an orientation of the fibers in the biocomposite simulates main directions of the meniscus fibers, and wherein the transverse / radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability2. The CP biocomposite of claim 1, comprising a quantitatively controlled amount and / or volume of fibers.

3. The CP biocomposite of claim 2, wherein the quantitatively controlled amount and / or volume of fibers comprises a total volume fraction (total VF) consisting of radial fibers volume and longitudinal fibers volume of at least about 10% of the laminate hydrogel matrix (v / v).

4. The CP biocomposite of claim 2 or 3, wherein the quantitatively controlled amount and / or volume of fibers comprises a total volume fraction (VF) consisting of radial fibers volume and longitudinal fibers volume of between about 15% and about 85% of the laminate hydrogel matrix (v / v).

5. The CP biocomposite of any one of claims 1-4, wherein the mechanical durability of the biocomposite is more similar to human or bovine meniscus as compared to a biocomposite lacking radial fibers, and / or wherein the mechanical durability of the biocomposite is enhanced compared to biocomposite lacking radial fibers.

6. The CP biocomposite of claim 5, wherein the mechanical durability comprises tolerance or resistance of a biomimetic model composite using a structure-function relationships of the human tissue, wherein the biomimetic of the structure-function relationships comprises the main direction(s) of the fibers of the human tissue, corresponding to the radial and / or longitudinal fibers of the cross-plied (CP) biocomposite.

7. The CP biocomposite of claim 6, wherein the structure-function relationships model of the human tissue comprises the meniscus in a knee joint model8. The CP biocomposite of any one of claims 1-7, wherein the mechanical durability comprises tolerance or resistance towards mechanically applied or computational simulated stretching, compression, and / or physiological moments.

9. The CP biocomposite of any one of claims 1-8, wherein the mechanical durability comprises at least one mechanical behavior / property selected from the group consisting of: structural width change (%), stress (MPa) vs. strain (mm / mm), Poisson’s ratio, tensile modulus (MPa), ultimate strains (mm / mm), UTS (MPa), toughness (MJ / m3), shear force (N), and rotation (degrees), or any combination thereof.

10. The CP biocomposite of any one of claims 1-9, wherein the hydrogel matrix comprises one or more material selected from a protein(s), a polysaccharide(s), a polypeptide(s), a polylipid(s), a synthetic polymer(s), a natural polymer, or any combination thereof.

11. The CP biocomposite of any one of claims 1-10, wherein the hydrogel comprises one or more of alginate hydrogel, Hyaluronic acid hydrogel, PEG hydrogel, cellulose hydrogel, Agar hydrogel, and Interpenetrating network (IPN) hydrogel, or any combination thereof.

12. The CP biocomposite of any one of claims 1-11, wherein the fibers comprise one or more type(s) of fiber(s) selected from woven fibers, twisted fibers, braided fibers, knitted fibers, tie fibers, and sutured fibers, or any combination thereof.

13. The CP biocomposite of any one of claims 1-12, wherein the fibers material comprises one or more material comprising a protein(s), a polysaccharide(s), a polypeptide(s), a polylipid(s), a synthetic polymer(s), natural polymer(s), or any combination thereof.

14. The CP biocomposite laminate of any one of claims 1-13, wherein the fibers comprise one or more fiber(s) comprising silk, silk-made fibroin fibers, collagen fibers, and cellulose fibers, or any combination thereof.

15. The CP biocomposite of claim 14, wherein the silk is derived from spider and / or silkworm.

16. The CP biocomposite laminate of any one of claims 1-15, wherein said fiber orientation comprises: a. the transverse / radial fibers of the implant projecting in a direction from about a center towards a perimeter of a c-shaped meniscus; and / or b. the longitudinal fibers of the implant projecting in a circumferential direction along the perimeter of the c-shaped meniscus.

17. The CP biocomposite laminate of claim 16, wherein the transverse / radial fibers of the implant orient essentially on a horizontal axis about perpendicular to a vertical femurtibia axis, projecting in a direction from about a center towards a perimeter of a c-shaped meniscus.

18. The CP biocomposite laminate of claim 16 or 17, wherein the longitudinal fibers of the implant orient essentially on a same horizontal axis, about perpendicular to the radial fibers, projecting in a circumferential direction along the perimeter of the c-shaped meniscus.

19. The CP biocomposite laminate of any one of claims 1-18, wherein the mechanical durability comprises reduction in width change and / or preservation of transverse deformation, under stretching relative to laminate without radial fibers.

20. The CP biocomposite laminate of any one of claims 1-19, comprising quantitatively controlled amount / volume of fibers; wherein said quantitatively controlled amount / volume of fibers comprises a total volume fraction (VF) consisting of radial fibers volume and longitudinal fibers volume of between about 20% and about 80% of the laminate hydrogel matrix (v / v), providing the biocomposite with similar mechanical durability / behavior to the mechanical durability / behavior of human meniscus under uniaxial tension / stretching.

21. The CP biocomposite laminate of any one of claims 1-20, comprising a quantitatively controlled amount / volume of fibers, wherein the quantitatively controlled amount / volume of fibers comprises a range of ratios of transverse / radial fibers volume to longitudinal fibers volume of between about 1 :3 (25%:75% transverse / radial: longitudinal vf(0°)) and about 4: 1 (80%: 20% transverse / radial: longitudinal vf(0°)), thereby providing mechanical resistance / shi elding, at least under stretching.

22. The CP biocomposite laminate of any one of claims 1-21, comprising a quantitatively controlled amount / volume of fibers, wherein the quantitatively controlled amount / volume of fibers comprises a range of ratios of transverse / radial fibers volume to longitudinal fibers volume of between about 4:6 (40%:60% transverse / radial: longitudinal vf(0°)) and about 9: 1 (90%: 10% transverse / radial: longitudinal vf(0°)), thereby providing mechanical resistance / shi elding, , at least under stretching.

23. The CP biocomposite laminate of claim 21 or 22, wherein the structural shielding comprises an increased ratio of transverse / radial: longitudinal fibers volume thereby reducing the change in average behavior of stress (MPa) vs. strain (mm / mm) under stretching.

24. The CP biocomposite laminate of any one of claims 1-23, wherein the structural shielding is characterized by a non-linear increase in tensile modulus (MPa) under stretching, compared to a linear increase of laminates lacking radial fibers, thereby providing mechanical durability / resistance under uniaxial tension / stretching.

25. The CP biocomposite laminate of any one of claims 1-24, wherein the biocomposite is characterized by decreased ultimate strain under stretching and / or an increased UTS (MPa) under stretching compared to laminates lacking radial fibers.

26. The CP biocomposite laminate of any one of claims 1-25, wherein the biocomposite is characterized by similar or increased toughness (MJ / m3) under stretching, compared to laminates lacking radial fibers, thereby providing mechanical durability.

27. The CP biocomposite laminate of any one of claims 23-26, wherein the mechanical durability is compared to laminates lacking radial fibers, and / or wherein a fiber volume of longitudinal fibers vf(0°) is within the range of about 10%-40% for a total fiber volume fraction (VF) of about 50%±5.0%.

28. The CP biocomposite laminate of any one of claims 1-27, characterized by reduced change in stress (MPa) vs. strain (mm / mm) under stretching compared with laminate lacking radial / transverse fibers, thereby providing mechanical durability / resistance.

29. The CP biocomposite laminate of any one of claims 1-28, wherein the transverse / radial fibers is configured to reduce pressure on an articular cartilage under compression and / or distribute stress within the implant / tissue.

30. An annulus fibrosus - nucleus pulposus (AF-NP) disc-shaped biocomposite construct tissue analogue / implant comprising a plurality of circumferential rings and an NP core made of hydrogel matrix reinforced with radial fibers for human tissue substitution / replacement of an intervertebral disc (IVD), wherein each circumferential ring comprises AF lamellae made of hydrogel matrix reinforced with angle-plied (AP) fibers, and wherein the radial fibers cross through the plurality of lamellae towards a center of the disc connecting the circumferential AF lamellae rings and the NP core together, thereby reinforcing the disc-shaped biocomposite; and wherein an orientation of the fibers in the biocomposite simulates main directions of the IVD fibers, and wherein the radial fibers provide the implant with structural shielding / integrity characterized by mechanical durability / (tolerance or resistance).

31. The AF-NP biocomposite of claim 30, comprising a quantitatively controlled amount and / or volume of fibers.

32. The AF-NP biocomposite of claim 31, wherein the quantitatively controlled amount and / or volume of fibers comprises a total volume fraction (total VF) consisting of radial fibers volume and longitudinal fibers volume of at least about 10% of a total volume fraction (total VF) consisting of radial fibers and circumferential AP fibers of at least about 10% of the AF lamellae hydrogel matrix (v / v).

33. The AF-NP biocomposite of any one of claims 30-32, wherein the quantitatively controlled amount and / or volume of fibers comprises a total volume fraction (VF) comprising radial fibers volume and circumferential AP fibers of between about 15% and about 85% of the AF lamellae hydrogel matrix (v / v).

34. The AF-NP biocomposite construct of any one of claims 30-33, wherein said fibers orientation comprises: a. the radial fibers of the implant projecting in a direction from about a center of the NP core towards a perimeter of the disc / AF rings; and / orb. the angle-plied (AP) fibers projecting in a circumferential direction relative to the horizontal axis.

35. The AF-NP biocomposite construct of any one of claims 30-34, wherein the radial fibers of the implant oriented essentially along a horizontal axis about perpendicular 90° to a vertical spinal axis, projecting in a direction from about a center of the NP core towards a perimeter of the disc / AF rings.

36. The AF-NP biocomposite construct of any one of claims 34-35, wherein the angle-plied (AP) fibers arranged essentially parallel to / along the vertical spine axis within the circumferential AF lamellae rings, about perpendicular to the radial fibers, projecting in a circumferential direction of about ±30° relative to the horizontal axis.

37. The AF-NP biocomposite construct of any one of claims 30-36, wherein the mechanical durability comprises increased stress (MPa) vs. strain (mm / mm) under compression.

38. The AF-NP biocomposite construct of any one of claims 30-37, wherein the mechanical durability comprises increased shear force (N) between the circumferential AF lamellae rings and the NP core under compression providing structural integrity / shielding.

39. The AF-NP biocomposite construct of any one of claims 30-38, wherein the structural shielding comprises reduced delamination between the AF lamellae, compared with biomemetic model lacking radial fibers, thereby providing mechanical durability.

40. The AF-NP biocomposite construct of any one of claims 30-39, wherein the mechanical durability comprises maintaining rotation (degrees) under at least one of flexion, extension, bending, and / or torsion, with respect to biomimetic without radial fibers.

41. A method for fabricating cross-plied (CP) biocomposite laminates / layered meniscal tissue analogue / implant, the method comprises: a. wrapping fibers around each of a plurality of 3D frame in longitudinal (0°) and / or transverse / radial (about 90°) orientation; b. stacking the plurality of 3D frames containing the longitudinally and / or radially oriented fibers in CP orientation consisting of alternating 0° and about 90° directions; c. placing the stack in a mold and filling the mold with hydrogel solution;d. cross-linking the hydrogel; thereby, obtaining a cross-plied (CP) biocomposite laminates comprising longitudinal, and transverse / radial fibers at orientation that simulates main directions of the meniscus tissue fibers.

42. The method of claim 41, wherein the CP biocomposite comprises a wedge shape.

43. The method of any one of claims 41-42, wherein the hydrogel matrix comprises one or more materials selected from a protein(s), a polysaccharide(s), a polypeptide(s), a polylipid(s), a synthetic polymer(s), a natural polymer(s) or any combination thereof.

44. The method of any one of claims 41-43, wherein the hydrogel comprises alginate hydrogel, Hyaluronic acid hydrogel, PEG hydrogel, cellulose hydrogel, Agar hydrogel, and Interpenetrating network (IPN) hydrogel, or any combination thereof.

45. The method of any one of claims 41-44, wherein the fibers comprise one or more type(s) of fiber(s) selected from woven fibers, twisted fibers, braided fibers, knitted fibers, tie fibers, and sutured fibers, or any combination thereof.

46. The method of claim 45, wherein the fibers one or more fiber(s) comprises silk, silk- made fibroin fibers, collagen fibers, synthetic fibers, cellulose fibers, or any combination thereof.

47. A method for fabricating an annulus fibrosus - nucleus pulposus (AF-NP) biocomposite construct intervertebral disc (IVD) tissue analogue / implant, the method comprising: a. wrapping fibers around a frame in angle-plied (AP) orientation, at an angle of about 30°, inserting said fibers to a 3D mold with hydrogel solution and crosslink said hydrogel, thereby obtaining a plurality of lamella made of hydrogel matrix reinforced with angle-plied (AP) fibers; b. locating the plurality of AP AF laminates comprising alternating orientations in the circumference of a 3D cylinder frame; c. suture the AF laminates using a fiber in a radial direction of the cylinder connecting the circumferential AP AF lamella rings and NP core, while maintaining the laminates hydrated;d. adding hydrogel into the AF rings construct center to obtain the NP core embedded with the radial fibers and surrounded by the AF rings; thereby obtaining a disc-shaped AF-NP construct / biocomposite comprising / reinforced with angle-plied fibers, being at an angle of about 30° relative to horizontal axis, and radial fibers, being at about 90° relative to vertical spinal axis, at orientation that simulates main directions of the IVD tissue fibers.

48. The method of claim 47, wherein the AF lamellae and the NP core are made of same or different hydrogel matrix.

49. The method of any one of claims 47-48, wherein the hydrogel matrix comprises one or more materials selected from a protein(s), a polysaccharide(s), a polypeptide(s), a polylipid(s), a synthetic polymer(s), a natural polymer(s) or any combination thereof.

50. The method of any one of claims 47-49, wherein the hydrogel comprises one or more of alginate hydrogel, Hyaluronic acid hydrogel, PEG hydrogel, cellulose hydrogel, Agar hydrogel, and Interpenetrating network (IPN) hydrogel, or any combination thereof.

51. The method of any one of claims 47-50, wherein the fibers comprise one or more type(s) of fiber(s) selected from woven fibers, twisted fibers, braided fibers, knitted fibers, tie fibers, and sutured fibers, or any combination thereof.

52. The method of claim 51, wherein the fibers one or more fiber(s) comprises silk, silk- made fibroin fibers, collagen fibers, synthetic fibers, cellulose fibers, or any combination thereof.

Images