The present application is a continuation-in-part of international application PCT/US2010/049111, “Artificial Meniscal Implants,” filed on Sep. 16, 2010, which international application claims priority to U.S. Application 61/243,660, “Artificial Meniscal Implants,” filed on Sep. 18, 2009. The present application also claims priority to U.S. Application 61/594,551, “Aligned Fibrous Materials With Spatially Varying Fiber Orientation And Related Methods,” filed on Feb. 3, 2012. All of the foregoing applications are incorporated herein by reference in their entireties for any and all purposes.
This work was supported by the National Institutes of Health (grant no. R01 AR056624) and by the Department of Veterans Affairs (grant no. I01RX000174). The government has rights in this invention.
The present disclosure relates to the fields of biocompatible implant materials and to biocompatible polymer fibers.
The menisci are crescent-shaped fibrocartilaginous tissues that function to transmit and distribute loads between the femur and tibia of the knee joint. As such, the meniscus experiences complex loads, including tension, compression, and shear. Meniscus function in tension arises from an organized microstructure—bundles of highly aligned collagen circumnavigate the tissue between insertion sites on the tibial plateau. These aligned collagen bundles endow the tissue with mechanical properties that are highly anisotropic, and highest in the primary collagen orientation. Existing meniscus replacement materials, however, lack this unique structure and organization.
It is also known that load bearing fibrocartilaginous tissues of the musculoskeletal system, including the knee meniscus and the annulus fibrosus (AF) of the intervertebral disc, are prone to failure and have a limited reparative capacity once damaged. Both tissues are ordered hierarchical laminates: the knee meniscus has a preponderance of circumferential collagen bundles with interspersed, perpendicularly directed, ‘tie’ fibers, while the AF consists of multiple alternating layers of oriented (+/−about 30°) collagen fibers that form an angle-ply structure. The mechanical function of both tissues arises at least in part from this underlying fibrous architecture.
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To address engineering the meniscus and other fibrous tissues, presented here are aligned nanofibrous scaffolds that can recapitulate this mechanical anisotropy. In natural tissues, fibers within the native tissue have a pronounced c-shaped, or otherwise angled, macroscopic organization. To replicate this macroscopic change in organization over the anatomic size of the meniscus, presented here is an electrospinning method that collects organized fibers on a spinning disc or other mandrel.
This disclosure presents aligned nanofibrous scaffolds (formable from a variety of polymers) that can mimic the order of these native tissues, and direct cell and matrix organization with in vitro culture. Also disclosed are constructed biologic laminates, in which scaffold layers are fused with one another through cell mediated matrix-deposition with appositional culture. In some embodiments, the tensile characteristics of the scaffold may replicate those of a mammalian knee menisus. Since the materials of construction in some cases exhibit non-linear stress responses to strain and/or are biodegrade or bioerode when subjected to physiological fluids under physiological conditions, and the scaffold may continue to provide tensile support during this period of biodegradation or bioerosion over a range of strain conditions, it is often useful to characterize the scaffold in terms of these parameters. That is, in various embodiments, a material (e.g., a scaffold) according to the present disclosure exhibits an overall circumferential modulus that is in the range of about 10 MPa to about 200 MPa, preferably at least about 20 MPa, more preferably at least about 40 MPa, still more preferably at least about 60 MPa, and most preferably at least about 80 MPa, at a strain region of about 10%, and/or an overall circumferential modulus in the range of about 5 to about 60 MPa, preferably at least 10 MPa, more preferably at least 20 MPa, and most preferably about 30-35 MPa, at a strain region of about 3%, and these properties are either retained or developed when the scaffold is subjected to physiological implant conditions for time sufficient to allow cell infiltration and meniscal healing, during and after which the components of the matrix are dissolved, bioeroded, or biodegraded into the patient. Preferably the modulus of the scaffold, after exposure to physiological fluids under physiological conditions, retains at least about 60% of its value after 7 days, and more preferably at least about 50% of its value after 60 days. In order to retain these modulus levels this invention also provides that the scaffolds have correspondingly, proportionately higher initial values. Unless otherwise stated herein, any reference to a specific target modulus is intended to reflect an initial value (i.e., before biodegradation or bioerosion and the changes in mechanical properties that develop as cells infiltrate and deposit new, load-bearing extracellular matrix within the scaffold substance). It should be understood that any and all mechanical characterizations or properties of materials set forth in international application PCT/US2010/049111 may apply to the materials disclosed herein.
Clinical application of these materials may, in some cases, benefit from implantation of already formed acellular multi-lamellar constructs. A ‘spot-welding’ method has been previously described in which method individual layers are bound together through local scaffold melting brought on by contacting at least one of the layers with a heated probe. This approach creates stable bi-layers, but can cause compression of the construct with insertion of the heated probes or arrays of probes.
An example of spot-welded layers is shown in FIG. 7. That figure illustrates insertion of a heated probe into two adjacent layers so as to fuse them together. The number of spot welds (lower left of figure) affects the mechanical properties of the final material, but the macroscopic structure of the spot-welded layers (lower left of figure) is affected by insertion of the heated probe. Here is presented a new method for forming nanofibrous laminates using light responsive materials, which materials may be polymeric fibers having nanoscale bodies (e.g., gold nanorods) disposed within. The nanoscale bodies effect controlled levels of heat with exposure to near-infrared (IR) light, which in turn allows for fusion of layers without physical contact from a probe or other instrument. This lack of contact in turn allows for layer fusion without the disruption of the layers' underlying structure that may result from contacting the layers with thermal probes or other implements.
In one embodiment, the present disclosure provides laminates (which may, in some places, also be referred to as compositions), the laminates comprising a first layer comprising a first population of polymeric fibers, at least some of the first population of polymeric fibers comprising nanoscale bodies disposed within; and a second layer comprising a second population of polymeric fibers, the first and second layers being bonded together at one or more locations.
The present disclosure also provides methods, the methods comprising irradiating a first fibrous layer comprising a first population of polymeric fibers having a first population of nanoscale bodies disposed within, the irradiating being performed so as to bond at least a portion of the first layer to a second fibrous layer comprising a second population of polymeric fibers.
Also provided are methods, the methods comprising electrospinning, from a polymeric fluid, a first population of polymeric fibers onto a first rotating surface of a mandrel, the electrospinning being performed such that at least a portion of the first population of polymeric fibers is aligned on the first surface in an arcuate (which may be characterized, in some cases, as being circumferential) fashion.
Further provided are compositions, the compositions comprising a first layer comprising a first population of polymeric fibers, the first population of polymeric fibers having an anisotropic alignment that varies spatially within the layer.
Additionally provided are biocompatible implants, the implants comprising a quantity of a composition according to the present disclosure, the quantity of material being shaped to as to approximate at least a portion of a knee meniscus, an annulus fibrosis, or any combination thereof.
Further provided are methods, the methods comprising seeding a composition according to the present disclosure with a population of cells.
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The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, there are shown in the drawings exemplary embodiments of the disclosure; however, the disclosure is not limited to the specific methods, compositions, and devices disclosed. In addition, the drawings are not necessarily drawn to scale. In the drawings:
FIG. 1 presents a schematic of nanofibrous scaffold containing AuNRs interspersed in PCL fibers (A). MSCs align on NRS (B) and SEMs show no difference between PCL (C) and NRS (D), scale bar: 10 μm. Average stress-strain profiles for PCL and NRS (E). A small decrease in both modulus (F) and yield stress (G) was noted in NRS compared to PCL scaffolds (n=6, *p<0.05).
FIG. 2 presents a schematic of laminate construction and testing (left) and quantification (right) of maximum interface strength for 1 and 2 welds produced through heated probe or IR laser exposure of NRS (n=6, *=p<0.05);
FIG. 3 presents A) Bright field images of fibers collected on slides (4×). B) Plot of mean fiber angle as a function of position from center of scaffold;
FIG. 4 presents fluorescent imaging of actin (green) and nuclei (blue) for MSCs seeded on linearly aligned (A) and circumferentially aligned (B) scaffolds (scale bar=100 μm);
FIG. 5 presents A) Schematic of specimens taken for tensile testing from circumferentially aligned (CA) mats. B) Modulus of CA and linearly aligned specimens with varying radii (3 cm, 5 cm), sample length (short, long) and region for strain analysis (center, edge). (*p<0.05 between short and long groups, +p<0.05 between scaffold region). C) Representative strain plots for LinAl and CircAl scaffolds with a central region strain of 3%;
FIG. 6 illustrates the annulus fibrosis and the meniscus, two fibrous tissues;
FIG. 7 illustrates an existing method of forming a multi-lamellar nanofibrous structure;
FIG. 8 illustrates a micrograph of an annulus fibrosis and also an image of a meniscus replacement material;
FIG. 9 illustrates exemplary parameters used in an experiment involving dispersion of gold nanorods in poly-caprolactone polymer;
FIG. 10 illustrates an exemplary process for fabricating multilamellar materials according to the present disclosure;
FIG. 11 illustrates exemplary results realized from fabricating multilamellar materials according to the present disclosure;
FIG. 12 illustrates caprolactone nanofiber morphology without (left) and with (right) inclusion of gold nanorods within the nanofibers;
FIG. 13 illustrates a less-magnified view of FIG. 12;
FIG. 14 illustrates the morphology of a fibrous material made from polycaprolactone fibers after spot welding (left) and nanorod-infrared welding (right);
FIG. 15 illustrates mechanical data obtained from testing fibrous scaffolds without (“PCL”) and with (“NRS”) nanorods;
FIG. 16 illustrates interface strength achieved by materials according to the present disclosure;
FIG. 17 presents cell viability and morphology results obtained on fibrous polycaprolactone scaffolds without (PCL) and with (NRS) nanorod incusion;
FIG. 18 depicts an exemplary method of fabricating anatomic meniscus structures according to the present disclosure; and
FIG. 19 depicts (A) a schematic showing electrospinning of fibers onto a rotating mandrel; (B) and (C) bright field images of strips of angled fibers with magnified images showing local alignment.
In a first embodiment, the present disclosure provides laminates. The laminates suitably include a first layer comprising a first population of polymeric fibers, with at least some of the first population of polymeric fibers comprising nanoscale bodies disposed within. The laminates suitably include a second layer comprising a second population of polymeric fibers, and the first and second layers are suitably bonded together at one or more locations.
The first population of polymeric fibers may comprise virtually any polymer. Polymers that are natural, synthetic, biocompatible, biodegradable, non-biodegradable, bioabsorbable, or any combination thereof are all suitable. It should be understood that in some embodiments, e.g., when an electrospun material is made of a single fiber (e.g. nanofiber), the fiber is folded thereupon, hence can be viewed as a plurality of connected fibers. It is to be understood that a more detailed reference to a plurality of fibers is not intended to limit the scope of the present disclosure to such particular case. Thus, unless otherwise defined, any reference herein to a “plurality of fibers” applies also to a single fiber and vice versa.
This disclosure is not limited by the thickness or shape of the fibers generated and used. Accordingly, the cross-sections of the fiber or fibers may be circular, oval, rectangular, square, or any shape which can be defined by the spinneret. Similarly, the fibers can have a cross-sectional dimension in the range of about 1 nm to about 10 microns, in the range of about 20 nm to about 1000 nm, in the range of about 100 nm to about 1000 nm, or in the range of about 1 micron to about 10 microns.
Fibers may be polymer fibers having diameters typically between 10 nm and 1000 nm. Exemplary sub-ranges contemplated by the present disclosure include between 100 and 1000 nm between 100 and 800 nm, between 100 and 600 nm, and between 100 and 400 nm. Other exemplary ranges include 10-100 nm, 10-200 nm and 10-500 nm. As mentioned, the fibers of the present disclosure are preferably generated by an electrospinning processes. In certain preferred embodiments, the first population of fibers, the second population of fibers, or both, has an average cross-sectional dimension in the range of from about 10 nm to about 10,000 nm.
As described herein, the various fibers may comprise materials that are natural, synthetic, biocompatible, biodegradable, non-biodegradable, and/or biosorbable, and unless specifically restricted to one or more of these categories, the fibers may comprise materials from any one of these categories.
The phrase “synthetic polymer” refers to polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. Examples include, but are not limited to, aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, polysiloxanes, and combinations thereof.
Suitable synthetic polymers for use according to the present disclosure may include biosynthetic polymers based on sequences found in collagen, elastin, thrombin, fibronectin, starches, poly(amino acid), poly(propylene fumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin, hyaluronic acid, polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene), polycarbonate, polypropylene and poly(vinyl alcohol), ribonucleic acids, deoxyribonucleic acids, polypeptides, proteins, polysaccharides, polynucleotides and combinations thereof.
The phrase “natural polymer” refers to polymers that are naturally occurring. Non-limiting examples of such polymers include, silk, collagen-based materials, chitosan, hyaluronic acid and alginate.
The phrase “biocompatible polymer” refers to any polymer (synthetic or natural) which when in contact with cells, tissues or body or physiological fluid of an organism does not induce adverse effects such as immunological reactions and/or rejections and the like. It will be appreciated that a biocompatible polymer can also be a biodegradable polymer.
The phrase “biodegradable polymer” refers to a synthetic or natural polymer which can be degraded (i.e., broken down) in the physiological environment such as by enzymes, microbes, or proteins. Biodegradability depends on the availability of degradation substrates (i.e., biological materials or portion thereof which are part of the polymer), the presence of biodegrading materials (e.g., microorganisms, enzymes, proteins) and the availability of oxygen (for aerobic organisms, microorganisms or portions thereof), carbon dioxide (for anaerobic organisms, microorganisms or portions thereof) and/or other nutrients. Aliphatic polyesters, poly(amino acids), polyalkylene oxalates, polyamides, polyamido esters, poly(anhydrides), poly(beta-amino esters), polycarbonates, polyethers, polyorthoesters, polyphosphazenes, and combinations thereof are considered biodegradable. More specific examples of biodegradable polymers include, but are not limited to, collagen (e.g., Collagen I or IV), fibrin, hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(Lactide-co-Glycolide) (PLGA), polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG), Collagen, PEG-DMA, alginate or alginic acid, chitosan polymers, or copolymers or mixtures thereof.
The phrase “non-biodegradable polymer” refers to a synthetic or natural polymer which is not degraded (i.e., broken down) in the physiological environment. Examples of non-biodegradable polymers include, but are not limited to, carbon, nylon, silicon, silk, polyurethanes, polycarbonates, polyacrylonitriles, polyanilines, polyvinyl carbazoles, polyvinyl chlorides, polyvinyl fluorides, polyvinyl imidazoles, polyvinyl alcohols, polystyrenes and poly(vinyl phenols), aliphatic polyesters, polyacrylates, polymethacrylates, acyl-sutostituted cellulose acetates, nonbiodegradable polyurethanes, polystyrenes, chlorosulphonated polyolefins, polyethylene oxides, polytetrafluoroethylenes, polydialkylsiloxanes, and shape-memory materials such as poly (styrene-block-butadiene), copolymers or mixtures thereof.
The phrase “biosorbable” refers to those polymers which are absorbed within the host body, either through a biodegradation process, or by simple dissolution in aqueous or other body fluids. Water soluble polymers, such as poly(ethylene oxide) are included in this class of polymers.
It will be appreciated that more than one polymer may be used to fabricate the scaffolds of the present disclosure. For example, the scaffold may be fabricated from a co-polymer. The term “co-polymer” as used herein, refers to a polymer of at least two chemically distinct monomers. Non-limiting examples of co-polymers which may be used to fabricate the scaffolds of the present disclosure include, PLA-PEG, PEGT-PBT, PLA-PGA, PEG-PCL and PCL-PLA. The use of copolymers or mixtures of polymers/copolymers provides a flexible means of providing the required blend of properties. In but one non-limiting example, functionalized poly(β-amino esters), which may be formed by the conjugate addition of primary or secondary amines with diacrylates, can provide a range of materials exhibiting a wide array of advantageous properties for this purpose. Such materials are described, for example, in Anderson, et al., “A Combinatorial Library of Photocrosslinkable and Degradable Materials,” Adv. Materials, vol. 18 (19), 2006, which reference is incorporated by reference in its entirety.
Additionally, individual polymers or co-polymers may be physically mixed and co-spun through the same spinneret. Similarly, according to this disclosure, a composition may be comprised of a mixture of simultaneously or sequentially delivered polymers and/or copolymers. This includes mixtures of at least two natural, synthetic, biocompatible, biodegradable, non-biodegradable, and/or biosorbable polymers and co-polymers.
Other embodiments of this disclosure provide that, where a composition comprises two or more fibers, that each may have a different biodegradation and/or biosorption profile in a physiological fluid, said fluids including water, saline, simulated body fluid, or synovial fluid.
Still other embodiments provide that the polymers, co-polymers, or blends thereof may be photolytically active, such that once electrospun, they may be made to crosslink on exposure to light, thereby improving the tensile characteristics of the scaffold, and increasing the diversity and range of properties available. See for example, Tan, et al., J. Biomed Matl. Res., vol. 87 (4), 2008, pp. 1034-1043, which is incorporated by reference in its entirety.
In some embodiments, the first layer, the second layer, or both, further comprises a porogenic material. At least part of the porogenic material may be present as fibers, particles, or any combination thereof. As used herein, the term “porogen” refers to sacrificial materials added during the production of a scaffold (for example, during electrospinning) and subsequently removed, whose purpose is to occupy space during the construction process, such that their subsequent removal results in what amounts to engineered porosity. In tissue engineering, materials such as inorganic salt like sodium chloride, crystals of saccharose, gelatin spheres or paraffin spheres are used to introduce particulate porosity. In the present disclosure, the use of porogen fibers provides, in some embodiments, porosity aligned with the remaining fibers.
The second population of fibers may include a population of nanoscale bodies disposed within. A nanoscale body may be organic, inorganic (e.g., metallic). A nanoscale body may also be a biological molecule, such as a growth factor, a protease, trypsin, and the like. A variety of dopants may be present within (or on) the fibers of the disclosed materials.
In one set of embodiments, these dopants include at least one therapeutic compound or agent, capable of modifying cellular activity. Similarly, agents that act to increase cell attachment, cell spreading, cell proliferation, cell differentiation and/or cell migration in the scaffold may also be incorporated into the scaffold. Such agents can be biological agents such as an amino acid, peptides, polypeptides, proteins, DNA, RNA, lipids and/or proteoglycans.
These agents may also include growth factors [e.g., a epidermal growth factor, a transforming growth factor-α, a basic fibroblast growth factor, a fibroblast growth factor-acidic, a bone morphogenic protein, a fibroblast growth factor-basic, erythropoietin, thrombopoietin, hepatocyte growth factor, insulin-like growth factor-I, insulin-like growth factor-II, Interferon-β, platelet-derived growth factor, a nerve growth factor, a transforming growth factor, a tumor necrosis factor, Vascular Endothelial Growth Factor, an angiopeptin, or a homolog or combination thereof], cytokines [e.g., M-CSF, IL-1beta, IL-8, beta-thromboglobulin, EMAP-II, G-CSF and IL-IO, or a homolog or combination thereof], proteases [pepsin, low specificity chymotrypsin, high specificity chymotrypsin, trypsin, carboxypeptidases, aminopeptidases, proline-endopeptidase, Staphylococcus aureus V8 protease, Proteinase K (PK), aspartic protease, serine proteases, metalloproteases, ADAMTS 17, tryptase-gamma, and matriptase-2, or a homolog or combination thereof] and protease substrates.
Suitable proteins which can be used along with the present disclosure include, but are not limited to, extracellular matrix proteins [e.g., fibrinogen, collagen, fibronectin, vimentin, microtubule-associated protein ID, Neurite outgrowth factor (NOF), bacterial cellulose (BC), laminin and gelatin], cell adhesion proteins [e.g., integrin, proteoglycan, glycosaminoglycan, laminin, intercellular adhesion molecule (ICAM) 1, N-CAM, cadherin, tenascin, gicerin, RGD peptide and nerve injury induced protein 2 (ninjurin2)].
Additionally and/or alternatively, the materials of the present disclosure may comprise an antiproliferative agent (e.g., rapamycin, paclitaxel, tranilast, Atorvastatin and trapidil), an immunosuppressant drug (e.g., sirolimus, tacrolimus and Cyclosporine) and/or a non-thrombogenic or anti-adhesive substance (e.g., tissue plasminogen activator, reteplase, TNK-tPA, glycoprotein IIb/IIIa inhibitors, clopidogrel, aspirin, heparin and low molecular weight heparins such as enoxiparin and dalteparin).
Examples of immunosuppressive agents which can be used to minimize immunosuppression include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF-α, blockers, a biological agent that targets an inflammatory cytokine, IL-1 receptor antagonists, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.
Cytokines useful in the present disclosure include, but are not limited to, cardiotrophin, stromal cell derived factor, macrophage derived chemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophage inflammatory proteins 1 alpha (MOP-1 alpha, 2, 3 alpha, 3 beta, 4, and 5, IL-, 11-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF-α, and TNF-β. Immunoglobulins useful in the present disclosure include but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGFs (nerve growth factors), PFGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.
It should be understood that the disclosed materials may include a single type of nanoscale body/dopant, or two or more types of nanoscale bodies/dopants.
A nanoscale body suitably has at least one cross-sectional dimension in the range of from about 1 nm to about 100 nm. A cross-sectional dimension is a length, width, diameter, or thickness. A nanoscale body suitably has an aspect ratio in the range of from about 1 to about 100. For example, a nanorod having a height of 10 nm and a diameter of 5 nm would be considered suitable. A nanorod having a diameter in the range of from about 2 to about 4 nm and a length in the range of from about 7 nm to about 9 nm is considered an especially suitable nanoscale body, particularly when the nanorod is made of gold.
One embodiment of the present disclosure provides for the selection of materials for the fibers of sufficiently high modulus such that as one of the fibers degrades, the scaffold retains the required modulus, for example at least 20 MPa, preferably at least 40 MPa, more preferably at least 60 MPa, and more preferably at least about 80 MPa, at a strain region of about 10%, and/or an overall circumferential modulus in the range of about 5 to about 60 MPa, preferably at least 10 MPa, more preferably at least 20 MPa, and most preferably at least about 30-35 MPa, at a strain region of about 3%, as defined herein, for sufficient time, for example over 10-20 weeks, under physiological conditions.
In other embodiments, a first fiber comprises a material characterized as having a yield strain at least about 1%, preferably at least about 4%, more preferably at least about 8% and most preferably at least 10%. This fiber may be biocompatible, but may or may not be biodegradable, though it is preferably so. Absolute tensile properties of this material may beless important than are those of the second fiber, since it is the combination of the moduli of the first and second fibers, especially as a function of time of exposure to physiological conditions, that are important, but to accomplish this, the modulus of the first fiber material should be at least about 20 MPa at lower (3%) strain levels. Higher values are preferred, for example, preferably at least about 100 MPa, and most preferably at least about 200 MPa, especially at higher (10%) strain levels. In one embodiment, the first fiber material comprises poly(caprolactone). In other embodiments, this first fiber comprises a poly(β-amino ester) or an acrylate terminated poly(β-amino ester). Such materials are described, for example, in Anderson, et al., “A Combinatorial Library of Photocrosslinkable and Degradable Materials,” Adv. Materials, vol. 18 (19), 2006, this reference being incorporated by reference in its entirety.
A second fiber may comprise a biodegradable material characterized as having a modulus in the range from about 10 MPa to about 500 MPa, when subjected to a strain in the range of from about 1% to about 10% and measured in the direction of the fiber alignment; in another embodiment, this modulus is in the range of about 20 MPa to about 500 MPa; in another, this modulus is in the range of about 300 MPa to about 500 MPa, especially at higher (10%) strain levels. In another embodiment, the second fiber comprises poly(glycolic acid). In yet another embodiment, the second fiber comprises a blend of poly(caprolactone) and poly(glycolic acid). In other embodiments, this second fiber also comprises a poly(β-amino ester) or an acrylate terminated poly(β-amino ester). Yield stress for this fiber material should be at least 1%, preferably at least about 4%, and most preferably at least about 8%.
One embodiment of the invention provides that the first and second fibers are be biodegradable, and that the rates of biodegradability of the two fibers are different, when subjected to similar or the same physiological conditions. In one embodiment, the second fiber biodegrades more quickly than the first. In this embodiment, when taken together, the relative rates of biodegradability (or biosorption or dissolution) of the first fiber, the second fiber, and the porogen fiber can be considered slow, medium, and fast. It is preferred that the relative lifetime of the second fiber in vivo is sufficiently long so as to provide a sustained basis for tissue regeneration—generally on the order of weeks under physiological conditions. The relative lifetime of the second fiber in vivo can be determined or approximated by measuring tensile properties or weight loss of the scaffold under simulated physiological conditions.
In one embodiment, the scaffolding contains a porogen fiber, co-spun with the first and the second fibers, comprising an amount in the of about 10 to about 80 weight percent based on the total weight of electrospun fibers, preferably in the range of about 20 to about 60 weight percent, more preferably in the range of about 30 to about 60 weight percent, and most preferably in about 40-55 weight percent, all with respect to the total weight of electrospun fibers. As used herein, the term “porogen” refers to sacrificial materials added during the production of a scaffold (for example, during electrospinning) and subsequently removed, whose purpose is to occupy space during the construction process, such that their subsequent removal results in what amounts to engineered porosity. In tissue engineering, materials such as inorganic salt like sodium chloride, crystals of saccharose, gelatin spheres or paraffin spheres are used to introduce particulate porosity. In the present invention, the use of porogen fibers provides porosity aligned with the remaining fibers.
In another embodiment, this porogen fiber has been removed, such that the resulting scaffold contains spacings defined by the absence of this porogen material. This removal can be accomplished in several ways, though the most usual way of doing so is by selective dissolution. Preferably, this porogen fiber is capable of selectively and substantially dissolving in physiological fluids, such as water, saline, meniscal fluid, simulated body fluid, or synovial fluid, such dissolution occurring within one hour, preferably within 30 minutes, and most preferably within 10 minutes of contact with the physiological fluid at ambient or physiological conditions. In such circumstances, one non-limiting example, the porogen fiber comprises poly(ethylene oxide). However, the means of removal is not limited to use of physiological fluids. For example, depending on the porogen, hydrocarbons or other organic solvents may also be used (ex vivo). Also the removal can be accomplished inside or outside the patient. One of the purposes of applying and then removing this porogen fiber material is to provide spaces within the matrix to expedite cellular ingress into the scaffold matrix, whose fiber densities otherwise inhibit this incursion. For this reason, one skilled in the art would appreciate that removing the porogen material from the matrix before seeding with, for example, cell populations, and implanting into the patient can be a desirable scenario.
Other embodiments describe the relative proportion of the first and second fiber. In one such embodiment, the first fiber comprises an amount in the range of about 20% by weight to about 80% by weight, relative to the combined weight of the first and second fiber. Other embodiments define the relative amount of the first fiber to be in the range of about 40% by weight to about 60% by weight, or about 50% by weight, each relative to the combined weight of the first and second fiber. The specific ratio of the two fibers will depend on the particular choice of fibers, and one skilled in the art would be able to understand the most appropriate ratio for a given set of fiber materials based on the teaching herein.
The invention teaches that the electrospun fibers may individually comprise the individual polymers or copolymers, or blends of polymers or copolymers or both. Within the scaffold and/or within the individual fibers, the first fiber material may be present in the range of about 1 to about 80 weight percent, and the second fiber may be present in the range of about 80 to about 1 weight percent, each with respect to the total weight of electrospun fibers.
Together, the first and second fibers may form a scaffold whose circumferential modulus in the range of about 10 MPa to about 100 MPa, preferably in the range of about 60 MPa to about 90 MPa, more preferably in the range of about 70 MPa to about 85 MPa, most preferably about 80 MPa. This is accomplished by combining the fiber materials, applied either as individual fibers or co-spun as blended materials. such that the weighted average of the materials according to their individual moduli provide the target scaffold circumferential modulus. One skilled in the art would be able to measure and/or calculate the combined modulus as a function of such a composite. In the simplest case, this relationship can be characterized according to the Rule of Mixtures equation:
Modulus of the composite=Σ[(φx*(modulus of material x)],
where φx represents the weight fraction of the xth component (strictly speaking, the rule of mixtures deals with volume fractions, but to a good approximation, and assuming polymers of comparable densities are used, use of weight fractions provides an equivalent means of characterization).
Tensile modulus is a property which is often defined in terms relative to the total cross-sectional area of the fiber or fiber bundle, or in this case, to the circumferential alignment of fibers. So as to maintain internal consistency, as described herein, whether the scaffold contains or has had removed the porogen fiber, the moduli are calculated and described so as to consider the cross-sectional area of the porogen fiber, but not to consider the tensile properties of that porogen fiber. For example, in but one non-limiting example, a mixture comprising 25% by weight (of the total polymer weight) of a first polymer, having a modulus of ca. 20 MPa, and 25% by weight a second fiber, having a modulus of 300 MPa, and 50% by weight of a porogen fiber, having a modulus of 100 MPa is described herein as having a composite modulus of 80 MPa for the composite (i.e., (25%×20 MPa)+(25%×300 MPa)+(50%×0 MPa)=(5+75+0)=80 MPa), and not 130 MPa (as would result if the modulus of the scaffold retained the contribution of the porogen; i.e., (25%×20 MPa)+(25%×300 MPa)+(50%×100 MPa)=(5+75+50)=130 MPa) or 160 MPa (as would result if the cross-sectional area of the porogen were ignored; i.e., (50%×20 MPa)+50%×300 MPa)=(10+150)=160 MPa. It should be appreciated that this definition provides a more rigorous requirement for tensile modulus for the scaffold than if the tensile contribution of the porogen material had been considered or if the cross sectional area of the porogen material had been ignored.
Other embodiments of this invention lift the constraint that the first fiber have a particular yield stress value, and allowing the second fiber to have a modulus lower in value than described above, replacing these requirements with one that the combination of first and second fibers maintain a mean circumferential scaffold modulus of at least about 40 MPa, preferably about 60 MPA, and most preferably at least about 80 MPa, at higher (10%) strain levels, when subjected to physiological fluids under physiological conditions for times sufficient to allow for cell ingress and proliferation, typically on the order of weeks. As described earlier, it is highly desirable that the scaffold maintain a minimum modulus during the time of this cell ingress and proliferation, corresponding to healing.
It is also understood that electrospinning provides fibrous solid bodies which contain a degree of porosity which can be affected by the materials of construction—both fibers and other incorporated materials—and the method of making. Accordingly, certain embodiments of this invention describe porous solids whose void volumes are on the order of about 5 to 99 volume percent; other embodiments describe porous solids with void volumes at the lower end of this range, e.g., in the range from about 5 volume percent to about 25 volume percent; still other embodiments describe porous solids with void volumes in the middle of this range, e.g., in the range from about 25 volume percent to about 75 volume percent; and still other embodiments describe solids with void volumes at the high end of this range, e.g., in the range from about 50 volume percent to about 95 volume percent. Other exemplary sub-ranges contemplated by the present invention include the range of about 80 to about 99 volume percent, the range of about 85 to about 95 volume percent, and the range of about 90 to about 95 volume percent.
In the disclosed compositions, at least a portion of the first population of fibers are suitably substantially aligned in a first direction. It should be understood that not all of the first population of fibers need be aligned in this first direction, which first direction may be characterized as being about circumferential to a hypothetical central axis, as illustrated in, e.g., FIG. 3b, FIG. 4b, and FIG. 19. It is preferable that more than 50% of the fibers be aligned in this first direction, but 50% should not be understood as being a particular threshold. Similarly, at least a portion of the second population of fibers are substantially aligned in a second direction. The first a second directions may be parallel to one another, although parallel directions are not a requirement. In some embodiments, at least a portion of the second population of fibers is aligned perpendicular to at least a portion of the first population of fibers. In this way, the perpendicular fibers may act as tie fibers in an artificial meniscus material.
In some preferred embodiments, at least a portion of the first population of fibers, at least a portion of the second population of fiber, or both, have an arcuate alignment. Arcuate should be understood as referring to fibers that are curved (as opposed to being straight). The curve may be a circular or circumferential one, although other curves (e.g., elliptical or other curves that are not based on a constant radius) are also suitable.
Illustrative fibers are shown in FIG. 3, which shows linearly aligned fibers (FIG. 3A) and circumferentially-aligned fibers (FIG. 3B). FIG. 3C illustrates the mean fiber angle (degrees) as a function of the fiber\'s position from the center of the mandrel. Data are shown for linearly-aligned fibers (which have an unchanging fiber angle) and for circularly-aligned fibers (which have an angle that changes as a function of the fiber\'s distance from the center of the mandrel).