Devices and methods for ameliorating implant-induced inflammation

Abstract

The present invention features methods and devices for ameliorating implant-induced inflammation and reducing the risk of fibrosis, including a substrate coated with P-15 peptide that is positioned at the implantation site. TIFF2025527336000021.tif89165

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of the XML created on August 10, 2023 is named "50517-031WO2_Sequence_Listing_8_10_23" and is 1,783 bytes in size. [Background technology]

[0002] Background of the Invention Surgical implantation of a medical device will initiate the development of a foreign body reaction (FBR), regardless of whether the implant is made of inert, nontoxic materials (see Bridges et al., J Diabetes Sci Technol. 2(6):984 (2008)). The combination of at least two events has been demonstrated to contribute to the host's induction of an inflammatory cascade: the immediate adsorption and binding of plasma proteins and other molecules to the implant surface, followed by microvascular and tissue damage during implantation and the associated release of histamine by local mast cells. The progression of the FBR, including leukocyte recruitment, differentiation into foreign body giant cells (FBGCs), and release of cytokines and matrix metalloproteinases (MMPs), is characteristic of the acute host inflammatory response to device implantation and can lead to implant-induced fibrosis and ultimately implant failure.

[0003] There is a need for methods to resolve the inflammatory events associated with implant-induced inflammation. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Bridges et al., J Diabetes Sci Technol. 2(6):984 (2008) Summary of the Invention

[0005] The present invention features a method for fusing two vertebral bodies of a subject, the method including: (a) providing a spinal fusion cage comprising (i) polyaryletherketone (PAEK) having an inner surface and an outer surface, and (ii) inorganic particles coated with P-15 peptide; and (b) positioning (i) the spinal fusion cage and (ii) the inorganic particles coated with P-15 peptide between a first vertebral body and a second vertebral body, wherein step (b) includes disposing the inorganic particles coated with P-15 peptide inside the spinal fusion cage and outside the spinal fusion cage.

[0006] In certain embodiments, the PAEK is polyether-ether-ketone (PEEK), polyetherketone (PEK), polyetherketoneketone (PEKK), polyetheretherketoneketone (PEEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK).

[0007] In some embodiments, the PAEK has a molecular weight (Mn) of 110-120 KDa, a molecular weight (Mn) of 100-110 KDa, or a molecular weight (Mn) of 80-100 KDa.

[0008] In certain embodiments, the PAEK has a glass transition temperature of 250° C. to 450° C. For example, the PAEK can have a glass transition temperature of 300° C. to 380° C., 340±20° C., 365±5° C., or 375±5° C.

[0009] In certain embodiments, the PAEK is a composite material that includes fibers (eg, carbon fibers) and / or a radiopaque agent (eg, barium sulfate).

[0010] The invention further features a method for fusing two vertebral bodies in a subject, the method including: (a) providing a spinal fusion cage comprising (i) polyetherketoneketone (PEKK) having an inner surface and an outer surface, and (ii) inorganic particles coated with P-15 peptide; and (b) positioning the (i) spinal fusion cage and the (ii) inorganic particles coated with P-15 peptide between a first vertebral body and a second vertebral body. In certain embodiments, the PEKK has a glass transition temperature of 250°C to 450°C (e.g., 300°C to 380°C, 340±20°C, 365±5°C, or 375±5°C). In some embodiments, the PEKK has a molecular weight (Mn) of 110-120 KDa, 100-110 KDa, or 80-100 KDa. In certain embodiments, the PEKK is a composite material that includes fibers (e.g., carbon fibers) and / or radiopaque agents (e.g., barium sulfate).

[0011] The invention also features a method for fusing two vertebral bodies in a subject, the method including: (a) providing a spinal fusion cage comprising (i) polyether-ether-ketone (PEEK) having an inner surface and an outer surface, and (ii) inorganic particles coated with P-15 peptide; and (b) positioning the (i) spinal fusion cage and (ii) inorganic particles coated with P-15 peptide between a first vertebral body and a second vertebral body. In certain embodiments, the PEEK has a glass transition temperature of 250°C to 450°C (e.g., 300°C to 380°C, 340±20°C, 365±5°C, or 375±5°C). In some embodiments, the PEEK has a molecular weight (Mn) of 110-120 KDa, 100-110 KDa, or 80-100 KDa. In certain embodiments, the PEEK is a composite material that includes fibers (eg, carbon fibers) and / or radiopaque agents (eg, barium sulfate).

[0012] The present invention further features a method for fusing two vertebral bodies of a subject, the method comprising: (a) providing a spinal fusion cage comprising (i) polyetherketone (PEK) having an inner surface and an outer surface, and (ii) inorganic particles coated with P-15 peptide; and (b) positioning the (i) spinal fusion cage and the (ii) inorganic particles coated with P-15 peptide between a first vertebral body and a second vertebral body. In certain embodiments, the PEK has a glass transition temperature of 250°C to 450°C (e.g., 300°C to 380°C, 340±20°C, 365±5°C, or 375±5°C). In some embodiments, the PEK has a molecular weight (Mn) of 110-120 KDa, 100-110 KDa, or 80-100 KDa. In certain embodiments, the PEK is a composite material including fibers (e.g., carbon fibers) and / or a radiopaque agent (e.g., barium sulfate).

[0013] The invention also features a method for fusing two vertebral bodies in a subject, the method including: (a) providing a spinal fusion cage comprising (i) polyetheretherketoneketone (PEEKK) having an inner surface and an outer surface, and (ii) inorganic particles coated with P-15 peptide; and (b) positioning (i) the spinal fusion cage and (ii) the inorganic particles coated with P-15 peptide between a first vertebral body and a second vertebral body. In certain embodiments, the PEEKK has a glass transition temperature of 250°C to 450°C (e.g., 300°C to 380°C, 340±20°C, 365±5°C, or 375±5°C). In some embodiments, the PEEKK has a molecular weight (Mn) of 110-120 KDa, 100-110 KDa, or 80-100 KDa. In certain embodiments, the PEEKK is a composite material that includes fibers (eg, carbon fibers) and / or radiopaque agents (eg, barium sulfate).

[0014] The invention further features a method for fusing two vertebral bodies in a subject, the method comprising: (a) providing a spinal fusion cage comprising (i) polyaryl-ether-ketone-ether-ketoneketone (PEKEKK) having an inner surface and an outer surface, and (ii) inorganic particles coated with P-15 peptide; and (b) positioning (i) the spinal fusion cage and (ii) the inorganic particles coated with P-15 peptide between a first vertebral body and a second vertebral body. In certain embodiments, the PEKEKK has a glass transition temperature of 250°C to 450°C (e.g., 300°C to 380°C, 340±20°C, 365±5°C, or 375±5°C). In some embodiments, the PEKEKK has a molecular weight (Mn) of 110-120 KDa, 100-110 KDa, or 80-100 KDa. In certain embodiments, PEKEKK is a composite material that includes fibers (eg, carbon fibers) and / or radiopaque agents (eg, barium sulfate).

[0015] In the above method, the inorganic particles can be selected from hydroxyapatite particles, dallite particles, tetracalcium phosphate particles, calcium pyrophosphate particles, tricalcium phosphate particles, calcium hydrogen phosphate particles, octacalcium phosphate particles, calcium fluoroapatite particles, and mixtures thereof. For example, the inorganic particles can be hydroxyapatite particles having a diameter of 250 microns to 425 microns, such as inorganic bone mineral coated with P-15 peptide.

[0016] In any of the above methods, the spinal fusion cage comprises a porous material, and the amount of P-15 peptide bound to the surface of the inorganic particles is 100-1500 ng of P-15 peptide per gram of inorganic particles, and / or the P-15 peptide-coated inorganic particles are suspended in a collagen hydrogel. In some embodiments, the weight ratio of the P-15 peptide-coated inorganic particles to collagen is 50:50-95:5. In certain embodiments, the amount of P-15 peptide bound to the surface of the inorganic particles is 200-1200 ng of P-15 peptide per gram of inorganic particles, and the weight ratio of the P-15 peptide-coated inorganic particles to collagen is 75:25-95:5.

[0017] In one embodiment of any of the methods of the present invention, placing the P-15 peptide in or around the spinal fusion cage reduces local inflammation between the two vertebral bodies.

[0018] In another embodiment of any of the methods of the present invention, placing the P-15 peptide in or around the spinal fusion cage reduces localized fibrosis between two vertebral bodies.

[0019] The present invention features a method for ameliorating implant-induced inflammation at an implantation site in a subject, the method including administering to the implantation site (i) an implantable medical device and (ii) a substrate coated with P-15 peptide, wherein the substrate is not a calcified substrate.

[0020] The invention further features a method for ameliorating implant-induced inflammation at an implantation site in a subject, the method comprising inserting into the implantation site (i) an implantable medical device and (ii) a substrate coated with P-15 peptide, wherein the implantation site does not contain bone tissue.

[0021] The invention also features a method for ameliorating implant-induced inflammation at an implantation site in a subject, the method comprising inserting into the implantation site (i) an implantable medical device comprising a biodegradable polymer and (ii) a substrate coated with P-15 peptide. In certain embodiments, the biodegradable polymer is selected from poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly(butylene succinate) (PBS), and sucrose acetate isobutyrate (SAIB).

[0022] The present invention features a method for ameliorating implant-induced inflammation at an implantation site in a subject, the method comprising inserting (i) an implantable medical device and (ii) a substrate coated with P-15 peptide into the implantation site, where the implantation site is soft tissue (e.g., muscle tissue, adipose tissue, connective tissue, organ tissue, subcutaneous tissue, ocular tissue, or brain tissue). In certain embodiments, the implantable medical device is implanted into a soft tissue selected from nervous tissue, vascular tissue, oral tissue, ocular tissue, nasal tissue, genitourinary tissue, gastrointestinal tissue, biliary tissue, auditory tissue, or subcutaneous tissue.

[0023] The invention further features a method of ameliorating implant-induced inflammation at an implantation site in a subject, comprising inserting an implantable medical device comprising a substrate coated with P-15 peptide into the implantation site, wherein the implantable medical device is a neurological device, a vascular device, a cardiovascular device, an oral device, an ocular device, a nasal device, a genitourinary device, a gastrointestinal device, a biliary device, an auditory device, a subcutaneous device, a plastic surgery device, a general surgery device, or a prosthetic device. In some embodiments, the implantable medical device is a membrane, a mesh, a sling, a tissue anchor, a tissue expander, a suture, or a gel. In some embodiments, the implantable medical device is an extracellular matrix (ECM). In some embodiments, the implantable medical device is a tissue. In some embodiments, the implantable medical device is a graft. In some embodiments, the implantable medical device is a dialysis device.In certain embodiments, the neural device is an electrode, a pulse generator, or a neurovascular catheter; the vascular device is a vascular stent or a vascular graft; the cardiovascular device is a pacemaker, a defibrillator, a coronary stent, a cardiovascular catheter, or a heart valve, optionally, the heart valve is a tricuspid valve, a pulmonary valve, a mitral valve, or an aortic valve; the oral device is a tracheostomy tube; the ocular device is an intraocular lens, an intrastromal corneal ring segment (ICRS), or an ophthalmic catheter; the nasal device is a nasal stent; the genitourinary device is a mesh, a contraceptive implant, a hernia mesh, a pelvic mesh, a urinary stent, an artificial urinary sphincter, or a urinary catheter; and optionally, the contraceptive implant is an intrauterine device (IUD) or a birth control device. the gastrointestinal device is a staple, a balloon, a sleeve, a band, a gastric stimulator, or a gastrointestinal catheter, and optionally the band is a LINX device, the biliary device is a biliary stent, the hearing device is a cochlear implant or an ear tube, the subcutaneous device is a drug delivery needle or a glucose sensor, the prosthetic device is an artificial eye, a breast implant, a prosthetic nose, a penile implant, or a cosmetic implant, or the breast implant is a saline breast implant or a silicone breast implant.

[0024] In any embodiment of the methods of the present invention, the implantable medical device comprises a polyaryletherketone (PAEK) (e.g., polyether-ether-ketone (PEEK), polyetherketoneketone (PEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK)).

[0025] In any embodiment of the methods of the present invention, implantation of the implantable medical device reduces local inflammation at the site (e.g., compared to implantation of an identical implantable medical device except that a P-15 coated substrate is administered to the site).

[0026] In another embodiment of any of the methods of the present invention, implantation of the implantable medical device reduces local fibrosis at the site (e.g., compared to implantation of an identical implantable medical device except that a P-15 coated substrate is administered to the site).

[0027] Various embodiments of any of the methods of the present invention optionally include one or more of the following features: (i) the substrate is not a mineralized substrate; (ii) the implantation site does not include bone tissue; (iii) the implantation site is soft tissue; and / or (iv) the implantable medical device comprises a biodegradable polymer.

[0028] In a related aspect, the invention features an implantable medical device comprising (i) a biodegradable polymer and (ii) a substrate coated with P-15 peptide. In a related aspect, the invention features an implantable medical device comprising a biodegradable polymer directly coated with P-15. In a related aspect, the invention features an implantable medical device comprising a biodegradable polymer comprising P-15. In some embodiments, P-15 is incorporated within the biodegradable polymer. In a related aspect, the invention features an implantable medical device comprising a hydrogel comprising P-15. For example, the biodegradable polymer can be selected from poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly(butylene succinate) (PBS), and sucrose acetate isobutyrate (SAIB).

[0029] In a related aspect, the invention features an implantable medical device that includes (i) a polymer selected from hyaluronic acid and carboxymethylcellulose, and (ii) a substrate coated with P-15 peptide.

[0030] In another aspect, the invention features an implantable medical device designed for implantation into soft tissue and comprising a substrate coated with P-15 peptide. For example, the implantable medical device can be a neurological device, a vascular device, a cardiovascular device, an oral device, an ophthalmic device, a nasal device, a genitourinary device, a gastrointestinal device, a biliary device, an auditory device, a subcutaneous device, or a prosthetic device. In certain embodiments, the neurological device is an electrode, a pulse generator, or a neurovascular catheter; the vascular device is a vascular stent; the cardiovascular device is a pacemaker, a defibrillator, a coronary stent, a cardiovascular catheter, or a heart valve, optionally, the heart valve is a tricuspid valve, a pulmonary valve, a mitral valve, or an aortic valve; the oral device is a tracheostomy tube; the ophthalmic device is an intraocular lens, an intrastromal corneal ring segment (ICRS), or an ophthalmic catheter; the nasal device is a nasal stent; and the genitourinary device is a mesh, a contraceptive implant, a hernia mesh, a pelvic mesh, a urinary stent, an artificial urinary sphincter, or a urinary catheter. and optionally the contraceptive implant is an intrauterine device (IUD) or a birth control implant; the gastrointestinal device is a staple, a balloon, a sleeve, a band, a gastric stimulator, or a gastrointestinal catheter, and optionally the band is a LINX device; the biliary device is a biliary stent; the hearing device is a cochlear implant or an ear tube; the subcutaneous device is a drug delivery device or a glucose sensor; the prosthetic device is an artificial eye, a breast implant, a prosthetic nose, a penile implant, or a cosmetic implant, or the breast implant is a saline breast implant or a silicone breast implant.

[0031] In any of the embodiments of the implantable medical device of the present invention, the implantable medical device comprises a polyaryletherketone (PAEK) (e.g., polyether-ether-ketone (PEEK), polyetherketoneketone (PEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK)).

[0032] In a related aspect, the invention features a vascular stent having a surface comprising a substrate coated with P-15 peptide. In certain embodiments, the stent comprises stainless steel, cobalt-chromium alloy, nickel-titanium alloy, platinum, or tantalum alloy coated with P-15 peptide.

[0033] In another aspect, the invention features a spinal fusion cage including a substrate coated with P-15 peptide, wherein the substrate is not a mineralized substrate. In some embodiments, the spinal fusion cage includes polyaryletherketone (PAEK) (e.g., polyether-ether-ketone (PEEK), polyetherketoneketone (PEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK)).

[0034] In some embodiments, the spinal fusion cage comprises acellular tissue, hi some embodiments, the acellular tissue comprises an extracellular matrix (ECM), such as acellular dermal matrix (ADM).

[0035] In some embodiments, the spinal fusion cage comprises a mesh.In some embodiments, the spinal fusion cage comprises a collagen mesh.

[0036] In some embodiments, the first vertebral body can be an implant or bone. In some embodiments, the second vertebral body can be an implant or bone.

[0037] definition To facilitate understanding of the present invention, a number of terms are defined below and throughout this disclosure. 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 invention belongs. While the terms herein are used to describe particular embodiments of the invention, their use does not limit the invention, except as outlined in the claims.

[0038] Terms such as "a," "an," and "the" are not intended to refer only to a singular entity, but include general classes within which specific examples can be used for illustration.

[0039] As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0040] As used herein, the term "mineralized substrate" refers to a substrate that includes calcium cations and phosphate or hydrogen phosphate anions.

[0041] As used herein, the term "P-15 peptide" refers to the peptide of SEQ ID NO: 1 It refers to Gly-Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val (SEQ ID NO: 1, "P-15").

[0042] As used herein, the term "reducing local inflammation" refers to the observed average reduction in one or more markers of inflammation (e.g., TNFα) at the implantation site following implantation according to the methods of the present invention compared to implantation of an otherwise identical implantable medical device administered without the P-15 peptide. Reduction of local inflammation due to incorporation of P-15 peptide can be measured as described in Example 1.

[0043] As used herein, the term "reducing local fibrosis" refers to the observed average reduction in fibrosis at the implantation site following implantation by the methods of the present invention compared to implantation of an identical implantable medical device administered without the P-15 peptide.

[0044] Other features and advantages of the invention will be apparent from the following detailed description, the drawings and the claims. [Brief explanation of the drawings]

[0045] [Figure 1]1A and 1B are graphs showing the immunomodulatory effects of P-15 peptide on BMDMs plated on PEEK in culture, as described in Example 1. As shown in FIG. 1A, TNFα production was significantly reduced in cells plated with P-15 compared to cells cultured on PEEK alone. As shown in FIG. 1B, differences in IL-1β production from MSCs in culture with P-15 compared to PEEK alone were also observed. [Figure 2] 2A and 2B are graphs showing ALP activity of BMDMs plated on PEEK and alone during culture, as described in Example 1 to measure osteogenic potential. As shown in FIG. 2A, ALP activity increased slightly in P15-L on PEEK after 7 days of incubation, but significantly in P-15 alone. As shown in FIG. 2B, ALP activity increased in P15-L on PEEK compared to the control and PEEK alone after 28 days of incubation. [Figure 3] Figures 3A and 3B are images showing devices implanted into the femurs of a rabbit with bilateral femoral defects. Figure 3A shows a control PEEK device, and Figure 3B shows a PEEK device filled with P-15. [Figure 4] 1 shows micro-CT data demonstrating increased bone deposition around implants filled with P-15 compared to implants not filled with P-15, positioned in the femurs of rabbits with bilateral femoral defects. [Figure 5] 1 shows the microCT methodology for three regions of control and P-15 filled implants placed in the femurs of rabbits with bilateral femoral defects. [Figure 6] Figures 6A and 6B are graphs showing bone mass in two regions around control and P-15-filled implants in rabbits with bilateral femoral defects identified by micro-CT. Figure 6A shows bone mass offset 1 mm from the implant surface. Figure 6B shows bone mass offset 0.5 mm from the implant surface. [Figure 7A]Figures 7A-F are graphs showing bone mass in three peri-implant regions for control and P-15-filled implants in rabbits with bilateral femoral defects identified by micro-CT. Figure 7A shows bone mass, and Figure 7B shows bone density in an ROI of 1000-500 μm. Figure 7C shows bone mass, and Figure 7D shows bone density in an ROI of 500-136 μm. Figure 7E shows bone mass, and Figure 7F shows bone density in an ROI of 136-0 μm. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 8] Figure 7A shows a statistical analysis of the micro-CT data shown in Figures 7A, 7C, and 7E. The results indicated a significant difference in the bone-implant interface between the P-15 constructs and the control samples. Samples containing P15 showed a significant increase in bone volume in the peri-implant space. The data provided strong evidence that P-15 promotes bone ingrowth and reduces the fibrous layer known to form around PEEK implants. [Figure 9] 9A-E show histological images of PEEK devices implanted in rabbits, in which the fibrous layer is evident. [Figure 10] Figures 10A and B show micro-CT imaging of a PEEK device implanted in a rabbit, in which the fibrous layer is evident. [Figure 11] Figures 11A and 11B show micro-CT images depicting PEEK implants in a first region of a first rabbit. Figure 11A shows a control PEEK implant without P-15, and Figure 11B shows a PEEK implant filled with P-15. [Figure 12]Figures 12A and 12B show micro-CT images depicting PEEK implants in the second region of the first rabbit. Figure 12A shows a control PEEK implant without P-15, and Figure 12B shows a PEEK implant filled with P-15. [Figure 13] Figures 13A and 13B show micro-CT images depicting PEEK implants in the third region of rabbit 1. Figure 13A shows a control PEEK implant without P-15, and Figure 13B shows a PEEK implant loaded with P-15. [Figure 14] Figures 14A and 14B show micro-CT images depicting PEEK implants in a first region of a second rabbit. Figure 14A shows a control PEEK implant without P-15, and Figure 14B shows a PEEK implant filled with P-15. [Figure 15] Figures 15A and 15B show micro-CT images depicting PEEK implants in a second region of a second rabbit. Figure 15A shows a control PEEK implant without P-15, and Figure 15B shows a PEEK implant filled with P-15. [Figure 16] Figures 16A and 16B show micro-CT images depicting PEEK implants in the third region of the second rabbit. Figure 16A shows a control PEEK implant without P-15, and Figure 16B shows a PEEK implant loaded with P-15. [Figure 17] Figures 17A and 17B show micro-CT images depicting PEEK implants in a first region of rabbit No. 3. Figure 17A shows a control PEEK implant without P-15, and Figure 17B shows a PEEK implant loaded with P-15. [Figure 18] Figures 18A and 18B show micro-CT images depicting PEEK implants in a second region of a third rabbit. Figure 18A shows a control PEEK implant without P-15, and Figure 18B shows a PEEK implant loaded with P-15. [Figure 19]Figures 19A and 19B show micro-CT images depicting PEEK implants in a third region of a third rabbit. Figure 19A shows a control PEEK implant without P-15, and Figure 19B shows a PEEK implant filled with P-15. [Figure 20A] 1 is a graph showing the concentration of pro-inflammatory cytokines in rabbit femurs containing control and P-15-loaded implants at 4 and 8 weeks from peri-implant core samples. Mean TNF-α concentration is shown. [Figure 20B] 1 is a graph showing the concentration of pro-inflammatory cytokines in the femurs of rabbits receiving control and P-15-loaded implants at 4 and 8 weeks, taken from graft samples inside the implant window. Mean TNF-α concentration is shown. [Figure 20C] 1 is a graph showing the concentration of pro-inflammatory cytokines in the femurs of rabbits receiving control and P-15-loaded implants at 4 and 8 weeks from peri-implant core samples. Mean IL-1β concentration is shown. [Figure 20D] 1 is a graph showing the concentration of pro-inflammatory cytokines in the femurs of rabbits receiving control and P-15-loaded implants at 4 and 8 weeks, taken from graft samples inside the implant window. Mean IL-1β concentration is shown. [Figure 20E] 1 is a graph depicting the changes in these cytokines over an 8-week period. FIG. 1 shows the changes in TNF-α and IL-1β concentrations in control implants. [Figure 20F] Figure 1 is a graph depicting the changes in these cytokines over an 8-week period. The graph shows the changes in TNF-α and IL-1β concentrations in P-15-loaded implants. The results show a sharp increase in both TNF-α and IL-1β concentrations in the femurs of rabbits with P-15-loaded implants, indicating increased osteoblast proliferation. [Figure 21A]1 is a graph showing the mean IL-6 concentrations in femurs of rabbits receiving control and P-15 loaded implants at 4 and 8 weeks from peri-implant core samples. [Figure 21B] 1 is a graph showing the mean IL-6 concentrations in the femurs of rabbits receiving control and P-15 loaded implants at 4 and 8 weeks from explant samples inside the implant window. [Figure 21C] The change in the concentration of IL-6 in the control implants is shown. [Figure 21D] Figure 1 shows the changes in TNF-α and IL-6 concentrations in P-15 loaded implants. The results show a rapid increase in IL-6 in the femurs of rabbits with P-15 loaded implants. [Figure 22] 22A and 22B are graphs showing the mean IL-4 concentrations in the femurs of rabbits receiving control and P-15-filled implants at 4 and 8 weeks, obtained from core samples around the implant (FIG. 22A) and explant samples inside the implant window (FIG. 22B). The results show a decrease in IL-4 in both the P-15-filled and control implants, with the decrease being more pronounced in the P-15-filled implants. [Figure 23] 23A and 23B are graphs showing mean IL-2 concentrations in rabbit femurs containing control and P-15-loaded implants at 4 and 8 weeks, obtained from peri-implant core samples (FIG. 23A) and explant samples inside the implant window (FIG. 23B). The results show that P-15-loaded implants significantly reduced IL-2 concentrations over time. [Figure 24] A schematic summary of the stages of bone healing and the temporal patterns of relative immune cell and cytokine / growth factor expression is shown. DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description of the Invention Foreign body reaction (FBR) and implant debris-induced biological reactivity / inflammation are peri-implant phenomena primarily driven by local innate immune cells (e.g., macrophages) that produce pro-inflammatory cytokines, such as tumor necrosis factor-α, among others.

[0047] Fibrosis is essentially unregulated tissue regeneration. It results from the increased production of collagens I and III, fibronectin, and proteoglycans due to the overproduction of TGF-β during inflammatory fibrosis. These components bind intramolecularly and intermolecularly, leading to the formation of collagen bundles. Furthermore, the simultaneous decrease in matrix-degrading proteases and the upregulation of protease inhibitors by TGF-β lead to an environment favoring ECM formation. Under the influence of TGF-β and PDGF released by macrophages, fibroblast-like cells differentiate and proliferate into myofibroblasts.

[0048] The present invention features methods and devices for ameliorating implant-induced inflammation and reducing the risk of fibrosis. The methods and devices of the present invention include a substrate coated with P-15 peptide that is positioned at the implantation site.

[0049] Interbody fusion cage Interbody fusion cages are implantable devices that are placed between the bodies of two adjacent vertebrae, typically after removal of the disc that occupies this space. Cages can be used to treat multiple diseases or disorders, including degenerative disc disease (DDD), spondylolisthesis, spinal tumors, spinal stenosis, or herniated discs. Interbody fusion cages can be placed in the cervical, lumbar, or thoracic spine.

[0050] The interbody fusion cage can be shaped to nest between a first intervertebral disc and a second intervertebral disc. For example, the interbody fusion device can include a first surface positioned in contact with the first intervertebral disc and a second surface positioned in contact with the second intervertebral disc. The interbody fusion cage can be cylindrical, round, rectangular, substantially flat, amorphous, or the shape of a human intervertebral disc.

[0051] Interbody fusion cages can be made of metal, polymer, ceramic, or a fusion of different materials. They can have a hollow center or contain orifices, which can be filled with bone growth-promoting materials such as beta-tricalcium phosphate, external organic bone material, or bone material harvested from the patient, such as from the patient's own hip during the same surgery as the fusion. Interbody fusion cages can be porous, allowing bone grafts to grow from one vertebra through the cage to the next. They can also have raised or textured surfaces.

[0052] The interbody fusion cage may include additional hardware, such as pedicle screws or rods, configured to maintain the placement of the interbody fusion cage.

[0053] PEEK interbody fusion cage modified with P-15 peptide High-performance organic polymers offer an emerging alternative to titanium-based orthopedic implants. The high stiffness of traditional metallic orthopedic devices creates a risk of premature implant failure and leads to bone degradation due to stress shielding resulting from elastic discontinuity between the implant and the surrounding bone. Polymer implants offer the possibility of an isoelastic implant-tissue interface, which significantly reduces the risk of stress shielding.

[0054] The polyaryletherketone (PAEK) polymer family is one group of emerging alternatives to titanium for the production of orthopedic implants and includes polyether-ether-ketone (PEEK), polyetherketone (PEK), polyetherketoneketone (PEKK), polyetheretherketoneketone (PEEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK).

[0055] PEEK, a member of the PAEK polymer family, is a promising candidate for next-generation orthopedic implant materials due to its bone-like mechanical properties and excellent thermal and chemical stability. PEEK offers stability at high temperatures (above 300°C), resistance to chemical and radiation damage, compatibility with many reinforcing agents (such as glass and carbon fibers), and strength superior to that of many metals. Furthermore, PEEK is radiolucent, allowing surgeons to inspect whether bone is filling the intervertebral space.

[0056] Implants made from PAEK polymers, including PEEK, are often encapsulated by fibrous tissue. Lack of bone integration can ultimately lead to implant subsidence and nonunion. Cells on PEEK have been shown to upregulate mRNA for chemokine ligand-2, interleukin (IL) 1β, IL6, IL8, and tumor necrosis factor. Cells on PEEK induce the formation of factors strongly associated with cell death / apoptosis, suggesting that the fibrous tissue surrounding PEEK implants arises from an inflammatory environment that promotes cell death via apoptosis and necrosis (see, e.g., Olivares-Navarrete et al., Spine, 40(6), 399-404 (2015)).

[0057] PEEK(-C6H4-OC6H4-O-C6H4-CO-) n , PEK(-OC6H4-CO-C6H4-) n , and PEKK(-C6H4-OC6H4-CO-C6H4-CO-) n The material is a semi-crystalline polymer. Exemplary PEEK materials that can be used in combination with the P-15 peptides described herein for implants are listed in Table 1. Specific medical grades of PEEK that can be combined with the P-15 peptides described herein are listed in Table 2.

[0058] [Table 1]

[0059] [Table 2]

[0060] PAEK materials for use in implants can be processed by injection molding, extrusion, compression molding, and / or powder coating methods.

[0061] Implantable grade P-15 modified PAEK polymers can also be incorporated into medical devices as PAEK fibers or PAEK films. Furthermore, P-15 modified PAEK polymers can be composites containing, for example, radiopaque agents (e.g., barium sulfate) or reinforcing fibers (e.g., carbon fibers such as ENDOLIGN®). In certain embodiments, PAEK polymer implants can be surface-modified to allow covalent attachment of P-15 peptides, for example, using low-temperature plasma, surface etching, or surface grafting methods. In yet other embodiments, the surface of a PAEK polymer implant can be coated with hydroxyapatite particles (using a plasma spray method), which are then subsequently coated with P-15 peptide. In other embodiments, the surface of a PAEK material can be coated using methods similar to those described in Examples 4 and 5.

[0062] Using the methods of the present invention, a substrate coated with P-15 peptide is positioned at the site of implantation (eg, implantation of an interbody fusion cage) to ameliorate implant-induced inflammation and fibrosis.

[0063] Biodegradable polymer implants modified with P-15 peptide Despite their many beneficial properties, biodegradable polyesters (i.e., poly(lactide) (PLA), poly(glycolide) (PGA), and poly(lactide-co-glycolide) (PLGA)) have not yet been adopted globally in clinical settings. Variable tissue responses to FBR and degradation rates caused by acidic by-products of implants have been well documented. For example, as PLGA degrades, lactic acid and glycolic acid monomers are released in the surrounding tissue. The resulting acidic environment significantly impacts the cytokine profile of inflammatory cells surrounding the implant. A decrease in pH has been demonstrated to alter the amount of angiogenesis after implantation. Furthermore, polymer implants with fast degradation times may also alter the amount of angiogenesis and implant integration.

[0064] Using the methods of the present invention, a substrate coated with P-15 peptide is positioned at the implantation site to ameliorate implant-induced inflammation due to changes in local pH that accompany the in vivo degradation of the biodegradable polymer.

[0065] Biodegradable polymers can be coated with P-15 peptide, for example, using methods similar to those described in Examples 4 and 5.

[0066] Vascular stents modified with P-15 peptide Since the first reported successful angioplasty of human coronary atherosclerotic lesions, restenosis has been confronted as a significant limitation to the long-term effectiveness of the procedure. Subsequent studies have supported the pivotal role of inflammatory cells in the restenosis process. Long-term indwelling stents have a significant impact on the inflammatory response and risk of restenosis in patients who receive them.

[0067] Using the methods of the present invention, vascular stents are coated with or contain P-15 peptide to ameliorate implant-induced inflammation and reduce the risk of restenosis after implantation.

[0068] Biodegradable polymers can be coated or mixed with P-15 peptide, for example, using methods similar to those described in Examples 3-6. [Example]

[0069] The following examples are presented to provide one of ordinary skill in the art with a description of how the devices and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0070] Example 1. In vitro immune response of PEEK devices with or without P-15 peptide Studying the relationship between the musculoskeletal and immune systems is becoming an increasingly important consideration in biomaterials research. In vitro investigations of immune responses provide insight into the effectiveness of currently engineered materials in spinal implant devices and also provide a potential clinical understanding of complications. When using bone graft materials, the probability of arthrodesis and bone apposition increases due to the selected material. The potential for adverse events, including fibrotic responses, presents challenges that must be addressed. In the context of bone biologics, studies involving pro-inflammatory cytokine responses provide mechanistic information about the fibrotic pathways that have been anecdotally reported in PEEK spinal interbody implants. In vitro cellular studies combined with larger animal studies facilitate understanding of factors associated with bone graft materials. Combining results from three different types of studies has the potential to develop a better understanding of PEEK fibrous encapsulation around retrieved implanted PEEK devices. It was hypothesized that the expression of IL-1β, IL-4, IL-6, and TNFα would differ between PEEK and expanded PEEK devices in the presence of a bone biologic containing P-15 peptide.

[0071] The aim of this study was to compare the in vitro pro-inflammatory cytokine response of PEEK as an implant material with or without P-15. Specifically, cytokine responses measured during culture using bone-derived MSCs by ELISA techniques provide information about the bone-implant interface.

[0072] A cell culture study measuring the pro-inflammatory response of PEEK substrates with and without P-15 was evaluated. Human mesenchymal stem cells (MSCs) were cultured on PEEK samples with and without P-15. Expression of key cytokines, including IL-1-β, IL-4, and TNF, was quantified from these cultures. The cytokines IL-4 and TNF were selected for their activity in the macrophage polarization process. IL-4 and TNF are known to induce polarization toward M2 and M1 phenotypes, respectively. IL-1β was quantified because this cytokine has been directly associated with the formation of peri-implant fibrous tissue. Quantification of alkaline phosphatase (ALP) levels was also performed to assess the osteogenic potential of P-15-supplemented cultures. Conventionally, MSCs grown on smooth PEEK do not typically express high levels of ALP in culture. Furthermore, fluorescent imaging was used to visualize the cell morphology and cell density of both cohorts. Finally, a macrophage polarization assay was performed using the RAW264.7 macrophage-like cell line to provide a predictor of in vivo outcomes comparing implanted PEEK dwells with and without P-15 in the internal inorganic particles, and bone growth and ingrowth were assessed by micro-CT.

[0073] Bone marrow-derived MSCs (ATCC, PCS-500-012, BMDM) were passaged in MSC basal medium (ATCC, PCS-500-030) according to standard procedures. Cells were seeded at a density of 2 x 10^4 cells / mL. Cell viability was assessed after 7 days of culture using the Cell Titer Glo Assay (Promega). Cytokine production was analyzed in cell-conditioned medium or cell lysates using ELISA. Culture plates were incubated at 37°C for 1, 4, 7, or 14 days. IL-1B (RAB0273) and TNFα (RAB1089) sandwich ELISA kits were used to analyze cell-conditioned medium and lysates, respectively. Cell lysates were collected in 1x RIPA buffer. ELISA was performed according to the recommended standard protocol. Statistical analysis was performed using one-way ANOVA with multiple comparisons.

[0074] BMDMs were cultured in PEEK cups with or without TCPS, P15-L, or P15-L alone for 7 or 28 days. After 7 days, cells were lysed with RIPA buffer. BMDM-conditioned medium was collected after 28 days, and ALP activity in both samples was assessed. A fluorometric ALP assay (ab83371) was performed according to the manufacturer's protocol. ALP activity toward 4-methylumbelliferyl phosphate disodium salt (MUP) results in the production of a fluorescent by-product (Ex / Em: 360 nm / 440 nm) that was analyzed on a BioTek Cytation. All statistical data analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA) with a significance threshold of p<0.05.

[0075] These data indicate that P-15 has a potential immunomodulatory effect on MSCs plated on PEEK during culture. Cells plated on tissue culture polystyrene, PEEK, or PEEK with added P-15 showed no significant differences in cell viability after 1 week of culture. When examining the production of inflammatory cytokines, ELISA data showed a significant decrease in TNFα (Table 3, Figure 1A) and IL-1β (Table 4, Figure 1B). TNFα production was significantly reduced after 4, 7, and 14 days of culture in cells plated on P-15 compared to cells cultured on PEEK alone (PEEK + P-15, PEEK (pg / mL)). Furthermore, a difference in IL-1β production from MSCs was observed on day 7 in cultures with P-15 compared to PEEK alone, and remained significant by day 14.

[0076] [Table 3]

[0077] [Table 4]

[0078] [Table 5]

[0079] ALP is an enzyme upregulated during osteoblast differentiation. Cells incubated in P15-L alone showed increased ALP activity in BMDM lysates or cell culture media at both days 7 and 28 (Tables 5 and 6). Although a slight increase in ALP activity was observed when cells were incubated in PEEK dishes filled with P15-L, cells incubated in P15-L alone increased ALP production (Figures 2A and 2B).

[0080] To evaluate the osteogenic differentiation potential of P15-L in vitro, alkaline phosphatase activity was assessed in BMDMs incubated in osteogenic medium. Cells incubated with P15-L alone, without PEEK, show increased ALP activity after 7 days of culture when compared with PEEK alone or PEEK cups filled with P15-L. After 28 days, ALP activity in cells incubated with P15-L on PEEK was increased when compared with the control or PEEK alone, but this data was not significant.

[0081] The development of biomedical implants is a hotly anticipated area of research due to the ongoing need for strong, durable, and immunomodulatory materials in some surgical procedures. PEEK has recently been developed and is being used in the field of orthopedics. While PEEK is a widely used material for orthopedic spinal implants, its inert and hydrophobic surface impairs cell adhesion, attachment, and proliferation, potentially leading to persistent inflammatory responses, fibrosis, and implant failure. Our results demonstrate that the addition of P-15 as a bone graft material has a significant immunomodulatory effect on osteoblast-like cells in the presence of PEEK in culture. We demonstrate no significant difference in cell viability of human MSCs cultured with P-15. Importantly, we observed a significant decrease in the expression of inflammatory cytokines after 7 or 14 days in culture with P-15. The data suggest that P-15-filled PEEK implants may be advantageous in suppressing the inflammatory response, leading to better bone formation adjacent to the PEEK.

[0082] [Table 6]

[0083] Example 2. In vivo immune response of devices with or without P-15 peptide The immune response to the P-15 peptide was tested in vivo in rabbits. Seventeen rabbits with bilateral femoral defects were implanted with P-15-loaded devices (Figures 3A-B). Figure 3A shows the implanted PEEK control device. Figure 3B shows the implanted PEEK device loaded with P-15. Three rabbits were euthanized immediately after surgery. Seven rabbits survived for 4 weeks, and seven rabbits survived for 8 weeks.

[0084] Cytokine analysis was performed using R&D Systems Rabbit Duo-Set ELISA kits for IL-2, IL-4, IL-6, TNF-α, and IL-1β. The manufacturer's protocol was followed for each cytokine analyzed.

[0085] MicroCT analysis was performed to study the effect of P-15 on bone deposition (Figure 4). Three rabbits were analyzed by microCT at 4 and 8 weeks. Preliminary analysis was limited to the central region of the implant to reduce the effects of implant placement and image artifacts. Quantification was performed in three regions (Figure 5): a region offset 1 mm from the implant surface (8000 μm ROI), a region offset 0.5 mm from the implant surface (7000 μm ROI), and the graft window. The 1 mm offset was intended to correlate with cytokine analysis and provide an assessment of bone growth surrounding the central dwell region. The 0.5 mm offset was intended to focus on bone growth in the peri-implant region, indicating the amount of bone-to-implant contact. The graft window was intended to indicate the amount of bone growth into the central graft window. The bone volume fraction (BV / TV) was quantified for 1 mm offsets (Figure 6A) and 0.5 mm offsets (Figure 6B) and correlated with the percentage of the total region of interest occupied by mineralized bone tissue. Preliminary micro-CT data demonstrate that the P-15 constructs exhibited greater overall bone deposition around the implant than the control specimens, which lacked mineral deposition, suggesting an inadequate bone repair and remodeling phase.

[0086] The secondary microCT analysis was performed to investigate the effects of P15-L on surrounding tissues while minimizing the effects of imaging artifacts and residual implant products. For this purpose, analysis was performed on regions of interest around the implant, with the central implant window excluded from the overall analysis. BV / TV was quantified for the 1000-500 μm ROI (Figures 7A and 7B), 500-136 μm ROI (Figures 7C and 7D), and 136-0 μm ROI (Figures 7E and 7F) and correlated with the percentage of the total ROI occupied by mineralized bone tissue. The largest of these ROIs, 1000-500 μm from the implant surface, was intended to provide additional context for immunogenicity assessment and promote a global understanding of bone growth around PEEK implants (Figures 7A and 7B). The second ROI, 500-136 μm, provided a more sensitive assessment of the effects of P15L on the surrounding microenvironment (Figures 7C and 7D). Results from this ROI showed a significant increase in bone growth from 4 to 8 weeks in the P15L cohort, but not in the control cohort. This may indicate a more favorable bone growth environment due to the presence of the P15-L product. This trend is supported by results from the smallest ROI. Within only 136 μm from the implant surface, this analysis focused on bone growth only within the periprosthetic region and was intended to correlate with the thickness of the fibrous encapsulation construct (Figures 7E and 7F). Results within this region again showed a significant increase in bone mass between 4 and 8 weeks in the P15-L cohort. A significantly higher bone mass fraction was also observed in the 8-week P15-L cohort compared to the control cohort. Furthermore, bone density observed at 4 weeks was significantly denser in the P15-L cohort compared to the control cohort. Taken together, these results indicate that P15-L promotes faster bone deposition and the transition from immature cancellous bone to mature mineralized tissue.

[0087] Figure 8 shows the statistical analysis of the percentage of bone mass in the peri-implant area at 8 weeks postoperatively. Secondary analysis results also demonstrated significant differences in the bone-implant interface between P-15 constructs and control samples. Samples containing P15 significantly increased bone mass in the peri-implant space. The observed effect of bone formation outside the P-15-filled implants was distant from the location of the P-15-coated inorganic particles. The data provided strong evidence that P-15 promotes device growth and reduces the fibrous layer known to form around PEEK implants. Figures 9A-E show histological images of PEEK devices implanted in rabbits, in which the fibrous layer is evident. Figures 10A-B show microCT images of PEEK devices implanted in rabbits, in which the fibrous layer is evident.

[0088] Figures 11-19 show additional microCT images of control and P-15-filled devices implanted in rabbits. Figures 11A-B show microCT images depicting a PEEK implant in a first region of a first rabbit. Figure 11A shows a control PEEK implant without P-15. Figure 11B shows a PEEK implant filled with P-15. Figures 12A-B show microCT images depicting a PEEK implant in a second region of a first rabbit. Figure 12A shows a control PEEK implant without P-15. Figure 12B shows a PEEK implant filled with P-15. Figures 13A-B show microCT images depicting a PEEK implant in a third region of a first rabbit. Figure 13A shows a control PEEK implant without P-15. Figure 13B shows a PEEK implant filled with P-15. FIGS. 14A-B show micro-CT images depicting a PEEK implant at a first region of a second rabbit. FIG. 14A shows a control PEEK implant without P-15. FIG. 14B shows a PEEK implant filled with P-15. FIGS. 15A-B show micro-CT images depicting a PEEK implant at a second region of a second rabbit. FIG. 15A shows a control PEEK implant without P-15. FIG. 15B shows a PEEK implant filled with P-15. FIGS. 16A-B show micro-CT images depicting a PEEK implant at a third region of a second rabbit. FIG. 16A shows a control PEEK implant without P-15. FIG. 16B shows a PEEK implant filled with P-15. FIGS. 17A-B show micro-CT images depicting a PEEK implant at a first region of a third rabbit. FIG. 17A shows a control PEEK implant without P-15. FIG. 17B shows a PEEK implant filled with P-15. FIGS. 18A-B show micro-CT images depicting a PEEK implant in a second region of a third rabbit. FIG. 18A shows a control PEEK implant without P-15. FIG. 18B shows a PEEK implant filled with P-15. FIGS. 19A-B show micro-CT images depicting a PEEK implant in a third region of a third rabbit.Figure 19A shows a control PEEK implant without P-15. Figure 19B shows a PEEK implant loaded with P-15. The images demonstrate that the P-15 loaded device shows improved bone ingrowth. The brackets show areas of visually noticeable differences in bone ingrowth.

[0089] Both pro- and anti-inflammatory cytokines were quantified in the tissue immediately surrounding the implant (core) and in the implant window (graft) in both the 4- and 8-week cohorts. The pro-inflammatory cytokines evaluated were IL-1β, IL-6, and TNF-α, while IL-4 and IL-2 were evaluated as anti-inflammatory cytokines (Figures 20-23 and Tables 7-16). Similar trends in concentrations were observed in both types of samples from 4 to 8 weeks.

[0090] While trends in cytokine expression were observed, it was clear that the P15L samples exhibited a more active cellular environment, as indicated by higher cytokine expression across all assays. We hypothesized that this increase in cytokine expression may indicate a shift from the repair phase to the remodeling phase between weeks 4 and 8. While the concentrations of most cytokines in the control treatment group remained unchanged from weeks 4 to 8, a shift in concentrations was observed between time points in the P15-L treatment group.

[0091] One subject was excluded from analysis due to an excessively high cytokine response. Femur samples were flash-frozen during autopsy and stored at -80°C. Two samples were isolated from each femur for analysis: the core, which is the bone surrounding the exterior of the implant, and the graft, which is the tissue within the central graft window of each implant. Tissue samples were then homogenized using a Fisher Bead Mill. IL-1β and TNF-α are pro-inflammatory cytokines required for remodeling as part of healing.

[0092] In both the implant and core samples, the concentration of TNF-α produced in the tissue increased from 4 to 8 weeks (Figures 20A and 20B, Tables 7 and 8). TNF-α is upregulated during the remodeling phase of healing and functions to promote osteoclast formation. Sustained and elevated levels of TNF-α expression in tissues can cause damage and reduce bone mass. As shown by micro-CT analysis, bone mass increased in the P15-L samples compared to controls. A decrease in TNF-α indicates increased osteoblast proliferation, while an increase in TNF-α indicates decreased osteoblast proliferation. Based on the mineral deposition shown in micro-CT, it was hypothesized that there would be a surge in TNF-α, indicating healthy bone healing associated with P-15, which was confirmed by analysis (Figures 20A and 20F). There was no surge in TNF-α in the control samples (Figures 20A and 20E). The sharp increase in bone mass and TNF-α at 8 weeks suggests that the body adjusts cytokine expression to induce the remodeling phase of healthy healing.

[0093] The second inflammatory cytokine involved in late remodeling is IL-1β. The data show that IL-1β concentrations decreased from 4 to 8 weeks in both control and P15-L samples in both core and graft samples (Figure 20C-F, Tables 9 and 10). There is a significant difference in IL-1β concentrations in graft samples at 4 weeks between control and P15-L. Additionally, in the P15-L cohort, IL-1β concentrations significantly decreased from 4 to 8 weeks in graft samples. In fracture models, IL-1β is produced by osteoblasts 3 weeks after injury to stimulate bone remodeling. Here, there was a significant difference in IL-1β concentrations between control and P15-L at 4 weeks. This data suggests that osteoblasts and other cell types increase IL-1β concentrations in bone tissue when P15-L is present, compared with controls. In control samples, there was no significant difference in IL-1β between 4 and 8 weeks, but in the presence of P15-L, concentrations were significantly reduced by 8 weeks, indicating that P15-L may regulate cytokine expression patterns to induce healing.

[0094] [Table 7-1] [Table 7-2]

[0095] IL-6 is a pro-inflammatory cytokine that is upregulated in the early inflammatory phase of healing in response to IL-1β stimulation. Our data showed no significant changes in IL-6 expression at 4 or 8 weeks in graft or core samples, control or P15-L samples (Figures 21A-D, Tables 11 and 12). In future studies, earlier analysis of IL-6 on days 1-5 will provide a better indication of how P15-L may regulate expression.

[0096] In bone healing, an increase in anti-inflammatory cytokines is required to transition from the inflammatory to the repair phase and reduce the risk of chronic inflammation, and IL-4 is a pro-healing and anti-inflammatory cytokine. IL-4 stimulates the differentiation of M2a macrophages, which are important in ECM formation and are required for the proliferative phase of wound healing. Based on the literature, a gradual decrease in anti-inflammatory cytokines was expected as healing progressed. However, this dataset shows no significant differences between IL-4 expression at weeks 4 or 8, or between control and P15-L samples (Figure 22, Tables 13 and 14). There was a trend toward a decrease in IL-4 expression in P15-L from weeks 4 to 8, further illustrating the hypothesized progression from the repair phase to the remodeling phase, although this was not significant.

[0097] Finally, the data show a significant difference in IL-2 expression at 4 weeks in the graft samples compared with the control P15-L samples (Figure 23, Tables 15 and 16). There is also a significant decrease in IL-2 expression from 4 to 8 weeks in the P15-L samples, while there is no change in the control samples. IL-2 is a cytokine with several distinct functions. IL-2 expression promotes local endothelial cell growth and angiogenesis, increasing vascularization to the wound, which is necessary for healing. IL-2 is also required for the development and function of T regulatory cells (Tregs). Tregs are a branch of the adaptive immune system required for controlling immune responses. While IL-2 stimulates the development of Tregs, Tregs also upregulate IL-2 receptors and function to capture additional IL-2, reducing its concentration in the wound environment. The upregulation of IL-2 in the presence of P15-L at 4 weeks may indicate increased Treg development and angiogenesis to the graft site. The subsequent decrease in IL-2 in P15-L samples from weeks 4 to 8 may be the result of sequestration by Tregs, allowing progression to the late remodeling stage of healing.

[0098] [Table 8-1] [Table 8-2]

[0099] Figure 24 shows a schematic summary of the stages of bone healing and the relative temporal patterns of immune cell and cytokine / growth factor expression. Bone healing can be considered as three biological phases (inflammation, repair, and remodeling), which can be further divided into six major substeps: hematoma, inflammation, soft callus formation, hard callus formation, remodeling, and bone healing. After fracture, immune cells, including PMNs, NK cells, mast cells, and platelets (platelets are not true cells because they lack nuclei), are activated in the early stages of inflammation, and the secreted cytokines / chemokines subsequently recruit and activate monocytes / macrophages, which play even more important roles throughout this process. Pro-inflammatory cytokines, including IL1, IL6, and TNFα, are essential signals in the early stages of fracture. Furthermore, TNFα increases again in the later repair phase, and several pro-inflammatory cytokines (e.g., IL1, IL6, and TNFα) are particularly expressed during the remodeling phase. The regulatory switch in expression patterns from pro- to anti-inflammatory responses (IL4, IL10, IL13) during the later stages of inflammation is crucial for fracture repair.

[0100] [Table 9]

[0101] [Table 10]

[0102] [Table 11]

[0103] [Table 12]

[0104] [Table 13]

[0105] [Table 14]

[0106] [Table 15]

[0107] [Table 16]

[0108] In summary, P15 constructs demonstrated increased bioactivity, including increased bone deposition and more pronounced cytokine activity, compared with control samples, and we evaluated the mechanisms underlying the clinical success of P15-L as a bone graft material. These data suggest that P15-L may function to regulate cytokine production upon interaction with cells. Changes in the expression patterns of both pro- and anti-inflammatory cytokines were identified in the presence of P15-L, but not in control samples. Cell surface interaction with biomaterials induces intracellular signaling cascades that direct cell differentiation, proliferation, and extracellular signaling. The data presented here demonstrate that P15-L regulates cytokine production, enabling increased bone deposition and overall enhanced bone healing during both the early inflammatory and late remodeling stages of healing.

[0109] Example 3: PEEK Spinal Fusion Cage Coated with P-15 As mentioned above, PEEK is often encapsulated by fibrous tissue, and the lack of bone integration can ultimately lead to implant subsidence and nonunion.

[0110] The surface of PEEK interbody fusion cages is chemically activated via treatment with cold plasma (see, e.g., Hubbell et al., Trends Polym. Sci. 2 (1) (1994) 20-25; Lopez et al., Desalination 200 (2006) 503-504; Tang et al., J Biomed Mater Res. 42 (1998) 156-163; and Jha et al., J. Appl. Polym. Sci. 118 (1) (2010)). The activated surface is then reacted with P-15 peptide. This treatment method can be used to modify different types of surfaces, including chemically inert ones, without affecting the bulk chemistry.

[0111] The resulting PEEK interbody fusion cage is coated with P-15 peptide and can be implanted into a subject's spine to replace a damaged spinal disc and promote spinal fusion.

[0112] The P-15 coated PEEK interbody fusion cage of the present invention can reduce local inflammation after implantation, reduce the risk of fibrous tissue formation, and reduce the risk of implant subsidence and non-fusion.

[0113] Example 4: PEEK / ABM-P-15 composite for spinal fusion cages As mentioned above, PEEK is often encapsulated by fibrous tissue, and the lack of bone integration can ultimately lead to implant subsidence and nonunion.

[0114] PEEK interbody fusion cages are coated with hydroxyapatite (HA). Regarding HA coatings, ISO Standard 13779-2 specifies requirements for hydroxyapatite coatings applied to surgical implants and also serves as a guideline for characterization (see ISO Standard 13779-2:2008, Implants for Surgery - Hydroxyapatite - Part 2: Coatings of Hydroxyapatite, International Organization for Standardization, Geneva, Switzerland, 2008). HA coatings can be fabricated using plasma spraying (see, e.g., Paital et al., NB Mater. Sci. Eng. R. Rep. 66 (2009) 1-70).

[0115] Once the HA-coated PEEK implant is formed, the surface HA is contacted with a solution of P-15 peptide and dried, resulting in an HA-coated PEEK surface that is itself coated with P-15 peptide.

[0116] The P-15 coated PEEK interbody fusion cage of the present invention can reduce local inflammation after implantation, reduce the risk of fibrous tissue formation, and reduce the risk of implant subsidence and non-fusion.

[0117] Example 5: Coating of titanium with P-15 This example demonstrates the successful coating of titanium discs with P-15.

[0118] Titanium foil discs were obtained from a commercial supplier. The discs were prewashed with PBS buffer. The discs were immersed in PBS buffer containing P-15. The discs were agitated overnight in the P-15 binding solution. The discs were washed six times with PBS and dried overnight in a freeze dryer.

[0119] The dried discs were placed in wells of a 24-well plate for P-15 ELISA testing. In parallel, uncoated discs were also tested using an ELISA method. The ELISA test is based on a currently validated method for measuring the amount of P-15 in inorganic bone mineral. The presence of bound P-15 peptide is demonstrated by an increase in the optical density (OD) of the marker.

[0120] Table 17 shows the OD values of the test and control discs for the presence of bound P-15 peptide.

[0121] [Table 17]

[0122] Using coating technology, we succeeded in binding the P-15 peptide to the surface of titanium.

[0123] Example 6: P-15 coated titanium bare metal vascular stent The widespread use of coronary stents has fundamentally altered vascular response to injury by inducing a more intense and prolonged inflammatory state. Traditional coronary stent materials include stainless steel (316L), cobalt-chromium alloy, nickel-titanium alloy (nitinol), platinum, and tantalum alloy, but they can also be made from biodegradable and non-degradable polymers. To address the problem of local inflammation at the implantation site and the risk of restenosis, vascular stents have been coated with antiproliferative agents (e.g., paclitaxel and rapamycin macrolide). Using the methods of the present invention, local inflammation can be reduced without antiproliferative agents by coating the surface of vascular stents with P-15 peptide before implantation.

[0124] A bare metal titanium stent is coated with P-15 according to Example 5. The titanium surface can also be coated with P-15 by attachment of a ligand or by rapid cooling of the coating solution. The stent can be implanted into a subject to treat a vascular stenosis.

[0125] The P-15 coated vascular stent of the present invention can reduce local inflammation after implantation and reduce the risk of restenosis in a subject.

[0126] Other embodiments While the invention has been described in connection with particular embodiments thereof, it will be understood that the invention is capable of further modifications, and that this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention in general, including departures from the invention which become known or customarily practiced in the art to which the invention pertains, as applicable to the essential features described above, and in accordance with the scope of the appended claims. Other embodiments are within the scope of the claims.

Claims

1. 1. A method for fusing two vertebral bodies of a subject, comprising: (a) (i) a spinal fusion cage comprising polyaryletherketone (PAEK) having an inner surface and an outer surface; and (ii) Inorganic particles coated with P-15 peptide and (b)(i) the spinal fusion cage; and (ii) Inorganic particles coated with the P-15 peptide between the first vertebral body and the second vertebral body; Including, The method, wherein step (b) comprises placing inorganic particles coated with the P-15 peptide inside the spinal fusion cage and outside the spinal fusion cage.

2. 2. The method of claim 1, wherein the PAEK is polyether-ether-ketone (PEEK).

3. 10. The method of claim 1, wherein the PAEK is polyetherketone (PEK).

4. 10. The method of claim 1, wherein the PAEK is polyetherketoneketone (PEKK).

5. 2. The method of claim 1, wherein the PAEK is polyetheretherketoneketone (PEEKK).

6. 2. The method of claim 1, wherein the PAEK is poly(aryl-ether-ketone-ether-ketoneketone) (PEKEKK).

7. 7. The method of any one of claims 1 to 6, wherein the PAEK has a molecular weight (Mn) of 110 to 120 KDa.

8. 7. The method of any one of claims 1 to 6, wherein the PAEK has a molecular weight (Mn) of 100 to 110 KDa.

9. 7. The method of any one of claims 1 to 6, wherein the PAEK has a molecular weight (Mn) of 80 to 100 KDa.

10. 10. The method of any one of claims 1 to 9, wherein the PAEK has a glass transition temperature of from 300°C to 380°C.

11. The method of any one of claims 1 to 10, wherein the PAEK is a composite material comprising fibers and / or radiopaque agents.

12. 1. A method for fusing two vertebral bodies of a subject, comprising: (a) (i) a spinal fusion cage comprising polyetherketoneketone (PEKK) having an inner surface and an outer surface; and (ii) Inorganic particles coated with P-15 peptide and (b)(i) the spinal fusion cage; and (ii) Inorganic particles coated with the P-15 peptide between the first vertebral body and the second vertebral body; Including, The method, wherein the PEKK has a glass transition temperature of 250°C to 450°C.

13. 13. The method of claim 12, wherein the PEKK has a molecular weight (Mn) of 110-120 KDa, the PEKK has a molecular weight (Mn) of 100-110 KDa, or the PEKK has a molecular weight (Mn) of 80-100 KDa.

14. 14. The method of claim 12 or 13, wherein the PEKK is a composite material including fibers and / or radiopaque agents.

15. 1. A method for fusing two vertebral bodies of a subject, comprising: (a) (i) a spinal fusion cage comprising polyether-ether-ketone (PEEK) having an inner surface and an outer surface; and (ii) Inorganic particles coated with P-15 peptide and (b)(i) the spinal fusion cage; and (ii) Inorganic particles coated with the P-15 peptide between the first vertebral body and the second vertebral body; Including, The method, wherein the PEEK has a glass transition temperature of 250°C to 450°C.

16. 16. The method of claim 15, wherein the PEEK has a molecular weight (Mn) of 110-120 KDa, the PEEK has a molecular weight (Mn) of 100-110 KDa, or the PEEK has a molecular weight (Mn) of 80-100 KDa.

17. 17. The method of claim 15 or 16, wherein the PEEK is a composite material comprising fibers and / or radiopaque agents.

18. 1. A method for fusing two vertebral bodies of a subject, comprising: (a) (i) a spinal fusion cage comprising polyetherketone (PEK) having an inner surface and an outer surface; and (ii) Inorganic particles coated with P-15 peptide and (b)(i) the spinal fusion cage; and (ii) Inorganic particles coated with the P-15 peptide between the first vertebral body and the second vertebral body; Including, The method, wherein the PEK has a glass transition temperature of 250°C to 450°C.

19. 19. The method of claim 18, wherein the PEK has a molecular weight (Mn) of 110-120 KDa, the PEK has a molecular weight (Mn) of 100-110 KDa, or the PEK has a molecular weight (Mn) of 80-100 KDa.

20. 20. The method of claim 18 or 19, wherein the PEK is a composite material including fibers and / or radiopaque agents.

21. 1. A method for fusing two vertebral bodies of a subject, comprising: (a) (i) a spinal fusion cage having an inner surface and an outer surface, the cage comprising polyetheretherketoneketone (PEEKK); and (ii) Inorganic particles coated with P-15 peptide and (b)(i) the spinal fusion cage; and (ii) Inorganic particles coated with the P-15 peptide between the first vertebral body and the second vertebral body; Including, The method, wherein the PEEKK has a glass transition temperature of 250°C to 450°C.

22. 22. The method of claim 21, wherein the PEEKK has a molecular weight (Mn) of 110 to 120 KDa, the PEEKK has a molecular weight (Mn) of 100 to 110 KDa, or the PEEKK has a molecular weight (Mn) of 80 to 100 KDa.

23. 23. The method of claim 21 or 22, wherein the PEEKK is a composite material comprising fibers and / or radiopaque agents.

24. 1. A method for fusing two vertebral bodies of a subject, comprising: (a) (i) a spinal fusion cage having an interior surface and an exterior surface, the cage comprising polyaryl-ether-ketone-ether-ketoneketone (PEKEKK); and (ii) Inorganic particles coated with P-15 peptide and (b)(i) the spinal fusion cage; and (ii) Inorganic particles coated with the P-15 peptide between the first vertebral body and the second vertebral body; Including, The method, wherein the PEKEKK has a glass transition temperature of 250°C to 450°C.

25. 25. The method of claim 24, wherein the PEKEKK has a molecular weight (Mn) of 110-120 KDa, the PEKEKK has a molecular weight (Mn) of 100-110 KDa, or the PEKEKK has a molecular weight (Mn) of 80-100 KDa.

26. 26. The method of claim 24 or 25, wherein the PEKEKK is a composite material including fibers and / or radiopaque agents.

27. The method according to any one of claims 1 to 26, wherein the inorganic particles are calcium phosphate particles.

28. 28. The method of claim 27, wherein the calcium phosphate particles are hydroxyapatite particles, anorganic bone mineral (ABM) particles, tricalcium phosphate particles, or an admixture of hydroxyapatite particles.

29. The method of any one of claims 1 to 28, wherein the spinal fusion cage comprises a porous material.

30. 30. The method of any one of claims 1 to 29, wherein the amount of P-15 peptide bound to the surface of the inorganic particles is 100 to 1500 ng of P-15 peptide per gram of inorganic particles.

31. The method of any one of claims 1 to 30, wherein the P-15 peptide coated inorganic particles are suspended in a collagen hydrogel.

32. 32. The method of claim 31, wherein the weight ratio of the P-15 peptide-coated inorganic particles to the collagen is 50:50 to 95:

5.

33. (i) The method according to any one of claims 30 to 32, wherein the amount of the P-15 peptide bound to the surface of the inorganic particles is 200 to 1200 ng of P-15 peptide per gram of inorganic particles, and the weight ratio of the P-15 peptide-coated inorganic particles to the collagen is 75:25 to 95:

5.

34. 34. The method of any one of claims 1-33, wherein placing P-15 peptide in or around the spinal fusion cage reduces local inflammation between two vertebral bodies.

35. 35. The method of any one of claims 1-34, wherein placing P-15 peptide in or around the spinal fusion cage reduces localized fibrosis between two vertebral bodies.

36. 1. A method for ameliorating implant-induced inflammation at an implantation site in a subject, comprising applying to the implantation site (i) an implantable medical device and (ii) a substrate coated with P-15 peptide, wherein the substrate is not a calcified substrate.

37. 1. A method for ameliorating implant-induced inflammation at an implantation site in a subject, comprising inserting into the implantation site (i) an implantable medical device and (ii) a substrate coated with P-15 peptide, wherein the implantation site does not contain bone tissue.

38. 1. A method for ameliorating implant-induced inflammation at an implantation site in a subject, the method comprising inserting into the implantation site (i) an implantable medical device comprising a biodegradable polymer and (ii) a substrate coated with P-15 peptide.

39. 38. The method of claim 37, wherein the biodegradable polymer is selected from poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly(butylene succinate) (PBS), and sucrose acetate isobutyrate (SAIB).

40. 1. A method for ameliorating implant-induced inflammation at an implantation site in a subject, the method comprising inserting into the implantation site (i) an implantable medical device and (ii) a substrate coated with P-15 peptide, wherein the implantation site is soft tissue.

41. 41. The method of claim 40, wherein the implantable medical device is implanted into soft tissue selected from nervous tissue, vascular tissue, oral tissue, ocular tissue, nasal tissue, genitourinary tissue, gastrointestinal tissue, biliary tissue, auditory tissue, or subcutaneous tissue.

42. 1. A method for ameliorating implant-induced inflammation at an implantation site in a subject, comprising inserting into the implantation site an implantable medical device comprising a substrate coated with P-15 peptide, wherein the implantable medical device is a neurological device, a vascular device, a cardiovascular device, an oral device, an ocular device, a nasal device, a genitourinary device, a gastrointestinal device, a biliary device, an auditory device, a subcutaneous device, a plastic surgery device, a general surgery device, or a prosthetic device.

43. the neurological device is an electrode, a pulse generator, or a neurovascular catheter; the vascular device is a vascular stent; the cardiovascular device is a pacemaker, a defibrillator, a coronary stent, a cardiovascular catheter, or a heart valve, optionally wherein the heart valve is a tricuspid valve, a pulmonary valve, a mitral valve, or an aortic valve; the oral device is a tracheostomy tube; the ocular device is an intraocular lens, an intrastromal corneal ring segment (ICRS), or an ophthalmic catheter; the nasal device is a nasal stent; the genitourinary device is a mesh, a contraceptive implant, a hernia mesh, a pelvic mesh, a urinary stent, an artificial urinary sphincter, or a urinary catheter, optionally wherein the contraceptive implant is an intrauterine device (IUD) or a birth control device.

43. The method of claim 42, wherein the gastrointestinal device is a staple, a balloon, a sleeve, a band, a gastric stimulator, or a gastrointestinal catheter, optionally wherein the band is a LINX device; the biliary device is a biliary stent; the hearing device is a cochlear implant or an ear tube; the subcutaneous device is a drug delivery needle or a glucose sensor; the prosthetic device is an artificial eye, a breast implant, a prosthetic nose, a penile implant, or a cosmetic implant, or the breast implant is a saline breast implant or a silicone breast implant.

44. The method of any one of claims 36 to 43, wherein the implantable medical device comprises polyaryletherketone (PAEK).

45. 45. The method of claim 44, wherein the PAEK is polyether-ether-ketone (PEEK), polyetherketone (PEK), polyetherketoneketone (PEKK), polyetheretherketoneketone (PEEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK).

46. 46. The method of any one of claims 36 to 45, wherein implantation of the implantable medical device reduces local inflammation at the site.

47. 47. The method of any one of claims 36 to 46, wherein implantation of the implantable medical device reduces local fibrosis at the site.

48. An implantable medical device comprising: (i) a biodegradable polymer; and (ii) a substrate coated with P-15 peptide.

49. 49. The implantable medical device of claim 48, wherein the biodegradable polymer is selected from poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly(butylene succinate) (PBS), and sucrose acetate isobutyrate (SAIB).

50. An implantable medical device designed for implantation into soft tissue, comprising a substrate coated with P-15 peptide.

51. 51. The implantable medical device of claim 50, which is a neurological device, a vascular device, a cardiovascular device, an oral device, an ocular device, a nasal device, a genitourinary device, a gastrointestinal device, a biliary device, an auditory device, a subcutaneous device, or a prosthetic device.

52. the neurological device is an electrode, a pulse generator, or a neurovascular catheter; the vascular device is a vascular stent; the cardiovascular device is a pacemaker, a defibrillator, a coronary stent, a cardiovascular catheter, or a heart valve, optionally, the heart valve is a tricuspid valve, a pulmonary valve, a mitral valve, or an aortic valve; the oral device is a tracheostomy tube; the ocular device is an intraocular lens, an intrastromal corneal ring segment (ICRS), or an ophthalmic catheter; the nasal device is a nasal stent; the genitourinary device is a mesh, a contraceptive implant, a hernia mesh, a pelvic mesh, a urinary stent, an artificial urinary sphincter, or a urinary catheter, optionally, 52. The implantable medical device of claim 51, wherein the contraceptive implant is an intrauterine device (IUD) or a birth control implant; the gastrointestinal device is a staple, balloon, sleeve, band, gastric stimulator, or gastrointestinal catheter, optionally wherein the band is a LINX device; the biliary device is a biliary stent; the hearing device is a cochlear implant or ear tube; the subcutaneous device is a drug delivery device or a glucose sensor; the prosthetic device is an artificial eye, breast implant, artificial nose, penile implant, or cosmetic implant, or the breast implant is a saline breast implant or a silicone breast implant.

53. 52. The implantable medical device of any one of claims 48 to 51, comprising polyaryletherketone (PAEK).

54. 53. The implantable medical device of claim 52, wherein the PAEK is polyether-ether-ketone (PEEK), polyetherketone (PEK), polyetherketoneketone (PEKK), polyetheretherketoneketone (PEEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK).

55. A vascular stent having a surface comprising a substrate coated with P-15 peptide.

56. 56. The vascular stent of claim 55, comprising stainless steel, cobalt-chromium alloy, nickel-titanium alloy, platinum, or tantalum alloy coated with P-15 peptide.

57. A spinal fusion cage comprising a substrate coated with P-15 peptide, wherein said substrate is not a mineralized substrate.

58. 58. The spinal fusion cage of claim 57, comprising polyaryletherketone (PAEK).

59. 59. The spinal fusion cage of claim 58, wherein the PAEK is polyether-ether-ketone (PEEK), polyetherketoneketone (PEKK), or poly(aryl-ether-ketone-ether-ketoneketone (PEKEKK).

60. 60. The spinal fusion cage of any one of claims 57 to 59, wherein the spinal fusion cage comprises polyaryletherketone (PAEK) coated with P-15 peptide.

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