Bioengineering approach to restoring inflammatory bone loss

Abstract

Disclosed herein are compositions and methods to create bone implants that are biocompatible and custom designed to implant in a bone lesion. The implant comprises a unique scaffolding customized to the bone lesion and combined with one or more specialized proresolving mediators (SPM). According to certain embodiments, the scaffolding matrix comprises an alloplastic material developed into a matrix and including one or more SPM including synthetic SPM and natural SPM wherein the SPM is released over time.

Description

Bioengineering Approach to Restoring Inflammatory Bone LossRELATD APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 713,477, filed October 29, 2024, which is incorporated herein by reference.STATEMENT REGARDING FEDERAL FUNDING

[0002] This invention was made with government support under grant no. 5R01DE032406- 02 awarded by the National Institutes of Health (NTH). The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Periodontal diseases, ranging from gingivitis to more severe forms of periodontitis, are initiated by a bacterial infection followed by a host response - inflammation- that may lead to a highly degenerative oral disease including tooth loss, bone loss and tissue damage. Several inflammatory mediators such as cytokines, chemokines and metalloproteases are associated with periodontal disease (Romanelli, R., et al. (1999) Infect. Immun. 67, 2319; Gainet, J., et al. (1998) Lab. Invest. 78, 755; Assuma, R., et al. (1998) J. Immunol. 160, 403). Other prominent mediators are the arachidonic acid derived products, including leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) (Offenbacher, S. et al. (1986) J. Periodontal Res. 21, 101). Indeed, many of the pathophysiological events that occur in periodontal diseases can be explained to a large extent by the activities of lipid mediators (Solomon, L. M., et al. (1968) J. Invest. Dermatol. 51, 280; Raisz, L. G , et al. (1974) Prostaglandins 8, 377; Klein, D. C., et al. (1970) Endocrinology 86, 1436; Crunkhorn, P., et al. (1969) Br. J. Pharmacol. 36, 216; Collier, J. G., et al. (1972) Br. J. Pharmacol. 44, 374). For example, LTB4, a well appreciated and potent chemoattractant, also initiates the accumulation of leukocytes within inflamed sites, stimulates the release of granule-associated enzymes (Borgeat, P., et al. (1990) Clin. Biochem. 23, 459) and was recently found to stimulate bone resorption.

[0004] Similarly, PGE2 is a very potent stimulator of bone loss, which is held to be a hallmark15243.001 WO 1of periodontal disease (Zubery, Y., et al. (1998) Infect. Immun. 66, 4158). PGE2 is also well appreciated for its ability to directly mediate vasodilation, increase vascular permeability, enhance pain perception by bradykinin and histamine, alter connective tissue metabolism, and enhance osteodastic bone resorption (Tsai, C.-C. et al. (1998) J. Dentistry 26, 97). The levels of PGE2 are significantly elevated in the crevicular fluid of patients with periodontal infections, especially localized juvenile periodontitis, when compared to healthy sites. These levels correlate with disease severity and aggressiveness and constitute a reliable indicator of ongoing clinical periodontal tissue destruction (Offenbacher, S., et al. (1984) J. Periodontal Res. 19, 1).

[0005] Pathophysiological responses that occur in periodontal diseases, including inflammatory cell recruitment, edema, pain, bone resorption and collagen destruction, can be mediated for the most part by effector molecules originating from the arachidonate cascade (Solomon, L. M. et al. (1968) J. Invest. Dermatol. 51, 280; Raisz, L. G., et al. (1974) Prostaglandins 8, 377; Klein, D. C., et al. (1970) Endocrinology 86, 1436; Crunkhorn, P., et al. (1969) Br. J. Pharmacol. 36, 216; Collier, J. G., et al. (1972) Br. J. Pharmacol. 44, 374). In particular, considerable evidence has demonstrated the importance of PGE2 in the pathogenesis of periodontal diseases. In vitro, PGE2 increases osteoclast numbers and bone resorption (Lader, C. S., et al. (1998) Endocrinology 139, 3157), decreases proteoglycan synthesis and increases metalloprotease production by cultured chondrocytes (Debrumfemandes, A. J., et al. (1996) Br. J. Pharmacol. 188, 1597). Bone resorption in vivo caused by three periodontal pathogens is mediated in part by PGE2, causing tooth attachment loss and bone loss (Zubery, Y., et al. (1998) Infect. Immun. 66, 4158). Prior to these findings, PGE2 was proposed as a reliable molecular indicator of ongoing periodontal tissue destruction that might be used to predict future acute periodontal attachment loss.

[0006] Inflammation is an essential biologic response. The first descriptions were recorded by the ancient Egyptian and Greek cultures. Five cardinal signs: redness, swelling, heat, pain and loss of organ / tissue function. Although the inflammatory response is protective, unresolved, continuous inflammation is detrimental to tissue function. In periodontitis, chronic inflammation promotes oral microbiome dysbiosis. Failure to resolve inflammation leads to many chronic diseases, including type 2 diabetes, cardiovascular diseases (CVDs), Alzheimer’s Disease, arthritis, osteoporosis and osteoarthritis and periodontal diseases.

[0007] Until recently, our understanding of inflammation was focused on initiation as an active25243.001 WO 1process, whereas termination (a.k.a. resolution) was thought to be a passive process with decay of proinflammatory mediators. New data has led to a paradigm shift in our understanding of inflammation termination. Resolution is an active process initiated when sufficient bioactive proresolution lipids are produced. Anti-inflammatory compounds block and suppress pathways that activate inflammation. Proresolution compounds are specific lipids that activate eicosanoid class switching from proinflammation to proresolution. Class switching is a process that occurs when the body transitions from inflammation to resolution by switching the class of lipid mediators from pro-inflammatory to pro-resolving. These proresolving mediators are referred to as specialized proresolving mediators (SPMs) and include lipoxins (derived from arachidonic acid, AA), resolvins (derived from docosahexaenoic acid, DHA and eicosapentaenoic acid, EP A), maresins (DHA and EP A), protectins (DHA), cysteinyl SPMs (DHA) and their docosapentaenoic acid (n-3DPA) analogs. With the exception of AA the remaining lipids are co-3 polyunsaturated fatty acids (PUFA)

[0008] 80% of Americans experience permanent alveolar bone loss resulting from oral diseases and consequent inflammation. Small defects spontaneously heal, larger defects do not. Current treatments include autografts, autografts and xenografts. Autografts are considered the gold standard but are limited due to resource limitation. Allograft and xenograft are also used but there is a high risk of immune rejection and possible disease transmission. Further, these treatments do not address the underlying inflammation that is a cause of the bone loss. Thus, there is a need for more predictable options.SUMMARY OF THE INVENTION

[0009] Disclosed herein are compositions and methods to fabricate bone implants that are biocompatible and custom designed to fit in a bone lesion. The disclosed implants may comprise a scaffold customized to the lesion and combined with one or more specialized proresolving mediators (SPMs) that can be embedded / impregnated in the implant and / or coat one or more surfaces of the implant. In some embodiments, the implant matrix comprises one natural SPM and one synthetic SPM.

[0010] Therefore, in some exemplary embodiments disclosed herein is a composition comprising a biocompatible 3D matrix scaffold; and one or more specialized pro-resolving mediators (SPMs) selected from lipoxins, resolvins, maresins, protein conjugates in tissue35243.001 WO 1regeneration, their n-3DPA analogs and synthetic mimetics thereof. In these embodiments, the biocompatible matrix scaffold comprises and alloplastic material, such as hydroxyapatite, whitlockite, chitin, hydrogel, polycaprolactone and other polymers, (FDM), tricalcium phosphate, or combinations thereof. In various embodiments, the matrix contains one or more SPM and is fabricated into a 3D scaffold. In other embodiments, the scaffold is an amorphous matrix implanted into the lesion.

[0011] In yet other exemplary embodiments, disclosed is a method of enhancing bone regeneration for treating a degenerative bone disease using a composition comprising a biocompatible matrix, combined with at least one specialized proresolving mediator (SPM) of inflammation resolution. In various exemplary embodiments the matrix comprises any alloplastic material. In these embodiments, the SPMs comprise lipoxins, maresins resolvins, protein conjugates in tissue regeneration, their n-3 DPA analogs, synthetic mimetics or combinations thereof.

[0012] In other exemplary embodiments disclosed is a method of fabricating implants for bone regeneration comprising imaging a bone lesion; fabricating a scaffold for the bone lesion with a biocompatible alloplastic matrix including one or more SPMs; and implanting the scaffold within the bone lesion; wherein the matrix comprises a substrate and one or more specialized proresolving mediators. In some embodiments the bone lesion results from periodontal inflammation. In various embodiments, the SPMs comprise lipoxins, maresins resolvins, protein conjugates in tissue regeneration, their n-3 DPA analogs, synthetic mimetics or combinations thereof. In various embodiments the scaffold is fabricated into a 3D model of the lesion for implantation. In yet other embodiments, the scaffold is amorphous and is implanted into the lesion.

[0013] In still other exemplary embodiments disclosed is a biodegradable 3D implant for bone regeneration characterized by the implant being designed and configured to be accommodated by a bone lesion; and having an alloplastic scaffold matrix containing one or more specialized proresolving mediators. In these embodiments the SPMs comprise lipoxins, maresins resolvins, protein conjugates in tissue regeneration, n-3 DPA analogs thereof, synthetic mimetics or combinations thereof. In yet other embodiments, the scaffolding is an amorphous matrix implanted into the lesion.45243.001 WO 1

[0014] These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.BRIEF DESCRIPTION OF DRAWINGS

[0015] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0016] Fig. 1, is a cartoon illustrating the stages of periodontitis.

[0017] Fig. 2, is an illustration showing the surgical procedure for implanting the alloplastic scaffold.

[0018] Fig. 3, are micrographs comparing the results of bone remodeling in the negative control (left panel), Whitlockite (the alloplastic material) only (middle panel) and Whitlockite with addition of two SPMs in the alloplastic material (right panel). Two of each treatment are presented.

[0019] Fig. 4, micrographs illustrating the replacement of graft material with bone. The middle panel is graft material without SPMs and the bottom panel is with SPMs. The insets higher magnification with the white areas in the middle panel unresorbed graft material. The bottom panel shows that most of the implant is resorbed, indicating better and faster replacement of graft material with new bone.

[0020] Fig. 5, micrographs illustrating bone regeneration and an area analysis comparing the amount of bone regeneration for each treatment.DETAILED DESCRIPTION

[0021] Disclosed herein are devices and methods to bioengineer permanent bone replacement implants using a biocompatible alloplastic scaffold impregnated with specialized proresolving55243.001 WO 1mediators, wherein the scaffold is tailored to each patients’ unique needs from a lytic or resorbed bone lesion.Definitions

[0022] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001, or Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N.Y. (1991).

[0023] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0024] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0025] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10,65243.001 WO 1100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring.

[0026] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0027] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0028] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges or combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units is also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third75243.001 WO 1and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0029] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number 2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, ..., 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above.

[0030] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0031] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For85243.001 WO 1example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0032] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open- ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0033] As used herein the term “amorphous” refers to a material or compound with no clearly defined shape such as sand or paste. The term “alloplastic” refers to synthetic, man-made materials used to replace or augment the body's own tissues, particularly in medicine and surgery. An alloplastic material is amorphous or may be molded or otherwise shaped into a specific 3D structure.

[0034] As used herein paracrine signaling refers to a molecule released from a cell or structure that acts on neighboring target cells and coordinates its functions. As used herein, paracrine signaling refers to the effects SPMs have on target cells to promote their function including the attracting macrophage, neutrophil, leukocytes, fibroblasts, platelets and the like, and encouraging class switching in its immediate environment.

[0035] As used herein, the term scaffolding refers to a matrix comprising a chemical and mechanical environment for cells to grow and form functional tissues. In embodiments the scaffold comprises an alloplastic substrate combined with bioactive molecules to comprise a biocompatible matrix. When implanted, the matrix provides a scaffold; a structural framework made of biomaterials that provides support for cells to adhere, migrate, and form functional tissues. In some embodiments the scaffold is pre-formed into a 3D image of the lesion. In other embodiments, the amorphous matrix is implanted in the lesion to provide the scaffold. The95243.001 WO 1scaffold is typically formed from a porous matrix that allows for nutrient transport and degradation over time.

[0036] In various aspects the alloplastic matrix substrate is a calcium phosphate material comprising crystal forming compositions including but not limited to calcium phosphate minerals including hydroxyapatite (HA), whitlockite (WH), fluorapatite (FA), tricalcium phosphate (TCP), calcium sulphate hemihydrate (POP), and compositions thereof. or polymers including polycaprolactone (PCL), polyglycolic acid (PGA) and the like. In some embodiments, the calcium phosphate can be mixed with other components to form the matrix.

[0037] Other tissue matrix substrates may comprise collagen, fibrinogen, silk, and alginate. Various tissue scaffolding may comprise hydrogels, alginic acid, chitosan, hyaluronic acid, dextran, thiolated polymers or decellularized tissues. Still yet other means of scaffold fabrication include melt electrospun polymer scaffolding, human amelogenins, chitin, chitin-poly(lactic-co- glycolic acid) (PLGA) / nanobioactive glass ceramic (nBGC) nanocomposite and the like.

[0038] In various embodiments, the matrix may comprise combinations of calcium phosphate materials, PCL and PGA polymers and various tissue matrix substrates including collagen, fibrinogen, silk, and alginate. Various tissue scaffolding may comprise hydrogels, alginic acid, chitosan, hyaluronic acid, dextran, thiolated polymers or decellularized tissues as well as electrospun scaffolding, amelogenins, chitin, chitin-poly(lactic-co-glycolic acid) (PLGA) / nanobioactive glass ceramic (nBGC) nanocomposites and the like.

[0039] Those of skill in the art will appreciate that printed, nano-spun scaffolding or similar may be used to create a custom implant to exact measurements of the lesion while other methods may include creating block scaffolding and milling it to exact measurements using, for example, a laser In other embodiments, the scaffold comprises an amorphous matrix of alloplast and SPM implanted into the lesion.

[0040] Imaging of bone lesions using current techniques is accomplished using microcomputerized tomography although other methods of imaging may be used however current technology means micro-CT requires minimal sample preparation. Studies measuring the effectiveness of Cone-beam computed tomography (CBCT) and micro-CT were compared for alveolar bone measurements using alveolar blocks from pigs and found that regions of thin bone tissue were not visible on CBCT but could be identified using micro-CT. Other forms of imaging105243.001 WO 1include MRI, X-ray and intraoral scanners which are capable of capturing thousands of images which can be converted into a digital model and used as a template for implant fabrication. However, it should be appreciated that optical forms of imaging cannot penetrate to internal bone lesions.

[0041] As used herein, the term alveolar bone refers to the jawbone i.e., membranous bone. As used herein membranous bone refers to bone developed from connective tissue membranes. Membranous bones include much of the skull, face, jaws, ribs and clavicles as well as vertebrae and spinal discs. However, those of skill in the art will appreciate that all types of bone are contemplated. Other bone types include long bones and short bones. Each of these bone types arise from ossification of specific tissues comprising intramembranous ossification which converts mesenchymal tissue into bone and endochondral ossification in which mesenchymal tissue is transformed into cartilage which is then replaced by bone. This process, osteogenesis, is similar in all types of bones and involves a constant remodeling of the bone by both osteoblasts and osteoclasts.

[0042] As used herein the term “lytic lesion” refers to a portion of damaged bone that is caused by a disease process such as a tumor or resorption due to diseases such as periodontal disease. In some instances, the lesion results in destroyed bone leaving a hole or depressions where resorption has occurred.

[0043] As used herein the term “analog” and “mimetic” are used interchangeably to refer to drugs or molecules that mimic the structure and function of a natural peptide or hormone.

[0044] As used herein the term specialized proresolving mediators (SPMs) refers to a class of cell signaling molecules formed in cells by the metabolism of polyunsaturated fatty acids (PUFA) by one or a combination of lipoxygenase, cyclooxygenase, and cytochrome P450 monooxygenase enzymes. SPMs have been found essential in orchestrating the resolution of inflammation. In various embodiments, the SPMs are naturally occurring. In various other embodiments the SPMs are synthetic mimetics or analogs of naturally occurring SPMs.

[0045] Specialized proresolving mediators (SPMs) are a class of polyunsaturated fatty acids (PUFAs) derived from either arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or n-3 docosapentaenoic acid (DPA, n-3DPA, DP An-3). Actions of SPMs include actively resolving the inflammatory response, promoting healing by limiting tissue115243.001 WO 1damage, promoting healing and alleviating pain. SPMs inhibit neutrophil migration by inhibiting neutrophils from entering inflamed tissue and releasing tissue damaging reactive oxygen species. SPMs have antibacterial properties by enhancing phagocytic activity of neutrophils and promoting apoptosis of activated neutrophils. SPMs include lipoxins, derived from AA; series D resolvins, derived from DHA; series E resovlins, derived from EP A, maresins, derived from DHA, and their structural analogs derived from n-3 docosapentaenoic acid (n-3 DPA). DHA, EP A, and n-3DPA are omega 3 PUFAS. It should be noted that, as various SPMs are produced by different cell types they also have slightly different effects. A compendium of SPMs, their synthesis and effects is found at Cayman Chemicals (www.caymanchem.com) hereby incorporated in its entirety by reference.

[0046] Molecular origins and synthesis of SPMs appear to be important to their effects on tissues. For example, lipoxin A4 (LXA4) is synthesized in neutrophils and is metabolized in platelets. LXA4 stimulates non-phlogistic phagocytosis of apoptotic leukocytes and inhibits apoptosis to prolong the phagocytotic phase. In neutrophils, it inhibits chemotaxis and transmigration and inhibits production of LTB4. Lipoxin B4 (LXB4) is a positional isomer of LXA4. LXB4 inhibits polymorphonuclear leukocyte (PMN) migration stimulated by leukotriene B4 (LTB4) and inhibits LTB4-induced adhesion of PMNs with an IC50 value of 0.3 nM. Resol vin Ei (RvEi) is a product of eicosapentaenoic acid in polymorphonuclear (PMN) neutrophils. RvEi activates chemokine-like receptor 1 and RvEi (20 ng / animal) inhibits increases in inflammatory exudate neutrophil infiltration in a mouse model of peritonitis. It increases survival and prevents decreases in colon length in a mouse model of TNBS-induced colitis, inhibits ovalbumin-induced increases in eosinophil and total cell numbers, as well as inhibiting IL-13 and IgE levels in bronchoalveolar lavage fluid (BALF) in an ovalbumin-sensitized mouse model of asthma. Resolvin Di (RvDl) is produced physiologically from the sequential oxygenation of DHA by 15- and 5-lipoxygenase. A 17(R)-epimer of RvDl can also be generated in aspirin-treated samples. Both RvDl and its 17(R)-configuration reduce human polymorphonuclear leukocyte (PMNL) transendothelial migration, the earliest event in acute inflammation, with EC50 values of ~30 nM. RvDl and its aspirin-triggered form also exhibit a dose-dependent reduction in leukocyte infiltration in a mouse model of peritonitis with a maximal inhibition of -35% at a 10-100 ng dose. In nature there is a series of SPMs synthesized which may be, in part, tied to the nutritional intake and kind of omega-3 fatty acids in the diet.125243.001 WO 1

[0047] Other SPM compounds comprise SPMs conjugated to amino acids termed PCTR (protein conjugates in tissue regeneration (PCTR). At the present time, amino acids found to conjugate to SPM are glutathione and cysteine.Bone loss

[0048] Bone loss is a hallmark of many diseases and inflammation has been found to be a hall mark of all diseases of bone loss including osteoarthritis, arthritis, osteomyelitis, osteoporosis Majeed syndrome, Cherubism and periodontal disease.Synthetic Analogs:

[0049] Benzo-lipoxin A4 is a synthetic analog or mimetic of LXA4. Replacement of the tetraene unit of native LXA4 with a benzo-fused ring system not only increases the thermal stability but also enables highly convergent and efficient syntheses of these analogs. In addition, they resist rapid catalysis and inactivation by eicosanoid oxidoreductase. Similarly, 17(R,S)-Benzo-resolvin Di (benzo-RvDi) is a derivative of the specialized pro-resolving mediator (SPM) RvDl reduces PDGF-BB-induced cytoskeletal changes in, and PDGF-induced migration of, isolated human vascular smooth muscle cells (VSMCs) when used at concentrations of 10 and 100 nM. Benzo- RvDl (10 nM) inhibits p65 nuclear translocation in human umbilical vein endothelial cells (HUVECs). It also increases RAW 264.7 phagocytosis of S. aureus and zymosan particles.

[0050] The temporal sequence and interplay among various SPM along with other physiological anti-inflammatory agents in resolving human inflammatory responses remain to be defined precisely. However, studies suggest that synthetic SPM that are resistant to being metabolically inactivated hold promise of being clinically useful pharmacological tools for preventing and resolving a wide range of pathological inflammatory responses along with the tissue destruction and morbidity that these responses cause. However, there is interest in identifying SPMs that can be synergistic when used together to enhance their pro-resolving and anti-pathological effects of inflammation.EXAMPLES

[0051] The present invention is further illustrated and supported by the following examples. However, these examples should in no way be considered to further limit the scope of the invention. To the contrary, one having ordinary skill in the art would readily understand that there135243.001 WO 1are other embodiments, modifications, and equivalents of the present invention without departing from the spirit of the present invention and / or the scope of the appended claims.Fabrication of Implants

[0052] Permanent alveolar bone loss is a common disease and 80% of Americans experience it throughout their life (Fig. 1). Although small infrabony bony defects heal spontaneously, larger ones require therapeutic grafting. To address the issue of bone lesions in clinical practice novel bone graft materials were investigated for effective bone regeneration. Accordingly, the development of an innovative bone graft scaffold comprising an alloplastic matrix that fills or occupies the space of the lesion is desirable. There are several useful strategies in creating such matrices. First, an amorphous paste could be used to fill the lesion. Alternatively, such scaffold could be preformed by laser milling, and electrospinning, combining degradable osteogenic alloplastic bone graft, in this case with whitlockite (WH), and at least two specialized pro-resolving mediators (SPMs) was investigated. WH, a prevalent crystal in human bone and teeth, possesses remarkable bone regeneration capabilities and mechanical strength. The SPMs contained within the matrix are slowly released as osteoblasts are attracted to and colonize the implant. The controlled release of SPMs aims to control inflammation, reduce infections, and stimulate bone growth (Fig. 2).Methods

[0053] The graft was produced using a 3D mold (FIG. 3). An implant containing whitlockite (WH) and specialized pro-resolving mediators (SPMs) benzo lipoxin A4 (5S,6R,7E)-methyl 5,6- dihydoxy-8-(2-((R,E)-3-hydroxyoct-l-enyl)phenyl)oct-7-enoate BLXA4 and Resolvin El (5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) at a concentration of 0.1 pg / pL) was employed. A critical-sized buccal bone defect (3x4x1 mm) was created in rat mandibles under general anesthesia (Fig. 4). The subjects were split into 3 groups (5 each): a negative control group (without graft), a WH graft group, and a WH+SPM graft group (Fig. 5). Bone regeneration was assessed at one month and two months after graft placement using Micro CT and histological techniques.145243.001 WO 1RvEi benzo Lipoxin A4Results

[0054] Data from Micro CT and histological analyses indicate superior bone regeneration in the WH+SPM graft group compared to the other conditions. Micro CT assessments demonstrated greater new bone formation in the WH+SPM group (BV / TV: 0.35) compared to the negative control group (BV / TV: 0.27) Fig. 6. Histological findings showed the percentage of new bone in the negative control group after 28 days (58.3%, SD: 0.02) and the WH graft after 28 days (58.7%, SD: 0.14) and the WH+SPM graft displayed significant improvement with 88.8% (SD: 0.03) new bone formation after 28 days. Fig. 5 shows the replacement of graft material with bone. The middle panel is graft material without SPMs and the bottom panel is with SPMs. The insets are higher power with the white areas in the middle panel showing unresorbed graft material. The bottom panel shows that most of the implant is resorbed, indicating better and faster replacement of graft material with new bone.

[0055] Previous experiments using RvEl alone in animal models with induced periodontal bone loss were performed with the assumption that treatment would facilitate a 50% bone regeneration. Those experiments showed that prophylactic RvEl treatment significantly prevents alveolar bone loss in the ligature-induced periodontitis model in the rat. Those data suggest that reduced osteoclast activity following RvEl application plays an important role in controlling alveolar bone loss in this model. It was previously shown that RvEl inhibits osteoclast differentiation in vitro and promotes osteoblast formation in vivo. In contrast, the instant results indicate surprising synergy in using two different SPMs in the scaffolding matrix to enhance bone formation in a nondisease lesion. Further, this synergy may be enhanced by the use of one naturally occurring SPM and a synthetic one. Without being bound by any theory, synthetic SPMs due to their increased stability may, in addition to their own effects, increase the effectiveness of native SPMs. Further, because the SPM are included in the matrix scaffold, they elute slowly and through paracrine155243.001 WO 1signaling may act as a “sink” encouraging colonization of the scaffolding by osteoblasts further promoting osteogenesis.

[0056] The following paragraphs enumerated consecutively from 1 through 27 provide for various additional aspects of the present invention. In one embodiment, in a first paragraph:

[0057] 1. A composition comprising: a biocompatible alloplastic matrix scaffold comprising: a substrate; and one or more specialized pro-resolving mediator (SPM); wherein the substrate comprises calcium phosphate, chitin, hydrogel, polymers or combinations thereof; wherein the one or more SPMs comprise lipoxins, resolvins, maresins, protein conjugates in tissue regeneration (PCTR), their n-3DPA analogs or resolvins, maresins or PCTR synthetic mimetics, or combinations thereof; wherein when the composition is in contact with bone experiencing inflammatory bone loss, the SPM is released over time during the resorption of the substrate and is colonized by osteoblasts and replaced by bone.

[0058] 2. The composition of paragraph 1, wherein the substrate comprises an alloplastic calcium phosphate mineral comprising hydroxyapatite, whitlockite, polycaprolactone, tricalcium phosphate or combinations thereof.

[0059] 3. The composition of any of paragraphs 1 or 2, wherein the alloplastic matrix scaffold comprises a 3D implant or an amorphous crystal scaffold.

[0060] 4. The composition of any of paragraphs 1-3, wherein more than one SPM is present in the matrix.

[0061] 5. The composition of any of paragraphs 1-4, wherein the scaffold matrix comprises one or more natural SPM and / or one or more synthetic SPM.

[0062] 6. The composition of any of paragraphs 1 -5, wherein the one or more SPM comprises benzo-lipoxin A4 and resolvin El.165243.001 WO 1

[0063] 7. A method of enhancing bone regeneration for treating a degenerative bone disease comprising contacting degenerative bone with an effective amount of a composition comprising: a substrate; and one or more SPM to form a biocompatible matrix; wherein the matrix is applied to a bone defect to form a scaffold; wherein the scaffold promotes bone regeneration

[0064] 8. The method of paragraph 7, wherein the substrate comprises the alloplasts calcium phosphate, hydrogel, chitin.

[0065] 9. The method of any of paragraphs 7-8, wherein the alloplast comprises one or more of whitlockite, hydroxyapatite, tricalcium phosphate, polycaprolactone or combinations thereof.

[0066] 10. The method of any of paragraphs 7-9, wherein the one or more SPM comprises lipoxins, resolvins, maresins, protein conjugates in tissue regeneration (PCTR), their n- 3DPA analogs and synthetic mimetics thereof.

[0067] 11. The method of paragraph 7-10, wherein the matrix comprises a natural SPM and a synthetic SPM.

[0068] 12. The method of paragraph 7-11, wherein the natural SPM is selected from lipoxin A4, lipoxin B4 and resol vin El .

[0069] 13. The method of any of paragraphs 7-11, wherein the synthetic SPM is selected from benzo-lipoxin A4, and benzo resol vin DI.

[0070] 14. The method of any of paragraphs 7-13, wherein the degenerative bone disease is osteoarthritis, arthritis, osteomyelitis, osteoporosis Majeed syndrome, Cherubism and periodontal disease.

[0071] 15. The method of any of paragraphs 7-14, wherein the inflammatory bone loss is periodontal bone loss.

[0072] 16. A method of fabricating implants for bone regeneration comprising:175243.001 WO 1creating an alloplastic scaffold for the bone lesion with a biocompatible matrix comprising a substrate and one or more SPM; and implanting the scaffold within the bone lesion.

[0073] 17. The method of paragraph 16, wherein creating the scaffold comprises one or more of 3D printing, laser milling, and electrospinning and an amorphous matrix.

[0074] 18. The method of any of paragraphs 16-17, further comprising imaging the lesion.

[0075] 19. The method of any of paragraphs 16-18, wherein the imaging comprises one or more of micro CT, photo imaging, MRI and CBCT.

[0076] 20. The method of any of paragraphs 16-19, wherein the alloplastic substrate comprises calcium phosphate, hydrogel, chitin.

[0077] 21. The method of any of paragraphs 16-20, wherein the calcium phosphate comprises one or more of hydroxyapatite, whitlockite, P-tricalcium phosphate or combinations thereof.

[0078] 22. The method of any of paragraphs 16-21, wherein the one or more pro-resolving mediators comprise lipoxins, resolvins, maresins, protein conjugates in tissue regeneration, n-3DPA analogs, synthetic mimetics or combinations thereof.

[0079] 23. The method of any of paragraphs 16-22, wherein the one or more SPM comprise a synthetic SPM and a natural SPM.

[0080] 24. The method of any of paragraphs 16-23, wherein the synthetic SPM comprises benzo-lipoxin A4 and benzo resolvin DI.

[0081] 25. The method of any of paragraphs 16-23, wherein the natural SPM comprise one or more of lipoxin A4, lipoxin B4 and resolvin El.

[0082] 26. A biodegradable implant for bone regeneration characterized by: the implant comprising an alloplastic material accommodated in a bone lesion; and having a calcium phosphate scaffold matrix comprising one or more specialized proresolving mediators.

[0083] 27. The matrix of any of the preceding paragraphs wherein the matrix comprises:185243.001 WO 1hydroxyapatite (HA), whitlockite (WH), fluorapatite (FA), tricalcium phosphate (TCP), calcium sulphate hemihydrate (POP), and compositions thereof, or polymers including polycaprolactone (PCL), polyglycolic acid (PGA) , collagen, fibrinogen, silk, and alginate, hydrogels, alginic acid, chitosan, hyaluronic acid, dextran, thiolated polymers or decellularized tissues, human amelogenins, chitin, chitin-poly(lactic-co- glycolic acid) (PLGA) / nanobioactive glass ceramic (nBGC) nanocomposites or combinations thereof.

[0084] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0085] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.195243.001 WO 1

Claims

What is Claimed is:

1. A composition comprising: a biocompatible matrix scaffold comprising: an alloplastic substrate; and one or more specialized pro-resolving mediators (SPMs); wherein the substrate comprises calcium phosphate, chitin, hydrogel, or combinations thereof; wherein the one or more SPMs comprise lipoxins, resolvins, maresins, protein conjugates in tissue regeneration (PCTR), their n-3DPA analogs, resolvins, maresins, protein conjugates in tissue regeneration (PCTR), synthetic mimetics thereof, or combinations thereof; wherein when the composition is in contact with bone experiencing inflammatory bone loss, the SPMs are released over time during the resorption of the alloplastic substrate and the alloplastic substrate is colonized by osteoblasts and replaced by bone.

2. The composition of claim 1, wherein the alloplastic substrate comprises hydroxyapatite, whitlockite, polycaprolactone, tricalcium phosphate, calcium sulphate hemihydrate (POP), polycaprolactone (PCL), polyglycolic acid (PGA) , collagen, fibrinogen, silk, and alginate, hydrogel, alginic acid, chitosan, hyaluronic acid, dextran, thiolated polymers, decellularized tissues, human amelogenins, chitin, chitin-poly(lactic-co-glycolic acid) (PLGA) / nanobioactive glass ceramic (nBGC) nanocomposites, or combinations thereof.

3. The composition of claims 1 or 2, wherein the matrix scaffold comprises an amorphous crystal scaffold.

4. The composition of any one of claims 1-3, wherein the scaffold matrix comprises one or more natural SPMs and / or one or more synthetic SPMs.

5. A method of enhancing bone regeneration for treating a degenerative bone disease comprising contacting degenerative bone with an effective amount of a composition comprising: an alloplastic substrate; and one or more specialized pro-resolving mediators (SPMs) to form a biocompatible matrix; wherein the matrix is applied to a bone defect to form a scaffold; and205243.001 WO 1wherein the scaffold promotes bone regeneration.

6. The method of claim 5, wherein the alloplastic substrate comprises hydroxyapatite, whitlockite, polycaprolactone, tricalcium phosphate, calcium sulphate hemihydrate (POP), polycaprolactone (PCL), polyglycolic acid (PGA) , collagen, fibrinogen, silk, and alginate, hydrogel, alginic acid, chitosan, hyaluronic acid, dextran, thiolated polymers, decellularized tissues, human amelogenins, chitin, chitin-poly(lactic-co-glycolic acid) (PLGA) / nanobioactive glass ceramic (nBGC) nanocomposites or combinations thereof.

7. The method of any one of claims 5-6, wherein the one or more SPMs comprise lipoxins, resolvins, maresins, protein conjugates in tissue regeneration (PCTR), their n-3DPA analogs and synthetic mimetics thereof.

8. The method of any one of claim 5-7, wherein the matrix comprises a natural SPM and a synthetic SPM.

9. The method of any one of claim 5-8, wherein the natural SPM is selected from lipoxin A4, lipoxin B4 and resolvin El .

10. The method of any one of claims 5-9, wherein the synthetic SPM is selected from benzolipoxin A4, and benzo resolvin DI.

11. A method of fabricating implants for bone regeneration comprising: creating a scaffold for the bone lesion with a biocompatible matrix comprising an alloplastic substrate and one or more specialized pro-resolving mediators (SPMs); and implanting the scaffold within the bone lesion.

12. The method of claim 11, wherein creating the scaffold comprises one or more of 3D printing; laser milling, and electrospinning and an amorphous matrix.

13. The method of any one of claims 11-12, wherein the substrate comprises hydroxyapatite, whitlockite, polycaprolactone, tricalcium phosphate, calcium sulphate hemihydrate (POP), polycaprolactone (PCL), polyglycolic acid (PGA) , collagen, fibrinogen, silk, and alginate, hydrogels, alginic acid, chitosan, hyaluronic acid, dextran, thiolated polymers, decellularized tissues, human amelogenins, chitin, chitin-poly(lactic-co-glycolic acid) (PLGA) / nanobioactive glass ceramic (nBGC) nanocomposites or combinations thereof.

14. The method of any one of claims 11-13, wherein the one or more proresolving mediators215243.001 WO 1comprise lipoxins, resolvins, maresins, protein conjugates in tissue regeneration, n-3DPA analogs or combinations thereof.

15. The method of claim 11, wherein the scaffold is block molded, electrospun, 3D molded, or amorphous.225243.001 WO 1

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