MUSCULOSKELETAL REGENERATION

REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Patent Application Serial No. 61/590,059, filed January 24, 2012, the entire content of which is incoiporated herein by reference.

GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant No. R01AR054937-01A2, awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] General aspects of the present invention relate to implant compositions, methods of their manufacture and use. Specific aspects of the present invention relate to nanotopographic features of implant compositions, methods of their manufacture and use.

BACKGROUND OF THE INVENTION

[0004] Impaired healing of bone transplants represents a significant clinical problem. Bone transplants undergo extensive chemical processing to decrease risk of disease transmission. These processing techniques alter the surface of the bone transplants and decrease the osteogenic potential of cells at the healing site. Similarly, successful implant of medical devices to treat musculoskeletal conditions can be impaired by inadequate integration of host cells and tissues. There is a continuing need for compositions and methods relating to bone implants and medical device implants.

SUMMARY OF THE INVENTION

[0005] Bone transplant compositions, soft musculoskeletal tissue implants, and orthopedic device implants are provided according to the present invention which include an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, more preferably 48-65 nm, inclusive, and still more preferably, 50-60 nm, inclusive.

[0006] Bone transplant compositions, soft musculoskeletal tissue implants, and orthopedic device implants are provided according to the present invention which include an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, more preferably 48-65 nm, inclusive, and still more preferably, 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive, and an average trough-to-crest height in the range of 4-10 nm, inclusive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1A is an image of an atomic-force micrograph showing nanotopography of uncoated bone;

[0008] Figure IB is an image of an atomic-force micrograph showing nanotopography of bone coated with a flat polymer film;

[0009] Figure 1C is an image of an atomic-force micrograph showing nanotopography of bone coated with a polymer biofilm with 45 nm islands;

[0010] Figure ID is an image of an atomic-force micrograph showing a hydroxyapatite coating characterized by nanoislands having a maximum trough-to-crest height of 17.1 nm and an average overall thickness of the coating of 10-15 nm;

[0011] Figure IE is an image of an atomic-force micrograph showing a hydroxyapatite coating characterized by nanoislands having a maximum trough-to-crest height of 8.8 nm and an average overall thickness of the coating of 25-30 nm;

[0012] Figure IF is an image of an atomic-force micrograph showing a hydroxyapatite coating characterized by nanoislands having a maximum trough-to-crest height of 6.5 nm and an average overall thickness of the coating of 50-60 nm;

[0013] Figure 1G is an image of an atomic-force micrograph showing a hydroxyapatite coating characterized by nanoislands having a maximum trough-to-crest height of 8.1 nm and an average overall thickness of the coating of 100-120 nm; [0014] Figure 2 is a graph showing results of culture of cells on bone surfaces coated with poly-L-lysine (PLLA) or hydroxyapatite having the indicated average overall thickness and characterized by the nanotopographic features described for each average overall thickness in Figures 1B-1G;

[0015] Figure 3A is an image showing a three-dimensional μ€Ύ reconstruction of an uncoated bone graft and shows that un-coated grafts remain poorly healed with limited callus formation;

[0016] Figure 3B is an image showing a three-dimensional μΟΤ reconstruction of an bone graft coated with flat poly-L-lysine (PLLA) coating and shows formation of a creeping callus that did not bridge the two sides of the host bone;

[0017] Figure 3C is an image showing a three-dimensional μΟΓ reconstruction of an bone graft coated with 45 nm poly-L-lysine (PLLA) coating and shows formation of a creeping callus that did not bridge the two sides of the host bone;

[0018] Figure 3D is an image showing a three-dimensional ^iCT reconstruction of an bone graft coated with an average overall thickness of 10-15 nm hydroxyapatite coating and shows formation of a creeping callus confined to the graft-host junction;

[0019] Figure 3E is an image showing a three-dimensional μϋΤ reconstruction of an bone graft coated with an average overall thickness of 50-60 nm hydroxyapatite coating and shows complete callus bridging at the center of the graft bone;

[0020] Figure 3F is a graph showing mineralized callus volume (MCV) for the grafts represented in Figures 3A-3E;

[0021] Figure 3G is a graph showing total callus volume (TCV) for the grafts represented in Figures 3A-3E;

[0022] Figure 3H is a graph showing mineralized callus volume/total callus volume (MCV/TCV) for the grafts represented in Figures 3A-3E;

[0023] Figure 4A is a graph showing results of biomechanical testing to determine ultimate torque of the grafts represented in Figures 3A-3E;

[0024] Figure 4B is a graph showing results of biomechanical testing to determine torsional rigidity of the grafts represented in Figures 3A-3E; and

[0025] Figure 5 is a schematic not-to-scale drawing to assist in illustrating the structural features of various aspects of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] Compositions and methods relating to bone implants, soft musculoskeletal tissue implants, and orthopedic device implants are provided according to the present invention. According to aspects of the present invention, compositions and methods relating to bone implants, soft musculoskeletal tissue implants, and orthopedic device implants are provided for use in subjects to promote osteogenesis at a site of implant.

[0027] Bone implants have numerous uses, including but not limited to, treatment of spine injuries, small bone defects, and structural replacement of damaged bone, fracture non-union and critical size defects. Bone for bone implants can be autograft bone, allograft bone and/or xenograft bone.

[0028] Medical device implants have numerous uses, including but not limited to, bone replacement, bone repair, joint replacement and joint repair.

[0029] The term "medical device implant" as used herein refers to any medical device that contacts a tissue of a subject in the course of treatment of the subject. According to aspects of the present invention a medical device implant is an orthopedic device. The term "orthopedic device" as used herein refers to any medical device in contact with a bone of a subject. Orthopedic devices provided according to aspects of the present invention include, but are not limited to, hip implants, knee implants and fixation devices including, but not limited to, bone pins and bone screws. Medical devices can be made of any of various materials, including but not limited to, metal, polymer, ceramic, rubber, textile, plastic, glass and composite.

[0030] Soft musculoskeletal tissue implants including, but not limited to tendon and ligament implants, have numerous uses, including but not limited to, repair of soft musculoskeletal tissue injury or disease.

[0031] Bone transplants, soft musculoskeletal tissue implants, and orthopedic device implants coated with specific-scale hydroxyapatite nanotopographies characterized by two-scale fractal distributions are provided according to embodiments of the present invention. Methods of treatment of a subject in need of a bone transplant soft musculoskeletal tissue implant, and/or orthopedic device implant are provided according to embodiments of the present invention.

[0032] According to particular aspects, bones for bone transplant coated with specific-scale hydroxyapatite nanotopographies characterized by two-scale fractal distributions are provided by the present invention. Methods of treatment of a subject in need of bone transplants are provided according to embodiments of the present invention.

[0033] Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; Engelke, D.R., RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K. Vintersten, R. Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; December 15, 2002, ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol. 2002; 185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 9780470151808.

[0034] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless explicitly state or the context clearly indicates otherwise.

[0035] Figure 5 is a schematic not-to-scale drawing to assist in illustrating the structural features of various aspects of the present invention. An implant composition 10 according to aspects of the present invention is shown having a bone, musculoskeletal tissue or orthopedic device having a surface 12 and an applied coating having an overall average thickness 14. The applied coating includes a continuous portion 15 which has a portion immediately adjacent the surface 12, a top portion 19 and a thickness 28. The applied coating is further characterized by nanotopographic features including a plurality of nanoislands 16 which protrude above the top portion 19 of the continuous portion 15. Each nanoisland has a crest 18. Between nanoislands 16 are troughs 20. The nanoislands have a maximum trough-to-crest height 30 and an average trough-to-crest height 24. Each nanoisland has a base portion adjacent the top portion 19 of the continuous portion 15, the base portion having a diameter. Each nanoisland has a top portion adjacent the crest 18, the top portion adjacent the crest having a diameter.

[0036] Bone transplant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive, and an average trough-to-crest height in the range of 4-10 nm, inclusive.

[0037] Bone transplant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0038] Bone transplant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150- 250 nm, inclusive.

[0039] The effective diameter of a two-dimensional region is the diameter of either the circumscribing circle or the inscribed circle or of a circle of area equal to the area of the region. [0040] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150- 250 nm, inclusive.

[0041] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive, and an average trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150- 250 nm, inclusive.

[0042] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive.

[0043] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45- 70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0044] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45- 70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive, and an average trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0045] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45- 70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive.

[0046] Coating thickness and nanotopographic features can be assessed using well- known techniques such as atomic force microscopy.

[0047] Generally, the exterior surface of the bone, tissue implant or medical device implant is sterile. Treatment of the exterior surface to render it sterile can be achieved by procedures including, but not limited to, treatment of the surface with ethanol. [0048] Bone implant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5-8 nm, inclusive and an average trough- to-crest height in the range of 5-8 nm, inclusive.

[0049] Bone implant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5-8 nm, inclusive, and an average trough-to-crest height in the range of 5-8 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 850-950 nm, inclusive, and a top portion having a top portion effective diameter in the range of 175-225 nm, inclusive.

[0050] Bone implant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5-8 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 850-950 nm, inclusive, and a top portion having a top portion effective diameter in the range of 175- 225 nm, inclusive.

[0051] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5- 8 nm, inclusive and an average trough-crest height in the range of 5-8 nm, inclusive.

[0052] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5- 8 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 850-950 nm, inclusive, and a top portion having a top portion effective diameter in the range of 175- 225 nm, inclusive.

[0053] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5- 8 nm, inclusive, and an average trough-crest height in the range of 5-8 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 850- 950 nm, inclusive, and a top portion having a top portion effective diameter in the range of 175- 225 nm, inclusive.

[0054] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5- 8 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 850-950 nm, inclusive, and a top portion having a top portion effective diameter in the range of 175-225 nm, inclusive.

[0055] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5- 8 nm, inclusive, and an average trough-to-crest height in the range of 5-8 nm, the nanoislands having a base portion having average effective diameter in the range of 850- 950 nm, inclusive, and a top portion having a top portion effective diameter in the range of 175-225 nm, inclusive.

[0056] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 48-65 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 5- 8 nm, inclusive, and an average trough-to-crest height in the range of 5-8 nm.

[0057] Bone implant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6-7 nm, inclusive and an average trough- to-crest height in the range of 6-7 nm, inclusive.

[0058] Bone implant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6-7 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 875-925 nm, inclusive, and a top portion having a top portion effective diameter in the range of 190- 210 nm, inclusive.

[0059] Bone implant compositions are provided according to the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6-7 nm, inclusive and an average trough- to-crest height in the range of 6-7 nm, the nanoislands having a base portion having average effective diameter in the range of 875-925 nm, inclusive, and a top portion having a top portion effective diameter in the range of 190-210 nm, inclusive.

[0060] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6- 7 nm, inclusive and an average trough-to-crest height in the range of 6-7 nm, inclusive.

[0061] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6- 7 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 875-925 nm, inclusive, and a top portion having a top portion effective diameter in the range of 1 0-210 nm, inclusive.

[0062] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6- 7 nm, inclusive and an average trough-to-crest height in the range of 6-7 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 875-925 nm, inclusive, and a top portion having a top portion effective diameter in the range of 190-210 nm, inclusive.

[0063] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6- 7 nm, inclusive and an average trough-to-crest height in the range of 6-7 nm, inclusive.

[0064] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6- 7 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 875-925 nm, inclusive, and a top portion having a top portion effective diameter in the range of 190-210 nm, inclusive.

[0065] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating is characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6- 7 nm, inclusive and an average trough-to-crest height in the range of 6-7 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 875-925 nm, inclusive, and a top portion having a top portion effective diameter in the range of 190-210 nm, inclusive.

[0066] Bone implant compositions are provided according to aspects of the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0067] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0068] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45- 70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0069] Bone implant compositions are provided according to aspects of the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0070] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0071] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45- 70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0072] Bone implant compositions are provided according to aspects of the present invention which include a bone having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive.

[0073] Orthopedic device implant compositions are provided according to the present invention which include an orthopedic device having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive.

[0074] Soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characteiized by an average overall thickness in the range of 45- 70 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4- 10 nm, inclusive and an average trough-to-crest height in the range of 4-10 nm, inclusive. [0075] Bone implant compositions, orthopedic device implant compositions and soft musculoskeletal tissue implant compositions are provided according to the present invention which include a tissue having an exterior surface, the exterior surface at least partially coated with an applied hydroxyapatite coating, the applied hydroxyapatite coating characterized by an average overall thickness in the range of 50-60 nm, inclusive, the applied hydroxyapatite coating further characterized by fractal nanotopographic features, the fractal nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 6- 7 nm, inclusive and an average trough-to-crest height in the range of 6-7 nm, inclusive.

[0076] Methods of treatment of a subject in need of an implant are provided according to aspects of the present invention which include providing an implant composition according to the present invention; and implanting the implant composition into the subject.

[0077] Methods of treatment of a subject in need of a bone implant, soft musculoskeletal tissue implant and/or orthopedic device implant are provided according to aspects of the present invention which include providing a bone implant, soft musculoskeletal tissue implant and/or orthopedic device implant composition according to the present invention; and implanting the bone implant, soft musculoskeletal tissue implant and/or orthopedic device implant composition into the subject.

[0078] The term "subject" refers to humans as well as other mammals, such as, but not limited to, non-human primates, cats, dogs, horses, cows, pigs, sheep, goats, rabbits and rodents.

[0079] Methods of making a bone transplant, soft musculoskeletal tissue implant and/or orthopedic device implant material are provided according to aspects of the present invention which include: providing a bone implant, soft musculoskeletal tissue implant and/or orthopedic device implant having a sterile exterior surface; subjecting the bone implant, soft musculoskeletal tissue implant and/or orthopedic device implant to physical vapor deposition of hydroxyapatite to generate a hydroxyapatite coating on at least a portion of the sterile exterior surface characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive, the nanoislands having a base portion having average effective diameter in the range of 800-1000 nm, inclusive, and a top portion having a top portion effective diameter in the range of 150-250 nm, inclusive.

[0080] Methods of making a bone transplant, soft musculoskeletal tissue implant and/or orthopedic device implant material are provided according to aspects of the present invention which include: providing a bone implant, soft musculoskeletal tissue implant and/or orthopedic device implant having a sterile exterior surface; subjecting the bone implant, soft musculoskeletal tissue implant and/or orthopedic device implant to physical vapor deposition of hydroxyapatite to generate a hydroxyapatite coating on at least a portion of the sterile exterior surface characterized by an average overall thickness in the range of 45-70 nm, inclusive, the applied hydroxyapatite coating further characterized by nanotopographic features, the nanotopographic features comprising a plurality of nanoislands, the nanoislands having a maximum trough-to-crest height in the range of 4-10 nm, inclusive and an average trough-to-crest height in the range of 4- lOnm, inclusive.

[0081] According to aspects of the present invention, methods of making a bone implant material are provided according to aspects of the present invention which include: providing a bone having a sterile exterior surface; subjecting the bone to physical vapor deposition of hydroxyapatite to generate a hydroxyapatite coating on at least a portion of the sterile exterior surface, wherein subjecting the bone to physical vapor deposition of hydroxyapatite includes at least two time periods of physical vapor deposition of hydroxyapatite onto the bone surface interspersed with a "fallow" time period during which no physical vapor deposition of hydroxyapatite takes place. Physical vapor deposition is a general technique which involves the generation of a vapor flux from a solid target material and the direction of that vapor flux onto the surface of an object to be coated. The vapor flux may be generated by one of several methods, including but not limited to the electrical heating of the boat containing the target material (thermal evaporation), the bombardment of the target material's surface by a beam of energized electrons (electron-beam evaporation), or the bombardment of the target material's surface by a beam of energized ions or other heavy particles (sputtering). Different target materials require different methods for the generation of the vapor flux, and some target materials may be susceptible to more than one method of generation of the vapor flux. Although hydroxyapatite is commonly deposited by sputtering, hybrid methods of generating a vapor flux of hydroxyapatite may involve combinations of thermal evaporation, electron-beam evaporation and/or sputtering.

[0082] According to aspects of the present invention, methods of making a soft musculoskeletal tissue implant material are provided according to aspects of the present invention which include: providing a soft musculoskeletal tissue implant having a sterile exterior surface; subjecting the soft musculoskeletal tissue implant to physical vapor deposition of hydroxyapatite to generate a hydroxyapatite coating on at least a portion of the sterile exterior surface, wherein subjecting the bone to physical vapor deposition of hydroxyapatite includes at least two time periods of physical vapor deposition of hydroxyapatite onto the soft musculoskeletal tissue implant surface interspersed with a "fallow" time period during which no physical vapor deposition of hydroxyapatite takes place.

[0083] According to aspects of the present invention, methods of making an orthopedic device implant material are provided according to aspects of the present invention which include: providing a orthopedic device implant having a sterile exterior surface; subjecting the orthopedic device implant to physical vapor deposition of hydroxyapatite to generate a hydroxyapatite coating on at least a portion of the sterile exterior surface, wherein subjecting the bone to physical vapor deposition of hydroxyapatite includes at least two time periods of physical vapor deposition of hydroxyapatite onto the orthopedic device implant surface interspersed with a "fallow" time period during which no physical vapor deposition of hydroxyapatite takes place.

[0084] For example, hydroxyapatite is deposited by physical vapor deposition for 30 minutes, followed by a fallow period of 90 minutes during which no hydroxyapatite is deposited, then followed by a second vapor deposition of hydroxyapatite for 30 minutes.

[0085] Physical vapor deposition of hydroxyapatite

[0086] For hydroxyapatite coatings deposited by physical vapor deposition according to aspects of the present invention, calcium phosphate tribasic or hydroxyapatite powder (Alfa Aesar, Ward Hill, MA) is pressure-compacted into a disk of 70 mm diameter and 2 mm thickness in a circular copper mold of 76 mm diameter. This assembly is mounted as a sputtering target in the vacuum chamber of a DC/RF-sputtering plant. Larger or smaller disks may be used as the target for sputtering in bigger or smaller vacuum chambers. The target bone implant, soft musculoskeletal tissue, or orthopedic device is mounted to a platform in the chamber. The distance between the sputtering target and the platform was fixed at a distance in the range of about 135 - 165 mm. For RF sputtering, the base pressure was maintained in the range of about 1.8 xlO ^"6 - 2.2xl0 ^"6 Ton- by a helium- compressor cryo pump, the sputtering target is cooled, and >99.99%-pure argon and <99.99 -pure oxygen are pumped into the chamber. Use of oxygen in the chamber is found to produce better stoichiometry of the hydroxyapatite film deposited on the target. The deposition parameters during sputtering are: argon flow rate in the range of about 125-155 seem, oxygen flow rate in the range of about 9-11 seem, chamber pressure in the range of about 5.8 x 10 ^~3 - 7.0 x 10 ^"3 Ton-, and input power level 125-155 W. The sputtering time is in the range of about 115-135 minutes for a coating having an average overall thickness of 50-60nm. The deposition rate is in the range of about 22-33nm/h. Denaturation of bone and soft musculoskeletal tissue targets during long-time sputtering is avoided by interspersing 25-35-min sputtering periods and 80-150-minute fallow periods, depending on the size of the bone implant, soft musculoskeletal tissue, or orthopedic device being coated.

[0087] Hydroxyapatite coatings according to aspects of the present invention optionally further include one or more additional therapeutic agents. Additional therapeutic agents included in compositions of the present invention include, but are not limited to, antibiotics, antivirals, analgesics, antipyretics, antihistamines, an ti osteoporosis agents, antiosteonecrosis agents, antiinflammatory agents, growth factors, hormones, steroids and vasoactive agents.

[0088] Hydroxyapatite coatings according to aspects of the present invention optionally further include one or more extracellular matrix proteins such as, but not limited to, fibronectin, collagens, and integrins as well non-proteinaceous compounds including, but not limited to, citric acids such as citrate.

[0089] Extracellular matrix proteins included in a hydroxyapatite coating according to aspects of the present invention may be generated by any of various techniques such as by isolation from natural sources, generation by recombinant methodology or chemical synthesis.

[0090] One or more therapeutic agents, one or more extracellular matrix proteins and/or citric acids, such as citrate may be applied simultaneously with the hydroxyapaptite according to aspects of inventive methods. Thus, for example, the selected one or more therapeutic agents, one or more extracellular matrix proteins and/or citric acids, such as citrate may be mixed with the hydroxyapatite and the mixture applied by physical vapor deposition.

[0091] One or more therapeutic agents, one or more extracellular matrix proteins and/or citric acids, such as citrate may be applied after application of the hydroxyapaptite coating according to aspects of inventive methods. Thus, for example, the selected one or more therapeutic agents, one or more extracellular matrix proteins and/or citric acids, such as citrate may be applied following physical vapor deposition of hydroxyapatite by any of various methods, illustratively including dip coating, brush application and/or spray application.

[0092] All or a portion of a bone transplant, soft musculoskeletal tissue implant and/or orthopedic device implant may be coated with a hydroxyapatite coating with or without one or more therapeutic agents, one or more extracellular matrix proteins and/or citric acids, such as citrate. According to preferred aspects, an entire a bone transplant, soft musculoskeletal tissue implant and/or orthopedic device implant is coated with a hydroxyapatite coating with or without one or more therapeutic agents, one or more extracellular matrix proteins and/or citric acids, such as citrate.

[0093] Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.

[0094] Examples

[0095] Bone Processing

[0096] Femurs were harvested from C57B16J female mice, soft tissue and bone marrow was removed. Bones were processed in ethanol, as described in Dumas A, et al., Biomaterials, 2006, 27(23):4204-l l. Bones were then coated with either a flat PLLA film, 45nm islands of PLLA, or hydroxyapatite nanotopographies having an average overall thickness between 10-120nm.

[0097] Polymer de-mixing and two-stage replication

[0098] For polymer deposition, polymer de-mixing was used to produce nanoscale topographies. Poly-l-lactic acid (PLLA, Polysciences) and polystyrene (PS, Sigma Aldrich) were mixed (70/30 w/w) in chloroform for a total polymer concentration of 2%. One-percent PLLA was used a flat control. Spin-casting of polymers on to 25 mm glass coverslips was performed at 4000 rpm for 30 s. 25-mm diameter glass coverslips were spin-coated at 4000 rpm for 30 s as described in Lim JY, et al., Biomaterials, 2007, 28(10): 1787-97. Two-stage replication using elastomeric negatives was used to coat processed bones with nanotopographic films. Following spin-casting, the coverslip was coated with PDMS to create a negative, and the PDMS mold was carefully peeled off the coverslip. Processed bones were briefly dipped in 1% PLLA in chloroform and the PDMS mold was wrapped around the bone to create a replica of the original flat or 45nm nanotopography. The PDMS negative was removed and bones were sterilized by ultraviolet light for one hour prior to cell culture.

[0099] Physical vapor deposition of hydroxyapatite

[00100] For hydroxyapatite coatings deposited by physical vapor deposition, calcium phosphate tribasic or hydroxyapatite powder (Alfa Aesar, Ward Hill, MA) was pressure- compacted into a disk of 70 mm diameter and 2 mm thickness in a circular copper mold of 76 mm diameter. This assembly was mounted as a sputtering target in the vacuum chamber of a pilot plant for DC/RF-sputtering plant custom-designed and manufactured by K. J. Lesker (Clairton, PA). Up to four murine femurs were mounted at their ends by vacuum tape to a planar steel platform of 90 mm diameter. The distance between the sputtering target and the platform was fixed at 150 mm. During RF sputtering, a physical vapor deposition technique, the base pressure was maintained at 2xl0 ^"6 Torr by a helium-compressor cryo pump, the sputtering target was cooled by chilled water, and 99.995%-pure argon and 99.994%-pure oxygen were pumped into the chamber. The oxygen was used for better stoichiometry of the hydroxyapatite film deposited on the murine femurs. The deposition parameters during sputtering were: argon flow rate 140 seem, oxygen flow rate 10 seem, chamber pressure 6.4 x 10 ^"" ' Torr, and input power level 140 W. The sputtering times were 30 min, 1 h, 2 h, and 4 h for the nanotopographies having an average overall thickness of 0-15 nm, 25-30 nm, 50-60 nm, and 100-120 nm, respectively. A dummy silicon substrate was attached on the substrate for thickness measurement done by Tencor 500 profilometer. The deposition rate was 25-30 nm/h. After one-sided deposition on the murine femurs, the bones were flipped for deposition on the other sides. The HAP nanotopographies produced on the femurs were characterized by a LEO 1530 field emission scanning electron microscope (FESEM). Denaturation of the murine femurs during long-time sputtering was avoided by interspersing 30-min sputtering periods and 90-minute fallow periods.

[00101] Surface geometry characterization [00102] Nanotopographies of bone and the various bone coatings are characterized using an atomic force microscope (Dimension 3100/VEECO). The 3-dimensional atomic-force micrographs with topographic information were obtained using a silicon cantilever (PPP-NCH/Nanosensors) under tapping mode at scan frequencies of 1-2 Hz with a typical resonance frequency around 250 kHz. The sharp tip on cantilever was scanned laterally across the sample surface, and the vertical movement of the tip was continuously recorded by a computer, which used the data to construct a quantitative 3- dimensional topographic map.

[00103] Altering the Bone Surface at the Nanoscale

[00104] Bone nanotopography is depicted in atomic-force micrographs (AFM) in Figure 1A. Nanotopographies of either PLLA or hydroxyapatite were created on the surface of processed bone. Coating bone with a flat polymer film results in a relatively flat surface at the nanoscale, with the native nanotopography of the bone slightly masked (Figure IB). Coating with a polymer biofilm with 45-nm islands results in a surface with easily identifiable nanoislands (Figure 1C) compared to the flat coating.

[00105] AFMs of the hydroxyapatite nanotopographies in Figures 1D-G show that the maximum trough-to-crest height is 17.1 nm for 10-15 nm average overall thickness hydroxyapatite deposit (Figure ID), 8.8 nm for 25-30 nm average overall thickness hydroxyapatite deposit (Figure IE), 6.5 nm for 50-60 nm average overall thickness hydroxyapatite deposit (Figure IF), and 8.1 nm for 100-120 nm average overall thickness hydroxyapatite deposit (Figure 1G). The anomalous height in Figure ID can be explained by the fact that the very thin hydroxyapatite deposit is unable to completely hide the rugosity of bone itself. Once the deposit is sufficiently thick, the maximum trough-to-crest heights in Figures 1E-G are not significantly different from one another.

[00106] However, top-surface geographies in Figures 1E-G appear very different from each other.

[00107] The 25-30nm thick hydroxyapatite deposit in Figure IE is made of islands of 900nm diameter.

[00108] These islands in the 50-60-nm thick hydroxyapatite deposit in Figure IF are decorated with secondary islands of about 200 nm in diameter. Tertiary islands, even smaller at about 40nm in diameter, appear in the 100-120-nm thick hydroxyapatite deposit in Figure 1G. Thus, doubling of the thickness of the hydroxyapatite deposit reduces small-scale geography by a factor of about 4.5, indicating that the multi-scale surface geography of sufficiently thick hydroxyapatite deposits has a fractal nature with a similarity dimension of about (log 4.5)/(log 2) = 2.17. The similarity dimension exceeds 2.0 but would be less than 3.0, indicating the fractal nature of the hydroxyapatite deposit. The similarity dimension could be different on different parts of the surface on which the hydroxyapatite is deposited.

[00109] Cell culture

[00110] Pre-osteoblastic MC3T3-E1, subclone 4 (ATCC #CRL-2593) cells were cultured in osteoblastic differentiation media (a-MEM, 10% FBS, 1% Penicillin/Streptomycin, 50ug/mL ascorbic acid, ΙΟηΜ Dexamethasone and lOmM β- Glycerol phosphate). One million cells per bone were cultured. Cells were cultured in 0.05% FBS growth media for 16 hours prior to culturing on bones. Processed bones were incubated in growth media containing 0.05% FBS for 3 hours prior to cell cultures. One million cells were then seeded on to each bone in growth media with 0.05% FBS for three hours. Following this incubation, cells/bones were cultured in complete osteogenic media. Data from un-coated bones, cells cultured in growth media and osteogenic media were collected from experiments involving both PLLA-coated bones, and hydroxyapatite- coated bones.

[00111] Alkaline Phosphatase Activity Assay

[00112] Alkaline phosphatase (AP) activity in MC3T3-E1 cells cultured on coated bones having a specified nanotopography and on uncoated bones that were processed in ethanol, as described in Dumas A, et al., Biomaterials, 2006, 27(23):4204-l l, were compared to MC3T3-E1 cells grown on tissue culture plates in non-osteogenic growth media. Alkaline Phosphatase activity was quantified by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Cells were removed from bones using 0.025% trypsin- EDTA, and lysed with Triton X. AP buffer (1: 1 0.75 M 2-amino-2-methyl-l-propanol (Sigma Aldrich) and 2 mg/ml p-nitrophenol phosphate (Sigma Aldrich) and 0.1N NaOH were added to cell lysates and incubated at 37 °C for 15 min. The absorption was measured at 410nm, and enzyme activity was estimated from a p-nitrophenol standard curve. AP activity was normalized to total protein concentration using a detergent compatible protein assay (Bio-Rad). [00113] All groups had significantly increased AP activity compared to cells on uncoated bones, except cells cultured in growth media. Cells cultured on bones with 45nm islands had a significant 2.5-fold increase in AP activity compared to cells cultured on bone with a flat polymer nanotopography (Flat: 0.44 ± 0.01 ; 45nm: 0.99 ± 0.1, p=0.0006), although this was not significantly different than cells in growth media. Cells cultured on bones with either 25-30nm islands or 50-60nm islands of hydroxyapatite had significantly higher levels of AP activity compared to all other groups, with a 4.75 and 6.15-fold increase respectively over growth media controls (p<0.0001), and a 10.8-fold and 14-fold increase over cells on un-coated bone, respectively. AP activity was not significantly different between cells on 25-30nm and 50-60nm hydroxyapatite nanotopographies (p=0.083). Interestingly, cells cultured on 100-120nm hydroxyapatite nanotopographies had significantly decreased AP activity compared to 25-30nm and 50- 60nm nanotopographies, and there was no significant difference between cells cultured on 100-120nm hydroxyapatite nanotopographies and cells cultured on a flat surface in osteogenic media (Figure 2).

[00114] Bone Isograft Surgery

[00115] Ten-week old female C57B16/J mice underwent femoral isograft surgery as described in Tiyapatanaputi P et al., J Orthop Res, 2004, 22(6): 1254-60. Grafts were processed and left un-coated or coated with polymer or hydroxyapatite nanotopographies as described above. Healing was assessed at 42 days by X-ray, μΟΓ, and biomechanical torsion testing.

[00116] Specific-Scale hydroxyapatite Nanotopographies Enhance Mineralized Callus Formation During Bone Graft Healing

[00117] Three-dimensional μΟΓ reconstructions of coated and uncoated bone grafts are shown in Figures 3A-3E. Figure 3A shows that un-coated grafts remain poorly healed with limited callus formation. Grafts coated with PLLA (Onm, 45nm, Figure 3B, 3C) displayed formation of a creeping callus that did not bridge the two sides of host bone, and grafts coated with 10-15nm hydroxyapatite also had callus formation that was confined to both graft-host junction (Figure 3D). In contrast, grafts coated with 50-60nm hydroxyapatite displayed complete callus bridging at the center of the graft bone (Figure 3E).

[00118] To quantify changes in new bone formation following bone transplantation, μθ ¹ images were semi-manually segmented to exclude graft and host bone, while including only mineralized tissue in the healing callus. No significant differences in mineralized callus volume (MCV), or total amount of mineralized tissue in the callus, were observed between un-coated bones (2.60 ± 0.283 mm ³ ), and bones coated flat PLLA (4.46 ± 0.41), 45nm PLLA (3.00 ± 0.29), or 10-15nm hydroxyapatite (3.42 ± 0.48). Bones coated with 50-60nm hydroxyapatite had a significant increase in MCV (5.47 ± 1.01, p=0.003) compared to un-coated bones (Figure 3F).

[00119] Total callus volume (TCV), a measure of both mineralized and non- mineralized tissue in the callus was also significantly increased in bones coated with 50- 60nm hydroxyapatite (17.76 ± 2.83 mm ³ ) compared to un-coated bones (8.92 ± 1.04, p=0.027), and bones coated with 10-15nm hydroxyapatite (10.11 ± 0.82, p=0.01). No changes in TCV were observed in bones coated with a flat PLLA nanotopography (10.65 ± 1.93), or 45 nm PLLA (11.92 ± 2.24) compared to any other group (Figure 3G). The MCV/TCV ratio represents proportion of total callus that is composed of mineralized tissue. No significant differences in MCV/TCV were found in any group at 42 days post-transplant, indicating that 50-60nm hydroxyapatite results in a larger callus with more mineralized tissue, however, proportion of mineralized tissue to total tissue area is not significantly different than any other group (Figure 3H).

[00120] Osteointegration of Bone Grafts is Improved by 50-60nm hydroxyapatite Nanotopographic Coating

[00121] Torsional biomechanical testing was done to determine the degree of osteointegration of graft bone. Ultimate torque was not significantly different between un-coated bones (6.19 ± 0.78 N*mm), both PLLA coated groups (8.3 ± 0.67 for Onm PLLA, 7.26 ± 0.94 for 45nm PLLA), and bones coated with 10-15nm hydroxyapatite (6.03 ± 0.88). Grafts coated with 50-60nm hydroxyapatite (12.42 ± 2.79 N*mm) had significantly increased ultimate torque compared to un-coated bones (p=0.024), and 10- 15nm hydroxyapatite (p= 0.039) (Figure 4A). Torsional rigidity, or graft stiffness, was significantly increased in bones coated with Onm PLLA (451.9 ± 33.6 N*mm ^" ), and 45nm PLLA (299.1 ± 67.14) compared to un-coated bones (102 ± 33.91), or bones coated with 10-15nm hydroxyapatite (135.4 ± 25.83). Torsional rigidity of bones coated with 50-60nm hydroxyapatite (238.5 ± 95.08) was not significantly different than any other group (Figure 4B).

[00122] Micro Computed Tomography [00123] Isografts within host bone were scanned at an isometric resolution of 10.5 μιη using a vivaCT 40 microCT (Scanco Medical AG, Briittisellen, Switzerland). The reconstructed images were segmented and analyzed using the Image Processing Library supplied by the manufacturer. Changes in total callus volume and mineralized callus volume were quantified for nanotopography coated isografts and un-coated controls. All slices containing isograft bone were included in the analysis. Slices were contoured to define the outer border of the callus, and to exclude cortical bone and the medullary cavity from the center of the callus. All voxels within the callus were used to calculate the Total Callus Volume (TCV). Images were thresholded and the voxels considered mineralized were used to calculate Mineralized Callus Volume (MCV).

[00124] Biomechanical Torsion Testing

[00125] Following harvest and removal of the intramedullary pin, femurs were potted in square aluminum pots using PMMA, and rehydrated in phosphate buffered saline for at least three hours prior to testing. Femurs were tested in torsion at a rate of 1 second until failure using a 177N-mm load cell. Torsional rigidity was calculated as the slope of the linear region of the rotational deformation (Radians) normalized to gage length (mm), versus torque (N*mm).

[00126] Statistical Analysis

[00127] Data are presented as mean ± standard error of the mean (SEM). Significant differences between groups were determined by non-parametric ANOVA. Differences were considered significant at p (probability value) <0.05.

[00128] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.

[00129] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.