THERAPEUTIC COMPOSITION AND ADMINISTRATION METHODS FOR INCREASING OSSEOINTEGRATION WITH DENTAL IMPLANTS, GRAFT MATERIALS AND PRF

Abstract

The composition and method of the invention relate to dental implant or orthognathic implant, graft materials and platelet-rich fibrin (PRF) applications in the field of dental and jaw health. The invention particularly relates to a therapeutic composition comprising at least one anti-sclerostin antibody and at least one anti dickkopf-1 antibody in combination for increasing osseointegration and accelerating healing in dental implantation and bone volume augmentation in filling of insufficient bone sites, and methods of local administration thereof.

Claims

1. A method for increasing the osseointegration of a dental implant or orthognathic implant, creating a healthy, quality and rapid bone formation in the field of dental and jaw health, comprising administering locally at least one anti-sclerostin antibody and at least one anti dickkopf-1 antibody to a patient in receipt of said implant, characterized in a total amount of said anti-sclerostin and anti dickkopf-1 antibodies per unit volume of void of extracted tooth of the patient or per unit volume of the implant to be applied is 0.001 to 50.0 mg/mm.sup.3, preferably 0.01 to 5 mg/mm.sup.3.

2. The method according to claim 1, characterized in that the ratio of said anti-sclerostin antibodies to said anti dickkopf-1 antibodies is 10% to 99%, preferably 50% to 90% by weight.

3. The method according to claim 1, characterized in that said local administration is achieved by coating said implant with said anti-sclerostin and anti dickkopf-1 antibodies.

4. The method according to claim 1, characterized in that said local administration is achieved prior to implantation by adding said anti-sclerostin and anti dickkopf-1 antibodies into a graft material and/or platelet-rich fibrin (PRF).

5. The method according to claim 1, characterized in that said anti-sclerostin and anti dickkopf-1 antibodies are applied locally to the patient's diseased gum area or problematic dental or periodontal tissues thereof.

6. A therapeutic composition for increasing the osseointegration of a dental implant or orthognathic implant, creating a healthy, quality and rapid bone formation in the field of dental and jaw health, comprising at least one anti-sclerostin antibody and at least one anti dickcopf-1 antibody in combination, administered locally to a patient in receipt of said implant, wherein a total amount of said anti-sclerostin and anti dickkopf-1 antibodies per unit volume of void of extracted tooth of the patient or per unit volume of the implant to be applied is 0.001 to 50.0 mg/mm.sup.3, preferably 0.01 to 5 mg/mm.sup.3.

7. The therapeutic composition according to claim 6, wherein the ratio of said anti-sclerostin antibody to said anti dickkopf-1 antibody is 10% to 99%, preferably 50% to 90% by weight in the said local administration.

8. The therapeutic composition according to claim 6, characterized by further comprising one or more bone enhancing therapeutics to be selected from the group consisting of platelet-rich fibrin (PRF), parathyroid hormone, teriparatide, bisphosphonate and RANKL antibody.

9. The therapeutic composition according to claim 6, wherein the dental implant or orthognathic implant is coated with at least one anti-sclerostin antibody and at least one anti dickkopf-1 antibody.

10. The therapeutic composition according to claim 6, wherein a gel, graft material, platelet-rich fibrin (PRF) or matrix comprises at least one anti-sclerostin antibody and at least one anti dickkopf-1 antibody.

11. A kit comprising at least one anti-sclerostin antibody and at least one anti dickkopf-1 antibody according to the therapeutic composition of claim 6, wherein the composition is used as a filler to the graft material and/or the platelet-rich fibrin (PRF), and the kit further comprises at least one of a dental implant or orthognathic implant and instructions for use.

12. Local use of administering at least one anti-sclerostin antibody and at least one anti dickkopf-1 antibody in preparation of a therapeutic composition for increasing osseointegration and providing a healthy and rapid bone formation, to a patient in need of dental implant or orthognathic implant application and/or increase in bone volume in fields of dental and jaw health, wherein a total amount of said antibody combination is 0.001 to 50.0 mg/mm.sup.3, preferably 0.01 to 5 mg/mm.sup.3, per unit volume of void of the extracted tooth of the patient or per unit volume of the implant to be applied.

13. The local use according to claim 12, characterized in that the ratio of said anti-sclerostin antibody to said anti dickkopf-1 antibody is 10% to 99%, preferably 50% to 90% by weight.

14. The 1 12, wherein said local administration is achieved prior to implantation by adding said anti-sclerostin and anti dickkopf-1 antibodies into a graft material and/or platelet-rich fibrin (PRF).

15. The local use according to claim 12, wherein said anti-sclerostin and anti dickkopf-1 antibodies are applied locally to the patient's diseased gum area or problematic dental or periodontal tissues thereof.

16. The local use according to claim 12, wherein the therapeutic composition is in the form of a gel, graft material, platelet-rich fibrin (PRF) or matrix.

17. The therapeutic composition according to claim 6, wherein the anti-sclerostin antibody (SCL-AB) is lyophilized and the anti dickcopf-1 antibody (DKK1-AB) is dried.

18. The therapeutic composition according to claim 17, wherein the dental implant is coated with lyophilized SCL-AB and dry DKK1-AB antibodies.

19. The therapeutic composition according to claim 18, wherein a total of about 0.005 mg to about 50 mg, preferably about 0.05 mg to about 30 mg, of the lyophilized SCL-AB antibody and dry DKK1-AB antibody combination may be used, wherein the weight ratio of SCL-AB to DKK1-AB antibody may range from 10% to 99%, preferably 50% to 90%.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0026] Dental Implant

[0027] Dental implants are used for fixing artificial teeth or prostheses to the jaw and can be produced from plastic, ceramic, bioglass, metal and alloys, boron or stem cells. The implants for use according to the invention can be selected from long or short prosthetic implants of 2 to 8 mm length, preferably from titanium, zirconium, ceramic or boron, known from the prior art, or patient's own tooth can be replaced again. Short but wide implants have been preferred recently, particularly in cases of anatomical constraints such as maxillary sinus and mental foramen, and in cases where bone loss is high.

[0028] In order to further increase osseointegration, well-known methods such as thermal etching, sandblasting and/or laser roughening to the implant surface may also be applied.

[0029] Dental Implant Coating

[0030] Various methods known from the prior art may be applied in coating the implant surface. For example, a thermally etched and/or sandblasted titanium implant surface can be modified with physiological serum. Again, the titanium implant may be coated with hydroxyapatite by using the sol-gel method and then coated with the composition of the invention via plasma spraying thereon.

[0031] Osseointegration

[0032] The term osseointegration refers to the integration of the dental implant, graft material and/or platelet-rich fibrin (PRF) with the surrounding bone tissue, especially in the field of dental, maxillofacial and orthognathic surgery and health. It is important to observe healthy and quality bone formation after implantation. Accordingly, the mineral density of the bone around the implant, the volume of the bone, the bone-implant contact area, the torque and force indicators required for the removal of the implant are taken into consideration and are measured by methods and devices frequently used in the prior art. For example, microCT (microcomputer tomography) or histomorphometry may be used for measuring bone mineral density, bone-implant contact area and bone volume.

[0033] Graft Material and Platelet Rich Fibrin (PRF)

[0034] Long-term success of osseointegrated dental implants depends largely on the quality and quantity of the available bone in the recipient site and gingiva. Pre-prosthetic procedures such as sinus floor elevation, bone base elevation (augmentation) and/or gingival grafting may be required to reconstruct the ideal bone and gingiva, as there may be atrophy in the jaw bones and thereby surrounding tissues following tooth extraction. In particular, bone grafts and PRF may be used as a filler and support in the field of dental and jaw (orthognathic) surgery and health to facilitate bone formation and promote wound healing according to the invention. These grafts and PRF act as a mineral reservoir that induces new bone formation, are biodegradable and exhibits no antigen-antibody reactions. There are many types of grafts according to the origin of bone powder (i.e. bovine, algae, porcine, beta-tricalcium phosphate (β-TCP), synthetic hydroxyapatite) and material structure. PRF, on the other hand, is derived in gel form from the centrifugation of the patient's own blood and then separation thereof following related techniques. Despite heterogeneous grafts are preferred in common, autogenous grafts may be used, as well. The therapeutic composition and method of use of the invention can be applied in both grafting systems.

[0035] Antibodies

[0036] Local administration of the anti-sclerostin antibody (SCL-AB) and the anti dickkopf-1 antibody (DKK1-AB) in combination is provided in the method and therapeutic composition according to the invention. In this context, (poly)peptides comprising a framework region from an immunoglobulin gene or fragments thereof providing that specifically recognizes and binds epitopes found on sklerostin and DKK1, respectively and separately; and thus all antibodies selected from monoclonal, chimeric, humanized and/or human antibodies can be applied.

[0037] For example, the anti-sclerostin antibody may preferably be selected from synthetic peptides (preferably KLH conjugated) between 12-42 amino acids from the N-terminal region of human SOST, ab63097 (Abcam), Romosozumab (AMG785, Amgen), Blosozumab (Eli Lilly), BPS804 (Novartis) and the anti-DKK1 antibody may preferably be selected from synthetic peptides between 68-83 amino acids from the N-terminal region of human DKK1, Boster and the like.

[0038] Tests

[0039] To investigate and compare experimentally the effects of SCL-AB and DKK1-AB -each alone and in combination-on bone regeneration and dental implant osseointegration, dental implantation together graft material and PRF were applied to rabbits, and then to pigs, after tooth extraction.

[0040] For in-vivo tests, thermally acid-etched and sandblasted titanium implants brought in the carrier liquid were selected among the commercially available ones. The composition of the present invention was sprayed thereon and coated by sol-gel method. As the graft material, Synergy® brand granulated bovine bone graft, and PRF obtained from the blood of the subjects were used.

[0041] For the first tests with graft material, seven (7) groups were assigned from totally 42 rabbits, and two comparative studies were conducted. After the extraction of two suitable teeth of each subject, the followings were applied to the each extraction sites (void volume), also reported in Table 1 below: empty socket (control) in the first group; selected bone graft in the second group; followed by SCL-AB only and DKK1-AB only, together with the graft material; and in the last three groups, the SCL-AB+DKK1-AB combination of the invention in varying proportions (50%, 75% and 90%, respectively, as the SCL-AB weight ratio in the total combination).

TABLE-US-00001 TABLE 1 # First Tooth Void (Test 1) Second Tooth Void (Test 2) Group 1 Empty Socket PRF (Control Group 1) Group 2 Graft Material PRF + Graft Material (Control Group 2) Group 3 Graft Material + PRF + SCL-AB SCL-AB Group 4 Graft Material + PRF + DKK1-AB DKK1-AB Group 5 Graft Material + PRF + SCL-AB + SCL-AB + DKK1-AB [I] DKK1-AB - I Group 6 Graft Material + PRF + SCL-AB+ SCL-AB + DKK1-AB [II] DKK1-AB - II Group 7 Graft Material + PRF + SCL-AB+ SCL-AB + DKK1-AB [III] DKK1-AB - III

[0042] Approximate volume of one extraction site (as the void left after a tooth extraction) was calculated as 8 mm.sup.3. The bone graft to be applied to this extraction site is fixed at 86.0 mg. In additive-free use on the one hand, the graft was diluted with 0.150 ml physiological serum. On the other hand, the followings were added separately into the each graft of the same amount: (1) 0.4-0.5 mg/mL SCL-AB solution with a concentration of 0.150 ml (=0.075 mg), (2) 0.010 mg DKK1-AB, and (3) the SCL-AB+DKK1-AB combination, total weight of which did not exceed 0.025 mg, wherein weight percentage of SCL-AB ranged from 50% to 90%.

[0043] In the second test study carried out together with the first one, the PRF was applied to the void of second extracted tooth of each rabbit, instead of the graft material. Thanks to the data obtained in both studies, similar tests were repeated in pigs by extracting premolar and molar teeth, opening nests into voids thereof in accordance with implant procedures and placing 3.3 to 4.8×10 mm titanium, zirconium, boron and ceramic implants, separately.

[0044] Following the said surgical operations, healing progress and bone formation around dental implants, graft materials and PRF were evaluated periodically according to the region in comparison with control group 1 (empty socket) and control group 2 (graft material and PRF). At the end of 4th, 6th and 8th weeks, subjects were sacrificed for tissue sampling and related measurements were carried out by using micro-computed tomography (microCT), histopathological methods, hematoxylin-eosin staining, histochemical staining, Masson's Trichrome and immunohistochemical staining (osteocalcin, osteopontin, alkaline phosphatase) methods.

[0045] Radiological Analysis

[0046] Jaw bones were prepared for micro-computed X-ray tomography (Micro-CT, Super Argus PET/CT, Sedecal, Spain) in a way that only bone structure will remain as soon as possible after sacrificing animals, volume assessment of the socket areas was performed via 3D reconstruction modeling of the micro-CT images. The processing of the target images was carried out with standard resolution, 0.12 mm slice thickness, 40 kV and 140 microA, and the crossectional images were separated into segments, and then volume analysis was performed by loading thereof into the analyze program (3D Slicer, 4.11.0 version GitHub, San Francisco) for evaluation.

[0047] In accordance with MicroCT analysis, average bone formation results of the first 4-week are reported for each group in Table 2 below. According to these results, new bone formation rates in the group of SCL-AB and DKK1-AB combination (Group 6), were observed as 20% at the end of the first week, 43% in the 2nd week, 70% in the 3rd week, and almost full in the 4th week. This formation was also preserved in the 6th and 8th weeks. Compared to the administration of SCL-AB alone and DKK1-AB alone together with the graft material, the SCL-AB and DKK1-AB combination of the invention achieves an additional bone formation rate of 19% and 63% respectively in the 3rd week, and 12% and 67% respectively in the 4th week and this is therefore considered as an achievement beyond expectations.

TABLE-US-00002 TABLE 2 1st 2nd 3rd 4th #/Bone Formation (%) Week Week Week Week Group 1 (Empty Socket) 8 13 18 22 Group 2 (Graft_GM) 11 22 37 41 Group 3 (GM + SCL-AB) 18 35 59 82 Group 4 (DKK1-AB) 15 32 43 55 Group 5 (GM + 18 33 45 66 %50 SCL-AB + %50 DKK1-AB) Group 6 (GM + 20 43 70 92 %75 SCL-AB + %25 DKK1-AB) Group 7 (GM + 21 39 67 74 %90 SCL-AB + %10 DKK1-AB)
Consistent with these findings, the 2-D bone fill percentages within the daily coronal osteotomy sites at the end of 4th week were highest in the said SCL-AB and DKK1-AB combination treatment groups.

[0048] Histological and Histomorphometric Analysis

[0049] The jaw bones removed for tissue sampling were fixed in 10% buffered formalin solution for 14 days and decalcified for 60 days at room temperature in 10% ethylenediaminetetraacetic acid in phosphate buffered saline solution or using osteosoft/osteomall (sigma). Samples were placed in cassettes, washed under running water for two hours, dehydrated through increasing ethanol, cleaned in xylene, and then embedded in paraffin. Sagittal sections of 3 pm thickness cut by a microtome were examined after they were applied Hematoxylin Eosin, Masson's Trichrome and immunohistochemistry stains (osteocalcin, osteopontin, alkaline phosphatase) and then placed on glass slides.

[0050] In the hematoxylin-eosin staining method, the sections taken from the test groups were kept at 60° C. for 60 minutes in an oven, and then they were removed from paraffin via treatment in xylol (or xylenes) for 3×10 minutes. After slides were passed through the decreasingly graded alcohol series (100%, 96%, 90%, 70%), respectively, and washed for 1 minute under running water, they were stained in Harris Hematoxylin for 2 minutes and then washed for 2×2 minutes under running water. They were immersed in 1% ammonia-water mixture and washed again under running water for 1 minute. The slides were kept in Eozin for 2 minutes, passed through increasingly graded alcohol series (70%, 80%, 96%, 100%), taken into xylol for 2×1 minutes and then mounted in Entellan®. In order to perform numerical evaluation of the complete area size in the tissue, data was created for each group by using a 4× objective for statistical application in the whole area. On the images taken, total tissue area in each section and mature bone tissue areas in the said total tissue area were measured. The newly formed bone tissue index was found by proportioning the mature bone tissue area/total tissue area in each section.

[0051] In the Histochemical Staining (Masson's Trichrome) method, the sections taken from the test groups were kept in 60° C. for 60 minutes in an oven, and then they were removed from paraffin via treatment in xylol for 3×10 minutes. After slides were passed through the decreasingly graded alcohol series (100%, 96%, 80%, 70%), respectively, and then washed for 1 minute under running water, they were stained in Weigert's Iron Hematoxylin for 5 minutes and washed for 3×1 minutes under running water. They were immersed in picric acid for 5 minutes and washed 3×1 minutes under running water. Slides were stained for 5 minutes in Biebrich Scarlet-Acid Fuchsin solution. They were kept in phosphotungstic/phosphomolybdic acid for 10 minutes. They were kept in Aniline Blue solution for 10 minutes, passed through increasingly graded alcohol series (70%, 80%, 96%, 100%), taken into xylol for 2×1 minutes and then mounted in Entellan® (Masson's Trichrome Stain Kit, Brand: Polysciences, Inc., Catalog Number: 25088-100).

[0052] According to the results of histological analysis, the findings of the 4th week are summarized in Table 3 below:

TABLE-US-00003 TABLE 3 Test Results (4th Week) Empty Socket Lengthened by showing osteoconductive activity towards the defect area of the bone tissue. The defect areas are in the form of fibrous connective tissue, and there are small, mature bone pieces inside. Osteoblasts located in the periphery of bone tissue fragments were observed. Graft Material Bone tissue showed osteoconductive activity towards empty space. It was observed that the area was mainly covered with fibrous connective tissue and there was also bone tissue formation. Within the fibrous connective tissue areas, there are immature bone tissue areas. PRF Osteoconductive activity exists in bone tissue, growth into empty space. Large, irregular, mature bone pieces formed throughout the extracting area. Areas other than bone tissue fragments have fibrous connective tissue. There is an increase in fibroblastic activity around bone tissue fragments Greft + SCL-AB Osteoconductive growth was observed. Large, irregular graft material areas covering the extraction area, mature and immature bone tissue fragments and fibrous connective tissue were observed. Significantly intense osteoblast and osteogenic activities were observed around the graft material areas and at the periphery of bone tissue fragments. Greft + DKK1-AB Osteoconductive growth was observed. Large, irregular graft material areas covering the extraction area, mature and immature bone tissue fragments and fibrous connective tissue were observed. Osteoblast and osteogenic activities were partly observed around the graft material areas and at the periphery of bone tissue fragments. Greft + Osteoconductive growth is available. Graft material areas were seen as SCL-AB (75%) + irregular large spaces. Around the graft material throughout the DKK1-AB (25%) extraction area, mature bone tissue formations in the form of lamellas and partly osteoid tissue areas were observed. The outside areas are in the structure of fibrous connective tissue. Osteoblasts are available at the periphery of bone tissue fragments.

[0053] Immunohistochemical Staining Method (Osteocalcin, Osteopontin, Alkaline Phosphatase)

[0054] From bone tissue blocks of each test groups, 3 μm thick sections were taken to place onto the slides. After the sections were kept at 60° C. for 1 hour in an oven, they were removed from paraffin via treatment in xylol for 3×10 minutes. The slides were then passed through the decreasingly graded alcohol series (100%, 96%, 80%, 70%) and rehydrated. Sections were passed through distilled water for 1 minute twice to purify alcohol. To remove the antigen mask, it was applied with 1:10 diluted EDTA Buffer (AP-9004-999 ThermoScientific) via a pressure cooker. After washing with distilled water for 3 minutes, endogenous peroxidase activity was blocked in tissues that were activated with 3% hydrogen peroxide (TA-125-HP ThermoScientific) for 10 minutes. Sections washed with PBS were protein-blocked (TA-125-PBQ ThermoScientific) for 10 minutes. Primary antibodies were incubated in humid environment for 2 hours (Osteocalcin (Abcam ab13420), Osteopontin (MS-1376-P0 LabVision/ThermoScientific), Alkaline Phosphate (Abcam ab95462), Amplifier Quanto (TL-125-QPB ThermoScientific) for 30 minutes. It was left in HRP Polymer Quanto (TL-125-QPH ThermoScientific) for 30 minutes. Carefully washed with PBS at each stage. In order to identify positive cells, (Osteocalcin, Osteopontin, Alkaline Phosphatase) stained with DAB, stained for 30 seconds in Harris Hematoxylin and washed for 2×1 minutes under running water. They were immersed in 1% ammonia-water mixture and washed again under running water for 1 minute. The stained glasses were passed through the increasingly graded alcohol series and kept in xylol for 5 minutes for having transparency after dehydration.

[0055] According to the results of histomorphometric analysis, it was observed that the bone-implant contact in this group was approximately 3.0-fold greater than the one in the control group in the same period, and that new bone formation area is measured as 2 mm.sup.2 at the end of the first week, 4-5 mm.sup.2 in the 2nd week, 7 mm.sup.2 in the 3rd week, and 8 mm.sup.2 in the 4th week. No inflammatory cell reaction was observed in any area.

[0056] Likewise, all findings obtained in the second tests performed with PRF application were compatible with the first tests in terms of demonstrating the success of the SCL-AB and DKK1-AB combination in bone formation and quality.

[0057] It was observed that the healing process was accelerated between 5-15% with applying PRF instead of graft material.

[0058] Moreover, following the teeth extraction of the pigs, the third and fourth tests were performed by placing dental implants with graft material and PRF. All findings obtained in these in-vivo tests are fully compatible with the previous ones. Although titanium and titanium alloys are the most commonly used implants, the interest in all-ceramic zirconia implants is increasing day by day. While titanium and titanium alloy implants have an edge over others, there are signs that similar successful results can also be achieved with other implant systems.

[0059] As the patients' expectation is to replace the missing teeth as soon as possible and the demand for satisfaction towards shortening the healing and treatment process and promoting quality bone tissue surrounding the dental implant, even in risky patient groups as in healthy individuals, the significance of the present invention increases apparently.

[0060] All these results clearly demonstrate that one-time local administration of the SCL-AB and DKK1-AB combination of the invention improves osseointegration and bone regeneration around dental implants in a healthy, quality and rapid manner. Considering that the earliest healing duration to be expected in the current conventional practice, following tooth extraction or conventional grafting and PRF treatments, in order to place dental implants in humans, is 8-12 weeks, and approximately 3-months more for awaiting the osseointegration thereof, it is a very critical achievement to reduce the said duration to 4-6 weeks in total, i.e. at least 50% less compared to current conventional practice, by applying the composition of the present invention.