METHOD AND APPARATUS FOR IMPROVING OSSEOINTEGRATION, FUNCTIONAL LOAD, AND OVERALL STRENGTH OF INTRAOSSEOUS IMPLANTS

Abstract

The present invention enables modification of an intraosseous implant device that is not only biologically non-inert, but can stimulate bone and vascular growth; decrease localized inflammation; and fight local infections. The method of the present invention provides a fiber with any of the following modifications: (1) Nanofiber with PDGF, (2) Nanofiber with PDGF+BMP2, and (3) Nanofiber with BMP2 and Ag. Nanofiber can be modified with other growth factors that have been shown to improve bone growth and maturationBMP and PDGF being the most common. Nanofiber can be applied on the surface of the implant in several ways. First, a spiral micro-notching can be applied on the implant in the same direction as the threads with the nanofibers embedded into the notches. Second, the entire surface of the implant may be coated with a mesh of nanofibers. Third, it can be a combination of both embedding and notching.

Claims

1. A method for improving osseointegration, functional load, and overall strength of intraosseous dental implants, the method comprising: providing a threaded endosseous dental device with a cylindrical shape; creating a laser-grooved surface consisting of microgrooves at the interspace between thread pairs of said dental device; activating said laser-grooved surface by applying tresyl chloride coupled with fibronectin (FN); coating said dental device threaded surfaces with a nanofiber matrix (NFM) comprising at least one of growth factor or antibiotic-modified polycapronlectron (PCL) Electrospun Nanofibers (ENFs), said PCL-ENF combined with at least any of PDGF, PDGF+BMP2, or BMP2 and Ag, and wherein, said NFM adheres to said threaded surfaces of said dental device.

2. The method of claim 1, wherein a rotary stage of a laser system is oriented according to the helix angle of said thread pairs to engrave said microgrooves between and at the root of said thread pairs to create said laser-grooved surface.

3. The method according to claim 2, wherein said microgrooves are engraved to exhibit approximately 50 m width, 5 m depth, and 150 m spacing between said grooves.

4. The method according to claim 3, wherein said NFM comprises at least 18 layers of fibers deposited along the direction of said microgrooves by circumferentially rotating said dental device until 18 layers of nanofibers are collected.

5. A threaded endosseous dental device coated with a nanofiber matrix (NFM) using the method of claim 1.

6. The dental device of claim 5, wherein a rotary stage of a laser system is oriented according to the helix angle of thread pairs on said device to engrave microgrooves between and at the root of said thread pairs to create said laser-grooved surface.

7. The dental device of claim 5, wherein said microgrooves are engraved to exhibit approximately 50 m width, 5 m depth, and 150 m spacing between said grooves.

8. The dental device of claim 5, wherein said NFM comprises at least 18 layers of fibers deposited along the direction of said microgrooves by circumferentially rotating said dental device until 18 layers of nanofibers are collected.

9. A threaded endosseous dental device coated with a nanofiber matrix (NFM) using the method of claim 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a non-limiting diagram showing microgrooves and nanofiber-assisted drug delivery on dental implant as provided by the present invention.

[0023] FIG. 2 is a non-limiting diagram showing the method of the present invention providing protein immobilization on Ti using nanofiber matrix as a functional coating.

[0024] FIG. 3 is a non-limiting diagram showing F1s, S2p, N1s and O1s spectra of the Ti, Tresyl/Ti, and FN/Ti surface by a XPS analysis.

[0025] FIG. 4 is a non-limiting diagram showing precision microgrooves on the flat surface of a Ti rod fromed by the method of the present invention.

[0026] FIG. 5 is a non-limiting diagram showing schematic representation of the processes for preparing an in vivo dental implant.

[0027] FIG. 6a is a non-limiting diagram showing a 3 mm diameter screw coated with 18 layers of PCL NFM.

[0028] FIG. 6b is a non-limiting diagram showing twisting of an NFM coated screw in to a pre-drilled hole (2.6 mm diameter) on a clear acrylic shows homogenous distribution of fiber along screw/acrylic interface.

[0029] FIG. 7a is a non-limiting image showing individual immobilization of CG and FN on PCL NFM.

[0030] FIG. 7b is a non-limiting graph showing that the individual immobilization of CG and FN on PCL NFM has no adverse effect on osteoblast cells adhesion and proliferation of PCL NFM, and significant increase of cell adhesion observed for FN-PCL-NFM when compared to PCL NFM (p<0.05).

[0031] FIG. 8a is a non-limiting graph showing a gradual increase of release of BMP2 for 28 days was observed for FN-Hep-BMP2/PCL samples.

[0032] FIG. 8b is a non-limiting image showing cell divisions after 48 hours of cell culture on FN-BMP2-PCL samples.

[0033] FIG. 9a is a non-limiting image showing PCL samples after Gram staining.

[0034] FIG. 9b is a non-limiting image showing CG-Ag-PCL samples after Gram staining.

[0035] FIG. 10 is a non-limiting diagram presenting the test results of time-dependent bone growth around the CG-PCL NFM-coated Ti implant and related in vivo pull-out tests to demonstrate mechanical stability of microgrooved-Ti.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0036] Our research demonstrates: (1) immobilization of ECM proteins (CG and FN) and bone growth factors (BMP2) with PCL NFM is possible, and such immobilization improves the in vitro cell viability of PCL NFM; (2) immobilization of antibacterial nanoparticles (Ag) with PCL NFM is possible, and such immobilization improves the in vitro antibacterial activity of PCL NFM; (3) direct attachment of FN on a dental implant material (Ti-6Al-4V) is possible using tresyl chloride activation method; and (4) microgrooving of a Ti implant followed by coating the microgrooves with CG-PCL NFM significantly improves in vivo mechanical stability and osseointegration.

[0037] Referring to FIG. 1, in a preferred embodiment the present invention 10 provides coating methods described above to dental implants with the goal of improving the osseointegration of implants. We use a laser pulse to create microgrooves 11 at the interspace between two threads 18 of a dental implant (Di) 12. Laser-induced microgrooves 11 were shown in our research to significantly influence the surface morphology, contact angle, surface roughness, and chemical composition of Ti that can influence the attachment of fibronectin on implants. A set of continuous microgrooves 11 with 50 m width, 5 m depth, and 150 m spacing between grooves are engraved at the root of the threads (0.5 mm) on a Di 12 using a laser system (e.g., a Galvo FP fiber marking) to produce a laser-microgrooved Di 13. A rotary stage of the laser system (not shown) is oriented according to the helix angle of the implant threads to produce the laser microgrooves 11 at the root 14 of the threads 18.

[0038] Referring to FIG. 2, in a preferred embodiment FN is attach on the laser-engraved implant 12 surface by tresyl chloride method 21, and coats the FN-immobilized implant surface 22 with BMP2- and Ag-immobilized PCL NFM as shown 23. Basic terminal hydroxyl groups of a pure titanium surface 22 react with tresyl chloride, which allows for further coupling with fibronectin (FN). Previous in vivo studies using a rabbit femur model found that immobilizing fibronectin (FN) onto cylindrical pure titanium implants enhanced bone regeneration around implants. However, pure titanium has limited applications in the biomedical industry due to its inferior mechanical and biological properties, compared to biomedical grade titanium alloys, such as Ti-6Al-4V (the most commonly used titanium alloy in medical devices). We examined whether human plasma FN can be attached to Ti-6Al-4V via the tresyl chloride activation method. Three groups of samples were prepared to test the FN attachment on Ti via the tresyl chloride activation process: (1) control, (2) tresyl chloride-activated Ti (referred to as Tresyl/Ti), and (3) tresyl chloride-activated Ti subsequently coupled with FN (referred to as FN/Ti). To prepare Tresyl/Ti, the top surface of a polished Ti-6Al-4V sample was treated with 2,2,2-Trifluoroethanesulfonyl chloride at 36 C. for 48 hours, then washed with water, water-acetone (50:50), and acetone. Samples were then dried and stored in a desiccator. To prepare FN/Ti, a Tresyl/Ti sample was treated for 24 hours at 37 C. with human plasma fibronectin diluted in phosphate-buffered saline (PBS) solution to a concentration of 0.1 mg/mL. X-ray photoelectron spectroscopy (XPS) analysis was conducted on all samples to determine the chemical state of Ti. The binding energy for each spectrum was calibrated against the C1s peak at 284.8 eV.

[0039] Referring to FIG. 3, XPS analysis found the presence of an amide group for FN/Ti, which confirms the surface activation by tresyl chloride and then direct coupling of FN with Ti. The N1s peak, derived from the amide bond of immobilized fibronectin, was detected around the binding energy of 400 eV for only FN/Ti samples. Therefore, this study suggested that direct attachment of FN is possible on a tresylated Ti alloy surface. Our proof of concept for the potential application of the treatment protocols on a Di led to the following: an ideal functional coating for a Di must reabsorb with time to allow and encourage new bone formation while maintaining its osteoconductive properties in vivo.

[0040] Referring to FIG. 4, the present invention provides laser engraving 41 to support attachment of FN on a dental implant (FIG. 1-12) and then coating the implant with BMP2 and Ag NP-immobilized PCL NFM. A laser pulse can be applied on the polished surfaces of Ti to create linear and continuous microgrooves on Ti implants. A laser capable of producing precision microgrooves (FIG. 1-11) on at least the flat surface of a Ti rod 42 can be used for this purpose. In our research we have used a Galvo FP fiber marking laser equipped with software for engraving a set of microgrooves (10 m width, 5 m depth, and 50 m spacing between grooves) on Ti. The reason for achieving 5 m-deep microgrooves on Ti is due to the fact that each groove of this size can accommodate at least 18 layers of nanofiber (average fiber diameter 300 nanometers). In the present invention, we use 18 layers of PCL NFM because our research shows that the porosity of a PCL NFM membrane comprising 18 layers of fibers is adequate for cells to migrate through the membrane. FN can be attached on laser engraved Ti implants (lgTi), where immobilization of FN with Ti is accomplished by the tresyl chloride method as provided by the present invention.

[0041] Referring to FIG. 5, aligned unidirectional PCL NFM can be collected using the methods disclosed in U.S. Pat. No. 9,809,906 by the present Applicant and illustrated in FIG. 5. The laser-grooved surface of lgTi is activated by tresyl chloride and then 18 layers of PCL NFM is deposited along the direction of the thread (clockwise) by rotating the tresylated Di 18 times until the implant collects 18 layers of fibers. The reason for adapting this coating method on a Di surface is due to fact that such a method should be able to maintain nanofibers along the Di/bone interface. FN, FN-Hep-BMP2 and CG-Ag complexes are gently splashed on the PCL coated Di samples to prepare FN-PCL/Di, FN-BMP-PCL/Di and CG-Ag-PCL/Di Di, respectively. All implants are prepared under sterile conditions and kept for 30 minutes in a portable ultraviolet sterilizer before surgery.

[0042] Referring to FIGS. 6a and 6b, in a method validation test we coated a M30.5 screw by PCL NFM using the method of the present invention (FIG. 5). We torqued the fiber-coated screw in to a pre-drilled hole (2.6 mm) on clear acrylic (FIG. 6b). We observed homogeneously-distributed fiber along the interface between the screw and the acrylic (FIG. 6b).

Experimental Aspects

Immobilization of Bone Morphogenic Protein-2 (BMP2) on Ti Using Fibronectin and PCL NFM.

[0043] Bone morphogenic proteins (BMPs) play important roles in in osteoblast and chondrocyte differentiation. Research shows that surface functionalization of Ti with BMP2 improves the osteoblast activities of Ti. Among BMP family members, BMP2 is a potent osteoinductive factor that plays key role during bone formation. Fibronectin (FN) is a multifunctional protein most abundantly found in the extracellular matrix (ECM) under dynamic remodeling conditions such as bone healing and development. Research shows that tethering of FN onto Ti effectively enhanced the bone regeneration around implants. Our preliminary studies show that FN-immobilized PCL NFM (referred as FN-PCL) has higher biocompatibility with osteoblast cells in comparison to PCL. FN contains binding domains for many bone growth signaling factors, including BMP2 and transforming growth factor-beta (TGF-). We have successfully immobilized BMP2 with PCL NFM using FN in our preliminary studies. The effect of BMP2-immobilized PCL NFM coating on the osteogenic functions of Ti is not known and thus it needs to be investigated.

Immobilization of Silver Nanoparticles (Ag NP) on Ti Using Collagen and PCL NFM.

[0044] Prolonged anti-bacterial activities of an implant are possible by tethering anti-bacterial molecules with the implant. Many studies reported that Ag NP inhibits bacterial growth, while retaining/promoting osteoblast viability. Among common antibacterial nanoparticles (Ag, CuO, ZnO), Ag NP shows the minimum toxicity to environmentally relevant test organisms and mammalian cells in vitro and in vivo. Since Ag NP dissolves in CG, it can be immobilized with CG-PCL NFM. Our in vivo and in vitro studies show that CG-PCL NFM coating enhanced biological functions of Ti. This is due to the fact that higher cell functions were created via better cell signaling arising from the cell-cell contact and the cell-NFM components in the case of the CG-PCL NFM-coated Ti samples than non-coated Ti samples. Our preliminary studies showed no antimicrobial activity of Ag NP-immobilized CG-PCL NFM towards Staphylococcus aureus in comparison to PCL NFM. The effect of Ag NP-tethered CG-PCL NFM on the osteogenic and anti-bacterial activities towards other common aerobic bacterial organisms on Ti implant is not known and thus needs to be investigated.

Effect of Immobilization of Fibronectin and Collagen on the Cellular Functions of PCL NFM

[0045] Fibronectin (FN) contains several active sites, known as the heparin-binding domains, collagen-binding domain, fibrin-binding domain, and cell-binding domain, that serve as platforms for cell anchorage. The goal of this preliminary study was to evaluate the effect of immobilization of collagen and plasma fibronectin with PCL NFM on the cellular functions of PCL NFM. The results (FIG. 7a and FIG. 7b) show that the individual immobilization of CG and FN on PCL NFM has no adverse effect on osteoblast cells adhesion and proliferation of PCL NFM, although a significant increase of cell adhesion was observed for FN-PCL-NFM when compared to PCL NFM (p<0.05). A significant improvement of cell adhesion and proliferation was observed for FN-CG-PCL NFM in comparison to PCL NFM (p<0.01). This is due to the fact that higher cell functions were created via better cell signaling arising from the cell-cell contact and the cell-NFM components in the case of FN-CG immobilized PCL NFM compared to PCL NFM.

[0046] Direct attachment of FN on a Ti implant surface is possible using a Tresyl Chloride-Activated Method (shown in Section C.5.). Since FN contains a CG binding domain, FN-immobilized Ti can therefore be polymerized into CG-PCL. The effect of the attachment of PCL NFM with Ti using CG and FN on the osteogenic functions of the implant is not known and needs to be investigated.

Immobilization of Human Bone Morphogenic Protein-2 (BMP2) with PCL NFM Using Fibronectin (FN).

[0047] The PCL NFM can be modified with heparin (Hep) and further immobilized with BMP2. The modified fibers showed the potential to effectively induce osteogenic differentiation of periodontal ligament cells. Since FN contains heparin-binding domains, PCL fibers can be modified with FN-Hep-BMP2 complex. The purpose of this preliminary study was threefold: (1) to immobilize BMP2 on PCL NFM using only FN-BMP2 and FN-Hep-BMP2 complexes, (2) to determine the amount of BMP2 release from the immobilized BMP2-PCL NFM, and (3) to compare the cell viability of BMP2-immobilized PCL NFMs with respect to PCL NFM (control). Immobilized BMP2 was released from the PCL NFMs in a sustained manner for 28 days, although the rates of release of BMP2 from FN-BMP2/PCL and FN-Hep-BMP2/PCL were different. A gradual increase of release of BMP2 for 28 days was observed for FN-Hep-BMP2/PCL samples (FIG. 8a). Rapid release of BMP2 for first 4 days, then gradual decline of release of BMP2 with time was observed for FN-BMP2/PCL samples. Cells displayed a well-extended morphology on all the BMP2-treated groups, when they were compared with the control group (8a). FIG. 8b depicts a representative image showing cell divisions after 48 hours of cell culture on FN-BMP2-PCL samples. In the image, blue color shows the attachment of cells on NFM. There was more than a 52% and 30% increase in the cell viability on FN-Hep-BMP2-PCL samples after culturing the cells for 72 hours compared to control and FN-BMP2-PCL. Both release and cell viability tests suggested an advantage of FN-Hep-BMP2 over FN-BMP2 complex for the immobilization of BMP2 with PCL NFM. FN-Hep-BMP2 immobilized PCL NFM has the potential to induce osteogenic differentiation of osteoblast cells on a Ti implant surface, which is not yet known. The effect of the treatment of Ti with FN by tresyl chloride method and then coating by FN-Hep-BMP2/PCL on the osteogenic functions needs to be investigated.

Attachment of Silver Nanoparticles (Ag NP) with PCL NFM Using CG

[0048] Silver nanoparticles (Ag NP) show promising anti-bacterial properties with biocompatibility and minimal toxicity. Ag NP-loaded collagen was immobilized with polymeric film to inhibit bacterial growth while promoting osteoblast cell viability. The anti-bacterial activities of PCL NFM can be improved by immobilizing Ag NP-loaded CG with PCL NFM. The purpose of this preliminary study was to examine the effect of immobilization of Ag NP-loaded CG on the anti-bacterial properties of PCL NFM. We succeeded in immobilizing Ag-loaded collagen with PCL NFM. The SEM and XRD analysis before and after 2 days of bacterial culture confirmed the presence of Ag with PCL. Our bacterial culture studies showed no sign of colonies growing on Ag-CG-PCL, whereas the presence of bacteria was observed in PCL. FIG. 9 shows PCL samples after Gram staining: (a) PCL and (b) CG-Ag-PCL. S. aureus that take up the Gram stain were present in PCL, as observed in the image by circular black shapes (pointed by arrows), while CG-Ag-PCL without S. aureus, did not stain and appears with a gray color. One reason for this might arise from an increased carrying capacity of Ag NP-loaded collagen by the nanofiber disc due to its unique surface-to-volume ratio.

In Vivo Evaluation of Coating a Titanium Implant with CG-PCL NFM

[0049] We have invented a method of coating a cylindrical metal implant with NFM that is made with CG-PCL (U.S. Pat. No. 9,809,906). Our invention implements a set of grooves that are created on Ti in a circumferential direction to increase the contact area between the implant and bone. CG-PCL NFM is subsequently coated along the sub-micrometer grooves on the Ti implant using our unique electrospinning process (U.S. Pat. No. 9,359,694). The goal of this research was to evaluate the effect of CG-PCL NFM coating on the mechanical stability and osseointegration of a Ti implant using a rabbit model. Our in vivo pull-out tests demonstrated that mechanical stability of microgrooved-Ti was significantly higher compared to non-grooved Ti. The mechanical stability (quantified by shear strength) of groove-NFM Ti/bone samples were significantly greater compared to other samples (p<0.05). The pull-out strength of groove-NFM-coated Ti was comparable to other functional coating-treated Ti reported in the literature. The types of new bone growth on Ti was different between groove and groove-NFM samples, which was observed from the stained images of histology-sectioned images (FIG. 10e). Histomorphometric results showed that the amount of BIC on Ti was higher for groove-NFM (12.180.94 mm, n=2) than groove (5.304.01 mm, n=2) samples. Due to the poor attachment of Ti with bone, Ti implants came out from their implant sites during histology preparation. Therefore, there was no result for any control sample. Both CT analyses (FIG. 10e and FIG. 10f) and blood serum (FIG. 10g) results confirmed the time-dependent bone growth around the CG-PCL NFM-coated Ti implant and determined that 6 weeks were required for sufficient bone growth around the implant.

[0050] Our in vivo pull-out tests demonstrated that mechanical stability of microgrooved-Ti was significantly higher compared to non-grooved Ti (FIG. 10c). The mechanical stability (quantified by shear strength) of groove-NFM Ti/bone samples were significantly greater compared to other samples (p<0.05). The pull-out strength of groove-NFM-coated Ti was comparable to other functional coating-treated Ti reported in the literature. The types of new bone growth on Ti was different between groove and groove-NFM samples, which was observed from the stained images of histology-sectioned images (FIG. 10e). Histomorphometric results showed that the amount of BIC on Ti was higher for groove-NFM (12.180.94 mm, n=2) than groove (5.304.01 mm, n=2) samples. Due to the poor attachment of Ti with bone, Ti implants came out from their implant sites during histology preparation. Therefore, there was no result for any control sample. Both CT analyses (FIG. 10e and FIG. 10f) and blood serum (FIG. 10g) results confirmed the time-dependent bone growth around the CG-PCL NFM-coated Ti implant and determined that 6 weeks were required for sufficient bone growth around the implant.

[0051] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.