WO2014087412A1

WO2014087412A1 - Nano surface modified metallic titanium implants for orthopaedic or dental applications and method of manufacturing thereof

Info

Publication number: WO2014087412A1
Application number: PCT/IN2012/000786
Authority: WO
Inventors: Deepthy DEEPTHY MENON SHANTIKUMAR V NAIR MANZOOR KOYAKUTTY DIVYA RANI VV VINOTHKUMAR LAKSHMANAN
Original assignee: Amrita Vishwa Vidya Peetham University
Priority date: 2012-12-03
Filing date: 2012-12-03
Publication date: 2014-06-12

Abstract

The present invention relates to a metallic implant product developed with surface nano features by means of wet hydrothermal technique, which provides better bio-compatibility and improved osteo-integration for specific use in orthopaedic and dental applications. Methods of creating nano features on surfaces of titanium di-oxide (Titania) on Ti implants and the corresponding improved implant behaviour as a consequence under in vivo conditions are demonstrated and proven in this invention.

Description

TH E PATENTS ACT, 1970

(39 of 1970) AND

THE PATENT RULES 2003

COMPLETE SPECIFICATION

(See Sections 10 rule 13) l. TITLE or TH E I N VENTION : ""The Art, Method and Manner of Nanosurface Modification of titanium implants for orthopedic or dental applications"³'

2.

2. APPLiCANT(S)

(a) Name : AMRITA YISHWAVIDYAPEETHAM represented by its Director, Amrita Centre of Nano Sciences, Dr. Shantikumar V. Nair

(b) Nationality : Indian.

(c) Address : "Elamakkara P.O., Cochin 682 041 , Kerala

3 PRE AMBLE TO THE DESCRIPTION

COMPLETE SPECIFICATION

The following specification describes the invention

The Invention relates to "The Art, Method and Manner of Nanosurface Modification of titanium implants for orthopedic or dental applications"

FIELD OF THE INVENTION

The present invention relates to a metallic implant product developed with surface nanofeatures by means of wet chemical hydrothermal technique, which provides better biocompatibility and improved osteointegration for specific use in orthopedic and dental applications. Methods of creating nano features on surfaces of titanium dioxide (titania) on Ti implants and the corresponding improved implant behaviour as a consequence under in vivo conditions are demonstrated and proven in this invention. BACKGROUND OF THE INVENTION

The success of metallic implants that are surgically inserted within living tissues is dictated by its ability to achieve and maintain an enduring bond between the confronting surface of the implant and the host tissue. Traditionally, titanium-based materials have been considered as durable and biocompatible implant materials for several implant applications, including vascular, orthopedic and neurological applications. The long term failure of metallic implants, especially those based on titanium, for orthopedic applications, has been attributed to insufficient bonding to bone. Aseptic loosening and periprosthetic osteolysis are the most commonly reported causes for revision surgery in total joint replacement. To address these issues, maximizing initial cellular adhesion and reducing inflammatory immune responses by altering intrinsic surface properties of titanium implants are considered important factors in the successful design of future implants. Titanium implants have a surface layer of titanium dioxide and this is responsible for the inertness of titanium-based implants within the human body. However, their cytocompatibility properties and long- term efficacy are limited without further surface engineering since the average functional lifetime of an orthopedic implant is only 10 to 15 years. Therefore, surface modification of titanium has been explored as a means to improve osteointegration. Studies have indicated that the surface topography of bio-implant materials influence cell response, including focal adhesion, cellular morphology, cytoskeleton rearrangements, cell proliferation and signalling as well as its gene expression [1-6]. In addition, the extracellular matrix (ECM) or substratum with which cells interact often includes topography at the nanoscale [7-9]. The influence of nanoscale topography on cellular behavior was revealed in various studies (10-13). Various surface modification techniques for providing varying roughness or topography to metallic surfaces have been suggested. Such approaches include the use of calcium phosphate or hydroxyapatite, a material suggested as a bone bonding mineral, or spraying of titanium particles to the titanium implant surface, grit blasting with fine particles to create a coarse roughened surface, acid etching, alkali treatment, anodization to create a nanotubular patterned titanium oxide layer on metallic titanium, lithography, chemical and physical vapor deposition and plasma spraying [14]. Etching the surface of titanium with acids has been a commonly adopted procedure for manufacturing dental implants. A series of US patents including US Patent No: 5,603,338; 5,876,543, 5,863,201; and 6,652,765 assigned to Implant Innovations Inc., detail the use of acids for etching, either individually or in a defined sequence to prepare Osseotite surfaces for dental implant applications [15-18]. A sequence of acid treatments wherein an initial etching with hydrochloric acid uniformly removes the oxide layer, and the subsequent use of hydrochloric and sulphuric acids to etch the exposed titanium surface have yielded commercial success. US Patent No: 5,307,594 describes a method for forming textured surface on orthopedic implants using a resilient mask with openings on the implant surface and subjecting it to high pressure blasting using an erosive blasting media such as metal oxide particles [19].

US Patent Application No: US2010_/0187172 describes the fabrication of vertically oriented, highly ordered nanotube titania (Ti0₂) arrays exhibiting lengths of 10-1000 μιτι formed by anodization of titanium thin or thick films [20]. However, anodization results in the formation of only uni-dimensional nanostructures of variable aspect ratios and is not effective for complex shaped implants. Recently, some of the authors of this invention published a process to produce controlled nanostructures of a variety of shapes using a simple scalable hydrothermal technique in the presence of NaOH [21] . The present invention applies this process for the development of a successful implant product that has the required tissue integration in vivo while maintaining the structural integrity of the implant.

SUMMARY OF THE INVENTION

In the present invention, we disclose a product based on metallic orthopedic or dental implants of Titanium with novel nanostructural surface features having controllable morphologies and uniformity with demonstrated in vivo applicability. In another important aspect of this invention, the surface modified titanium implants were tested both in vitro and in vivo, providing confirmed osteoblast cell response through enhanced cellular adhesion, proliferation as well as differentiation. Enhanced osteointegration was proven in vivo.

BRIEF DESCRIPTION OF DRAWINGS

Fig. la gives a diagrammatic sketch of the hydrothermal chamber used in the study for implant surface modification and lb shows the photograph of the chamber, la gives the details of the necessary components of the setup for hydrothermal processing, i.e., 1 - furnace, 2 - heating coils, 3 - stainless steel (SS) chamber, 4 - SS lid, 5 - screw locks, 6 - Teflon chamber, 7 - Teflon lid, 8 - implant holder, 9 - reaction medium, 10 - implant screw.

Although the attached figure shows that the main implant body is a screw-type with a tapered end, this is just one of the many variations of implant designs and the present invention is not to be limited to a particular type of implant. The present invention relates directly to nanosurface modification of implant products for any possi ble design alterations as well as metal biomaterials.

F ig. 2 gives the schematic of the Ti implant (screw in this case) and the representation of the n anostructures generated on this implant by hydrothermal modification . 1 - head diameter, 2- screw length, 3 - pitch of screw, 4 - Structure A, 5 - pore diameter in Structure A, 6 - spacing between pores in Structure A, 7 - Structure B₍ 8 - wall thickness in Structure B, 9 - void site in Structure B, 10 - Structure C, 11 - . nanostructure diameter, 12 - distance between nanostructures in Structure C.

F ig. 3 gives the representative SEM images of (a) Ti implant surface before hydrothermal treatment; and hydrothermally modified Ti implants with nanostructural features (b) Structure A; (c) Structure B; and (d) Structure C

Fig. 4 Graph showing cellular proliferation analysis of primary osteoblast cells using Aiamar blue on nanomodified titanium implants in comparison to nanopolished titanium. Statistical significance was assessed relative to control nanopolished Ti for each nanostructure with * and * * denoting p-<0.05 and p<0.01 respectively.

Fig. 5 Gives the SEM image of cellular proliferation of primary osteoblast cells cultured on nanomodified Ti after a) 24 hours b) 72 hours. Lane 1, 2, 3 and 4 represents control, Structure A, structure B and Structure C respetively F ig. 6 depicts the results of osteoblast specific gene expression analysis carried out using RT-PCR on primary osteoblast cells cultured on nanomodified Ti implants after 7 and 14 days of incubation, (a) alakline phosphatse (b) Osteocalcin (c) Collagen (d) Decorin and (e) RunX_2.

Fig. 7 represents the in vivo implantation of nanomodified Ti implants surgically implanted into the left femur condyle of a Sprague Dawley rat. (a) photograph of the implanted nanomodified Ti screw (shown in circle) and (b) X-ray image of the same.

Fig. 8 gives the results of the in vivo osseointegration study carried out by implanting various nanosurface modified Ti screws in the left femur condyle of Sprague Dawley rats. The images of qualitative histological analysis for (a) 2^nd (b) 8^th and (c) 12^th weeks after implantation are shown with the percentage of bone contact for the corresponding time points given in the inset.

Fig. 9 depicts the inflammatory response to nanomodified Ti implantation after (a) 2^nd, (b) 8^th and (c) 12^th week, in SD rats studied through cytokine analysis from blood serum using flow cytometry.

DEFINITIONS:

As used herein, any component that is intended for long or short-term contact with biological tissues and also which does not induce any adverse biological response of the tissue is encompassed by the term "biocompatible component" or "biocompatibility" of the material. Example of such a biocompatible component is an implant such as orthopedic, dental or cardiovascular implants.

As used herein, the term "implant" includes within its scope any device that is intended to be implanted into a human body and that can serve the purpose of replacing the anatomy and/or restoring any normal function of the body.

As used herein, the term "nanosurface modification" refers to the process of surface modification wherein the metallic surface is treated chemically by one or many means to achieve a homogeneous/uniform surface topography with structural features in the nanoscale with dimensions ranging from 1 - 500 nm.

As used herein, the term "hydrothermal treatment" refers to a chemical technique of surface modification of the metal, wherein the metals are treated in a sealed autoclave at elevated temperature and pressure, in a chemical environment offered by alkaline solution and in certain cases a combination with suitable chelating agent, thereby providing a roughened nanotexture to the implant surface.

As used herein, the term "osteointegration" refers to the capability of any implant to integrate well with bone tissues without inducing any fibrous encapsulation as well as inflammatory response. DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to. nanosurface modification of titanium based metal implants. It is the primary objective of the present invention to produce a biocompatible implant of metallic titanium having nanoscale roughness which is substantially uniform over the entire area of the implant that is intended to bond to the tissue or bone in a much improved fashion compared to existing implants where the surfaces are not modified.

It is an additional object of the present invention to prove that nanosurface modification, of the kind produced by the hydrothermal process described, provides substantially improved biocompatibility, with improved cellular functions, when tested in vitro with primary osteoblast cells.

Another objective of the invention is to develop a product with a particular nanostructure on the metallic implant surface that would enhance in vivo biocompatibility by promoting improved osteointegration in comparison to unmodified metallic surfaces.

This aim and these and other objects, which will become apparent hereinafter, are achieved by a surface modification technique on metal implants, particularly titanium and its alloys, for medical, surgical and implant applications, resulting in a modified metallic oxide layer, which is explained under the following steps.

The following are the steps used to create the new implant product. Several modifications of the steps below may be made to suit a particular implant, for example, type of surface cleaning, mechanical polishing etc., or the omission of step 1 or 2 if the implant is already of suitable quality:

Step 1 - Mechanically polishing commercially available pure titanium implants. This may be done using 600 grit silicon carbide to a uniform coarseness. This can be done manually using grit paper or automated using grit blasting

Ste 2 - Surface cleaning of the coarsened implant. This may be done ultrasonically in acetone and successive ultrasonic cleaning in distilled water.

Step 3 - Cleaned polished Ti implants are immersed in an autoclave (Fig. lb) containing sodium hydroxide.

Step 4 - Hydrothermal treatment of the Ti implant samples placed in the autoclave in a programmable temperature controlled furnace (Fig. 1) whose temperature is set to different temperature settings in the range 100 - 300 °C for a period varying from 1-10 hours, followed by ultrasonic or other cleaning action.

Step 5 - Drying of the hydrothermaily treated Ti implant samples in a medically sterile environment.

Step 6- Medically sterile sealing of the implant in plastic or other container.

The above said processes fabricated on a metallic screw (1) resulted in nanostructures with variable morphology as shown in Fig. 2a, which are labelled as Structure A (2), Structure B (3) and Structure C (4) as shown in Fig. 2. These nanostructures are compared with conventional polished surfaces labelled as Control. Figure 3 gives the electron micrographs of the varying surface features of Ti screw.

Structure A obtained through hydrothermal processing has a mesh-like porous architecture with interconnected pores having diameters in the range of 164.5 ± 83.52nm (5) and a pore-to-pore distance of 251.73 ± 115.616 nm (6). Structure B reveals a leafy architecture haying thick irregular ridges of wall thickness 20 ± 5nm (7) and voids of dimensions varying from 249.05 ± 64.08nm (8). Structure C shows 1-D needular features with diameter ranging from 122.88 ± 14.45(9), and intern needular distance in the range of 248.454 ± 85.22 nm (10).

For the purposes of proving the efficacy of the implant, the following studies have been done:

In vitro evaluation of primary osteoblast cell proliferation, functional analysis and in vivo osteointegration studies on surface modified implants and its comparison with control polished implant.

A distinct feature of the implants was revealed through the cellular proliferation studies, wherein all the developed nanostructure modified Ti implants enhanced cellular adhesion and proliferation significantly than polished Ti implant (Figure 4). Cellular proliferation rate of primary osteoblast cells cultured on nanofeatured titanium implants after 3, 5 and 7 days of incubation were assessed using alamar blue assay. Enhanced proliferation was observed on Structure B in comparison to control polished titanium and other nanostructured implants. Figure 5 a-b shows the SEM images of primary osteoblast cells adhered and proliferated after 24 and 72 hours respectively, depicting these differences. In relation to the above analysis, inventors further investigated the gene expression of ostoeblast specific genes such as Alkaline phosphatase, osteocalcin, collagen, decorin and RunX₂ after 7 and 14 days of growth of primary osteoblast cells using Real Time PCR. F igure 6a-e revealed that implants with surface topography as in Structure B induces a 15-35 fold higher osteoblast specific mRNA production of osteoblast cells in comparison to control polished titanium implant, suggesting the relevance of nano surface modification in promoting osteointegration. In an in vivo demonstration of the above mentioned details, Ti screws surface modified to generate nanopatterns were implanted into the femur condyle of Sprague dawley rats (Figure 7). Thirty SD rats divided into 3 groups of 10 each for study periods of 2, 8 and 12 weeks were used for the experiment. Histopathological analysis at the end of the study duration revealed that, of all the modified implants, the percentage of bone contact around the implant was maximally enhanced for Structure B after 2^nd, 8^th and 12^th week of surgical implantation (Figure 8 a-c). A quantitative analysis using ImagePro Express software also confirmed this proposition with ~ 90% bone contact after 12^th week of implantation (Inset of Figure 7 a-c). In yet another important aspect of the present invention, inventors examined whether nanostructuring of implants induced any in vivo inflammatory response upon implantation. A quantitative analysis of serum cytokine levels using flow cytometry after 2^nd, 8^th and 12^th week of implantation (Figure 9) revealed that none of the hydrothermally modified nanostructured implants induced any acute as well as chronic cytokine levels in comparison to the serum cytokine level of animal with no implant. References

1. Balu H M and Ba ltimore D 1991 /. Cell Biol. 112-781

2. Za mir E and Geiger B 2001 /. Cell Sci. 114 3577

3. Diener A, Nebe B, Lu then F, Becker P, Beck U, Neumann H G and Rychly J 2005

D ioma t nnis 26 383-92

4. Martin K H, Slack J K, Boerner S A, Martin C and Parsons J T 2002 Science 296 1652-3

5. Schwa rtz M A and GinsbergM H 2002 Nat. Cell Biol. 4 E65-8

6. How A, Aplin A E, Alahari S K and Juliano R L 1998 Curr. Opm. Cell Biol. 84 345-57

7. Ta l Dvir, Brian P. Timko, Daniel S. Kohane and robert Langer nature nanotechnology VOL 6 JANUARY 2011

8. Dalby J, Gadegaard N, Tare R, Andar A, Riehle M O, Herzyk P, Wilkinson C D W and Oreffo R O C.Nat. Mater 2007; (6) :997-1003

9. Stevens M M and George J H 2005 Science 310 1135-8

10. Kunzler T P, Huwiler C, Drobek T, Voros J, Spencer N D. Systematic study of osteoblast response to nanotropgraphy by means of nanoparticle-density gradients. Biomaterials, 28, 2007, 5000-5006.

11. William C. Clem, Shafiul Chowdhury, Shane A. Catledge, Jeffrey J. Weimer, Faheem M. Shaikh, Kristin M. Hennessy, Valery V. Konovalov, Michael R. Hill, Alfred Waterfeld Susan L. Bellis, and Yogesh K. Vohra. Mesenchymal stem cell interaction with ultra smooth nanostructured diamond for wear resistant orthopaedic implants, Biomaterials. 2008 ; 29(24- 25): 3461-3468.

12. Balasundaram G, Sato M,Webster TJ. Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD. Biomaterials 2006;27:2798-805

13. Matthew J Dalby. Cellular response to low adhesion nanotopographies. International journal of Nanomedicine 2007:2(3), 373-381

14. Xuanyog Liu, Paul K Chu, Chuanxian Ding. Surface modification of titanium, titanium alloys and related materials for biomedical application. Material science and Engineering R 47 (2004) 49-121

15. United States Patent 5603338 , Keith D, Beaty, Gardens, P B, Fla et al, Implant surface preparation utilizing acid treatment Feb 18, 1997 16. United state patent 5,876,543, Kinpara S, Ohsumi H, Sagisaka Method of finishing board with thick resin film for making under layer invisible, 14-8-1997

17. United State patent 5,863,201, Lazzara, Richard J, Heylmun, Thomas S, Beaty, Keith D, Infection-blocking dental implant, 26-1-1999

18. United state patent, 6,652,765 , Beaty, Keith D, Implant surface preparation, 25-11-2003.

19. US Patent No: 5,307,594, Panchison, clearence, Method for forming textured surfaces on an orthopedic implant, 14-12-1992

20. US Patent No: US2010/0187172, Paulose, Maggie, Shaaker, Karthik, Prakasam, Haripriya E, Yoria,Sorachon, Varghese, Oomman K, Grimes, Craig. A highly-ordered titania nanotube arrays, 25-1 -2010

21. Divya Rani V V, Manzoor K, Deepthy Menon, Selvamurugan N, Shantikumar V Nair. The design of novel nanostructures on titanium by solution chemistry for an improved osteoblast response. Nanotechnology 2009; 20: 195101-11

Claims

1. Titanium metallic implant surface modified to generate nanoscale topography by hydrothermal method which provides better osteointegration than unmodified surfaces and thus is appropriate for application as a biomaterial implant for use in orthopedic and dental implant surgery.

2. The nanomodfied metallic implant in claim 1 generated by a non-toxic aqueous hydrothermal technique in alkaline conditions at low temperature and time duration.

3. Titanium implant surfaces modified as in claim 1 containing a unique surface nano- morphology.

4. The product in claim 1 that is developed as in claim 2 with use of individual or combinations of various alkaline solutions of sodium hydroxide (NaOH), potassium hydroxide ( OH), ammonium hydroxide (NH₄OH), Lithium hydroxide (LiOH), Magnesium Hydroxide (Mg(OH)₂), Calcium hydroxide (Ca(OH)₂), or all possible water soluble hydroxides having pH in the range of 10-15. The alkaline solutions mentioned herein aids in the controlled dissolution of titania species to form specific nanofeatures.

5. The Ti product as in claim 3 having nano-patterned architectures for bone, dental or cranial implant applications.

6. Claim 5 with or without the incorporation of drugs within the nano-patterned surface.

7. The product as in claim 1 that is non-toxic to human cells, tissue or organs.

8. Nanostructured product as in claim 1 generated by hydrothermal modification that is stable under physiological conditions even after the product is subjected to harsh sonication conditions and incubation in body fluids.