BONE IMPLANT WITH A MAGNESIUM PHOSPHATE XEROGEL COATING

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit, under 35 U.S.C. § 119(e), of U.S. provisional application Serial No. 63/380,411, filed on October 21 , 2022. All documents above are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

[0001] The present invention relates to coating for bone implants. More specifically, the present invention is concerned with coating of a magnesium phosphate xerogel that enhance the bone-forming ability and rapid bone integration of the implant.

BACKGROUND OF THE INVENTION

[0002] As biocompatible material, titanium and its alloys are broadly used for clinical purposes in various medical fields related to bone repair due to their good biocompatibility, mechanical properties, and machinability ^{1 3} . However, because of the inert native surface oxide layer formed on the surface of titanium-based implants, its poor and long- lasting osseointegration remains a significant clinical challenge ⁴ . Despite their intensive use in such applications, sustained efforts have been made to improve their surface bioactivity and compatibility. These efforts have been devoted to reducing the time of chemical bonding between the Ti-based implant and the surrounding bone, by modifying the surface of Ti-based implants prior to implantation surgeries. This could result in promote osseointegration, increase their biological activity and thus faster healing ⁵⁷

[0003] To achieve these objectives, researchers have investigated a variety of methods such as mechanical and chemical treatments that provide an appropriate texture and/or new chemical functional groups on the surface that improve the tissue bonding. Additionally, Surface blasting and etching to activate the metal surface bioactivity ^{8 11} ; and Coating metal surfaces with a suitably bioactive material, such as hydroxyapatite (HAp), where HAp layer provides an osteoinductive surface for tissue fixation with the surrounding bone ¹² . Both approaches have been used in commercial implants of Ti but a complete solution to this problem is still far from being achieved.

[0004] On the other hand, most of the commercially coated implants utilize a plasma spraying technique to bind HAp to the metal surface. Unfortunately, these coatings have limited clinical success due to cracking, delamination, decomposition of HAp at the interface between the implant and the bone tissue. In addition, the plasma spray technique involves a high temperature which associated with disadvantages such as inhomogeneous phase and phase transformations that result in implant instability and degradation. These limitations increase the demand to look for an alternative option. SUMMARY OF THE INVENTION

[0005] In accordance with the present invention, there is provided:

1 . Use of a magnesium phosphate xerogel as a coating for a bone implant, wherein the magnesium phosphate xerogel comprises phosphate ions (PO4 ³ ), a divalent cation, and a monovalent cation at mole fractions of about 0.32 to about 0.44 (preferably about 0.33 to about 0.44), about 0.03 to about 0.09, and about 0.48 to about 0.63, respectively, wherein the divalent cation is magnesium (Mg ²⁺ ) or a mixture of magnesium and one or more of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , or Ca ²⁺ , wherein the mixture comprises up to a total of 30% by weight of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , and Ca ²⁺ based on the total weight of the mixture, and wherein the monovalent cation is sodium (Na ⁺ ), lithium (Li+) or a mixture thereof.

2. A coating for a bone implant comprising (preferably consisting of) a magnesium phosphate xerogel, wherein the magnesium phosphate xerogel comprises phosphate ions (PO4 ³ ), a divalent cation, and a monovalent cation at mole fractions of about 0.32 to about 0.44 (preferably about 0.33 to about 0.44), about 0.03 to about 0.09, and about 0.48 to about 0.63, respectively, wherein the divalent cation is magnesium (Mg ²⁺ ) or a mixture of magnesium and one or more of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , or Ca ²⁺ , wherein the mixture comprises up to a total of 30% by weight of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , and Ca ²⁺ based on the total weight of the mixture, and wherein the monovalent cation is sodium (Na ⁺ ), lithium (Li+) or a mixture thereof.

3. A bone implant, wherein a surface of the implant is covered by a coating comprising (preferably consisting of) a magnesium phosphate xerogel, wherein the magnesium phosphate xerogel comprises phosphate ions (PO4 ³ ), a divalent cation, and a monovalent cation at mole fractions of about 0.32 to about 0.44 (preferably about 0.33 to about 0.44), about 0.03 to about 0.09, and about 0.48 to about 0.63, respectively, wherein the divalent cation is magnesium (Mg ²⁺ ) or a mixture of magnesium and one or more of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , or Ca ²⁺ , wherein the mixture comprises up to a total of 30% by weight of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , and Ca ²⁺ based on the total weight of the mixture, and wherein the monovalent cation is sodium (Na ⁺ ), lithium (Li+) or a mixture thereof.

4. The use, coating and implant of any one of embodiments 1 to 3, wherein the magnesium phosphate xerogel comprises the phosphate ions (PO4 ³ ), the divalent cation, and sodium ions (Na ⁺ ) as the monovalent cation at mole fractions of about 0.33 to about 0.44, about 0.03 to about 0.09, and about 0.48 to about 0.63, respectively, wherein the divalent cation is magnesium (Mg ²⁺ ) or a mixture of magnesium and calcium (Ca ²⁺ ), the mixture comprising up to 30% by weight of calcium based on the total weight of the mixture. The use, coating and implant of any one of embodiments 1 to 4, wherein the xerogel comprises phosphate, the divalent cation, and the monovalent cation at mole fractions of about 0.36 to about 0.42, about 0.05 to about 0.08, and about 0.50 to about 0.57, respectively. The use, coating and implant of any one of embodiments 1 to 5, wherein the xerogel comprises the phosphate ions, the divalent cation and monovalent cation at mole fractions of:

0.36, 0.07, and 0.57, respectively,

0.39, 0.08, and 0.53, respectively,

0.41 , 0.05, and 0.54, respectively, or

0.42, 0.08, and 0.50, respectively. The use, coating and implant of any one of embodiments 1 to 6, wherein the xerogel comprises phosphate, magnesium, and sodium ions at mole fractions of about 0.39, about 0.08, and about 0.53, respectively. The use, coating and implant of any one of embodiments 1 to 7, wherein said mixture comprises a total of about 10 % to about 30% by weight of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , and Ca ²⁺ based on the total weight of the mixture. The use, coating and implant of any one of embodiments 1 to 8, wherein said mixture comprises magnesium and calcium only. The use, coating and implant of any one of embodiments 1 to 9, wherein the divalent cation is said mixture. The use, coating and implant of any one of embodiments 1 to 9, wherein the divalent cation is magnesium only. The use, coating and implant of any one of embodiments 1 to 11 , wherein the monovalent cation is sodium (Na ⁺ ) only. The use, coating and implant of any one of embodiments 1 to 11 , wherein the monovalent cation is lithium only (Li ⁺ ) or a mixture of sodium and lithium. The use, coating and implant of any one of embodiments 1 to 13, wherein, when observed by transmission electron microscopy (TEM), the xerogel appears to comprise nanosheets. The use, coating and implant of embodiment 14, wherein the nanosheets are made of magnesium phosphate (with some monovalent cation). The use, coating and implant of embodiment 15, wherein the magnesium phosphate comprises between about 10 and about 20% of hydration water by weight. 17. The use, coating and implant of any one of embodiments 1 to 16, wherein, when there is no calcium in the xerogel, the xerogel further comprise up to 200% by weight, preferably between about 10% and about 20% by weight, of pyrophosphate (P2O7 ⁴ ), based on the weight of the phosphate.

18. The use, coating and implant of any one of embodiments 1 to 17, wherein the xerogel further comprises chloride (Cl ) ions.

19. The use, coating and implant of any one of embodiments 1 to 18, wherein the xerogel further comprises one or more additives.

20. The use, coating and implant of embodiment 19, wherein the additives are selected from: corn oil (for example in a concentration varying between about 0.1 and about 1.5 % based on the total weigh of the gel), sodium metaphosphate or pyrophosphate (for example in a concentration varying between about 0.125 and about 0.5 % based on the total weigh of the gel), sodium citrate (for example in a concentration varying between about 0.1 and about 10% based on the total weigh of the gel), xantham gum (for example in a concentration varying between about 0.1 and about 1.5 % based on the total weigh of the gel), sodium alginate (for example in a concentration varying between about 0.1 and about 1.5% based on the total weigh of the gel), carboxylate salts, such as sodium glycolate and sodium tartrate (for example in a concentration varying between about 0.1 % and about 5 % based on the total weigh of the gel), carboxylic acids, such as glycolic acid and tartaric acid (for example in a concentration varying between about 0.1 and about 5 % based on the total weigh of the gel), and chitosan (for example in a concentration varying between about 0.1 and about 1 .5% based on the total weigh of the gel).

21 . The use, coating and implant of any one of embodiments 1 to 20, wherein the bone implant is a dental, implant, a craniofacial implant, or an orthopedic implant.

22. The use, coating and implant of any one of embodiments 1 to 21, wherein the coating covers the entirety of the implant surface. 23. The use, coating and implant of any one of embodiments 1 to 22, wherein the bone implant is a titanium bone implant, a cobalt chromium bone implant, a stainless-steel bone implant, or a polymer bone implant (such as a polyester bone implant); preferably a titanium bone implant.

24. The use, coating and implant of any one of embodiments 1 to 23, wherein the implant is a titanium bone implant, and the coating has an X-ray photoelectron spectrum that is free of titanium peaks.

25. The use, coating and implant of any one of embodiments 1 to 24, wherein the coating has an X-ray photoelectron spectrum comprising: a Mg1s peak at about 1304.58 eV, and a P2p peak at about 133 to about 134 eV, an O1s peak at about 530 to about 535 eV, and optionally, when the surface of the implant has been pretreated with NaOH, a Na1s peak at about 1071.88 eV.

26. The use, coating and implant of embodiment 24, wherein the P2p peak can be deconvoluted into two peaks at about 133.5 and about 134.4 eV.

27. The use, coating and implant of embodiment 24 or 25, wherein the O1s peak can be deconvoluted into three different components peaks at about 531 .2, about 532.98, and about 535.3 eV.

28. The use, coating and implant of any one of embodiments 1 to 26, wherein the coating comprises H2O at an atomic concentration of about 0% to about 15%, P-0 at an atomic concentration of about 65% to about 85%, and P-OH an atomic concentration of about 15% to about 35%.

29. The use, coating and implant of any one of embodiments 1 to 27, wherein the coating has a FTIR spectrum comprising a band at about 997 cm ¹ and a band at about 1060 cm ¹ .

30. The use, coating and implant of any one of embodiments 1 to 28, wherein the coating has a Raman spectrum comprising a band at about 967 cm ¹ .

31 . The use, coating and implant of any one of embodiments 1 to 29, wherein the coating has an average surface roughness of about 30 nm or more.

32. The use, coating and implant of any one of embodiments 1 to 30, wherein the surface of the implant has an average roughness parameter (Ra) of about 45nm or more. 33. The use, coating and implant of any one of embodiments 1 to 31, wherein the implant is a titanium bone implant and has a surface sodium titanate layer, preferably a layer of sodium titanate gel, underneath the xerogel.

34. The use, coating and implant of embodiment 32, wherein the sodium titanate layer is an amorphous sodium titanate layer with a gradient structure.

35. The use, coating and implant of embodiment 32, wherein the sodium titanate layer is a crystalline layer with phases of sodium titanate and rutile.

36. The use, coating and implant of any one of embodiments 32 to 34, wherein the sodium titanate layer has a porous network structure with an interconnected pore network.

37. A method of providing the coating/implant of any one of embodiments 1 to 35. This method comprises the steps of: a) providing a gel as described above, b) providing a bone implant as described above, c) coating a surface of the bone implant with the gel, d) allowing the gel to dry thus yielding the xerogel.

38. The method of embodiment 36, wherein step a) comprises: i. providing a first aqueous solution comprising sodium hydroxide (NaOH), ii. providing a second solution comprising phosphoric acid (H3PO4) or monomagnesium phosphate (Mg(H ₂ PO ₄ )2),

Hi. dissolving magnesium hydroxide (Mg(OH) ₂ ) or trimagnesium phosphate (MgsfPO^), and optionally calcium chloride or calcium hydroxide, in the second solution, and iv. mixing together the first solution and the second solution, thereby providing the gel.

39. The method of embodiment 37, wherein the time between steps Hi and iv is no more than 10 minutes.

40. The method of embodiment 37 or 38, wherein the method comprises after step iv, the step v of aging the gel, for example overnight.

41 . The method of any one of embodiments 36 to 39, wherein the method further comprises at step b, sand-blasting the surface of the bone implant. 42. The method of any one of embodiments 36 to 40, wherein the method further comprises at step b, pretreating the surface of the bone implant.

43. The method of any one of embodiments 36 to 41 , wherein the pretreating comprises adding a plasma coating or chemically treating the surface with a base, an acid, or an oxidizing agent.

44. The method of embodiment 42, wherein the base is an NaOH aqueous solution.

45. The method of embodiment 42 or 43, wherein the surface that has been chemically treated is further heat- treated before carryout step c).

46. The method of any one of embodiments 42 to 44, wherein the heat treatment comprises heating at a temperature of about 50°C to about 500°C for up to 5 hours.

47. The method of any one of embodiments 42 to 45, wherein the surface that has been chemically treated is washed and dried before the heat treatment or, in the absence of heat-treatment, before step c).

48. The method of any one of embodiments 36 to 46, wherein, in step c), the gel is coated on the surface of the bone implant by spin-coating, dip-coating, spray coating, dry lamination, or electrodeposition, preferably by spincoating.

49. The method of any one of embodiments 36 to 46, wherein, in step d), the gel is allowed to dry to form the xerogel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] In the appended drawings:

[0007] Figure 1. Schematic representatives of the modifications applied on Ti-substrates. (a) Schematic diagram showing the structural changes of Ti surface (Ti); untreated, (CT); subjected to 5.0M NaOH at 60°C for 24 hr., (CTT); subjected to 5.0M NaOH at 60°C for 24 hr. and then thermally treated at 500 °C for 5 hr., (b) Schematic representatives of Ti-substrates after magnesium phosphate coating (Ti-MgPi); untreated coated with MgPi hydrogel, (CT-MgPi); 5.0M NaOH at 60°C for 24 hr. and coated with MgPi hydrogel, and (CTT-MgPi); 5.0M NaOH at 60°C for 24 hr. then heat-treated at 500 °C for 5 hr. followed by coating with MgPi gel.

[0008] Figure 2. (a, b) SEM micrographs; (c) FTIR spectrum; (d) XRD pattern; I NMR spectrum for MgPi hydrogel used for coating the Ti-substrates. (a); Thixotropic MgPi hydrogel, (b and c); SEM micrographs of the dried MgPi hydrogel, (d); 2D structure of the MgPi hydrogel, (e); FTIR spectrum of the dried MgPi hydrogel; (f) NMR spectrum of the dried MgPi hydrogel, (g); XRD pattern of the dried MgPi hydrogel.

[0009] [0010] Figure 3. (a) ATR-IR; (b) Raman spectra; (c) GA-XRD patterns for MgPi coated and uncoated Ti- substrates. (Ti: titanium, R: rutile, SHT: sodium hydrogen titanate, ST: sodium titanate.)

[0011] Figure.4. (a) X-ray photoelectron spectroscopy (XPS) survey spectra of 2D nanocrystalline MgPi coated and uncoated Ti-substrates. (b) Curve-fitted Ti2p, O1s and P2p for the MgPi coated and uncoated Ti-substrates.

[0012] Figure 5. SEM images (left) and corresponding 2D micrographs (center) and 3D AFM micrographs (right) of (a) bare Ti, (b) uncoated-CT substrate, (c) uncoated-CTT substrate, (d) MgPi- coated Ti substrate, (e) MgPi- Coated CT substrate, and (f ¹ ) MgPi-Coated CTT substrate. In contrast to the more porous surface observed on the surface of NaOH-treated substrates (CT), the thermally treated Ti surface (CTT) is denser in the SEM micrographs of the uncoated substrates. The coated substrates' surface displayed irregular MgPi coatings.

[0013] Figure. 6. (a) High magnification (20000x) SEM images, (b) Mg ²⁺ concentration (ppm) released from Ti- MgPi, CT-MgPi, and CTT-MgPi coatings during biodegradation in comparison to other groups, the MgPi coating on the chemically treated Ti substrate, (CT-MgPi) degrades more slowly; and (c) bar graph comparing the critical load required to detach the MgPi coatings, The outcome shows that the both Ti-MgPi and NaOH-thermally treated Ti substrate (CTT-MgPi) require less force to remove the MgPi coating than the NaOH treated substrate (CT-MgPi).

[0014] Figure 7. Florescence microscopy showing stained MC3T3-E1 cells on the MgPi coated and uncoated Ti- substrates. Scale bar =200um. The results showed more cells on the surface of the MgPi coated Ti substrates

[0015] Figure 8. (a,b) Proteomics mass spectrometry analysis, the coated sample (Ti-MgPi) showed higher plasma protein adsorption capacity (97 proteins) compared to the uncoated control (Ti) (76 proteins), (c) Number of adhered cells on MgPi-coated and uncoated Ti substrates, The proliferation of MC3T3-E 1 cells increased on coated samples (Ti-MgPi, CT-MgPi, and CTT-MgPi) after four days of culture in comparison to uncoated Ti-substrates (Ti, CT, CTT).

[0016] Figure 9. Schematic illustration of the modifications made on Ti-substrates. (Ti); untreated, (CT); immersed in 5.0M NaOH at 60°C for 24 hr., (CTT); immersed in 5.0M NaOH at 60°C for 24 hr. followed by thermal treatment at 500 °C for 5 hr., (Ti-MgPi); bare Ti-substrate coated with MgPi hydrogel, (CT-MgPi); CT-substrate coated with MgPi hydrogel, and (CTT-MgPi); CTT-substrate coated with MgPi gel.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Turning now to the invention in more details, there is provided the use of a specific magnesium phosphate xerogel as a coating for bone implants.

[0018] There is also provided a coating for a bone implant comprising (preferably consisting of) the magnesium phosphate xerogel as well as a bone implant, wherein a surface of the implant is covered by a coating comprising (preferably consisting of) the magnesium phosphate xerogel.

Advantages/performances of the invention

[0019] As reported in Example 1 below, the coating of the invention presented irregular rough surface features and good adhesion to the underlying Ti substrate.

[0020] Moreover, an in-vitro study showed that the coating enhanced fibronectin adsorption compared with the uncoated Ti-substrates. Fibronectin is an extracellular matrix (ECM) glycoprotein that plays an important role in interacting with cell surface integrin receptors, mediates cellular migration and adhesion ³⁴ . Fibronectin (FN) is believed to support cell adhesion, growth, differentiation, or survival of osteoblasts and osteogenic cell responses in vitro ³⁵ . In addition, the accumulation of this protein is essential during wound healing to the migration, adhesion, and aggregation of various cell types including platelets, fibroblasts, and endothelial cells ³⁶ . The coated titanium showed improvement of protein adsorption in comparison to the uncoated samples. A higher number of plasma proteins were able to adsorb to coated substrate compared to uncoated control (Ti). Furthermore, our finding demonstrated a significantly higher score of fibronectin protein in case of coated samples compared to uncoated counterparts.

[0021] The coating increased cell proliferation compared to uncoated titanium substrates (Ti). The cell proliferation and adhesion on the coating were favorable and compatible, while examination of initial cell attachment to the coating showed that the cells grew more effectively and spread well. The improved cell adhesion to coated Ti- substrates is likely due to the affinity of the coating for fibronectin adsorption and to the differences in the surface roughness. Since both proliferation and adhesion of osteoblast is a prerequisite for their deposition of bone, these results suggest greater long-term functions of osteoblast on MgPi coated titanium.

[0022] The osteoconductive coating enhanced the bone-forming ability and rapid osseointegration of bone with metallic implants, due to an improved initial osseointegration rate.

[0023] The coatings degraded completely after 7 days of immersion in plasma proteins. This is highly advantageous compared to prior art implant coatings that would crack and then harbor bacteria.

[0024] The above results support that the surface functionalization using the coating of the invention can be used to enhance osseointegration of Ti implants. Hence, such coatings are expected to improve the success rates of titanium implants.

[0025] It was also shown that the proliferation of MC3T3-E1 cells could be significantly increased 9hermosrmo- chemical treatment of the Ti surface before coating.

The Magnesium Phosphate Xerogel

[0026] Herein, “xerogels” are solids formed by drying a gel with unhindered shrinkage. The magnesium phosphate xerogel in the present invention is a xerogel formed from a gel comprising water as its dispersing phase.

[0027] The magnesium phosphate xerogel is in embodiments, in the form of a membrane, such as a translucent membrane.

[0028] The magnesium phosphate xerogel comprises phosphate ions (PO4 ³ ), a divalent cation, and a monovalent cation at mole fractions of about 0.32 to about 0.44 (preferably about 0.33 to about 0.44), about 0.03 to about 0.09, and about 0.48 to about 0.63, respectively, wherein the divalent cation is magnesium (Mg ²⁺ ) or a mixture of magnesium and one or more of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , or Ca ²⁺ , wherein the mixture comprises up to a total of 30% by weight of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , and Ca ²⁺ based on the total weight of the mixture, and wherein the monovalent cation is sodium (Na ⁺ ), lithium (Li+) or a mixture thereof.

[0029] In embodiments, the magnesium phosphate xerogel is as described in WO 2013/142996, which is incorporated herein by reference. This document describes a xerogel comprising the phosphate ions (PO4 ³ ), the divalent cation, and sodium ions (Na ⁺ ) as the monovalent cation at mole fractions of about 0.33 to about 0.44, about 0.03 to about 0.09, and about 0.48 to about 0.63, respectively, wherein the divalent cation is magnesium (Mg ²⁺ ) or a mixture of magnesium and calcium (Ca ²⁺ ), the mixture comprising up to 30% by weight of calcium based on the total weight of the mixture.

[0030] The magnesium phosphate xerogel in WO 2013/142996 was disclosed as having a unique combination of four desirable properties: bioadhesion, thixotropy, bioresorption, and biocompatibility, which are expected to be shared by all the xerogels used in the present invention.

[0031] Preferably, the gel/xerogel comprises phosphate, the divalent cation, and the monovalent cation at mole fractions of about 0.36 to about 0.42, about 0.05 to about 0.08, and about 0.50 to about 0.57, respectively. More preferred examples of such gels/xerogels include gels/xerogels comprising the phosphate ions, the divalent cation and monovalent cation at mole fractions of:

• 0.36, 0.07, and 0.57, respectively,

• 0.39, 0.08, and 0.53, respectively,

• 0.41 , 0.05, and 0.54, respectively, and

• 0.42, 0.08, and 0.50, respectively.

It will be readily apparent to the skilled person that, as the above amounts of phosphate, divalent cation and monovalent cation are given as mole fractions, the sum of these three mole fractions should be 1 (give or take the rounding errors). This is indeed the standard definition of mole fraction in the art: “In chemistry, the mole fraction is defined as the amount of a constituent divided by the total amount of all constituents in a mixture. The sum of all the mole fractions is equal to 1". Herein, the mole fractions take only the divalent cation, phosphate and monovalent cation into account. Water and optional additives that can be added to the gel/xerogel are not considered.

[0032] In preferred embodiments, the divalent cation is magnesium only. In alternative embodiments, it is a mixture of magnesium and one or more of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , or Ca ²⁺ , the mixture comprising up to a total of 30% by weight of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , and Ca ²⁺ based on the total weight of the mixture. In embodiments, the mixture comprises a total of about 10 % to about 30% by weight of Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , and Ca ²⁺ based on the total weight of the mixture. In preferred embodiments, the mixture comprises magnesium and calcium only.

[0033] In preferred embodiments, the monovalent cation is sodium (Na ⁺ ) only. In alternative embodiments, it is lithium only (Li ⁺ ) or a mixture of sodium and lithium. [0034] In embodiments, the gel/xerogel comprises phosphate, magnesium, and sodium ions at mole fractions of about 0.39, about 0.08, and about 0.53, respectively (that corresponds to the gel identified as .75 0.15 1 in the Examples of WO 2013/142996).

[0035] The amount of water (as a dispersing phase) in the gel (prior to the formation of the xerogel) is typically about 50% or more, for example 70% or more by weight based on the total weight of the gel. In embodiments, the gel may comprise more than about 90% of water, for example between about 92 and 98% or between about 92 and 96% of water as the dispersing phase. Obviously, the xerogel, resulting from drying this gel, comprises little-to-no water as a dispersing phase.

[0036] When observed by transmission electron microscopy (TEM), in embodiments, the gel appears to comprise thin nano-plates or nanosheets. More specifically, these nanosheets can be about 200 nm wide, very thin (e.g., about 10 nm thick) and up to 1pm long. As seen by TEM, these nanosheets agglomerate, and form interconnected planes (see FIG 15 of WO 2013/142996). Without being bound by theory, these nanosheets are believed to be crystalline (because of their appearance and of their X-ray diffracted pattern when dried). However, as discussed in the Examples of WO 2013/142996, the hydrated gels of the invention appear to be amorphous when analyzed by X- ray diffraction (see FIGS 5 and 6 of WO 2013/142996). Herein, the te“m "amorph”us", as in “amorphous gel” means that the gel is only weakly diffracting X-rays in a standard powder X-ray diffraction equipment, giving patterns similar to amorphous materials, small particle sized materials or poorly crystalline materials without clearly defined diffraction peaks. It does not mean that the gel may not comprise any crystalline material.

[0037] The nanosheets are made of magnesium phosphate (with some sodium). This magnesium phosphate contains magnesium bi- and tri-phosphate. This magnesium phosphate contains hydration water. For example, it may contain between about 10 and about 20% of hydration water by weight.

[0038] A distinction should be drawn between water as a dispersing phase and hydration water. Water as a dispersing phase is the medium in which the nanosheets are dispersed. This water can be removed by drying the gel at a relatively low temperature, for example a temperature below the boiling temperature of water, such as 80°C (See the section entitled “Water Content” in Example 1 of WO 2013/142996). This process will produce a product that looks and feels dry, but that still contain hydration water. Hydration water consists in molecules of water that are bonded or somehow associated with a solid (for example entrapped within it). These molecules are typically only removed from the solid by heating the solid above the boiling temperature of water, often well above this temperature, for example between 100 and 250°C (See the section entitled “Thermogravimetry” in Example 2 of WO 2013/142996).

[0039] When there is no calcium in the gel/xerogel, the gel/xerogel may further comprise up to 200% by weight of pyrophosphate (P2O7 ⁴ ), based on the weight of the phosphate. In embodiments, the gel/xerogel may comprise between about 10% and about 20% by weight of pyrophosphate based on the weight of the phosphate. The presence of pyrophosphate makes the gel/xerogel more acidic and thereby tends to improve its resistance to acidic media. [0040] The gel/xerogel may also comprise chloride (Cl ) ions. These may be provided by one of the compounds used for making the gel/xerogel, for example calcium chloride, when it is present.

[0041] Additives can also be added to the gel/xerogel. For example, these additives can aim at improving the resistance of the gel/xerogel to dissolution in acidic media. Such additives include:

• corn oil (for example in a concentration varying between about 0.1 and about 1 .5 % based on the total weigh of the gel),

• sodium metaphosphate or pyrophosphate (for example in a concentration varying between about 0.125 and about 0.5 % based on the total weigh of the gel),

• sodium citrate (for example in a concentration varying between about 0.1 and about 10% based on the total weigh of the gel),

• xantham gum (for example in a concentration varying between about 0.1 and about 1.5 % based on the total weigh of the gel),

• sodium alginate (for example in a concentration varying between about 0.1 and about 1 .5% based on the total weigh of the gel),

• carboxylate salts, such as sodium glycolate and sodium tartrate (for example in a concentration varying between about 0.1 % and about 5 % based on the total weigh of the gel),

• carboxylic acids, such as glycolic acid and tartaric acid (for example in a concentration varying between about 0.1 and about 5 % based on the total weigh of the gel), and

• chitosan (for example in a concentration varying between about 0.1 and about 1 .5% based on the total weigh of the gel).

[0042] The gel/xerogel can be loaded with a variety of substances, including bioactive substances, depending of the desired properties and its end use.

The Implant and the Coating

[0043] As noted above, the magnesium phosphate xerogel is used for coating bone implants.

[0044] In embodiments, the bone implant is a titanium bone implant, a cobalt chromium bone implant, a stainless- steel bone implant, or a polymer bone implant (such as a polyester bone implant). In most preferred embodiments, the bone implant is a titanium bone implant.

[0045] Herein, “titanium bone implants” are bone implant with a surface comprising surface titanium atoms. Note that many such implant, e.g., a majority of orthopedic “titanium implants” are, in fact, alloys. These alloys are typically proprietary blends - differing from manufacturer to manufacturer. Common implantable titanium alloys include: ASTM F67 Unalloyed (Commercially Pure) Titanium ASTM F136 Ti-6AI-4V-ELI, ASTM F1295 Ti-6AI-7Nb, and ASTM F1472 Ti-6AI-4V. [0046] Furthermore, the surface of the bone implants (preferably titanium bone implants) can be chemically and/or heat treated as described herein below.

[0047] Herein, a “bone implant” are implants that replace missing bone tissue, support damaged bone tissue, or enhance an existing bone tissue. Non-limiting examples of bone implants include dental and craniofacial implants as well as orthopedic implants. Orthopedic implants help alleviate issues with the bones and joints of the body. T’ey're used to treat bone fractures, osteoarthritis, scoliosis, spinal stenosis, and chronic pain. Examples include a wide variety of pins, rods, screws, and plates used to anchor fractured bones while they heal.

[0048] The thickness of the coating of the invention is not particularly limited. A minimum thickness is dictated by the size of the magnesium phosphate nanosheets it contains. As such, a minimum thickness of 10 nm or 20 nm could be envisioned.

[0049] In preferred embodiments, the coating covers the entirety of the implant surface.

[0050] In preferred embodiments, the implant is a titanium bone implant, and the coating has an X-ray photoelectron spectrum that is free of titanium peaks. This indicates the continuous nature of the coating.

[0051] In more preferred embodiments, the X-ray photoelectron spectrum comprises: a Mg1s peak at about 1304.58 eV, and a P2p peak at about 133 to about 134 eV, an 01 s peak at about 530 to about 535 eV, and optionally, when the surface of the implant has been pretreated with NaOH, a Na1s peak at about 1071.88 eV.

The Mg1s peak position corresponds to Mg (HPO^ from the deposited layer ³¹ . Preferably, the P2p peak can be deconvoluted into two peaks at about 133.5 and about 134.4 eV. These peaks can be assigned respectively to PO4 ³ and HPO4 ² , thus confirming the presence of two different phosphate anions. Preferably, the O1s peak can be deconvoluted into three different components peaks at about 531 .2, about 532.98, and about 535.3 eV. These peaks correspond to (P-0 bond of PO4 ³ ), phosphorus hydroxide (hydroxyl groups of P-hydroxide (P-OH)), and H2O respectively ³² . Optionally, a peak at 535.2 eV can be present. This corresponds to H2O, which originates from the xerogel on the surface.

[0052] In embodiment, the coating has H2O at an atomic concentration of about 0% to about 15%, P-0 at an atomic concentration of about 65% to about 85%, and P-OH an atomic concentration of about 15% to about 35%.

[0053] In embodiments, the coating has a FTIR spectrum comprising a band at about 997 cm ¹ and a band at about 1060 cm ¹ . These bands can be assigned to symmetrical (vi)) and antisymmetrical (V3) stretching vibrations of P-0 bond in PO4 ³ ' respectively ¹³

[0054] In embodiments, the coating has a Raman spectrum comprising a band at about 967 cm ¹ . This band can be assigned to the stretching mode of PO4 ^3- group ²⁴ . [0055] In embodiments, the coating has a Raman spectrum comprising

[0056] Results in the literature indicated that rough implant surfaces as well as coated implant surfaces could increase osseointegration ³³ . In embodiments, the coating has an average surface roughness of about 30 nm or more.

Optionally Pretreated Bone Implants

[0057] As will be explained below, the surface of the bone implant can optionally be pretreated prior to the deposition of the xerogel.

[0058] In embodiments, the implant is a titanium bone implant and has a surface sodium titanate layer (produced by NaOH treatment), preferably a layer of sodium titanate gel, underneath the xerogel.

[0059] In embodiments, the sodium titanate layer is a crystalline layer with phases of sodium titanate and rutile.

[0060] In embodiments, the sodium titanate layer has a porous network structure with an interconnected pore network.

[0061] In embodiments, the sodium titanate layer is an amorphous sodium titanate layer with a gradient structure.

[0062] In embodiments, the surface of the bone implant has an average roughness parameter (Ra) of about 45nm or more.

Method of manufacturing the gel, coating and implant

[0063] There is also provided a method of manufacturing the coating/implant of the invention. This method comprises the steps of: a) providing a gel as described above, b) providing a bone implant as described above, c) coating a surface of the bone implant with the gel, d) allowing the gel to dry thus yielding the xerogel.

Step a) providing the gel

[0064] The gel can be prepared as described in 2013/142996, incorporated herein by reference.

[0065] Typically, the phosphate ions for the gel are provided by a solution of phosphoric acid (H3PO4) or monomagnesium phosphate (Mg^PCU ) in water used to make the gel. The magnesium ions are typically provided by magnesium hydroxide (Mg(OH)2 - a solid) or trimagnesium phosphate (MgsfPO^ - another solid) that is added to the abovementioned solution. The calcium is typically provided by calcium hydroxide or calcium chloride that is also added to that solution. The sodium ions are typically provided by a solution of sodium hydroxide (NaOH) that is mixed with the magnesium-containing solution.

[0066] In embodiments step a) comprises: i. providing a first aqueous solution comprising sodium hydroxide (NaOH), ii. providing a second solution comprising phosphoric acid (H3PO4) or monomagnesium phosphate (Mg(H ₂ PO ₄ )2),

Hi. dissolving magnesium hydroxide (Mg(OH) ₂ ) or trimagnesium phosphate (MgsfPO^), and optionally calcium chloride or calcium hydroxide, in the second solution, and iv. mixing together the first solution and the second solution, thereby providing the gel.

[0067] The gel forms within seconds of mixing the solutions together at step iv.

[0068] For better results, the time between steps Hi and iv should be no more than several minutes, for example 10 minutes.

[0069] The concentration and quantity of solutions and solutes used to make the gel are chosen so that the quantity of phosphate, magnesium (and optional calcium), and sodium in the gel respects the mole fractions and the water content discussed in the previous sections.

[0070] In embodiments, the method comprises after step iv, the step v of aging the gel, for example overnight.

Step b)

[0071] At step b) the implant is provided. The implant should be clean and sterile.

Optional Surface Pretreatment

[0072] In embodiments, the method further comprises at step b, sand-blasting the surface of the bone implant.

[0073] In further embodiments, the method further comprises at step b, pretreating the surface of the bone implant. This pre-treatment modifies the surface chemistry of the implant.

[0074] This can be achieved by adding a plasma coating or chemically treating the surface with a base, an acid, or an oxidizing agent.

[0075] In more preferred embodiments, the chemically treated surface is further heat-treated before carryout step c).

[0076] Non-limiting examples of alkaline pretreatment include immersing the implant is an NaOH aqueous solution.

[0077] Non-limiting examples of heat treatment include heating at a temperature of about 50°C to about 500°C for up to 5 hours.

[0078] Preferably, the chemically treated surface is then washed and dried before the heat treatment or, in the absence of heat-treatment, before step c). Step c)

[0079] The gel as provided above is solid, but is also thixotropic, which means that it reversibly liquefies with shear stress. Hence, it is possible to coat a surface of the bone implant with the gel in step c).

[0080] In embodiments, in step c), the gel is coated on the surface of the bone implant by spin-coating, dip-coating spray coating, dry lamination, or electrodeposition, preferably by spin-coating.

Step d)

[0081] In step d), the gel is simply allowed to dry to form the xerogel. Any drying temperature below 100°C can be used.

[0082] In embodiments, this drying can be done by simply leaving the coated implant in air at room temperature for a few minutes.

Definitions

[0083] The use of the terms “a” and “an" and “the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0084] The terms “comprising, “havincf, “including, and “containing are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. In contrast, the phrase “consisting of” excludes any unspecified element, step, ingredient, or the like. The phrase “consisting essentially of” limits the scope to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the invention.

[0085] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0086] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0087] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0088] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0089] Herein, the term “about' has its ordinary meaning. In embodiments, it may mean plus or minus 10% or plus or minus 5% of the numerical value qualified.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0091] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0092] The present invention is illustrated in further details by the following non-limiting examples.

Example 1 - Enhancing Titanium osseointegration using 2D Nanocrystalline Magnesium Phosphate Coating

Summary

[0093] The lack of bioactivity of titanium (Ti) implants is one of the main drawbacks for its application as orthopedic implants since it can reduce its osseointegration and osteoconductive capacities. One strategy to overcome this limitation and to enhance osseointegration is the coating of Ti with a bioactive layer. Therefore, the objective of this study was to fabricate two-dimensional (2D) nanocrystalline Magnesium phosphate (MgPi) coatings on various pre-treated Ti-substrates and to investigate its biological characteristics, mechanical properties, and osteogenic performance. Prior to the deposition of the MgPi layer, an alkaline thermal oxidation pretreatment has been applied to ensure an increase in the bioactivity of the Ti-substrates and to improve coating adhesion to the substrate. The MgPi coatings were deposited on Ti-substrates using the spin-coating technique. The coated and uncoated Ti-substrates were characterized in terms of morphology using scanning electron microscopy (SEM), crystallinity using X-ray diffraction (XRD), roughness using Atomic Force Microscope (AFM), functional groups identification using Infrared (ATR-IR), Raman, and composition using X-ray photoelectron spectroscopy (XPS). In addition, the coating adhesion was mechanically evaluated using the nano scratch test. Finally, the biocompatibility of these surfaces was characterized by evaluating blood plasma protein adsorption, coating degradation in blood plasma, and osteoblast cell proliferation.

[0094] The FTIR, SEM, XPS, and FT-R measurements verified the surface treatment as well as successful coatings fabrication. The novel MgPi coating presented irregular rough surface features and good adhesion to the underlying Ti substrate. Moreover, the in-vitro study showed that the MgPi coating enhanced fibronectin adsorption compared with the uncoated Ti-substrates. Most importantly, the proliferation of MC3T3-E1 cells was significantly increased on 17hermos-chemically coated surfaces, compared to uncoated control, (Ti) (p <0.005) and chemically treated coated samples, (CT-MgPi) (p < 0.005). Together, these results support that the surface functionalization using the MgPi coating as a promising candidate for enhancing osseointegration of Ti implants. Introduction

[0095] Pure titanium and its alloys are the most attractive materials for permanent applications as orthopedic implants. However, poor osseointegration is one of their drawbacks. Here we demonstrated that coating Ti- substrates with 2D nanocrystalline Magnesium Phosphate hydrogel (MgPi) could improve the cell response include adhesion and proliferation that are ultimately helpful for the tissue healing and bone regeneration process.

[0096] Indeed, we propose the use of hydrogel coatings composed of 2D nanocrystalline magnesium phosphate (MgPi). MgPi hydrogels are promising alternatives to replace traditional inorganic scaffolds for tissue engineering ¹³ . Indeed, Tamimi et al. reported that MgPi nanosheets accelerated bone healing and osseointegration by enhancing collagen formation, osteoblasts differentiation, and osteoclasts proliferation ¹³ . This type of hydrogel presented extreme thixotropy, injectability, biocompatibility, and bioresorpability.

[0097] We hypothesized that coating Ti-implants with MgPi could help address the issue of poor osseointegration, by providing surface roughness and bioactivity during the earliest osseointegration stages. Additionally, we hypothesized that this MgPi layer would acts as an intermediary layer between the Ti surface and surrounding tissue, encouraging in vivo bone formation along the implant surface. To date, these MgPi materials have been developed and used as hydrogel only for its thixotropy, injectability that helped the bone healing, and osseointegration.

[0098] To the best of the inventor’s knowledge, this is the first time that MgPi hydrogel has been used as a coating on Ti implants by using a 2D nanocrystalline MgPi hydrogel, in combination with chemical modification of the titanium surface by an alkaline thermal oxidation pretreatment. These coatings are easy to be prepared either on titanium surfaces or modified titanium surfaces due to precursor mixture at the molecular level in solution.

Materials and Methods

[0099] The fabrication process of the MgPi coating on untreated and alkaline treated Ti-substrates is shown in Figure 9. The following series of experiments were verified: 1) Alkaline thermal oxidation pretreatment of Ti- substrates; 2) the MgPi coating deposition on treated and untreated Ti-substrates using spin coating technique; 3) the coating composition, composition, microstructure and roughness of the coated implants; 4) the in vitro cellular responses of the coatings.

Titanium substrates

[00100] Commercially pure titanium (cp TI, grade 5, McMaster Carr), (n=48), 0.9 mm in diameter and 0.5 mm thickness were abraded on successive finer silicon carbide abrasive 320, 600, 1200 and 2000 #, ultrasonically cleaned in dd-H2O, acetone then EtOH for 20 min each, and then dried in an oven at 40°C for 12 hr.

Pretreatment of titanium implants

[00101] In the past, an alkaline pretreatment has been an important method to form a bioactive sodium titanate layer on the surface of Ti-substrates by immersing them in thermal NaOH solution and subsequent heat treatment. Prior to the deposition of the MgPi layer, an alkaline thermal oxidation pretreatment has been applied to ensure an increase in the bioactivity of the Ti-substrates. The alkaline pretreatment was performed by immersing dried Cp Ti specimens (16 Ti discs) in 100 ml of 5.0M NaOH aqueous solution at 60 °C for 24 hr. After the incubation in NaOH solution, the Ti-substrates were gently washed with dd-H2O to remove the weakly bonded Na ⁺ ions and then dried at 40 °C for 24 hr.

[00102] Herein, the NaOH treated samples are named chemically treated (CT) half of these treated samples were further thermally treated (8 Ti discs) at 500 °C for 5 hr. in an air atmosphere at a heating rate of 5°C / min. these samples are named thermo-chemically treated (CTT).

Hydrogel Preparation

[00103] 2D nanocrystalline Magnesium phosphate (MgPi) hydrogel was prepared by adding a solution of 1 .5 M phosphoric acid, (2.5 ml), (H3PO4, sigma Aldrich) to 0.09 grams magnesium hydroxide, (Mg (OH)2, Fisher, Belgium) whilst continuously stirring the solutions at room temperature. Then, a 1 .5 M solution of sodium hydroxide (4.2ml), (NaOH, Fisher, Belgium) was added. MgPi hydrogel was obtained when the solution was shook by hand for 20 seconds and then left to age overnight. The molar ratios were: 0.32 PO4, 0.54 Na and 0.13Mg.

Coating using Spin-Coater

[00104] The MgPi (250 pL), was deposited on the Ti-substrates using a WS-400 Spin Coater (Laurell Technologies Corporation, USA), at 3000 rpm for 30 s. The coated Ti-substrates were then kept at room temperature (to dry) for 24 hr. for further analysis. The different modifications applied on the Ti samples could be seen in Figure 1 .

Samples characterization

[00105] To identify the surface functionalization, attenuated total reflectance infrared spectroscopy (ATR-IR) measurements of the MgPi coated and uncoated Ti-substrates were taken using a Bruker Tensor 27 IR spectrometer (Bruker Optics Ltd., Coventry, UK) equipped with a deuterated triglycine sulfate pyroelectric detector with an accumulation of 16 scans in the range of 400-4000 cm ^-1 at a resolution of 4 cm ^-1 .

[00106] Also, the micro-Raman spectra of the coated and uncoated Ti-substrates were recorded using a SENTERRA-BRUKER German spectrometer in the range of 100-1000 cm ^-1 with a wavelength of 780 nm, which had been excited from a HeNe ion laser.

[00107] The phase analysis (structural identification) of coated and uncoated Ti-substrates was performed by Grazing Angle X-ray diffraction (GAXRD) (AXS GmbH diffractometer, Bruker, Germany), using a Cu K _a radiation source (AKO =1 .5406 A), generated at 40 kV and 40 mA, grazing incidence at 3° within the 10 to 60° range in 20.

[00108] The chemical composition and the binding state of elements in the MgPi coated and uncoated Ti-substrates were characterized by X-ray photoelectron spectroscopy (Thermo Scientific K _a spectrometer) using the Mg K _a X-ray source and pass energy of 50 eV. All the spectra were calibrated at the C1s binding energy (284.8 eV). The survey spectra in the range of 0-1100 eV were recorded in 1 eV step for each sample followed by high-resolution spectra over different element peaks in 0.1 eV steps. To prevent surface charging, samples were hit with a flood gun shooting. The atomic concentrations were calculated from the measured peak areas using sensitivity factors. The peak-fitting procedures were performed using the software Thermo Avantage (version 5.96).

[00109] The morphology of the coated and uncoated Ti-substrates was observed using an lnspect-50 field emission scanning electron microscope (SEM) (FEI, Japan), at 10 kV operating voltage.

[00110] Atomic force microscopy (AFM) was used to examine the changes in surface roughness induced by different treatments or MgPi coating of the Ti-substrates. The surface roughness measurement was performed at ambient conditions using an AFM instrument: a Nanoscope Multimode 8 equipped with a Nanoscope V controller (Bruker, Santa Barbara, CA). The topographies were acquired in peak force mode (Bruker ScanAsyst mode and Nanoscope 8.15r3 software). In peak force mode, silicon nitride cantilevers (ScanAsyst-Air Bruker) with a nominal spring constant of 0.4 N/m, a nominal resonance frequency of 50-90 kHz, and a nominal tip radius of 2 nm were used for imaging in air. AFM image analysis was performed using WSxM,69 Nanoscope Analysisl .4, and lAPro- 3.2.1 software.

Adhesion strength of the coating

[00111] The adhesion strength between the coating and the Ti-substrates was compared through nano scratch tests using a nanoindenter (Ubi3, Hysitron, US). A stylus with a diamond conical tip with a 2 pm radius was used for scratch testing. A ramp load from 0 to 10 pN was applied over 10 pm at a scratch rate of 0.66 pm/s. Five measurements were performed at room temperature for each sample. The average value of the critical load, and the lateral force at the critical load of coated samples were compared. The critical load is defined as the normal load where the coating is fractured or detached ¹⁴ .

In vitro degradation and protein adsorption of the coating

[00112] The in-vitro degradation of the MgPi coating was assessed by soaking the coated Ti samples in 50 ml of rat plasma diluted to a concentration of 1% using phosphate buffer solution (PBS) ¹⁵ . The specimens were immersed in separate plastic containers, and then incubated at 37 °C. A 1 ml sample of the diluted rat plasma, (Innovative research, Ml, USA), was taken from each container after 1, 3, 5, and 7 days and was stored frozen until being analyzed by inductively coupled plasma (ICP) technique using a Thermo Scientific iCAP 6500 dual view, UK.

[00113] SEM was also applied to study the surface morphologies changes of MgPi coatings after degradation in diluted plasma.

[00114] The affinity to protein adsorption was studied during immersion of coated and coated Ti-substrates in plasma proteins. For protein adsorption test, a set of coated and uncoated Ti-substrates were separately incubated for 3h at room temperature in equal volumes of rat plasma (Innovative research, Ml, USA) diluted with PBS to 1% at 37 °C. Then, the samples were rinsed with PBS to remove any weakly adhering proteins. The incubated samples were then subjected to a mass spectrometry analysis. The database search results were loaded onto Scaffold Q+ Scaffold_4.4.8 (Proteome Software Inc., Portland, Oregon, USA) for spectral counting, statistical treatment, and data visualization.

In-vitro cellular proliferation

[00115] The ring culture technique was used to culture MC3T3 cells on top of MgPi coated and uncoated Ti- substrates to facilitate the attachment of the cells. Briefly, constant diameter (5 mm) plastic cylinders were attached to the disk surface. Disks were sterilized and polystyrene cloning cylinders (Sigma) were attached to the disks using vacuum grease. MC3T3-E1 cells from ATCC (Manassas, VA, USA) were cultured in alpha-MEM (Invitrogen, Carlsbad, CA, USA). Culture media were supplemented with 10% FBS (PAA, Etobocoke, Ontario, Canada) and 100 U/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were grown at 37 °C under 5% CO2 in a humidified incubator. MC3T3 cells were cultured on top of Ti-substrates which were treated with 6 different conditions (Ti, CT, CTT, Ti-MgPi, CT-MgPi, CTT-MgPi) as described above. The medium was changed every 2 days. Cells were stained with calcein (Sigma-Aldrich, Saint Louis, MO, USA) and incubated for 20 min inside the incubator, after that cells were washed three times and were fixed with 4% paraformaldehyde and incubated for 5 min at room temperature. After washing three times in PBS, H33258 nuclear staining was performed and then washed once in PBS. Cells grown on the surface of Ti-substrates were imaged using an inverted fluorescent microscope (EVOS FL, Thermo Fisher Scientific) and cell nuclei were counted. Five titanium disks were analyzed per group and three regions of interest were selected randomly on each disk.

Statistical analysis

[00116] The data were evaluated for normal distribution using the Shapiro-Wilk test. For data following normal distribution, one-way ANOVA with post-hoc using Tukey HSD test was done and pairwise comparison was then applied to detect significant differences among groups. The statistical significance level was set at p < 0.05. 1 BM_ SPSS_ (v. 17, IBM Corp.; New York; USA) software was used for data analysis, and graphs were generated using GraphPad Prism version 6.01). To assist protein adsorption to Ti-substrates, Scaffold_4.4.8 software was used to analyze the proteomic data using Fisher’s exact test.

Results and Discussion

[00117] Immediately upon preparation at room temperature (25 °C), the prepared MgPi was in the sol state (Fig. 2a. To examine the flow, the vials were flipped upside down after the overnight incubation. The hydrogel formulation showed thixotropic shear-thickening behavior and could maintain gel state. As indicated by the SEM images (figure 2b and c), the analysis of the morphology of the dried MgPi powder revealed irregular rough surface (irregular 2D crystals). A schematic diagram of the MgPi hydrogel sandwich system (Na3PO4 + / MgHPO4-/ Na3PO4 +) is shown in Fig. 2d. The powder is composed mainly of the polyhedrons of PO4 ³ ' in the structure of the nanocrystalline MgPi as confirmed by FTIR, XRD, and NMR results (figure 2 d-f). Results in the literature indicated that rough implant surfaces as well as coated implant surfaces could increase osseointegration.

[00118] The ATR-IR technique was carried out to detect the surface pretreatment as well as the presence of MgPi coating on the surface of Ti-substrates. The ATR-IR spectra of the MgPi coated and uncoated Ti discs are shown in Figure 3a. All spectra showed tiny bands observed in the range of 650-800 cm ¹ , which is related to stretching Ti-0 and Ti-O-Ti vibrations, with pronounced lattice vibrations of TiC>2 at about 794 erm ^{1 16} . The relatively small band at 831 erm ¹ corresponds to the Ti-O-Ti asymmetric stretch, belonging to oxygen-bridged titanium (IV) oxides ¹⁷ . The band at 1353 erm ¹ in the spectrum of the CT substrate, which may be assigned to various kinds of Na-titanate compounds, confirms the formation of sodium titanate hydrogel layer on the Ti surface because of surface treatment using NaOH.

[00119] In the spectra of coated Ti-substrates, (Ti-MgPi, CT-MgPi, and CTT-MgPi), two obvious bands are observed in the range of 997-1060 erm ¹ which is assigned to symmetrical (vi)) and antisymmetrical (V3) stretching vibrations of P-0 bond in PO4 ³ ' at 997 erm ¹ and 1060 erm ¹ respectively ¹⁸ . These bands indicate the reordering of the polyhedrons of PO4 ³ ' in the structure of the deposited MgPi layer ¹⁸ The ATR-IR results were supported by measurements of Raman spectroscopy.

[00120] Raman spectroscopy measurements were used to support the ATR-IR findings. Figure 3b shows the Raman spectra of MgPi coated and uncoated titanium discs. In the spectrum of the untreated sample, (Ti), no crystalline peaks were observed due to the amorphous nature of the passive oxide layer ¹⁹ . The spectrum of the NaOH-treated Ti-substrates (CT) shows a set of four peaks. The peak at 440 erm ¹ is assigned to Ti-0 bending vibration involving three-fold oxygen; peaks at 682 and 806 erm ¹ are assigned to Ti-0 bending and stretching vibrations involving twofold oxygen; a peak at 901 erm ¹ is assigned to Ti-stretching vibration involving non-bridging oxygen, some of which are coordinated with Na ⁺ ions ²⁰ .

[00121] These peaks indicate that sodium hydrogen titanate (SHT), Nax^-xTisO? was formed on the surface of the chemically treated Ti-substrates as a result of the reaction with NaOH solution. These results are in good agreement with earlier reports on hydrogen titanate (HT) ^{21 23} . The Raman spectra of the coated titanium discs (Ti-MgPi, CT- MgPi, and CTT-MgPi) showed a band assigned to stretching mode of PO4 ^3- group developed at 967 erm ^{1 24} . The above results are clear evidence that MgPi has been deposited successfully on untreated and alkaline treated titanium surfaces.

[00122] Grazing angle X-ray diffraction (GAXRD) was chosen to probe the phase assemblage of the surface phases. Figure 3c summarizes the GAXRD data of MgPi coated and uncoated Ti-substrates. Ti peaks are observed at 20, 38.95, 35.02, 40.66, and 53.70° in the spectrum of the bare, (Ti) sample. Additionally, small peaks of sodium hydrogen titanate phase (SHT) were observed at 49.25, 31 .43, and 28.69° in 20 for the (CT) sample. These peaks arise from the surface porous network layer formed by the NaOH treatment ²⁵ . In the diffractogram of thermos- chemically treated sample (CTT), sodium titanate (ST) phase was observed at 20 49.71 °. Thus, during thermal treatment, the (SHT) is transformed to (ST) phase ²⁶ . For the coated samples include (Ti— MgPi), (CT-MgPi), and (CTT-MgPi) samples, the intensity of the main crystallized peak at 38.95, 35.02, 40.66, and 53.70° decreased after coating deposition. This reflects the nanocrystalline nature of the deposited MgPi layer which masked the phases from the underlying Ti-substrates.

[00123] Figure 4a shows the XPS survey spectra of the MgPi coated and uncoated Ti-substrates. The survey spectra show the presence of Ti, O, and C signals in the bare sample (Ti) while Ti, O, C and Na are observed in Ti- substrates that have been chemically or thermo-chemically treated, ((CT) and (CTT)). Ti, O, C, Na, P, and Mg signals are observed in the coated samples (Ti-MgPi), (CT-MgPi), and (CTT-MgPi). The presence of C peak was related to surface contamination due to exposure to air before the XPS analysis. The relative chemical composition of the prepared samples is tabulated in Table 1. For the uncoated Ti-substrates, (Ti), the dominant signals are only Ti, 0, and C.

[00124] The photoelectron peak for Ti2p appears clearly at binding energy, Eb, of 459.08 eV, O1s at Eb = 531.08 eV, and C1s at Eb = 284.8 Ev ²⁷ After NaOH and thermos-chemical treatment include both (CT) and (CTT) samples, Ti, O, C and Na signals were observed. This indicates that the formed layer is sodium hydrogen titanate. It has been observed that Na amount increases in the NaOH treated samples in comparison with bare (Ti) sample, that after NaOH treatment, Na is also found as a peak at 1070.98 eV ²⁸ . The presence of this peak together with the increase in the intensity and broadening of the Ti-0 peak at 530.4 indicate that a sodium titanate layer is formed on the surface of Ti as a result of NaOH treatment.

[00125] The XPS of O1s core levels of the MgPi coated and uncoated Ti-substrates (Figure 4b), are broad in nature that could be curve-fitted into several component peaks. After deconvolution, the binding energy peaks at 529.6 and 531 .5 eV are attributed to a combination of the oxygen from the titanium dioxide (Ti— 0 bond of TiO2), titanium hydroxide (hydroxyl groups of Ti-hydroxide (Ti— OH)), respectively.

[00126] The compound at binding energy peak 535.2 eV corresponds to H2O, which originated from the physically adsorbed H2O ²⁸ The bare, (Ti) sample, exhibited O1s peak at 529.6 eV due to the thin passive layer of titanium oxide on its surface. However, the NaOH and NaOH-thermally treated Ti-substrates, (CT) and (CTT) samples, show a clear 01s peak, ascribed to the Ti-0 bond, because of the sodium titanate and rutile on their surfaces as a result of NaOH and NaOH -thermal treatment, respectively.

[00127] Table 1. Surface Atomic concentration percentage of the MgPi coated and uncoated Ti-substrates obtained by the XPS.

[00128] The atomic concentration of the Ti— OH group increases from 24.3 % in the (Ti) sample to 34.6 % in the (CT) sample. This increase could be attributed to the chemical reaction of Ti and NaOH (Table 2). The high-resolution Ti2p of the (Ti), reveals that the Ti2p spectrum (Figure 4), is a doublet with Ti2p3/2 and Ti2p1/2 at 458.2 and 463.9 eV respectively, indicating the presence of Ti ³⁺ (Ti— 0) ²⁹ .

[00129] The presence of this surface oxide layer is confirmed by the Ti-0 peak at 530.5 eV in the high-resolution O1s spectral as shown in (Figure 4b). Another doublet with Ti2p3/2 and Ti2p1/2 at 463.98 and 455.48 e is assigned to peaks from Ti ²⁺ (Ti— O) ²⁹ . A third doublet observed at 453.48 eV (Ti2p3/2) and 459.38 eV (Ti2p1/2) is assigned to peaks from metallic Ti indicating Ti— Ti bonding is observed only on the uncoated Ti sample ²⁹ . After NaOH or NaOH- thermal treatment (CT) and (CTT) Ti-substrates, the remaining dominant peaks have been identified as being Ti ⁴⁺ (Ti— 0) 2p3/2 at 458.3 eV and Ti4+ (Ti— 0) 2p1/2 at 464.6 eV. This is consistent with the Raman, GAXRD, and ATR- IR results which showed the presence of sodium titanate and rutile as main phases on the surface (CT) and (CTT) samples. For the coated samples, significant differences were observed after deposition of the MgPi coating. While Na1s, Mg1s and, P2p, 01s, and C1s were identified, no titanium peaks from the underlying Ti-substrates were detected indicates the continuous nature of the deposited layer. All the spectra show the Na1s (1071.88 eV), Mg1s (1304.58 eV), and P2p (133.3 eV) peaks which originate from the deposited sodium MgPi layer ³⁰ . The Mg1s peak position corresponds to Mg (HPO^ from the deposited layer ³¹ .

[00130] High-resolution XPS spectra of P2p confirmed the presence of two different phosphate anions, and its deconvolution into two peaks at 133.5 and 134.4 eV were assigned respectively to PO^and HPO4' ² both of which could be identified as the signal from MgPi coating. The corresponding 01s spectra could be fitted with three different components from those detected on the uncoated Ti-substrates (Ti). These include signals with binding energy at 531 .2,532.98, and 535.3 eV corresponding to (P-0 bond of PO4 ³ ), phosphorus hydroxide (hydroxyl groups of P-hydroxide (P-OH)), and H2O respectively ³² . The peak at 535.2 eV corresponds to H2O, which originates from the 2D nanocrystalline MgPi hydrogel layer deposited on the surface. The presence of two distinct phosphate anions was confirmed by high-resolution XPS spectra of P2p, and its deconvolution into two peaks at 133.5 and 134.4 eV was assigned to PC>4 ³ ' and HPCU ² ', respectively, both of which could be recognized as the signal from MgPi coating.

[00131] Table 2. Atomic concentration percentage of the TiOz Ti-OH, H2O peaks obtained by deconvoiuting the XPS 01s spectra of the MgPi coated and uncoated Ti-substrates.

[00132] Figure 5 (a-c) shows the scanning electron images of the uncoated Ti-substrates.

[00133] It can be observed from micrographs that the surface of the bare titanium (Ti) sample is smooth with uniform patterns that form during the polishing process. The NaOH treated Ti-substrates, (CT) shows the formation of a porous network structure of the Ti-substrates surface with an interconnected pore network. The porous network structure on the NaOH - thermally treated Ti surface (CTT) is slightly denser after the heat treatment when compared with those before the heat treatment, (CT).

[00134] Figure 5 a-c shows the SEM micrographs and 2D and 3D AFM micrographs of the surfaces of the uncoated Ti-substrates. Figure 5 d-f shows the SEM micrographs and 2D and 3D AFM micrographs of the surfaces of the coated Ti-substrates.

[00135] The surface of the bare (Ti) Ti-substrates shows scratches due to mechanical polishing were visible in its surface (Figure5a). The SEM images of modified titanium surfaces are depicted in (Figure 5b). There is no obvious difference between the two images showing the morphology of only the NaOH-treated sample (Figure 5b) and the sample with additional heat treatment at 600 °C (Figure 5 c) In both cases, the modifications led to the formation of a rough surface with similar micro-sized craters. A uniform three-dimensional porous network structure with a size of about 300-350 nm is formed on the surface of Ti after NaOH treatment, as shown in (Figure 5b, c). It has been confirmed that this layer with a network structure is sodium titanate gel ¹⁶ , and its formation is as follows. The surface of Ti is covered by a thin layer of TiO ₂ formed spontaneously. When it is placed in NaOH solution 60 °C, TiO ₂ reacts with NaOH to form sodium titanate gel. Subsequently, the exposed Ti substrate will also react with NaOH to form sodium titanate gel.

[00136] Thermal treatment at 600 °C slightly changed the morphology of the surface decreasing its roughness. As we can see in Figure 5c, pores are smoother, and the surface appears to be denser. This might be due to after high- temperature treatment at 500 °C, the sodium titanate gel is transformed to a more stable amorphous sodium titanate layer with a gradient structure. [00137] The morphological differences were further assessed by the characterization of the surfaces’ topography by AFM. (Figure 5 d-f) shows the surface topographies of the coated Ti-substrates (Ti), (CT), and (CTT). The three types of uncoated samples that were modified under different conditions (Ti, CT, and CTT) were also examined for surface roughness. The AFM results showed that Ti surface modified with NaOH showed a significantly rougher surface (Ra = 53.95 ± 1.1 nm) than the one with subsequent thermal treatment at 500 °C (Ra = 48.33 ± 1 .09 nm) and untreated control Ti-substrate (Ra = 45.56 ± 1 .42 nm).

[00138] (Figures 5 a-c), show the morphology of the 2D nanocrystalline MgPi coated Ti-substrates investigated by SEM. A significant difference is observed in the microstructure of the coated and uncoated Ti-substrates (Ti). Scratches due to mechanical polishing disappeared in the bare Ti substrate suggesting that the coating was deposited successfully. The uneven and rough surfaces of the MgPi coated Ti-substrates (Ti-MgPi), (CT-MgPi), and (CTT-MgPi) were clearly visible. According to studies, coated and rough implant surfaces can both encourage osseointegration (Galli, Naito et al. 2014).

[00139] It was clear that the surface of the MgPi coated Ti-substrates (Ti-MgPi), (CT-MgPi), and (CTT-MgPi) was irregular and rough. Results in the literature indicated that rough implant surfaces as well as coated implant surfaces could increase osseointegration ³³ . The average surface roughness Ra values of the uncoated Ti-substrates (Ti), (CT), and (CTT) surfaces are 45.56 ± 1.42, 53.95 ± 1.1 , and 48.33 ± 1.09 nm respectively. The average surface roughness of the coated Ti-substrates (Ti-MgP), (CT-MgP), and (CTT-MgP) 36.20 ±0.47, 37.45 ± 2.60, and 32.50 ± 0.44 nm respectively. It is evident from the Ra values that the deposition of the MgPi coatings affects the surface roughness and resulted in significantly smoother surfaces.

[00140] To compare the coating adhesion to the Ti-substrates, scratch tests using the same testing parameters were performed on Ti-MgPi, CT-MgPi, and CTT-MgPi coatings. For Ti-MgPi and CTT-MgPi coatings, the average critical load was 5650 pN and 8270 pN respectively. A critical load associated with coating detachment or fracture was not observed in the CT-MgPi coating when a ramping load from 0-10mN was applied, indicating the critical load is higher than 10 mN. This signifies that the adhesion strength of the pre-treated coatings (CT-MgPi and CTT-MgPi) is greater than the baseline Ti coated sample (Ti-MgPi). The greater adhesion strength of the pre-treated samples could be due to the increased roughness and porosity network on the Ti-substrates surface, which allow for better mechanical interlocking between the coating and Ti-substrates, resulting in stronger adhesion. Similarly, the lateral force of the CT-MgPi during the scratch tests was consistently higher than the other two coatings, indicating the CT- MgPi coating has greater scratch resistance than the other two coatings.

[00141] In-vitro immersion tests were conducted to evaluate the degradation behavior of the MgPi coatings in terms of morphological changes.

[00142] (Figure 6a) shows the SEM micrographs of the coated Ti-substrates (Ti-MgPi), (CT-MgPi), and (CTT-MgPi) during different times of immersion in rat plasma. For all coated Ti-substrates, the surface morphology became more irregular and continuously degraded with time (Figure 6a). All coatings degraded completely after 7 days of immersion in plasma proteins, giving way to reveal the topology of the underlying Ti-substrates. During the immersion test, changes in concentration of the Mg ²⁺ ions were detected for variable immersion time by using inductively coupled plasma technique (ICP) (Figure 6b). For all coated Ti-substrates there was an increase in Mg ²⁺ concentration accompanied by a degradation of the coating as shown in SEM micrographs. The degradation rate of the coated Ti-substrates (CT-MgPi) was slower than that for both (Ti-MgPi) and (CTT-MgPi) (Figure 6b).

[00143] Pre-osteoblastic MC3T3-E1 cells were plated on each Ti-substrates (3000 cells/disk) for 4 days, (figures 7 and 9). Quantification of cells after nuclear staining by H33258 revealed an increased number of cells on the coated Ti- substrates (Ti-MgPi), (CT-MgPi), and (CTT-MgPi) compared to uncoated Ti-substrates (Figure 8). Furthermore, 2D nanocrystalline magnesium phosphate coatings on Ti-substrates, (Ti) sample significantly, (p < 0.05), increased cell proliferation compared to uncoated titanium substrates (Ti), (CT) and (CTT) (Figure 8).

[00144] Measurement of cell viability is taken by the increase of calcein staining. After 4 days of culture, MC3T3-E1 cells further supported an increase of cell proliferation on the coated samples (Ti-MgPi, CT-MgPi, CTT-MgPi) compared to uncoated Ti-substrates (Ti, CT, CTT). On the other hand, in the absence of MgPi coating, thermal treatment (CTT) significantly increased cell proliferation compared to the untreated substrate (Ti), (p < 0.05) (Figure 8). The biological properties of the MgPi coatings, examined as in vitro cellular responses were found to be superior to those of the Ti-substrates. The cell proliferation and adhesion on both MgPi coatings and treated Ti-substrates were favorable and compatible, while examination of initial cell attachment to the MgPi coatings showed that the cells grew more effectively and spread well (Figure 7). The improved cell adhesion with 2D nanocrystalline MgPi coated Ti-substrates compared to the uncoated Ti-substrates can be attributed to the affinity of the MgPi coatings for fibronectin adsorption and to the differences in the surface roughness between the coatings. Since both proliferation and adhesion of osteoblast is a prerequisite for their deposition of bone, these results suggest greater long-term functions of osteoblast on MgPi coated titanium.

[00145] Using Proteomics analysis, the affinity of the MgPi coatings to protein adsorption was studied after immersion of the bare (Ti) and coated (Ti-MgPi) samples in rat plasma for 3 hours at 37 °C (figure 8a). Our results revealed that a higher number of plasma proteins were able to adsorb to coated sample (Ti-MgPi) (i.e., 97 proteins) compared to uncoated control (Ti) (i.e. 76 proteins). Furthermore, our finding demonstrated a significantly higher score of fibronectin protein in case of coated samples compared to uncoated counterparts. Scores of this protein as found in coated and uncoated Ti-substrates exposed to plasma proteins are presented in table 3.

[00146] Cellular migration and adhesion are mediated by the glycoprotein fibronectin, an important ingredient of the extracellular matrix (ECM), which interacts with integrin receptors on cell surfaces (Hsiao, Cheng et al. 2017). Fibronectin (FN) is believed to promote osteoblast and osteogenic cell responses in vitro, particularly cell adhesion, proliferation, differentiation, and survival (Petrie, Reyes et al. 2009). Additionally, the accumulation of this protein is necessary for the migration, adhesion, and aggregation of numerous cell types during wound healing, including platelets, fibroblasts, and endothelial cells (Moretti, Chauhan et al. 2007). According to our study's findings, the coating layers' composition has an impact on the protein adsorption affinity. When compared to the uncoated samples. [00147] Table 3: Mass spectrometry analysis result of the fibronectin.

MgPi-Ti coated substrate showed a significantly higher fibronectin protein score compared to uncoated Ti substrate, Significance was found when p < 0.05.

[00148] The fibronectin, an extracellular matrix (ECM) glycoprotein that plays an important role in interacting with cell surface integrin receptors, mediates cellular migration and adhesion ³⁴ . Fibronectin (FN) is believed to support cell adhesion, growth, differentiation, or survival of osteoblasts and osteogenic cell responses in vitro ³⁵ . In addition, the accumulation of this protein is essential during wound healing to the migration, adhesion, and aggregation of various cell types including platelets, fibroblasts, and endothelial cells ³⁶ . In our study, the protein adsorption affinity is affected by the composition of the coating layers that the MgPi coated titanium showed improvement of protein adsorption in comparison to the uncoated samples.

[00149] Conclusion

[00150] Herein, we have demonstrated the possibility of using MgPi hydrogel as a bioactive coating.

[00151] Ti and its alloys are essential elements for dental and orthopedic implants. The absence of osseointegration, however, commonly results in the failure of a Ti implant. The biodegradable/resorbable MgPi- coated Ti substrate was fabricated using spin-coating techniques. On the MgPi coated titanium substrates as opposed to the Ti substrate, a significant amount of proteins were adsorbed. Additionally, compared to the uncoated titanium substrate, more osteoblast cells proliferated and adhered to the MgPi coated titanium substrate.

[00152] The osteoconductive coatings enhanced the bone-forming ability and rapid bone interaction of metallic implants, due to an improved initial osseointegration rate. Our results support the surface functionalization using the MgPi coating as a promising candidate for enhancing osseointegration thereby the MgPi coating is expected to improve the success rates of titanium implants.

[00153] This study offers concrete evidence that using MgPi coatings can improve Ti osseointegration. The MgPi coatings have the potential to replace conventional calcium phosphate coatings in the production of novel biomaterials for biomedical applications due to their excellent biocompatibility and biodegradability.

[00154] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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