Deep Shankar PPT _NITK _BIOIMPLANTS.pptx

The work describes innovative research in the development of bioimplants. This work produces a coating for metal implants beneficial for humans and can survive inside the body without any major inflammatory effects.
The implants were tested with human blood and showed no major inflammatory reactions. This work is an attempt to produce a biocompatible, durable, and mechanically strong orthopedic implant.

1. By , Under the guidance of , Deep Shankar Dr. Sudhakar C. Jambagi Research Scholar Associate Professor Reg: 187128ME019 Dept. of Mechanical Engg . NITK, Surathkal NITK, Surathkal 1 Improvement in the properties of thermally sprayed hydroxyapatite coating reinforced with carbon nanotube for orthopaedic applications Ph.D. Thesis Defense and Viva-Voce Presentation August- 2024

2. Contents Need for the study Introduction Literature Review Research Gaps Research Objectives Experimental Process & Methodology Results and Discussion Conclusions Future Scope, limitation, and Challenges Publications References 2

3. 13-08-2024 3 Need for the study

4. Aseptic loosening is responsible for ~21.9% of Total Knee Arthroplasty (TKA) failures in Germany alone ( Schiffner et al. 2020). Non-hemocompatibility is believed to be responsible for about 31 % of medical device failures in the US alone ( Kalathottukaren et al. 2018). As of 2021, the market value for orthopedic implants in the US is valued at $45.30 billion, and it is anticipated to reach $64.18 billion by 2028 (Pimentel et al. 2023). 13-08-2024 4 Source: Jafari et al. 2010 Need for the study

5. 13-08-2024 5 Need for the study Source: Lukas et al. 2023

6. 6 Source: DataM Intelligence Analysis (2023)

7. 13-08-2024 7 Introduction

8. Introduction 13-08-2024 8

9. Currently used implants 13-08-2024 9 Metal implants- 316 Stainless Steel, Co-Cr alloys, Titanium-based alloys Atmospheric Plasma Sprayed (APS)- Hydroxyapatite (HA) (FDA-approved) metal implants. Limitations of APS-HA implants: Phase changes Low degree of crystallinity Low adhesion strength Poor wear resistance Poor biocompatibility Fig. 1:  a) Dental implant, b) Total hip implant system. a) b)

10. 13-08-2024 10 Bioceramics Type of Bio-ceramics

11. 13-08-2024 11 Characteristics APS (FDA-approved) Flame spray (FS) HVOF Operating temperature 6000 and 15000 °C 2500-3500 °C 1800-2600 °C Velocity 150-600 ms −1 100-300 ms -1 400-1000 ms -1 Coating splat Fully melted, well-flattened lamellae, lower unmelted particles, Fully melted, well-flattened lamellae, Unmelted well- flattened lamellae Unmelted well- flattened lamellae Crystallinity Low Moderate to high High Table 1: Comparison of different thermal spray techniques (Shankar et al. 2024) Thermal spray techniques used for producing HA-based implants

12. 12 Biomaterials assessment

13. 13-08-2024 13 Literature Review

14. 13-08-2024 14 Author/Title/Year Materials & Methods Findings Hussain et al. (2023) Characterization and tribological behaviour of Indian clam seashell-derived hydroxyapatite coating applied on titanium alloy by plasma spray technique Different Hydroxyapatite powders (HA-700, HA-800, HA-900, and HA-1000) was prepared by hydrothermal method using the Indian clam seashell as a starting material. Titanium alloy (Ti6Al4V-ELI) – Substrate The atmospheric plasma spray system (Oerlikon Metco , Switzerland) was used to spray HA feedstocks. All the HA coatings have mostly stable HA phases and a low amount of TCP and CaO phases. HA coatings become more crystalline with increasing synthesis temperature from 700 to 900 ◦C and are Ca-rich. As the HA synthesis temperature rises from 700 to 1000 °C, surface roughness and porosity of the deposited coatings decreased while the melting and spreading of HA particles increased. The adhesion strength of HA coating increased at higher synthesis temperatures (700-900 °C). HA-900 powder had a high degree of melting, leading to strong coating adhesion with the substrate. Increasing the synthesis temperature of HA from 700 to 900 °C significantly increased the coating hardness. However, the hardness of the HA-1000 coating decreased due to relatively lower crystallinity. HA coatings fail in wear tests due to adhesive and brittle fracture. HA-900 was the most wear-resistant due to its hardness and toughness. Literature Review

15. Author/Title/Year Materials & Methods Findings Rattan et al. (2022) Wear studies on plasma-sprayed pure and reinforced hydroxyapatite coatings HA pure and HA+15 wt-% Al 2 O 3 – Feedstocks Titanium (cp-Ti) (Grade 2) – Substrate Plasma spray (Anode Plasma Limited) - Kanpur, India The XRD analysis indicated the presence of amorphous phases (TCP, TTCP) and traces of CaO in both coatings. During the wear test, the HA pure coatings exhibited an average friction coefficient of 0.21 ± 0.04, while the HA+15 wt % Al 2 O 3 coatings had a higher value of 0.4 ± 0.01. The reason for the lower friction coefficient in HA pure coatings is the removal of small coating particles from their outer surface during the test. These particles act as solid lubricants, reducing the friction between the contact surfaces. The wear studies revealed that the HA+15 wt -% Al 2 O 3 coatings exhibit superior wear resistance compared to the HA pure coatings. The wear rate for the former was 4.9x10 3 mm 3 /N-m, while the latter was 8.4x10 3 mm 3 /N-m. The reason behind was attributed to the presence of a dense microstructure, enhanced bond strength, and alumina, which all contribute to the high wear resistance of the HA+15wt% Al 2 O 3 coatings. 13-08-2024 15

16. Author/Title/Year Materials & Methods Findings Kowalski et al. (2022) Plasma-Sprayed Hydroxyapatite Coatings and Their Biological Properties HA coatings were deposited from XPT-D-703 powder (Sulzer Metco, Salzgitter, Ger- many) by a plasma-spraying process on grade 2 titanium. The plasma-spraying process was performed by axial powder injection on an Axial III system (Northwest Mettech Corp., Surrey, Canada). The process was carried out at different SOD: 100 (HA1), 120 (HA2), and 140 mm (HA3). Analysis of the phase composition did not showed significant degradation of HA due to changing the spray distance. HA2 coating showed the highest values of the surface geometry parameters (Sa=23.2 µm). However, HA1 coating displayed the lowest surface geometry (Sa=18.8 µm), which could be explained by the shorter spray distance, which resulted in a higher temperature of the sprayed substrate and a greater degree of deformation of the powder grains that hit it. None of the sprayed-HA coating methods affected the biocompatibility of L939 cells and the activation of the NF- κB signaling pathway. HA2 coating showed a slight increase in the biological properties tested. Moreover, HA coatings sprayed at different distances were not cytotoxic and did not stimulate the NF-kB. Bare titanium was less susceptible to colonization by staphylococcus aureus than HA-coated surfaces 13-08-2024 16

17. Author/Title/Year Materials & Methods Findings Widantha et al. (2021) Effect of hydroxyapatite/alumina composite coatings using HVOF on immersion behavior of NiTi alloys NiTi alloy ( 55.8% Ni and 44.2% Ti ) - Substrate HA powder ( 63 μ m ) derived from femur bones, Alumina ( 63 μ m ) HA and HA+15% Alumina were coated using TECKNOTHERM HVOF 2007 with a Hipojet-2700 spray gun T he crystallinity of the coated powders had changed from initial powders of 85.27% to 81.13% and 78.41%, respectively for HA and HA/alumina coatings. The microhardness of the HA and HA/alumina coatings obtained in this study were 226.8 HV and 250.6 HV. This higher hardness of HA/alumina coating is predicted due to the role of alumina as a reinforcing agent compensate for the difference in heat expansion between HA and the substrate. The results showed nearly 18-fold and 2-fold increase in the corrosion resistance on NiTi alloy through HA/alumina coating in PBS and Ringer’s solution. HA/alumina coating showed bioactive properties through HA precipitation on the coating surface. 13-08-2024 17

18. Author/Title/Year Materials & Methods Findings Vilardell et al. (2020) In-vitro comparison of hydroxyapatite coatings obtained by cold spray and conventional thermal spray technologies HA powder, Plasma- Biotal Ltd ( Captal 30, Derbyshire, UK) for HVOF and Plasma coatings. Agglomerated nanocrystalline HA (NC-HA) powder from Medicoat (France) for cold spray coatings. Ti-6Al-4V- Substrate An increase of HA crystallinity (APS < HVOF < CS) can be observed with the decrease of the operating temperature of the technique since higher temperatures, as well as fast cooling rates, promote HA decomposition. Regarding porosity, an amount in the ranges of 21–23% and 11–15%, respectively, for APS and HVOF coatings. Higher contact angles were measured on APS HA coatings with values of α1= 32.4 ± 6.9° and α2= 29.8 ± 5.3°, followed by HVOF HA and CS HA coatings, with values of α1= 10.8 ± 2.5° and α2= 13.8 ± 2.6°, respectively, and 0° (both angles) for CS HA coatings. Different types of HA coatings showed different cell morphology at 1 day of cell culture. Osteoblastic morphology was observed in cells attached to APS HA coatings, whereas cells attached to CS HA coatings appeared rounded. HVOF HA coatings showed good cell adherence but did not exhibit extended filopodia-like cells on APS HA coatings. APS HA coatings had surface micro-features that suggested higher stimulation of cell anchoring, which was favored by moderate wettability. The amount of crystallinity in HVOF and CS HA coatings was found to be associated with an increase in cell proliferation and differentiation. 13-08-2024 18

19. Author/Title/Year Materials & Methods Findings Munoz et.al (2019 ) Influence of HVOF parameters on HAp coating generation: An integrated Approach using process maps HA (25-45 μ m ) sprayed onto stainless steel with TiO 2 as a bond coat using HVOF (Sulzer metco 2700). Optimization of process parameters (2 3 ) with F/O ratio, SOD (cm) PFR (g/min) as the main parameters. No secondary phases of HA observed. SOD=20 cm, F/O ratio=0.14 , and PFR=16 g/min: Disk-like splat morphology, homogeneous surface, high crystallinity, and Ca/P ratio (within the range). Cracks and holes were observed on the cross-section of the coating. SOD (10 cm) and the highest F/O ratio (0.27): F aster and colder particles with a short residence time in the flame, leading to coating delamination. Dune apatite layer covered the entire coating after 14 days of immersion in SBF. No cytotoxic response. 13-08-2024 19

20. Author/Title/Year Materials & Methods Findings Kaur et al. (2018) Characterization of Thermal-Sprayed HA and HA/TiO 2 Coatings for Biomedical Applications HA and HA/TiO 2 coating was deposited by high-velocity flame spray (CERAJET, 2700°C) (MEC, Jodhpur) technique onto 316LSS. Titania (5- 22 µm ) was used as a bond coat, and HA (~30 µm ) as a top coat. HA/TiO 2 coating shows major peaks of HA and minor peaks of TCP and calcium phosphate hydrate. The average coating thickness of HA coating was found to be 181 microns, and for HAP/TiO 2 coating, it comes to be 230 µm. The average HA coating thickness was 181 µm , and for the HA/TiO 2 coating, it was 230 µm . The bond coating exhibited lesser porosity and higher microhardness and surface roughness than HA coating. Average microhardness of the HA-coated sample was found to be 254 Hv, and that of the HA/TiO 2 coating samples was found to be 280 Hv. 13-08-2024 20

21. Author/Title/Year Materials & Methods Findings Facca et al. (2011) In Vivo Osseointegration of Nano -Designed Composite Coatings on Titanium Implants HA nanorods (length: 100-325 nm, diameter:25-50 nm, MWCNTs (95% purity, 25-50 nm O.D., length: 0.5-2 μ m. Ti-6Al-4V alloy (beads & rods) with and without coatings were coated by a plasma spray (Plasma Forming Lab. USA). These sets of coated implants were inserted in the rodent’s femoral bone: uncoated titanium, (ii) HA coating, and (iii) HA-4 wt.% CNT coating. The coating thickness was 100-150 μm . Histological observations showed that implants (groups 1&2) from mice and rats were fully removed from the bone. It was difficult to detach group 3 implants as compared to groups 1 and 2. No adverse effect or cytotoxicity of CNT addition on bone tissues and cells was observed. Normal bone growth was observed around HA-CNT-coated implants. Unpurified CNTs have been observed by TEM in the cytoplasm and the nucleus. CNTs were able to enter the cell through the phenomena of endocytosis . CNTs may also cause incomplete phagocytosis in vivo and may result in oxidative stress and cell death. 13-08-2024 21

22. Author/Title/Year Materials & Methods Findings Balani et al. (2007) Plasma-sprayed carbon nanotube reinforced hydroxyapatite coatings and their interaction with human osteoblasts in vitro HA powder (10-50 μ m), blended with 4wt% MWCNTs (95%+ pure, OD = 40–70 nm, length= 0.5-2 mm) in a jar mill for 18 hours. Powder feedstock was sprayed onto Ti-grade 5 alloy using plasma spray (Praxair SG-100 gun). Cell culture was studied with human osteoblasts hFOB 1.19 cells. HA coating showed crystallinity of 53.7%, whereas it increases to 80.4% for HA–CNT coating, still much lower than HVOF spraying of HAp (93.81%) as reported by Stokes (2010). The presence of phosphate-rich needle phases implies the transformation of HA to secondary phases, ensuring reduced crystallinity. Vickers indentation toughness improvement of up to 56% was observed for HA–CNT coating (0.6170 ± 0.09MPam 1/2 ) compared to (0.397 ± 0.09MPam 1/2 ) for HA coating. Transverse cracking was observed in both coatings because of differential thermal quenching between splats and deposited coating. Thermal stresses usually build up in the plasma sprayed coatings owing to extreme cooling rates (in the order of 10 5 –10 8 K/s). Unrestricted human osteoblasts hFOB 1.19 cell growth and proliferation during cell culture studies demonstrate non-toxicity of HA–CNT coating. 13-08-2024 22

23. Author/Title/Year Materials & Methods Findings Lima et.al. (2005) HVOF spraying of nanostructured hydroxyapatite for biomedical applications HA ( Captal 30, Plasma Biotal , UK) powder was HVOF-sprayed (DJ2700-hybrid, Sulzer- Metco , USA) on a grit-blasted Ti- grade5 substrate. SBF test for in-vitro assessment of coating. Average particle velocity was 638±82 m/s, whereas the average particle temperature was 1826±346°C. The bond strength of the HVOF-sprayed HA coating was 24±8MPa (n =5), much higher than bond strengths of HA coatings deposited via air plasma spray (4-14MPa). High crystallinity levels (84%) and low degradation, without the presence of TCP, TTCP or CaO phases. High density and microstructural uniformity. The Almen strip exhibited a compressive stress. This characteristic is desirable as it inhibits crack propagation throughout the coating microstructure. Uniform layer of apatite (~35 microns) was formed on the HA coating after 7 days of incubation in SBF which is much higher than plasma spray (~20 microns). 13-08-2024 23

24. Literature based on hemocompatibility assessment of biomaterials The ISO guidelines for hemocompatibility evaluation are divided into many categories, out of which hemolysis test, whole blood clotting time measurement, platelet adhesion, & activation, and coagulation assays – Prothrombin time (PT) and aPTT are selected for the current study for biocompatibility assessment. In the current study, all procedures have been performed following the Declaration of Helsinki and have been approved by the ethical committee of Sanjay Gandhi Institute of Trauma and Orthopaedics, Bangalore, India, dated 12.01.2021. Informed consent was obtained for experimentation with human blood from the patients. 13-08-2024 24

25. Hemolysis assessment Hemolysis study of HA and Fe-doped HA with increasing Fe concentration (0Fe, 0.01Fe, 0.05 Fe, 0.1Fe). All the samples were blood-compatible, with a less than 5% value. 13-08-2024 25 Fig. 2: Source: Chandra et al. 2012

26. Irrespective of the particle size, all NDs showed no sign of RBC destruction at a concentration as high as 400 mg/ mL. In contrast, the membrane of the RBCs incubated with GOs was substantially damaged, leading to the release of free hemoglobin in the supernatant at a dose higher than 25 mg/ mL. 26 Fig. 3: Hemolysis studies of GOs and oxidized NDs of different size with human RBCs. (a) Hemolysis percentages measured at the concentration range of 25–400 mg/mL for GOs and 4 different ND samples (35, 100, 250, and 500 nm in diameter) incubated with RBCs at 25uC for 2 h. GOs served as the positive control. (b) Photographs of human RBCs treated with GOs and NDs of different sizes at the concentration range of 25 –400 mg/ mL. The red color of the solution is due to the release of hemoglobin from the damaged RBCs, and the red pellets at the bottom of the Eppendorf tubes are intact RBCs precipitated by centrifugation. PBS and DDW served as the negative (2) and positive (1) controls, respectively. The experiments were repeated in triplicate. (Source: Chen Li et al. 2013 )

27. The % hemolysis of all the samples are within the acceptable values - Highly Biocompatible. The explanation behind the hemocompatibility was not explored. 13-08-2024 27 Sample % Hemolysis HA 0.343 HA–CNT (0.5%) 0.958 HA–CNT (1%) 1.150 HA–CNT (2%) 1.753 HA–CNT (5%) 2.853 Table 2: Hemolysis % for HA-CNT pellets ( Mukherjee et al. 2014).

28. Thrombogenicity of Co-polymer PVA/ Dx (Polyvinyl alcohol hydrogel with Dextran) was studied using whole blood clotting time method with glass beads and polypropylene membrane as positive and negative control respectively. 13-08-2024 28 Thrombogenicity (Blood clot) Fig. 4: Whole blood clotting time for PVA/Dx. The positive control ( + ) used were glass beads, and for the negative control (-), polypropylene membranes were used. Samples were tested in triplicates. PVA/Dx-Polyvinyl alcohol hydrogel/dextran ( Alexandra et al. 2014 ). Polypropylene Glass PVA/Dx

29. Blood clot time measurement was studied for HAP and HAF powder ( Ooi et al., 2019). The degree of blood clotting on the HAP and HAF is relatively low as compared to reference materials as the absorbance value is inversely proportional to the blood clot formed on the sample. 13-08-2024 29 Fig. 5: Blood clotting time measurement for nanoporous hydroxyapatite, glass, and baseline samples (Ooi et al. 2019).

30. All the test tubes were added with Platelet Rich Plasma (PRP) and incubated for 60 min in a water bath-shaker at 37 °C. Phosphate buffered saline (PBS) was added into the tubes (to remove non-adhere platelets), kept for 20 min and counted for platelet count in the pure PRP. 13-08-2024 30 Platelet adhesion Fig. 6: Percentage of platelet adhesion on collagen-coated glass, silicone tube, HAP, and HAF samples (Ooi et al. 2019)

31. 13-08-2024 31 Research gaps

32. 13-08-2024 32 Most of the orthopedic implants available in the market are produced by APS, the only FDA-approved method for applying HA coatings onto metal implants. However, the high temperature of APS possesses many limitations. Fracture toughness of HA (1MPa.𝑚 0.5 ) is significantly lower than the minimum reported value for cortical bone (2MPa.𝑚 0.5 ). Poor wear resistance of HA is another limiting factor. Hence, it has to be improved with the addition of reinforcements like alumina/CNT. CNT has high surface energy and tends to agglomerate in the composite. Inhomogeneous dispersion of CNT results in weak anchoring between the splats. Research gaps

33. For the longer life of the implant, the mechanical strength, wear resistance, and bond strength of the coating need to be improved. It is important to assess the toxicity of the wear debris generated from the implant’s surface for its safe use. Implants must function in the presence of blood, so it is crucial to study the compatibility of human blood and coated implants for potential adverse effects. There has been very limited research work for the in-vivo compatibility of the newly developed bioimplants. 13-08-2024 33

34. 13-08-2024 34 Research Objectives

35. To prepare the heterocoagulated alumina (19.5-18wt%)/carbon nanotube (CNT) (0.5-2wt%) powder followed by ball mixing with HA to study the retention and degree of dispersion of CNT in HA/alumina matrix. Hemocompatibility assessment of carbon nanotube doped hydroxyapatite worn debris of low-temperature thermally sprayed implants. A Comparative study of plasma spray, flame spray, and HVOF sprayed-HA coatings based on their physiochemical, mechanical, and biocompatibility. Biocompatibility assessment of the reinforced implants obtained with a suitable thermal spray process. To determine and study the wear resistance of the best-performing reinforced coating using the ball-on-flat method. In-vivo osseointegration and toxicity assessment of bio-ceramics coated titanium rods in rabbit femoral bone. 13-08-2024 35 Research Objectives

36. 13-08-2024 36 Experimental Process & Methodology

37. 13-08-2024 37 Coating Feedstocks Substrate used Raw materials used Experimental Process & Methodology

38. Bio-ceramics composite preparatory method Heterocoagulation of the powders (Alumina/CNT) was performed according to the procedure reported by Jambagi et al. (2015) (Figure 14). The heterocoagulated powder obtained after drying was ball-mixed with HA at 300 rpm in alcohol media for 12 hours. The resultant composite obtained with varying concentrations is presented in Table 3. 13-08-2024 38 Fig. 7: Homogenous dispersion of CNT shows its functionalization

39. Physicochemical & Mechanical Characterization 13-08-2024 39 Field Emission Scanning Electron Microscope (FESEM) (7610F PLUS, Jeol , Japan) Energy dispersive spectroscopy (EDS) equipped with FESEM Transmission Electron Microscopy (TEM) operation at 200 kV (JOEL 2010 F, Tokyo, Japan). X-ray Diffractometer (XRD), Empyrean 3rd Gn , Malvern PANalytical , Netherlands, with CuK α radiation (wavelength of 1.54 A°) operating at 45 kV and 40 Ma (Chandra et al. 2012)

40. 13-08-2024 40 Compact Raman Spectrometer, Renishaw, UK, operating in 180° backscattering geometry. . A sessile drop method by Kyowa contact angle meter ( DME-211, Kyowa Interface Science Co., LTD., Japan). . The zeta potential (ZP)/surface charge of the HAp powder was measured in physiological saline solution at pH-7.4, using Anton- Paar , Litesizer TM 500, Austria, equipped with a 40 mW semiconductor laser (λ = 658 nm). 3D- Profilometer ( ST400, NANOVEA, USA )

41. 13-08-2024 41 Progressive scratch tester (TR-101, DUCOM, India). Three scratches were made under a progressive load of 2 to 100 N with a scratch velocity of 0.1 mm/sec and a stroke length of 10 mm . The scratch tester had a Rockwell C-type conical indenter with an apex angle of 120° and a tip radius of 100  μm . TRB3 Anton Paar , Austria Sliding reciprocating wear test (6 mm ф–Stainless steel ball, speed–2.5 cm/s, sliding distance–100 m, Load–5 N) on the HA and CNT doped HA coatings on titanium coupons. The polished cross-sectional area of the coating was subjected to an indentation load of 200 g (1.96 N) and a dwell period of 10 s using a Vickers microhardness tester (OMNITECH, S-Auto, India).

42. Fig. 8 : Experimental set-ups for tribological wear test (a) schematic illustration to show wear test mechanism under SBF environment, and (b) wear test under dry and physiological (SBF) conditions. Tribological study

43. 13-08-2024 43 Different biocompatibility assessment methods used

44. 44 Fig. 9: The pictograph shows the procedure for conducting a hemolysis test for HAp and its based composites. % Hemolysis = ( OD test - OD negative )/( OD positive - OD negative ) OD= Optical density/Absorbance value

45. 13-08-2024 45 Fig. 10: Pictograph depicts the procedure for conducting a whole blood clot test.

46. 46 Fig. 11: The pictograph shows the procedure for generating PRP from whole blood, followed by the platelet adhesion test methodology. % Platelet adhesion = ( PC initial - PC sample )/(PC initial )×100 PC= Platelet count

47. 13-08-2024 47 Fig. 12: The pictograph depicts the procedure for conducting a coagulation assay with PPP. (a) PT test, (b) aPTT test.

48. In-vivo study 48 Fig. 13: Different stages involved in the in vivo implantation of HA-based implants in New Zealand white rabbits.

49. 13-08-2024 49 Results and Discussion

50. 13-08-2024 50 Objective - 1 To prepare the heterocoagulated alumina (19.5-18 wt %)/carbon nanotube (CNT) (0.5-2wt%) powder followed by ball mixing with HA to study the retention and degree of dispersion of CNT in HA/alumina matrix.

51. 51 Fig. 14: Alumina suspended in DW with SDS surfactant at pH- 8. b) FCNTs suspended in DW with CTAB surfactant at pH- 8. c) Heterocoagulated composite precipitated at the bottom of the container. Heterocoagulation of Alumina and CNT Objective 1: Results and Discussion

52. 13-08-2024 52 Table 3: Powder designation with its processing description powder designation.   SL No.   Designation   Description 1. HA Hydroxyapatite 2. AC0.5 Heterocoagulated 19.5 wt.% alumina and 0.5 wt.% CNTs (20 wt.%) 3. AC1 Heterocoagulated 19 wt.% alumina and 1 wt.% CNTs (20 wt.%) 4. AC2 Heterocoagulated 18 wt.% alumina and 2 wt.% CNTs (20 wt.%) 5. HAC0.5 AC0.5 blended with 80 wt.% HA (alcohol media) in ball mill 6. HAC1 AC1 blended with 80 wt.% HA (alcohol media) in a ball mill 7. HAC2 AC2 blended with 80 wt.% HA (alcohol media) in ball mill.

53. 13-08-2024 53 Fig. 15: FESEM morphology for (a) HAp, (b) Alumina, (c) TEM image for FCNT, (d) FESEM image for AC2 Composite, (e) Dot mapping for AC2, (f) Binary image for AC2 dot map shows the degree of dispersion of CNTs, (g and h) TEM image for HAC1 and HAC2, respectively showed different junctions formed by CNTs, and ( i ) schematic illustration to understand the electrostatic interaction between HAp/Alumina/CNTs during powder preparation.

54. 13-08-2024 54 Fig. 16: XRD spectra for (a) HAp , (b) AC2, (c) HAC2, Raman spectra for (d) HAp, (e) Alumina, (f) FMWCNT (g) HAC2.

55. 13-08-2024 55 Fig.17: (a) Variation of zeta potential with pH for Alumina, Alumina with SDS, CNTs, CNTs with CTAB, and heterocoagulated sample (AC1 and AC2) (b) HA and HAC composites, n=3.

56. 13-08-2024 56 Hemocompatibility assessment of carbon nanotube doped hydroxyapatite worn debris of low-temperature thermally sprayed implants. Objective - 2

57. 13-08-2024 57 Fig. 18: A bar graph shows the percentage hemolysis of different powders with varying concentrations for (a) HAp, (b) HAC0.5, (c) HAC1, and (d) HAC2, colorimetric image to show RBC damage by (e) HA, (f) HAC0.5, (g) HAC1, and (h) HAC2. The white and light red colored tubes represent non-hemolytic, and the dark red colored solution depicts hemolytic behavior. Objective 2 : Results and Discussion

58. 13-08-2024 58 Fig. 19: (a) Absorbance vs. Time graph for whole blood clotting time measurement for the samples and the control, n=5, and (b) schematic diagram to illustrate clot formation when triggered by foreign material, and negative charge for HAC composites repels adjacent blood components, hinders clot formation.

59. 13-08-2024 59 Fig. 20: (a) Percentage of platelet adhesion on HAp-based composites, negative and positive control, n=5, (b) schematic diagram that shows the change in platelet morphology when in contact with foreign materials, FESEM images of platelets adhered on (c) Negative control, (d) HAp, (e) HAC0.5, (f) HAC1, (g) HAC2, and (h) Positive control.

60. 13-08-2024 60 Fig. 21 : Coagulation assays; (a) PT and (b) aPPT coagulation time of PPP incubated with different concentrations of HAp-based composites at 5, 20, and 80 mg, n = 5.

61. 13-08-2024 61 A Comparative study of plasma spray, flame spray, and HVOF sprayed-HA coatings based on their physiochemical, mechanical, and biocompatibility. Objective - 3

62. 13-08-2024 62 APS   FS   HVOF   Parameters Values Parameters Values Parameters Values Argon flow ( scfh ) 80-90 Oxygen flow ( slpm ) 45 Oxygen flow (scfh) 30-34 Hydrogen flow ( scfh ) 15-18 Acetylene flow ( slpm ) 55 Hydrogen flow (scfh) 55-60 Current (A) 500 N 2 Carrier gas ( scfh ) 20 PFR (g/min) 45-50 Voltage (v) 65.7 PFR (g/min) 35 - - PFR (g/min) 35 - - - - SOD (cm) 10 SOD (cm) 20 SOD (cm) 20 scfh = standard cubic feet per hour, slpm = standard litres per minute. Table 4: Deposition parameters for Atmospheric Plasma Spray (APS) ( Heydari et al. 2017), Flame Spray (FS) (Singh et al. 2014), and High-velocity oxy-fuel (HVOF) ( Vilardel et al. 2020) -HA coatings. Objective 3 : Results and Discussion

63. 13-08-2024 63 Fig. 22: FESEM micrographs (a) HA powder, (b) nanoporous structure of individual HA particle. FESEM top surface morphology for (c) APS, (e) FS, and (g) HVOF-HA coatings. Cross-sectional microstructure of (d) APS, (f) FS, and (h) HVOF-HA coatings. The white arrow represents partially melting, the black arrow shows the unmelting , and the yellow arrow depicts the complete melting of HA. ( i ) The top surface microstructure showed a nanoporous structure of the HVOF coating, similar to HA feedstock. (j) Histogram shows particle size distribution for HA powder fitted with a log-normal distribution function (solid black line). Coating thickness: APS: 252 ± 23.68 μ m FS: 113 ± 13.22 μ m HVOF: 55.36 ± 2.05 μ m Porosity: APS: 11.062 ± 1.42 % FS: 13.204 ± 2.34 % HVOF: 3.046 ± 0.45 % Ca/P ratio: APS: 1.56 FS: 1.51 HVOF: 1.69

64. 13-08-2024 64 Fig. 23: 3d surface profiles of surface roughness (Ra) for (a) APS, (b) FS, and (c) HVOF-HA coatings.

65. 13-08-2024 65 Fig. 24: (a) XRD spectra of feedstock powder and all coated samples with corresponding ICDD file numbers for different phases of HA. (b) Raman spectral analysis shows different HA phases for feedstock and coatings. (c) The surface roughness (Ra) value for Ti and coated samples was obtained using a 3D profilometer (n = 5). (d) Contact angle and corresponding front-view photographs show hydrophilicity and hydrophobicity behavior for titanium and coatings (n = 5). The error bar represents the standard deviation. 67.8% 81.2% 89.7% 99.2%

66. 13-08-2024 66 Fig. 25: Optical micrographs of scratch tracks of as-deposited HA coating for APS (a) scratch track, (b) First cracking, (c) first delamination, (d) Total delamination, Flame spray (e) Scratch track, (f) First cracking, (g) First delamination, (h) Total delamination, HVOF spray ( i ) Scratch track, (j) First cracking, (k) First delamination, (l) Total delamination. (m) Bar graph shows the adhesion strength of the different HA coatings (n = 3; *p-values < 0.05). The error bar represents the standard deviation. Optical microscope image of the Vickers hardness indentation for (n) APS, (o) FS, and (p) HVOF-HA coating, where D 1 and D 2 are the horizontal and vertical diagonal lengths of the indentation. HV=282.08 ± 10.65 HV= 232.27 ± 16.72 HV=369.06 ± 13.37

67. 13-08-2024 67 Fig. 26: 3D profile shows the coated samples' scratch track, (a-c) APS, (d-f) FS, and (g- i ) HVOF-HAp coatings.

68. 13-08-2024 68 Fig. 27: FESEM images show the change in apatite morphology on the surface of different HA coatings obtained after 15,30 and 60 days of immersion in SBF (a-d) APS, (e-h) FS, and ( i -l) HVOF-HA coatings. (m) % weight gain by HA coatings as a function of the time immersed in SBF (n = 3). The error bar represents the standard deviation. (n) XRD pattern shows the change in apatite growth observed over time on HA coatings. Ca/P ratio after 30 days APS:2.03 FS: 2.25 HVOF: 2.3 Ca/P ratio after 60 days APS:2.05 FS: 1.97 HVOF: 2.12

69. 13-08-2024 69 Fig. 28: (a) Bar graph shows the percentage hemolysis for titanium and HAp coatings, n=5. All samples displayed less than 5% hemolysis, indicating their non-hemolytic behavior. (b) Colorimetric images showing RBC damage for positive control-DW, negative control-saline, and samples. The white-colored tube represents no rupture of RBCs, and the red-colored tube shows a complete rupture of RBCs. Microscopic stained images show the change in RBCs morphology for (c) APS, (d) FS, (e) HVOF coatings, and (f) Positive control.

70. 13-08-2024 70 Fig. 29: (a) Bar graph shows the absorbance value of the whole blood clot study at different time points (15, 30, 45 minutes) for titanium and HA coatings (n = 5; *p-values < 0.05). The error bar represents the standard deviation, and the dotted line represents the absorbance of free hemoglobin in unclotted blood. A significant increase in free hemoglobin is present on all coated surfaces compared to the uncoated titanium at respective time intervals. Colorimetric images of the samples after (b) 15 minutes, (c) 30 minutes, and (d) 45 minutes. The dark red tube shows the release of free hemoglobin , indicating that the samples are non-thrombogenic. On the other hand, the light red tube in color depicts the thrombogenicity of the samples.

71. 13-08-2024 71 Fig. 30: (a) The bar graph represents the percentage of platelets adhered to titanium and coated samples (n=3). The error bar represents the standard deviation. FESEM morphology of platelets adhered on the surfaces of (b) Negative control, (c) Titanium, (d) APS, (e) FS, and (f) HVOF-HAp coatings. (g) Schematic illustration to show the role of implant surface chemistry on platelet adhesion and the mechanism underlying different stages involved in the change in platelet morphology and clot formation.

72. 13-08-2024 72 Fig. 31: (a) A bar graph shows the coagulation time for different samples and a control incubated with PPP for 1 hour, n=5. The error bar represents the standard deviation. (b) A schematic illustration depicts the mechanism underlying the role of implant surface chemistry in protein adsorption.

73. 13-08-2024 73 Fig. 32: Agar diffusion test for the titanium and HAp coatings with (a) Gram-negative E. Coli and (b) Gram-positive S. aureus shows no inhibition zone formation around the discs. The dotted circle represents the no-inhibition zone formation around the discs, indicating the non-microbial property of HAp

74. 13-08-2024 74 Table 5 : Comparative analysis of HA coatings acquired through APS, FS, and HVOF thermal spray techniques based on their in vitro results. In vitro studies Surface properties influencing   FS HVOF SBF apatite growth (60 days) Crystallinity, Wettability   +24.24% +30.90% Hemolysis Roughness, Crystallinity   -37.11% +86.59% Blood clot (45 minutes) Surface Charge, Roughness, and Porosity   +1.75% +21.05% Platelet adhesion Surface Charge, Roughness, Porosity, Wettability   -1.93% +18.35% PT Wettability, Porosity -8.63% +2.87% APTT Wettability, Porosity -5.61% +2.04% + represents the increment, - represents the decrement. All the percentage (%) values are with respect to plasma sprayed HA coating.  

75. 13-08-2024 75 Biocompatibility assessment of the reinforced implants obtained with a suitable thermal spray process. Objective - 4

76. 13-08-2024 76 Fig. 33: Apatite formation at 0 th , 7 th , 15 th , and 30 th day for pure HA (a-e), HAC1 coating (f-j). The FESEM images show the apatite layer precipitation on the HA and HAC1 coatings, which testifies to the non-toxicity of CNT. Percentage weight gain by HA coatings as a function of the time immersed in SBF (n = 2). The error bar represents the standard deviation. k) Ca/P ratio-15 days HA: 1.28 HAC1: 1.36 Ca/P ratio-30 days HA: 1.72 HAC1: 1.86 Objective 4 : Results and Discussion

77. 13-08-2024 77 Fig. 34: In-vitro blood compatibility assessments (a) colorimetric images show the non- hemolytic behavior of HAC1 coating similar to the negative control, FESEM morphology of the platelets adhered on to (b) HAC1 coating surface, and (c) negative control surface. The dotted yellow circle indicates round platelets adhered to coatings similar to the negative control, indicating no sign of platelet activation. HAC1 coating displayed a hemolysis ratio of 0.24±0.03, n=3. The HAC1 coating showed a significantly lower percentage of platelets adhered (~4.72%) than the negative control (polypropylene ~13.5%), highlighting its non-thrombogenicity.

78. 13-08-2024 78 To determine and study the wear resistance of the best-performing reinforced coating using the ball-on-flat method. Objective - 5

79. 13-08-2024 79 Fig. 35: FESEM micrograph (a) spherical morphology for HA powder, (b) Angular and blocky structure for alumina particle, (c) Kink and bulges represents functionalization of CNT, (d) Heterocoagulated alumina/CNT shows CNT gets adsorbed and completely engulfed alumina particles, (e and f) TEM images showed various junctions of CNT attached to HA/alumina particles due to electrostatic interaction between the ions, FESEM micrograph (g) Plain smooth titanium surface, (h and i ) Low and high magnification images showed top surface microstructure for HA coating displayed smooth surface with unmelted and partially melted HA particles, (j) Back scattered micrograph for HA coating with corresponding EDS analysis and dot mapping, (k and l) Low magnification images showed top surface microstructure for HAC1 coating, (m) High magnification image for HAC1 coating shows crack arresting, hook structure and homogenous dispersion and retention of CNT in HA matrix, and (n) Back scattered micrograph of HAC1 coating with corresponding EDS analysis and dot mapping, indicates no impurities were added during thermal spraying. Density: 97.2% Ra~ 7 µm Density: 96% Ra ~ 4 µm Objective 5 : Results and Discussion

80. 13-08-2024 80 99.16 % 88.62 % 91.23 % 99.72 % Fig. 36: X-ray diffraction spectra of the feedstocks and their corresponding coatings. The graph displays the retention of HA phases along with reinforcement elements

81. Fig. 37: Raman spectra for feedstock powder and their HVOF-sprayed coating. The graph shows the retention of HA phases and D and G peaks for CNT in the coatings and corresponding wear tracks.

82. 13-08-2024 82 Fig. 38: (a) Optical micrographs of scratch track for HA coating show different critical load of failures, (b-d) FESEM image of scratch track for HA coating shows critical loads and its failure mechanism, (e) EDS mapping shows the presence of Ti, Al, V along the scratch track for HA coating, indicates the penetration of indenter into the coating, (f) Optical micrographs of scratch track for HAC1 coating shows different critical load of failures, (g- i ) FESEM image of scratch track for HAC1 coating shows different critical loads and bridging mechanism by CNT, (j) EDS mapping shows the presence C, Ca, P, Al, along the scratch track for HAC1 coating, indicates the toughening mechanism of CNT in the HA matrix, and Optical microscope image of the Vickers hardness indentation for (k) HA (l) HAC1 coating, where D 1 and D 2 are the horizontal and vertical diagonal lengths of the indentation. L c1 HA: 6±0.7 N HAC1: 10±0.49N L c2 HA: 9.45±0.77 N HAC1: 22±1.41 N HV= 469.60±16.9

83. 13-08-2024 83 Table 6: Microstructural, mechanical, and bioactivity properties of as-deposited coatings.

84. 13-08-2024 84 Fig. 39: Wear profile for titanium alloy in the dry condition (a) FESEM images of wear track, (b) low magnification image shows scars on the wear track, (c) High magnification images show debris cracks and crater generated, (d-f) 3d profile images depicting the depth profile and the volume loss from the wear track, (g and h) wear debris size and its composition, ( i ) dot mapping with elemental analysis of wear track. Wear profile for HVOF-HA coating in dry condition (a) FESEM images of wear track, (b) low magnification image shows scars on the wear track, (c) High magnification images show debris cracks and crater generated, (d-f) 3d profile images depicting the depth profile and the volume loss of the wear track, (g and h) wear debris size and its composition, and ( i ) dot mapping with elemental analysis of wear track.

85. 13-08-2024 85 Fig. 40: Wear profile for HVOF-HAC1 coating in dry condition (a) FESEM images of wear track, (b) low magnification image shows scars on the wear track, (c) High magnification images show debris cracks and crater generated, (d-f) 3d profile images depicting the depth profile and the volume loss of the wear track, (g and h) wear debris size and its composition, ( i ) dot mapping with elemental analysis of wear track. Wear profile for HVOF-HAC1 coating with SBF as lubricant (a) FESEM images of wear track, (b) low magnification image shows scars on the wear track, (c) High magnification images show debris cracks and crater generated, (d-f) 3d profile images depicting the depth profile and the volume loss of the wear track, (g and h) wear debris size and its composition, ( i ) dot mapping with elemental analysis of wear track.

86. 13-08-2024 86 Fig. 41: FESEM images show (a-d) wear debris size distribution for Titanium substrate, HA, and HA/Alumina/CNT (HAC1) coatings in dry and SBF environments, and (e) Box plot shows the wear debris size distribution for the samples., and SBF environments.

87. 13-08-2024 87 Fig. 42: Bar graph shows a) Coefficient of friction generated during wear, b) the Roughness of the samples before and after wear, c) the width of the wear track generated after wear, d) the Depth of the wear track, 3D profile images shows the wear of the counter-ball to determine the volume loss for (e-h) Titanium, ( i -l) HA coating, (m-p) HAC1 coating, q) Bar graph shows wear volume of the corresponding balls and r) Roughness of the counter-ball generated during wear. The error bar represents the standard deviation (n=3).

88. 13-08-2024 88 Table 7. Characterization from a ball-on-disc test of Titanium and HA-based HVOF-sprayed samples under dry and wet (SBF) environments. Samples Volume loss (mm 3 ) Wear Rate (mm 3 . N -1 . m -1 ) Wear Resistance Titanium 0.1483±0.003 2.965*10 -4 ±0.071 Base Titanium-SBF 0.127182±0.005 2.543*10 -4 ±0.112 14.23% HA 0.063709±0.004 1.274*10 -4 ±0.080 57.03% HA-SBF 0.06007±0.001 1.201*10 -4 ±0.022 59.49% HAC1 0.052448±0.002 1.048*10 -4 ±0.050 64.65% HAC1-SBF 0.040704±0.002 0.814*10 -4 ±0.050 72.54%

89. 13-08-2024 89 In-vivo osseointegration and toxicity assessment of bio-ceramics coated titanium rods in rabbit femoral bone. Objective - 6

90. 13-08-2024 90 Type of implants Left Femur (Test item) Right Femur (Control) Total No. of Implants HA 3 3 6 HA 3 3 6 HA 4 4 8 HAC1 3 3 6 HAC1 3 3 6 HAC1 4 4 8 Table 8: List of different types of implants embedded in the femoral bone of the rabbits. Objective 6 : Results and Discussion

91. 13-08-2024 91 Fig. 43: Histopathology images (a) HA coating, (b) HDPE (control), (c) HAC1 coating, and (d) HDPE (control) implanted in the femur bone of rabbits (n=10). The figure shows different inflammatory responses to the coatings. CNT-based implants showed ~ 11.4% improvement in the histological values (such as Polymorphonuclear cells, lymphocytes, macrophages, plasma cells, fibrosis, necrosis, fatty infiltration, neovascularization, and giant cells ) from the microscopic images of the implant sites. Implants Weight gain % (1 month) HA 11.23±0.47% HAC1 12.45±0.35% Table 9 : Weight gain % by rabbits.

92. 13-08-2024 92 Conclusions

93. 13-08-2024 93 Objective Conclusions Objective 1: To prepare the heterocoagulated alumina (19.5-18wt%)/carbon nanotube (CNT) (0.5-2wt%) powder followed by ball mixing with HA to study the retention and degree of dispersion of CNT in HA/alumina matrix. The different concentrations of CNT (0.5-2wt%) and alumina were successfully produced by heterocoagulation , followed by ball milling with HA (80 wt %). The dispersion index of the composite powder containing 2 wt % CNTs was 1.20, thereby suggesting that the matrix had a uniform CNT distribution. XRD results showed improvement in the crystallinity % for increased to 1 wt % and decreased for 2 wt % CNT. The same trend was observed for crystal size. The decrease in crystal size at high concentrations can be attributed to the reinforcements. The zeta potential for HA, HAC.5, HAC1, and HAC2 was found to be ~ -2.1 mV, -10 mV, -11 mV, and -21 mV, respectively, a ttributed to the increase in CNT concentration.

94. 13-08-2024 94 Objective Conclusions Objective 2: Hemocompatibility assessment of carbon nanotube doped hydroxyapatite worn debris of low-temperature thermally sprayed implants. HA, HAC0.5, and HAC1 composites up to 80 mg showed no hemolytic activity, with a hemolysis percentage of <10%. However, HAC2 showed little hemolytic behavior (>10%). The absorbance values for HAC1 (80 mg) and HAC2 (80 mg) were 0.927±0.038 and 1.184±0.128, respectively, higher than HA and negative control, therefore can be considered as non-thrombogenic. The percentage of platelet adhered to the HAC1 sample (~5.67%) showed a ~2.5-fold decrement to the clinically used negative control (~13.73%). M icroscopic images showed that none of the samples showed any platelet activation, except for HAC2 at 80 mg, which showed some pseuodopodial extensions, indicating platelet activation. These findings suggested that the 1 wt % CNT composite outperformed the others and can be considered an ideal candidate for coating metal implants.

95. 13-08-2024 95 Objective Conclusions Objective 3: A Comparative study of plasma spray, flame Spray, and HVOF sprayed-HA coatings based on their physiochemical, mechanical, and biocompatibility. HVOF coating displayed a ~10% and ~32% increase in crystallinity % compared to FS and APS coating , attributed to its low temperature and high velocity. Raman spectra showed a broad and wide band near 962 cm -1 and a left shoulder at 950 cm -1 for APS and FS coatings due to amorphous phases, indicating their reduced crystallinity, unlike narrower peaks for HVOF coating similar to feedstock, indicating its higher crystallinity. All the coatings showed negative surface charge and hydrophilic behavior ( θ CA <90º), owing to nanoporous HA coating on the titanium substrate. HVOF coating displayed a ~34% and ~120% improvement in adhesion strength than APS and FS coatings, respectively. The increased adhesive behavior for HVOF coating was attributed to its low porosity (~3%), high kinetic energy involved in the process, and low coating thickness.

96. 13-08-2024 96 Objective Conclusions Objective 3: A Comparative study of plasma spray, flame Spray, and HVOF sprayed-HA coatings based on their physiochemical, mechanical, and biocompatibility. HVOF coating exhibited a ~31% and ~59% improvement in hardness compared to APS and FS coating, respectively, owing to the dense microstructure (~97%) and low coating thickness (~55 µm). The scratch adhesion and the Vickers hardness test results indicate that the HVOF coating possesses better mechanical strength and, therefore, has the potential to mitigate the issues associated with the aseptic loosening of implants when used clinically. A capsule-like apatite structure over all the coatings was observed after 60 days of SBF immersion, and a considerable weight gain was measured over time, indicating their bioactivity. A Ca-rich apatite on the HVOF coating (Ca/P 60 days = 2.12) at the end of 60 days was witnessed due to its high degree of crystallinity and moderated hydrophilicity ( θ CA ~ 85º).

97. 13-08-2024 97 Objective Conclusions Objective 3: A Comparative study of plasma spray, flame Spray, and HVOF sprayed-HA coatings based on their physiochemical, mechanical, and biocompatibility. The HVOF-HA coating demonstrated exceptional blood compatibility compared to APS and FS coating, with significantly lower hemolysis rates of ~8 and ~11 times, respectively. This decrease in hemolysis % for HVOF coating could be due to their smooth surface (Ra = ~4 µm) and higher crystallinity. On the other hand, the release and dissolution of secondary HA phases into the blood might have resulted in increased hemolysis for APS and FS coatings. HVOF-HA coating showed a much higher absorbance value during each time point compared to other samples, suggesting its non-thrombogenicity. The non-thrombogenicity of HVOF coating is due to the synergy effect of coating properties like low porosity, smoothness, negative surface charge, and hydrophilicity. The coagulation cascade and the WB clotting time assays showed a delayed clotting time for HVOF coating compared to others.

98. 13-08-2024 98 Objective Conclusions Objective 3: A Comparative study of plasma spray, flame Spray, and HVOF sprayed-HA coatings based on their physiochemical, mechanical, and biocompatibility. The platelet adhesion % for the HVOF-HA coating exhibited ~1.25-fold decrement compared to the APS and FS coatings. HVOF coating and titanium exhibited round platelet morphology, similar to the control. APS and FS coatings displayed dendritic and spread dendritic platelet structures, indicating minimal activation may be attributed to the combined effect of high porosity, rough surface, and electrostatic interaction between the protein molecule (COOH - /NH 3 + ) and HA ions (Ca 2+ /PO 4 -3 /OH - ). Bare t itanium and coated samples showed poor antimicrobial properties due to their high affinity for organic substances, which promotes bacterial adhesion and replication. HVOF-HA coatings can be the best alternative to other commonly used thermal sprayed coatings to increase biomedical devices’ bioactivity, blood compatibility, and lifespan.

99. 13-08-2024 99 Objective Conclusions Objective 4: Biocompatibility assessment of the reinforced implants obtained with a suitable thermal spray process. Osteoblast cells and filopodia-like apatite structure over the HAC1 coatings were observed after 30 days of SBF immersion, and a considerable weight gain was measured over time, indicating their bioactivity. A Ca-rich apatite on the coating (Ca/P 30 days = 1.86) was witnessed due to its high degree of crystallinity and moderated hydrophilicity ( θ CA ~ 88º ). HAC1 coating exhibited a hemolysis ratio of 0.24±0.03, n=3. HAC1's non-hemolytic behavior was due to its high degree of crystallinity (91.23%). HAC1 coating exhibited a considerably lower percentage of platelets adhered (~4.72%) than the negative control polypropylene (~13.5%) and round-shaped platelets on its surface, indicating its non-thrombogenicity . The hydrophilic nature (~88º), dense microstructure (~97%), and negative surface charge (-11 mV) of the HAC1 coating result in low platelet adhesion and round platelet morphology, similar to the negative control

100. 13-08-2024 100 Objective Conclusions Objective 5: To determine and study the wear resistance of the best-performing reinforced coating using the ball-on-flat method HA and HA+19wt.%Al 2 O 3 +1wt.%CNT coatings' deposition on Ti-6Al-4V substrates was achieved using low-temperature-operated HVOF spraying. HVOF-sprayed HAC1 coating exhibited a very high crystallinity level (91.23%) compared to HA coating (88.62%) without major secondary phases attributed to the process’s low temperature, high velocity, and difference in thermal conductivity of reinforcements. Raman's spectrum displayed distinct HA, D, and G peaks for CNT in HA and HAC1 coatings. The peaks were located in the same positions as the feedstocks and had the same width, indicating similar phases. HAC1 coating displayed improvement in adhesion strength by ~62% and hardness by ~45% compared to the HA coating, owing to the uniform distribution of CNT, high degree of crystallinity (91.2%), dense microstructure (97.2%), and good interfacial bonding, resulting in exceptional tribomechanical properties.

101. 13-08-2024 101 Objective Conclusions Objective 5: To determine and study the wear resistance of the best-performing reinforced coating using the ball-on-flat method HAC1 coating demonstrated 1.16 times and 1.48 times reduction in CoF value compared to HA coating in dry and wet conditions, respectively, owing to the lubrication offered by the peeled-off graphite layers from the CNT surface. Adding reinforcements to HA coatings significantly improved the wear resistance of HAC1 coating by ~32% and ~17% under SBF and dry conditions, respectively, attributed to the decrease in the coefficient of friction and strengthening of splats by CNT bridging. HA coating displayed slightly higher CoF than blank titanium alloy under both conditions. This can be attributed to bare titanium’s very smooth surface texture. It can be concluded that the HVOF-sprayed HAC1 coating has successfully improved the wear resistance of the substrate. This is due to the increased hardness and adhesion strength attributed to the synergistic role of reinforcements (CNT/alumina), heterocoagulation effect, and the use of low temperature involved.

102. 13-08-2024 102 Objective Conclusions Objective 6: In-vivo osseointegration and toxicity assessment of bio-ceramics coated titanium rods in rabbit femoral bone. Coated titanium rods were implanted inside the distal femoral bone of rabbits for one month to determine their clinical implications using histopathological assessments. Titanium rods had two types of coatings: ( i ) HA coating and (ii) HAC1 coating. The coating thickness was 50-62 μ m. Throughout the study, all the rabbits displayed typical mobility with no signs of inflammation, infection, or allergic reactions at the surgical site. The weight gain by HA and HAC1 implanted animals measured at the end of 29 days were 11.23±0.47% and 12.45±0.35%, respectively, indicating good healthy behavior during the test period. Femoral bones were fully restored, and sound healing was achieved for all bone groups after 29 days of implantation. Good osseointegration, with no osteolysis or periostal reactions around the implants in the rabbits, was observed.

103. 13-08-2024 103 Objective Conclusions Objective 6: In-vivo osseointegration and toxicity assessment of bio-ceramics coated titanium rods in rabbit femoral bone. CNT-based implants showed an ~ 11.4% improvement in the histological values (such as polymorphonuclear cells, lymphocytes, macrophages, plasma cells, fibrosis, necrosis, fatty infiltration, neovascularization, and giant cells) from the microscopic images of the implant sites. These positive results are mainly due to CNTs and alumina, which accelerate bone growth, promote biomineralization, and inhibit osteoclastic bone resorption. CNTs stimulate osteoblast proliferation and adhesion, leading to faster bone repair. In sum, HVOF-sprayed HAC1 coating effectively improves wear resistance by enhancing hardness and adhesion strength. Furthermore, the in vitro study with SBF solution indicates its bioactivity, and various studies with human blood indicate its hemocompatibility. Additionally, the in vivo studies of the HAC1 implants with rabbits demonstrated its potential for human use.

104. 13-08-2024 104 Future Scope, limitation, and challenges

105. Future Scope, limitation, and challenges Hemocompatibility study with different genders and age groups is needed. Post-surface modification process/smart bioactive coating. With the increasing demand for HA in biomedical applications, exploring natural and renewable alternatives for its production is essential - Animal bones and scales. In-depth in-vivo studies with bigger animals like dogs are required before clinical implementation for a longer period. Anti-bacterial assessment with the reinforced implants is further needed. FDA approval is needed for new thermally sprayed implants. 13-08-2024 105

106. 13-08-2024 106 Publications

107. 13-08-2024 107 Publications

108. 13-08-2024 108 References

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