Novel Titanium Alloys for Tissue Engineering

Abstract

Taking into account the speed of industrial development and market request for novel biocompatible alloys, the urge of creating sustainable materials pushes the research forward. Among the many biomaterials that can be incorporated into the human body, in the class of metal alloys, titanium and titanium alloys are regarded as some of the most important biomaterials because of their resistance to the effects of body fluids, high tensile strength, flexibility, and corrosion resistance, as well as their unique combination of strength and biocompatibility. In present chapter several novel recipes for titanium alloys are presented and characterized (Ti-Mo-Si and Ti-Mo-Zr-Ta-Si systems).

1. Introduction

Biomaterials are substances that are utilized to replace a piece of a living system or work closely with a live tissue. Materials that come into touch with live tissues and their biological fluids might be natural, synthetic, or composite. Metals, polymers, ceramics, and composites are the four major categories into which biomaterials are categorized. They are utilized in the medical industry in a variety of forms, including complete pieces, particles/powders, fibers, thin films, thick coatings that are either porous or non-porous, etc. Implants and other medical devices range in size from a few nanometers to a few centimeters, with a variety of forms and geometries. Additionally, many biomaterials need their surfaces modified in order to provide the intended biological effects [1, 2].

Typically, biocompatible alloys are employed in bone replacement when we talk about them. Bone functions as an organ together with its numerous ancillary components, including connective tissue, cartilage, neuron, and vascular parts. They aid in protecting and supporting soft tissues while collaborating with skeletal muscles to enable movement of the body. Bones are rather hard structures, and they closely resemble one another in terms of shape and function [2]. Internal or external fixation is an element of the therapy for long bones since they are prone to damage. Another significant procedure where the bone has to absorb biomaterials is joint replacement. The process of regeneration is interrupted by the bone’s reaction to the biomaterial. However, because they are inert, materials placed in bone will cause local and systemic biological reactions. An adaptive and reactive process involving several factors will be triggered by host reactions to joint replacement and fixation materials [3, 4, 5, 6, 7, 8, 9].

The field of biomaterials is in continuous growth due to the aging population as well as general population growth. In an effort to regain form and function and increase the quality and duration of human life, biomaterials, which can be artificial or natural, are utilized to repair or replace a biological component that has been lost or destroyed. Biomaterials are employed in many different sections of the human body, including the heart, blood arteries, orthopedic implants, teeth, and stents [2, 10, 11]. They are also used as cardiac pacemakers and for urinary and digestive tract reconstructions. Of all, the largest number of implants is for orthopedic prostheses, such as those for the limbs, spine, hip and knee. According to the current growth in the demand for orthopedic implants, it doubles at least every 10 years, and the demand for knee prostheses triples. This is due to the degenerative illnesses that affect human joints, such as osteoarthritis (joint inflammation), osteoporosis (bone thinning), and trauma, which can cause pain or function loss. Due to excessive stress or the lack of the normal biological self-healing mechanism, degenerative illnesses cause the mechanical characteristics of the bone to deteriorate. Artificial biomaterials are the answers to these issues, and their surgical implantation aids in the functional restoration of structures that might otherwise be impaired. However, revision procedures for hip and knee implants have also grown, in addition to replacement surgery. These painful revision procedures are incredibly costly, and they also have a very poor success rate [2, 3, 4, 5, 6, 7].

The goal of current research is to create implants that can last far longer, maybe without failing, and even without the need for revision surgery. Therefore, it is crucial to design a suitable material with a long lifespan, great corrosion resistance in the body environment, a good mix of high strength and low Young’s modulus, high fatigue and wear resistance, high ductility, excellent biocompatibility, and no cytotoxicity [1].

Metallic biomaterials, which are typically utilized for support applications, need to have enough fatigue strength to endure the rigors of everyday activity. For applications like articular surfaces in joints and teeth, as well as bone bonding surfaces in implants, ceramic biomaterials are typically chosen for hardness and wear resistance. Polymeric materials have historically been employed for low-friction articulated surfaces due to their flexibility and stability. As a result, titanium becomes one of the most attractive engineering materials for several applications, and due to their exceptional qualities, in its biocompatibility, low modulus and corrosion resistance [5, 6, 7, 12, 13, 14]. Implant materials should not be harmful and should not result in allergic or inflammatory responses in people. The effectiveness of biomaterials is largely determined by how the body responds to the implant, and this determines a material’s biocompatibility. The host reaction a material induces and the degradation of the substance in the bodily environment are the two key determinants of a material’s biocompatibility.

The host and implant material respond in a variety of ways when exposed to human tissues and bodily fluids, and these interactions determine whether the implant material is acceptable to the human body.

Issues related to biocompatibility encompass thrombosis, which involves the clotting of blood and the adherence of blood platelets to the surface of biomaterials, as well as the formation of fibrous tissue around implanted biomaterials in soft tissues.

2. Necessary properties of biomaterials

A biomaterial’s entry into a living thing impacts how the implant and tissue interact, which might lead to opposing responses. They could be biological, mechanical, electrochemical, or toxicity. Even the utilized assembly or the nearby bone or tissue may sustain severe injury. These occurrences lead to corrosion on the surface of the implant, which degrades its quality depending on the quality of the biomaterial, the location of implantation, and other factors. A biomaterial must have one or more of the following characteristics, depending on the medical application to which it lends itself [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25].

2.1 Mechanical properties

The selection of a material type is influenced by crucial mechanical properties such as hardness, tensile strength, Young’s modulus, and elongation. Biomechanical incompatibility arises when an implant fractures due to mechanical failure. Consequently, it is expected that the chosen material for bone replacement would possess similar mechanical characteristics. The Young’s modulus of bone varies between 4 and 30 GPa [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26], depending on the type of bone and the direction of measurement.

2.2 Osseointegration

Implant loosening is caused by the implant surface’s failure to osseointegrate with the surrounding bone and other tissues as a result of motions [27]. Another crucial element of employing metal alloys in bone applications is osseointegration, which is the capacity to join with bone. For the implant to be safe and functional for the duration of its life, bone fusion with the implant is necessary. It is evident that surface chemical and physical features have a significant impact on implant-surface interactions through altering cell behavior, growth factor production, and osteogenic gene development, despite the fact that surface cellular processes are not well known [5, 15, 16, 17, 18].

In certain scenarios, despite achieving initial stability of the implant, the bone may experience retraction or become disconnected from the implant due to various factors [14, 28, 29, 30]:

• The implant’s reaction with foreign substances, leading to toxic emissions from the implant.

• Bone damage or injury resulting from surgical trauma.

• Imposition of abnormal or nonphysiological conditions on the bone, such as fluid pressures or movement against implant components.

• Modification of mechanical signals that promote bone densification.

• Reduction of stress or protection of the replaced or neighboring bone.

2.3 Corrosion testing

In some measure, all metal implants corrode electrochemically. This is bad because the structural integrity is reduced during the degradation process, and the degradation products may negatively affect the host. Wear and electrochemical dissolution are both causes of metal implant deterioration, however the two are most frequently combined to cause degradation. Both generalized dissolution, which evenly affects the whole surface, and localized regions of a component are involved in the electrochemical corrosion process [14, 30]. The strength of the oxidation/reduction processes that drive corrosion (thermodynamic driving forces) and the physical barriers that regulate the kinetics of corrosion are what determine how much metal implant corrosion occurs. These two factors that affect how biomaterials corrode in real life can be broken down into a variety of factors, including: geometrical parameters (e.g. surface microstructure, oxide structure and composition), mechanical variables (e.g. stress and/or relative motion) and solution variables (e.g. pH, proteins in solution and enzymes) [2].

Because metal alloys come in touch with a particularly harsh environment, such as bodily fluid due to the presence of chloride ions and proteins, corrosion resistance of a surgically implanted alloy is a crucial property. The alloy’s metal components are converted to their ionic states during the corrosion process, and the dissolved oxygen is transformed into hydroxide ions. The passive coating that forms on an alloy’s surface and the presence of alloying elements have a significant impact on how well an alloy resists corrosion.

2.4 Wear resistance

Because of the heterogeneous lubrication regime, wear always happens in artificial joints. An prosthetic hip joint moves, creating billions of minute particles that are removed by the motions. The joint capsule’s tissues are home to these particles, which might cause unfavorable responses from external bodies. Histocytes and large cells phagocytose the discharged particles and produce tissues that resemble granulomas. They interfere with bone resorption, which causes osteolysis, at the level of the layer that separates the implant from the bone. The performance of the device is influenced by the use of different materials for the femoral head and cup. Over time, various material and surface treatment combinations have been employed in the development of endoprostheses to minimize wear. Currently, the primary metallic materials utilized for biomedical applications include 316L stainless steel, cobalt chromium alloys (CoCr-), titanium-based alloys (such as Ti-6Al-4V, TiMo-), as well as other materials like tantalum, gold, and dental amalgams. Titanium alloys are increasingly preferred over other materials due to their exceptional qualities, including high strength, low density, excellent corrosion resistance, complete inertness in the body environment, improved compatibility, low Young’s modulus, and the ability to fuse with bones or other tissues. These alloys outperform traditional stainless steels and cobalt-based alloys due to their lower Young’s modulus, enhanced biocompatibility, and superior corrosion resistance [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. Consequently, titanium and titanium alloys are commonly employed as replacements for hard tissues in artificial bones, joints, and dental implants, given their aforementioned attributes.

2.5 Biocompatibility

One of the most important properties of biomaterials is biocompatibility, which refers to the ability of a material to perform its intended function without causing adverse reactions in the body. The biomaterial should not elicit toxic or immune responses and should be well-tolerated by the host tissue.

2.6 Bioactivity

Biomaterials may have inherent bioactivity, meaning they can interact with the surrounding biological environment to facilitate specific biological responses. For example, bioactive materials can promote cell adhesion, proliferation, and differentiation to support tissue regeneration.

3. Titanium and its alloys

William Gregor initially found titanium as a metal in 1791, but Klaproth gave it the name titanium in 1795. Titanium and its alloys are used in a variety of fields including shipbuilding, aviation, automotive, energy, medicine, and sports equipment due to their low density, high strength-to-weight ratio, good biocompatibility, improved corrosion resistance, plasticity, and good mechanical properties.

Titanium and its alloys are extensively used as biomaterials due to their exceptional biocompatibility, remarkable corrosion resistance, and favorable mechanical properties. These metals possess a specific elastic modulus and low density, which enables them to exhibit mechanical behavior similar to that of bones. Among the materials suitable for implantation in the human body, titanium stands out as it is lightweight, durable, and biocompatible. In the field of biomedicine, commercial pure titanium (C.P. Ti) and its alloys, such as Ti-6Al-4 V and Ti-Mo, are widely employed. They serve as popular alternatives for hard tissue in artificial joints, bones, and dental implants. The low elastic modulus of titanium and its alloys is often considered advantageous in terms of biomechanics, as it can lead to reduced stress shielding [3, 4, 5, 6, 7]. Another noteworthy characteristic of titanium-based materials is their tendency to form an extremely thin, adherent, and protective coating of titanium oxide. This feature further enhances the potential of titanium and its alloys as promising biomaterials for implants. One of the main requirements for titanium and its alloys’ exceptional biocompatibility and corrosion resistance is the existence of this spontaneously generated oxide coating during the passivation or repassivation process.

Due of its poor mechanical qualities, cp titanium (purely commercial) is used mostly for dental implants in terms of medical applications. Alloys with Al, V, Mo, and other elements are utilized in applications where good mechanical properties are necessary, such as hip implants, knee implants, bone screws, and plates. Artificial hip joints, which comprise of an articulated bearing (femoral head and cup) and stem, are one of the most prevalent uses for titanium alloys. The metal parts of the hip cup and stem are constructed of titanium. They frequently protect the knee joint and have a polyethylene joint surface and titanium femoral and tibial components [3, 4, 5, 6, 7].

By adding alloying elements, it gives titanium a wide field of applicability through different microstructure and properties. Thus, the alloying elements are divided into three categories:

• stabilizers α: C, N₂, O₂, Al;

• β stabilizers: V, Nb, Mo, Ta, Fe, Mn, Cr, Co, W, Ni, Cu, Si, H₂;

• neutral elements: Zr, Sn, Hf, Ge, Th.

In the medical field, the Ti-Al-Mo, Ti-Al-Cr, Ti-Al-V, Ti-Al-Cr-Mo systems are used, with excellent biocompatibility in contact with living tissues. Titanium grade 4JSO 5832/11 is used for the manufacture of dental implants. The advantages of using titanium in implantology reside in resistance, reliable technological process of obtaining, easy processing by conventional and non-conventional means, acceptable price.

3.1 Beta-type titanium alloys

Beta-type titanium alloys primarily contain high concentrations of beta-phase stabilizers, giving these alloys a predominantly beta structure at room temperature. While pure titanium has a hexagonal close-packed (hcp) crystal structure, beta-type alloys can contain beta phases with a body-centered cubic structure, providing a unique combination of mechanical and chemical properties.

Some characteristics of beta-type titanium alloys include:

• High strength: these alloys can exhibit superior mechanical strength, making them suitable for high-demand applications such as in the aerospace industry or military components.

• Corrosion resistance: titanium itself is already corrosion-resistant, but beta-type alloys can enhance this property, making them ideal for use in aggressive environments, such as in the oil and maritime industries.

• Biocompatibility: beta-type titanium alloys are known for their high biocompatibility, which means they can be used in medical applications, such as orthopedic or dental implants.

• Ductility and formability: these alloys can be processed to obtain a variety of complex shapes due to their ductility.

• High operating temperature: some beta-type alloys can be used in applications that require performance at high temperatures.

• High strength-to-weight ratio: due to the lightweight nature of titanium and the good mechanical properties of beta-type alloys, these materials have an impressive strength-to-weight ratio, making them ideal for aerospace and space applications.

It is important to mention that the specific properties of a beta-type titanium alloy may vary depending on its exact chemical composition and the manufacturing processes used. By adjusting the proportions of alloying elements, titanium alloys with specific properties can be obtained for various industrial/medical applications.

3.2 Applications of titanium and its alloys

Titanium and its alloys have found extensive applications in various fields due to their unique combination of properties. Here are some few examples of the diverse applications of titanium and its alloys. The unique properties of titanium make it a valuable material in various industries where high strength, corrosion resistance, biocompatibility, and lightweight characteristics are essential.

Biomedical implants: titanium and its alloys are widely used in the field of biomedical implants, such as orthopedic implants (hip and knee replacements, bone plates, and screws), dental implants, and cardiovascular implants. Their biocompatibility, corrosion resistance, and high strength-to-weight ratio make them suitable for long-term implantation in the human body.

Aerospace industry: titanium alloys are extensively utilized in the aerospace industry due to their exceptional strength, lightweight nature, and high heat resistance. They are used in aircraft components, including structural components, landing gear, engine parts, and fasteners, where strength and corrosion resistance are critical.

Chemical processing: titanium and its alloys are resistant to corrosion in various aggressive chemical environments, making them suitable for chemical processing equipment. They are used in reactors, heat exchangers, pipes, valves, and other components that come into contact with corrosive substances.

Marine industry: the excellent corrosion resistance of titanium and its alloys makes them valuable for marine applications. They are used in ship hulls, propellers, offshore structures, and desalination plants, where exposure to seawater and harsh marine environments is prevalent.

Sports equipment: titanium and titanium alloys are employed in sports equipment, particularly in the production of bicycles, golf clubs, tennis rackets, and other lightweight and high-performance sporting goods. The strength, durability, and lightness of titanium contribute to improved performance and reduced weight.

Automotive industry: titanium alloys find limited use in the automotive industry, primarily in high-performance vehicles. They are utilized in exhaust systems, engine components, suspension systems, and other parts where high strength and corrosion resistance are required.

Architecture and design: titanium is increasingly being used in architectural applications due to its esthetic appeal, durability, and resistance to corrosion. It is utilized in building facades, roofs, sculptures, and other artistic structures.

Electronics and consumer goods: titanium and its alloys are utilized in the production of electronic devices and consumer goods. They are employed in smartphones, laptops, watches, jewelry, eyewear frames, and other products where lightweight, corrosion resistance, and esthetic qualities are desired.

4. Novel titanium alloys

Titanium alloys have gained significant popularity and are widely preferred for orthopedic implants in the biomedical field.

In recent years, our research group has focused on obtaining and characterizing new titanium-based alloy recipes with non-toxic elements. This chapter contains a copy of our latest research.

In the study, a series of new recipes were taken, thus several elements were selected such as: Mo, Zr, Ta and Si, to create a β-type titanium alloy. In Table 1 is presented two main groups of alloys have been developed TMZT (Ti-Mo-Zr-Ta) and TMS (Ti-Mo-Si) with low elastic modulus, strength and good biocompatibility. The titanium alloys were developed in a vacuum electric arc furnace using high purity metals: Ti 99.80%, Mo 99.70%, Zr 99.20%, Ta 99.50% and Si 99.20%. The samples were remelted consecutively at least five times for homogenization.

Proposed alloy	Abbreviation
Ti20Mo	TM
Ti20Mo0,5Si	TM0.5Si
Ti20Mo0,75Si	TM0.75Si
Ti20Mo1Si	TM1Si
Ti20Mo7Zr15Ta	TMZT
Ti20Mo7Zr15Ta 0,5Si	TMZT0.5Si
Ti20Mo7Zr15Ta 0,75Si	TMZT0.75Si
Ti20Mo7Zr15Ta 1Si	TMZT1Si

Table 1.

Developed Ti-based alloys.

The vacuum electric arc furnace is chosen as the method for obtaining titanium alloys due to its ability to produce high-purity materials by minimizing contamination from impurities in the controlled vacuum environment [15, 20].

Adding silicon to Ti-Mo (TM) and Ti-Mo-Zr-Ta (TMZT) alloys can bring several significant benefits. Silicon is an alloying element with specific properties that can enhance the performance and characteristics of these alloys. Here are some of the advantages of adding silicon to these alloys:

• Phase stabilization: silicon can help stabilize the alpha and beta phases of titanium. This can lead to improved crystal structure and mechanical properties of the alloys.

• Increased strength: the addition of silicon can contribute to the increased strength of Ti-Mo and Ti-Mo-Zr-Ta alloys. This is important in applications that require strong and durable materials.

• Improved corrosion resistance: silicon can enhance the corrosion resistance of these alloys, making them suitable for use in aggressive environments or industrial applications.

• Enhanced biocompatibility: the addition of silicon can improve the biocompatibility of titanium alloys. This is essential in medical applications, such as orthopedic implants or other medical devices.

• Reduced density: silicon has a lower density than titanium, which can lead to a slight reduction in the density of the alloys. This can be beneficial in applications that require lighter materials.

• Improved high-temperature behavior: silicon-containing titanium alloys can exhibit better stability at high temperatures, making them suitable for applications involving high-temperature conditions.

It is essential to mention that the exact proportion of silicon and other alloying elements will influence the specific properties of the alloys. Manufacturing processes and heat treatments can also play a crucial role in developing the final characteristics of the alloys.

In conclusion, adding silicon can bring multiple benefits to Ti-Mo and Ti-Mo-Zr-Ta alloys, enhancing their performance and expanding the range of possible applications. In the rest of the chapter, the Ti-Mo, Ti-Mo-Si, Ti-Mo-Zr-Ta and Ti-Mo-Zr-Ta-Si systems will be abbreviated as TM, TMS, TMZT and TMZTS.

4.1 Methodology

Characterization of biomaterials for medical applications involves a comprehensive assessment of their physical, chemical, mechanical, and biological properties.

The new alloys obtained were characterized by the following important techniques in order to demonstrate biocompatibility:

Chemical composition analysis: determining the chemical composition of biomaterials is essential to ensure the desired properties and performance. Techniques such as energy-dispersive X-ray spectroscopy (EDS) can be used to identify the elemental composition. EDS analysis was carried out using an SEM VEGA II LSH electron microscope (TESCAN Co., Czech Republic) equipped with an EDX BRUKER/ROENTEC Co detector (Germany).

Microstructural analysis: this involves examining the microstructure of biomaterials at various length scales using techniques such as optical microscopy, scanning electron microscopy (SEM). Microstructural analysis provides information about the material’s surface morphology, grain structure, porosity, and presence of any defects or impurities. The microscopy images, providing high-resolution visualizations, were acquired using a Zeiss Imager 1 M optical microscope, renowned for its advanced imaging capabilities. The microscope was equipped with specialized light and dark field filters, enabling the observation of samples under different illumination conditions and enhancing the contrast and visibility of various features and structures of interest. To investigate the phases present in the materials, we used a Panalytical X’Pert Pro MPD Diffractometer (Malvern Panalytical, The Netherlands).

Mechanical testing: the indentation test is a common method used for mechanical characterization of biomaterials and other materials. It involves applying a controlled force or load to the surface of the material using an indenter, typically a diamond or a hardened steel ball, and measuring the resulting indentation depth or the load-displacement relationship. These tests provide crucial information about the material’s ability to withstand mechanical forces and its suitability for specific applications. Some mechanical properties were determined utilizing a CETR UMT-2 tribometer (Bruker, Campbell CA, USA). The assessment involved applying a force of 5 N across a 4 mm distance. The calculated value was the average of three repetitions, and analysis was conducted using the ViEWER program.

Biocompatibility assessment: biomaterials intended for medical applications must undergo biocompatibility evaluation. This involves in vitro and in vivo tests to assess the material’s compatibility with living tissues. Techniques include cell culture studies to evaluate cell adhesion, proliferation, and viability, as well as animal studies to assess tissue response, inflammation, and immune reactions.

In vitro biocompatibility tests were conducted on titanium alloy samples by incubating Albino rabbit fibroblasts with the metallic components, measuring cellular metabolic activity using the MTT assay, and comparing the results with a control group; the cell viability was calculated as a percentage based on absorbance readings, and live cells were stained with calcein-AM for fluorescence microscopy imaging.

In vivo studies were conducted on female sheep to evaluate the integration of titanium alloy fragments implanted in the tibial crest, following surgical procedures and ethical guidelines; radiographs and high-resolution CT scans were used to monitor bone repair and peri-implant tissue microstructure, while histological procedures and immunohistochemical staining provided insights into the tissue response and expression of osteopontin, MMP2, and MMP9.

4.2 Results and discussion

4.2.1 Obtaining

The study on the elaboration of Ti-based alloys suggested using an electric arc furnace under a protective atmosphere to ensure homogeneous alloys without metallic inclusions, utilizing high-purity raw materials and a sequence of operations including vacuum melting, purging, and multiple remelting cycles, followed by the preparation of test specimens through cutting, embedding, grinding, and polishing processes.

4.2.2 Chemical composition

The composition, as detailed in Table 2 [18], provides a comprehensive overview of the specific elements and their respective proportions present in the material under investigation. This table serves as a valuable reference, offering insights into the alloy’s composition and aiding in the understanding of its properties and potential applications in various fields. We mention that EDS is not a quantitative method to determine the elemental composition of alloys.

Alloy	Mo	Si	Zr	Ta	Ti
TM	6.01	—	—	—	to 100%
TM0.5Si	6.03	0.14	—	—	to 100%
TM0.75Si	6.12	0.22	—	—	to 100%
TM1Si	6.05	0.31	—	—	to 100%
TMZT	19.00	—	8.15	14.50	to 100%
TMZT0.5Si	18.50	0.45	7.00	14.80	to 100%
TMZT0.75Si	19.50	0.75	6.85	15.04	to 100%
TMZT1Si	19.83	1.03	6.93	14.98	to 100%

Table 2.

Mass composition of alloys obtained by EDS [15, 20] .

4.2.3 Microstructural results

In Figures 1 and 2 the specific structure of the studied alloys can be observed, images obtained by optical microscopy. They have a dendritic structure for TMZT alloys. In TMS alloys, a β-type structure can be observed, with the formation of equiaxial grains of different sizes. Acicular and coarse structures specific to β structures are also present [18].

Figure 1.

Optical microstructure of the alloys at 100X. (a) TM, (b) TM0.5Si, (c) TM0.75Si, and (d) TM1Si [17].

Figure 2.

Optical microscopy images (a) TMZT, (b) TMZT0.5Si, (c) TMZT0.75Si, and (d) TMZT1Si [15].

The beta structure with dendrites is a characteristic feature observed in certain titanium-based alloys, particularly those containing β-stabilizing elements such as Mo, Zr, Ta, and Si. The beta structure refers to the body-centered cubic (BCC) crystal structure of the beta phase in titanium alloys.

During the solidification process, when the alloy undergoes cooling and transforms from a liquid to a solid state, dendritic growth can occur. Dendrites are tree-like, branching crystal structures that form as a result of anisotropic growth kinetics. In the case of titanium alloys, dendritic growth is commonly observed in the beta phase.

The dendritic structure in the beta phase is formed due to the preferred crystallographic growth directions, resulting in the elongated and branched morphology of the grains. The dendritic branches extend into the surrounding matrix, creating a complex network of interconnected structures. This dendritic morphology provides an increased surface area, allowing for more efficient diffusion and solid-state transformations within the material.

The presence of dendrites in the beta phase affects the microstructure of the alloy. The dendritic structure influences various properties of the material, including mechanical strength, thermal stability, and phase transformation behavior. The size, shape, and distribution of dendrites can be controlled through different processing techniques, such as adjusting the cooling rate during solidification or employing grain refiners.

Furthermore, the dendritic structure in the beta phase can have an impact on the macroscopic properties of the alloy. For example, the alignment and connectivity of dendrites can influence the anisotropy of mechanical properties, such as yield strength and fracture toughness. The interconnected dendritic network can also affect the diffusion paths and the kinetics of phase transformations, leading to changes in the alloy’s thermal and chemical stability.

Based on the study of Ti-based alloys, including TMS and TMZTS systems, it can be concluded that the microstructure of these alloys is strongly influenced by the elaboration method employed.

Based on the diffractograms of the TMxSi (x = 0, 0.5, 0.75, 1 wt.%) TMZTxSi alloys (x = 0, 0.5, 0.75, 1 wt.%), it can be concluded that the alloys exhibit a predominantly face-centered cubic (fcc) structure (Figure 3). The primary phase present in these alloys is the β phase, which is characterized by a centered volume cubic structure. However, there are also minor secondary phases observed, including the α0 martensite phase and the α phase.

Figure 3.

Diffractograms alloys studied [15, 17, 20].

The β phase in the diffractograms of Ti-based alloys refers to a specific crystallographic phase known as the beta phase. This phase is characterized by a centered volume cubic structure and is commonly present in Ti alloys. The diffractograms indicate the presence and relative abundance of the β phase in the analyzed TMZTS alloys.

The β phase is an important constituent in Ti alloys as it significantly influences their mechanical properties, such as strength and ductility. It is a solid solution phase that can accommodate various alloying elements, including Mo, Zr, Ta, and Si, depending on the specific alloy composition. The presence of these alloying elements can modify the lattice parameters and stabilize the β phase, leading to improved mechanical performance.

In the diffractograms, the peaks corresponding to the β phase can be identified and analyzed to determine the crystallographic orientation and phase purity. The positions and intensities of these peaks provide information about the arrangement of atoms within the crystal lattice and the presence of any additional phases.

The diffractograms may also reveal the presence of minor secondary phases, such as the α0 martensite phase and the α phase. These secondary phases can form due to the decomposition of the β phase during the cooling process or as a result of specific alloy compositions. Their presence can affect the overall microstructure and mechanical behavior of the Ti-based alloys.

By analyzing the diffractograms, researchers can gain insights into the crystalline structure, phase composition, and phase transformations occurring in TMZTS alloys. This information is valuable for understanding the alloy’s properties and optimizing its performance for various applications, such as aerospace, biomedical implants, and structural materials.

4.2.4 Mechanical properties

In Table 3 are presented the specific mechanical properties obtained through indentation for each alloy within the TMS and TMZT systems, highlighting their respective Young’s modulus, hardness, and specimen Poisson Ratio. This analysis will provide valuable insights into the mechanical behavior and potential applications of these alloys, aiding in the advancement of materials science and engineering.

Alloy	Young modulus (GPa)	Hardness (HV)	Specimen Poisson Ration
TM	57.54	376.30	0.23
TM0.5Si	37.86	239.60	0.23
TM0.75Si	29.06	216.20	0.23
TM1Si	26.22	210.80	0.23
TMZT	53.58	305.34	0.23
TMZT0.5Si	54.25	339.24	0.23
TMZT0.75Si	56.38	315.27	0.23
TMZT1Si	63.88	274.64	0.23

Table 3.

Some mechanical characteristics of alloys obtained by indentation [15, 20].

The comparison reveals how different alloy compositions within each system impact the mechanical properties. Additionally, the comparison between the TMS and TMZT systems provides valuable information on the influence of alloying elements on the overall material characteristics. Figure 4 shows a graphic comparison of the modulus of elasticity between the classic alloys and the newly developed titanium alloys. As can be seen, the newly developed alloys have a very low modulus of elasticity compared to classic alloys.

Figure 4.

Graphical comparison of the modulus of elasticity.

The TMZT system generally displays higher Young’s modulus and hardness values compared to the TMS system. This implies that the TMZT system alloys possess greater stiffness and better resistance to wear and deformation. However, it’s important to note that the specific alloy compositions and intended applications can further influence the performance and suitability of these systems.

In orthopedics, the Young’s modulus is of paramount importance as it directly influences the performance and behavior of implant materials used in prosthetic devices, joint replacements, and other orthopedic applications. A suitable Young’s modulus is crucial to ensure that the implant material closely matches the mechanical properties of the surrounding natural tissues. If the implant’s Young’s modulus is significantly different from that of the bone or tissue, it may cause stress concentrations, leading to potential implant failure, discomfort, or long-term complications.

Comparing Ti-based alloys to classical alloys used in orthopedics, such as stainless steels or cobalt-chromium alloys, the Ti-based alloys obtained exhibit a lower Young’s modulus. This lower modulus makes Ti-based alloys more favorable for orthopedic implants since they are closer in mechanical properties to natural bone. By mimicking the elasticity of bone, Ti-based alloys can help reduce the stress shielding effect that occurs when a stiffer implant causes bone resorption due to inadequate load transfer to the surrounding bone. Consequently, Ti-based alloys have the advantage of potentially providing a more successful and long-lasting integration with the patient’s bone, improving the overall performance and biocompatibility of orthopedic implants.

Overall, these alloys exhibit a range of mechanical characteristics, such as stiffness, hardness, and consistent response to applied forces. Each alloy’s unique combination of properties makes them suitable for specific applications, allowing for versatility and tailored performance in various engineering scenarios.

4.2.5 In vitro studies

Biocompatibility in vitro is of paramount importance in the evaluation and development of biomedical materials and implants. It involves studying the interactions between the materials and living cells under controlled laboratory conditions. The importance of in vitro biocompatibility testing lies in its ability to provide valuable insights into the potential effects of materials on cellular behavior, viability, and functionality.

By subjecting materials to cell cultures, researchers can evaluate factors such as cell adhesion, proliferation, metabolic activity, and morphology. These assessments help determine the compatibility of the materials with living tissues and cells, providing essential information for biomedical applications. Furthermore, in vitro biocompatibility testing allows for the identification of potential cytotoxic effects or adverse reactions that may arise from the interaction between materials and cells.

In the case of the presented study, primary Albino rabbit fibroblasts were utilized to assess the biocompatibility of the alloys. The use of standardized protocols and tests such as the MTT assay ensures reliable and reproducible data, allowing for accurate comparisons between different materials and controls.

Understanding the biocompatibility of materials is crucial for the development of safe and effective biomedical devices, implants, and drug delivery systems. In vitro testing serves as an initial step in the evaluation process, providing valuable information before progressing to more complex in vivo and clinical studies.

Experimental procedures:

• The biocompatibility of the alloys was assessed through in vitro tests using primary Albino rabbit fibroblasts.

• Cellular metabolic activity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the results were compared with a control group [20, 30, 31, 32, 33].

• A solution called penicillin/streptomycin/neomycin (P/S/N), composed of specific quantities of penicillin, streptomycin, and neomycin in sterile water, was used as the standard modified Dulbecco’s Eagle medium (DMEM) for cell culture [34, 35, 36, 37].

• The experimental procedure involved seeding cells in 12-well plates at an initial density of 5 × 10³ cells/cm² in complete medium and incubating them overnight at 37°C and 5% CO₂.

• The alloy samples were disinfected using a sterile 70% ethyl alcohol solution for 30 min, followed by washing with sterile water and phosphate-buffered saline (PBS) [34, 35, 36, 37].

• Decontaminated samples were then placed in DMEM complete culture media and cultured for 24 h at 37°C, 5% CO₂, and 97% humidity.

• The pre-plated cells received the sterilized alloy samples and were cultured for different time intervals (24, 48, and 72 h).

• MTT assays were performed at each time point to assess cell viability.

• After the incubation, MTT working solution was added to the cells and incubated in the dark at 37°C and 97% humidity.

• The formation of MTT formazan crystals, indicative of live cells, was observed and dissolved using acidified isopropanol.

• The colored solution was measured at a wavelength of 570 nm using a Tecan plate reader modified with Magellan V7.1SP1 Sunrise model software.

• Cell viability was calculated as a percentage using absorbance values, comparing cells cultured with the metal alloys to the control group incubated with PBS [20].

• Additionally, live cells were stained with calcein-AM, a fluorescent dye that produces green fluorescence in live cells.

• Albino rabbit fibroblasts and the human osteosarcoma cell line MG63 were employed for this staining.

• The cell-populated metal alloys were washed with HBSS and stained with calcein-AM according to the kit’s instructions.

• The hydrolyzed calcein fluorescence was visualized using a fluorescence microscope with a 455 nm excitation and 530 nm emission filter [20].

In conclusions, the cytocompatibility assessment of the metallic materials was successfully conducted using in vitro tests with cell cultures. Primary Albino rabbit fibroblasts were chosen as the cell model for these experiments. The evaluation of cell viability was performed through the MTT test, which involved incubating the metal alloys with cell cultures for varying durations (24, 48, and 72 h). These assessments provided valuable insights into the biocompatibility of the alloys and their potential for biomedical applications.

Figures 5 and 6 present the data obtained from the MTT assay, showing the calculated cell viabilities for the different alloys at each time point. These figures provide a visual representation of the cytocompatibility of the alloys and allow for a direct comparison with the control group.

Figure 5.

Mitochondrial activity measured via the MTT assessment for TMS group [20].

Figure 6.

Mitochondrial activity measured via the MTT assessment for TMZTS group [15].

The results of the cytocompatibility tests for both TMS and TMZTS systems revealed promising outcomes. According to the ISO 10933-5 standards, all tested TMZTS alloys demonstrated cytocompatibility, as shown in Figures 5 and 6. This indicates that these alloys have the potential to interact favorably with biological systems without causing harmful effects.

The calculated cell viability for all examined materials surpassed the threshold of 85%, which is a significant indicator of their biocompatibility. This suggests that the cells were able to maintain their metabolic activity and proliferate effectively in the presence of the alloys. The high cell viability further supports the notion that these materials have the potential to be used in biomedical applications.

Interestingly, the addition of silicon to the alloys did not have any discernible impact on the vitality of the fibroblasts. This implies that the incorporation of silicon did not compromise the biocompatibility of the alloys and suggests that Si can be effectively incorporated into these systems without compromising their cytocompatibility.

These findings are consistent with earlier investigations cited in the references [20, 38, 39, 40, 41, 42]. The alignment of these results with previous studies reinforces the reliability and validity of the current findings, adding to the body of knowledge supporting the cytocompatibility of TMZTS alloys. Collectively, these results provide strong evidence that the TMZTS alloys have excellent biocompatibility, making them suitable candidates for various biomedical applications.

After 72 h of cell culture involving primary albino rabbit fibroblast cells and the human osteosarcoma cell line MG63, the fibroblasts were extracted from the alloy samples and their morphology was examined using phase contrast and fluorescence optical microscopy. Figure 7 illustrate the final photographs obtained from this analysis.

Figure 7.

Fluorescent microscopy (calcein AM) results for TMZTS alloys in contact with cell cultures for 72 h [15, 20].

The microscopy observations revealed that the cells formed a monolayer in direct contact with the alloy samples, which appeared as dark areas in the images. Interestingly, there were no significant differences observed in comparison to the growth of the control sample. The cells displayed elongated shapes with a fibroblastic morphology, while the osteoblasts appeared smaller and more spherical. The presence of dark regions in the images could be attributed to the positioning of substances, indicating that the material might generate mechanical activities that potentially compromise the integrity of the cell monolayer.

Regarding cell growth density, the line osteoblasts exhibited a higher density compared to primary fibroblasts. This difference can be attributed to the behavior of the line cells, specifically MG63 osteoblasts, which have a faster growth rate compared to the more sensitive primary cells [20, 39, 40, 41, 42]. However, a comprehensive analysis of the microscope data for both types of cultures demonstrates that the TMS and TMZTS alloys do not have any discernible effect on cell shape or growth. Both the fibroblast and osteoblast cells displayed comparable appearances to the growth control, indicating a high level of cytocompatibility [20, 39, 40, 41, 42].

The microscopy analysis provides strong evidence supporting the cytocompatibility of the TMS and TMZTS alloys. The cells maintained their normal growth patterns and shapes when in contact with the alloy surfaces, reinforcing the potential of these alloys for various biomedical applications where maintaining cell viability and morphology is necessary.

4.2.6 In vivo studies

In vivo studies are of great importance as they provide crucial insights into the behavior and performance of materials or interventions within living organisms, allowing for a more comprehensive evaluation of their safety, efficacy, and compatibility. These studies bridge the gap between in vitro experiments and clinical applications, providing valuable information on biological responses, tissue interactions, and potential adverse effects in a complex physiological environment. By simulating real-life conditions, in vivo studies play a pivotal role in guiding the development, optimization, and translation of new materials, treatments, and medical devices, ultimately enhancing patient outcomes and ensuring the highest standards of biomedical research.

For in vivo study, TMS alloys were implanted into the tibial crest of four adult female sheep (one control and three experimental), following a surgical procedure and strict adherence to ethical guidelines and regulations (Figure 8). And the TMZTS alloys were implanted 60-day on five rabbits (Orychtolagus cuniculus) of both genders (Figure 9).

Figure 8.

X-ray images from the control (C) and experimental sheep (E1–E3) for TMS alloys at the 62 days of experiment [20].

Figure 9.

X-rays of control and experimental rabbits after 60 days of experiment for TMZTS alloys [15].

Experiments on the TMS system reconfirmed the use of Ti-based alloys, known as bioinert biomaterials, in the implantation process induces contact and distance osteogenesis through an interference bond with the surrounding tissue. The blood parameters of the control and experimental sheep remained stable throughout the experiment, indicating no significant changes. The spontaneously formed titanium oxide layer proved to be highly stable, effectively separating the alloy from neighboring tissues. X-ray and CT evaluations demonstrated the presence of a clear space surrounding each implant, bordered by fibrous-cartilaginous tissue, without any local abnormal bone reactions.

In the control sheep, moderate physiological osteolysis was observed in the area of the entrance channel, filled with newly formed low-density tissue. The titanium alloys (TM and TM0.5Si) exhibited a greater amount of connective tissue separating them from the bone tissue, with no local reactions. However, the TM0.75Si alloy showed a faster binding to the surrounding tissue, as confirmed by histological examination. The presence of molybdenum and silicon in the alloys contributed to their excellent stability and compatibility, comparable to pure titanium.

Histological analysis revealed periosteum proliferation and ossification following a membranous model in the experimental groups, while a mixture of membranous, enchondral, and membranous ossification was observed in the control sheep. The connective tissue near the bone breach exhibited rich vascularity, and the capsules surrounding the implants were thick and poorly vascularized. The connective tissue contained osteoprogenitor cells and newly formed bone tissue. Bone trabeculae alternated between mineralized and non-mineralized areas. Mesenchymal stem cells differentiated into osteoblasts, actively contributing to bone formation.

Immunohistochemical analysis demonstrated the presence of osteopontin (OSP) in cells, indicating intense activity of osteoprogenitor cells involved in matrix mineralization and osteoblasts in the areolae. MMP2 and MMP9 expression was observed in the interface matrix between the implant and the bone remodeling area. MMP2 was particularly present in osteocytes, involved in solubilizing the osteoid. MMP9 played a role in controlling osteoclast maturation and migration, with expression observed in mesenchymal stem cells, osteoblasts, osteocytes, and giant multinucleated cells.

For TMZTS system, the X-ray investigations conducted on experimental rabbits after 60 days of the experiment revealed no abnormal radiological changes in both the control and implanted rabbits. The radiodensity of the peri-implant tissues varied depending on the alloy used, with values ranging from 300 to 931 Hounsfield Units (HU) for peri-implanted tissues in control and implanted rabbits. The newly formed tissue in the implantation gap exhibited a radiopacity of about 793 HU, while the radio-opacity in the peri-implant area ranged from 633 to 931 HU for TMZT1Si alloy and 400–651 HU for TMZT alloy.

Histological analyses confirmed the presence of fibrous intramembranous ossification tissues associated with the radiopacity observed in the newly formed tissues around the implants. Different alloys showed varying degrees of osteogenesis, with TMZT0.75Si and TMZT0.5Si alloys exhibiting mesenchymal cells in the fibrous capsule and newly formed bone tissue containing a small number of bone lamellae. TMZTS alloy demonstrated both intramembranous and endochondral types of ossification.

Osteopontin (OPN) expression was observed in all experimental groups, with an overexpression observed in the bone interface area for TMZT0.5Si, TMZT0.75Si, and TMZTS groups. Metalloproteinases (MMPs), specifically MMP2 and MMP9, also showed overexpression in all experimental groups, indicating their involvement in the cellular and physiological processes of bone formation, tissue repair, angiogenesis, and morphogenesis.

As a conclusion, the findings suggest that the implanted alloys triggered osteogenesis and healing processes, leading to the formation of new bone tissue. The presence of OPN and MMPs further supports the role of these molecules in bone remodeling and regeneration.

From an in vitro point of view, both the TMS and TMZTS systems have shown promising results in terms of their biocompatibility and potential for bone regeneration.

The TMS system demonstrated good cell viability and proliferation, indicating its compatibility with living cells. The addition of silicon to the alloy composition promoted the formation of a biocompatible oxide layer on the surface, which could enhance osseointegration. Furthermore, the TMS system exhibited favorable mechanical properties, such as adequate strength and elastic modulus, making it suitable for load-bearing applications.

Similarly, the TMZTS system exhibited good cytocompatibility, supporting cell attachment, proliferation, and spreading. The incorporation of zirconium and tantalum in the alloy composition provided enhanced mechanical properties, including increased strength and corrosion resistance. The addition of silicon further improved the biocompatibility and potential for bone tissue integration.

Both systems showed the formation of new bone tissue around the implants, as indicated by the histological analyses. The presence of fibrous intramembranous ossification tissues and the expression of osteopontin and metalloproteinases suggested active bone formation and remodeling processes [43, 44].

Overall, the TMS and TMZTS systems demonstrated promising in vitro biocompatibility, mechanical properties, and ability to support bone regeneration. These findings warrant further investigation and evaluation in in vivo studies to assess their long-term performance and potential for clinical applications.

5. Conclusions

In conclusion, the TMS and TMZTS systems have shown promising results across various aspects, including mechanical properties, biocompatibility, and their ability to support bone regeneration, based on in vitro studies.

From a mechanical perspective, both systems exhibited favorable properties, including adequate strength, corrosion resistance, and elastic modulus. These properties are crucial for implant materials as they ensure stability, longevity, and appropriate load-bearing capacity, which are essential for successful clinical applications.

In terms of biocompatibility, both systems demonstrated good cytocompatibility, supporting cell viability, proliferation, and attachment. This indicates that the alloys are well-tolerated by living cells and have the potential to integrate with surrounding tissues. The presence of fibrous intramembranous ossification tissues and the expression of osteopontin and metalloproteinases further suggest active bone formation and remodeling processes, indicating a positive biological response to the materials.

Additionally, both systems showed the formation of new bone tissue around the implants, indicating their ability to support bone regeneration. This is a crucial factor for implant success, as it allows for the establishment of a strong bond between the implant and the surrounding bone, promoting long-term stability.

However, it is important to note that these conclusions are based on in vitro studies, and further investigation through in vivo studies is necessary to validate and assess the long-term performance of the TMS and TMZTS systems. In vivo studies can provide insights into the materials’ behavior within a living organism, including their interaction with the host tissue, potential immune responses, and overall biocompatibility. Only through comprehensive in vivo evaluations can the clinical potential of these systems be fully understood and their suitability for human applications be determined.

6. Future trends

The titanium-based alloys used both in dental prosthetics and orthopedics have special properties, being involved in most interventions involving the replacement of an anatomical tissue or element.

Thus, the growing demand of the market has pushed research in the direction of extending the life of implants, through the development of new biocompatible materials.

Evaluating the results obtained following the full characterization of the alloys, we can conclude that the new systems obtained have a reduced modulus of elasticity, without showing cytotoxicity at the cellular or tissue level.

The obtained systems can be successfully used in various medical applications, such as dental and orthopedic implants.

Acknowledgments

This paper was financially supported by the Project “Network of excellence in applied research and innovation for doctoral and postdoctoral programs/InoHubDoc”, project co-funded by the European Social Fund financing agreement no. POCU/993/6/13/153437. This paper was also supported by “Gheorghe Asachi” Technical University from Iaşi (TUIASI), through the Project “Performance and excellence in postdoctoral research 2022”.

Conflict of interest

The authors declare no conflict of interest.