Enhanced knee joint treatment using a hybrid hyaluronic acid-alginate filler reinforced with hydroxyapatite-titanium nanoparticles for sports-related injuries

2.1 Preparation of polymer solution

This article develop a porous nanocomposite scaffold using gelatin, hyaluronic acid, and Ti-NP via the freeze-drying method to enhance the speed and quality of current treatment methods. Additionally, Ti-NP with a particle size less than 50 nm is utilized to accelerate the antibacterial properties of bone filler, reduce the risk of infection, and expedite the treatment process. The resulting scaffold exhibits favorable chemical and biological properties. Specifically, in this study, a homogeneous solution of hyaluronic acid and HA is prepared by stirring HA in deionized water at 450 rpm for 12 h at a temperature of 45 ± 0.5 °C. The hyaluronic acid purchased from Sigma USA with a molecular weight of 848.8 g/mol for high purity is used, along with HA nanoparticles from a density of 0.038 g/ml and Ti-NP with a density of 4.23 g/cm³ and a molar mass of 79,866 g/mol from Sigma Company. The resulting solution is divided into four containers and different amounts of Ti-NP (0 wt%, 5 wt%, 10 wt%, and 15 wt% labeled S1, S2, S3, and S4, respectively) are added and magnetically stirred at 450 rpm for 2 h to obtain a homogeneous solution while maintaining the functional groups of the polymers at a temperature of 45 ± 0.5 °C. The solution was initially frozen at −80 °C in the refrigerator before being transferred to the freezer dryer where it was left to prepare the scaffold under a pressure of 0.5 tor for 24 h. Once the scaffold was prepared, a series of tests were conducted to characterize it, which were divided into three main sections.

2.2 XRD analysis

The crystal structure and chemical composition were determined using X-ray diffraction (XRD) with a BRUKER D8-Advance model from Germany in which the CuKα beam was utilized to prepare diffraction patterns with a scanning speed of 0.0058°/s in the range of 10–90°. The identification of the patterns was accomplished by employing phase-detection reference cards from JCPDS.

2.3 FTIR analysis

To study the chemical agents and bonds in the samples, an infrared spectrum was taken in the wavelength range of 400–4000 cm⁻¹. To investigate, the powdered samples were mixed with KBr in a ratio of 1:100 and then the infrared spectrum was taken from them. The obtained numbers are collected in Excel software and their diagrams are drawn and then the comparison between the diagrams is done.

2.4 SEM analysis

The microstructure of the samples is examined by SEM. For this purpose, a coating of gold is created on the samples and then the microstructure may be seen by a scanning electron device. The porosity of the samples may be evaluated using Image-J software. In this study, 20 porosities are measured from SEM images and the porosity is reported as an average. Porosity analysis involves quantifying the void spaces or pores within the scaffold, which is a critical parameter impacting cell infiltration, nutrient transport, and waste removal capacity. Porosity, typically expressed as a percentage, can be determined through methods such as SEM or micro-CT image analysis or liquid displacement techniques. Conducting a morphology study and porosity analysis provides researchers with valuable insights into the scaffold's physical structure, pore characteristics, and overall architecture. This information is vital for understanding how the scaffold interacts with cells, tissues, and biofluids, and evaluating its suitability for cell attachment, proliferation, differentiation, and the diffusion of nutrients and oxygen. Morphology and porosity analyses offer quantitative and qualitative data that contribute to the comprehensive assessment of scaffold performance, assisting researchers in optimizing scaffold design and fabrication techniques specific to tissue engineering applications.

2.5 Compressive strength test

The Hounsfield machine is employed for measuring compressive strength. The compressive strength test is carried out on the sample by applying pressure and tension at a rate of 0.2 mm/min. After placing the samples in the machine, pressure is exerted, and the resulting data is recorded in Excel, followed by graphing. The determination of tensile and compressive strength is crucial for bone scaffolds and can be calculated. The mechanical properties of bone scaffolds are significantly influenced by various factors, including the chemical structures, presence of additives, radiographic materials, porosity, sterilization methods, and monomer and polymer mixing techniques (Martínez-Vázquez et al., 2010).

2.6 Biodegradability test

To investigate the biodegradability of scaffolds, a methodology was employed wherein the porous scaffolds were submerged in a solution of phosphate buffer saline for a period of four weeks. The phosphate buffer saline solution was prepared by dissolving 0.988 g of the powder in 100 ml of water. The biodegradability of the samples was evaluated by measuring their dry weight (W1), followed by immersing them in a falcon containing 30 ml of phosphate buffer saline and placing it in a water bath set at 37 °C and 50 rpm. After a week, the scaffolds were removed from the solution, and their weight was measured again (W2). Subsequently, the samples were re-immersed in the same PBS solution. The biodegradability of the scaffolds was determined using Eq. (1).

2.7 pH concentration changes

The experiment analyze the pH fluctuations that occur during the degradation process of the samples. The samples were incubated at 37 °C in phosphate buffer saline and their solutions were subsequently measured for pH after removal from the incubator. The pH measurements were used to plot graphs that illustrate the pH changes for each group over the specified period. In the pH experiment, a specific pH value (e.g., pH 7.4) will be utilized. The scaffolds will be incubated at this pH for a designated incubation time (e.g., 24 h) to evaluate their performance under physiological conditions. By including these details, we can provide a clearer understanding of the experimental setup and ensure reproducibility.

2.8 Release of calcium ions

This investigate the degradation of hyaluronic acid/HA samples containing varying amounts of Ti-NP, with a focus on measuring and comparing the release of calcium ions into the surrounding environment and its potential impact on the healing process. The samples were incubated in phosphate buffer saline at a weight-to-volume ratio of 2.5 mg/ml and at 37 °C to carry out the investigation. The concentration of calcium ions was measured using an inductively coupled plasma atomic emission spectroscopy (ICP-AES), and the data was plotted over time to obtain a concentration curve. Furthermore, the study examined the formation of a HA layer on the scaffolds by immersing five samples in simulated body fluid (SBF) for 14–21 days, with weekly solution changes. The scaffolds were then analyzed using a TESCAN SEM machine to examine their microstructure and morphology, with a focus on the formation of two types of pores in the bone scaffold: large cavities with a diameter of more than 1 mm and small cavities with a diameter between 0.1 and 1 mm. The scaffolds were incubated in a soluble SBF solution for two weeks with a pH maintained between 7.42 and 7.45. After each stage of removal, the samples were washed with deionized water and dried in a vacuum oven.

2.9 Contact angle test (hydrophilicity)

A crucial aspect of this study was to investigate and compare the hydrophilicity of scaffolds that incorporated hyaluronic acid and HA with differing weight percentages of Ti-NP. To achieve this objective, four samples of each scaffold were cut to dimensions of 3 × 3 cm². A droplet of water was deposited on the surface of each scaffold, and after 30 s, a photograph was taken using a device designed by the Faculty of Medical Engineering at Advance Research Center. The average contact angle of water on each scaffold was then calculated and recorded.

2.10 Cell survival assay

Measurement of viability and cell proliferation form the basis of many methods for studying the cellular response to external factors. At present, tetrazolium salt reduction is a safe and low-cost method for studying cell proliferation and analyzing the effects of substance toxicity on cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Tetrazolium yellow, is regenerated by metabolically viable cells and dehydrogenases to form regenerative equivalences such as nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH. Intracellular purple formazan can be measured by spectrophotometer. In this work, the source of the cells prepared from Pasteur cell Bank as mesodermal progenitors were passaged when 80–90 % confluence was reached, until the 5th passage. To investigate ossification and toxicity, osteoblasts and toxicology cells are used. Using these cells, the effects of hyaluronic filler with HA-TiNPs on bone formation and its toxicity can be investigated. In this work, the culture medium was Dulbecco's Modified Eagle Medium (DMEM) containing 10 % fetal bovine serum, 1 % antibiotic–antimycotic which purchased from Adeb Knowledge based company. In this way, the rate of cell proliferation can be measured, and conversely, when metabolic events leading to programmed cell death and necrosis, the slow rate of cell proliferation can be measured. In this method, the number of work steps to increase the test speed is minimized.

This study investigate the effectiveness of UV radiation and 70 % EtOH for sterilizing porous scaffolds, albeit with an incomplete sterilization outcome. The MTT reagent is utilized for accurately measuring cell proliferation rate by taking advantage of the linear relationship between cell numbers and generated signals. It is worth noting that various methods are available for measuring cell proliferation rate, and in this study, the MTT test is conducted using human osteoblast cells. The test procedure involves specific steps for cell suspension preparation and incubation. The presence of purple spot dots is examined using an inverted microscope, and if observed, Dimethylsulfoxide (DMSO) solution is added to dissolve the sediment. The absorption of wells is measured using a spectrophotometer, and the results are recorded to compare cell growth.

In this study, we assessed the impact of composite scaffolds on cell viability and proliferation over time. Osteoblast cells were cultured and seeded onto scaffolds composed of a hybrid hyaluronic acid-alginate filler with hydroxyapatite-titanium nanoparticles. After sterilization, the cell-seeded scaffolds were incubated for cell attachment. Experimental groups with osteoblast-seeded scaffolds and control groups without cells were compared. Cell viability assays were conducted at 24, 48, and 72 h using MTT or resazurin assays, and absorbance or fluorescence intensity measurements were obtained to assess cell viability and proliferation. Statistical analyses including one-way ANOVA or t-test were performed to determine significant differences between the experimental and control groups. Graphical representations were generated using GraphPad Prism software. This study provide a comprehensive understanding of how the composite scaffolds influence cell behavior over time, offering valuable insights into their potential to promote cell viability and proliferation.

2.11 Statistical analysis

In this study, the data and results were presented as mean standard deviation (SD), with all tests being repeated at least thrice to ensure accuracy. Statistical significance of the results was determined using SPSS software and one-way analysis of variance, with a p-value of less than 0.05 being considered significant. The freeze-drying method was chosen as the scaffold fabrication technique due to its cost-effectiveness, widespread use and simplicity for incorporating Ti-NP in tissue engineering.

3.1 Materials characterization

3.1.1

3.1.1 X-ray diffraction spectrum analysis

The X-ray diffraction (XRD) spectrum was used to evaluate the crystalline and amorphous phases of the samples. Fig. 2(a-b) illustrates the XRD results of pure HA samples composite, which contain HLA-Alginate/-HA and various amounts of Ti-NP. The peaks at 26.93°, 31.37°, 39.81°, 45.15°, and 51.01° in the HA diagram correspond to the crystal plates 002, 211, 310, 222, and (3 2 0), respectively. As it is known, HA exhibits a crystalline phase. The sample containing 5 wt% by weight of Ti-NP did not show Ti-NP peaks due to the low ratio of Ti-NP to the components of the titanium peak. However, the Ti-NP peaks were observed in samples containing Ti-NP at 25.17°, 37.73°, 48.03°, 54.64°, 62.95°, 69.26°, and 69.83°, corresponding to the pages of crystalline (1 0 1), (0 0 4), (2 0 0), (2 1 1), (2 2 0), (2 0 4), (3 0 1), and (1 1 2), representing the anatase and rutile phases of Ti-NP. The addition of higher amounts of Ti-NP increased the crystalline phase of the samples, and the amorphous peak of hyaluronic acid was not observed. The main peaks of HA are within the range of 20-60°. A comparison of the obtained pattern with the existing standard pattern revealed a slight displacement of the peaks towards higher angles, indicating the replacement of calcium ions with a smaller ion radius instead of ions.

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Fig. 2

XRD pattern a) sample containing different amounts of TiNPs, and b) of HA nanoparticles.

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3.1.2

3.1.2 X-ray diffraction spectrum analysis

The lattice of a material becomes less constant as the size of its constituent ions decreases, leading to a shift in the Bragg angle of its peaks. The Williamson-Hall method was employed to determine the crystallite size of HA powder, yielding an average size of 40–80 nm, confirming the powder's nanostructure. Analysis of functional groups and chemical interactions was performed using the FTIR spectrum in Fig. 3. Absorption bands at around 900 cm⁻¹ indicate phenolic C—O tensile vibrations of hyaluronic acid. However, the presence of hyaluronic acid's 650 cm⁻¹ bandwidth precludes the identification of phosphate peaks in this region due to overlap. A broad absorption band centered at 3425 cm⁻¹ corresponds to the tensile vibrations of hydroxyl groups in all three materials. The peak at 3064 cm⁻¹ is attributed to HA, displaying a wide absorption band in this region. Peaks at 2930 and 2861 cm⁻¹ correspond to the tensile vibrations of CH and CH₃ in hyaluronic acid, respectively. At 1641 cm⁻¹, the carbonyl acid and amide groups of hyaluronic acid exhibit tensile vibrations, overlapping with Ti-OH-related tensile vibrations. Additionally, the peak at 1532 cm⁻¹ is associated with the tensile vibrations of carbonate in apatite, while the 1406 cm⁻¹ band reflects the C—O tensile vibration in the COO^– group. Peaks at 1237 cm⁻¹ and bands below 600 cm⁻¹ correspond to the tensile vibrations of P⚌O. An absorption band at 1068 cm⁻¹ indicates the C—O tensile vibration in alcohols, with the shoulder in this band arising from the C—O—C tensile vibration. As previously mentioned, phosphate peaks cannot be identified in this region due to overlap with hyaluronic acid's 650 cm⁻¹ bandwidth.

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Fig. 3

FTIR spectrum of samples containing various amount of TiNPs as S2, S3 and S4.

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3.2 Morphology study and porosity analysis

The microstructure of scaffolds with different amounts of Ti-NP was analyzed using SEM images, as shown in Fig. 4(a–c). The SEM images indicate the presence of a porous structure that meets the acceptable range for bone tissue engineering, with elliptical and interconnected cavities. These features play a crucial role in bone tissue growth and penetration into the scaffold. Interestingly, the porous structure emerged spontaneously, without any added porosity, and was the result of material dissolution in water. The SEM images provide a clearer view of the pore structure, which becomes more defined as the percentage of Ti-NP less than 50 nm increases, forming spherical particles and indicating better mechanical strength. Furthermore, the SEM images reveal interconnected pores that trap Ti-NP and enable internal communication. The pore walls consist of HLA-Alginate nanocomposites with CaP nanoparticles reinforced with Ti-NP. The quantity of nano Ti-NP directly affects the scaffold morphology, with porosity size and internal cohesion of pores being vital for cell growth, migration, nutrient flow, angiogenesis, and spatial organization for cell growth and ECM production. It is worth noting that porosities less than 100 µm do not facilitate sufficient transport of oxygen and nutrients, leading to limited osteoblast survival and bone formation, as shown in Fig. 4(a–d). The microstructure of scaffolds with varying Ti-NP amounts was examined through SEM images depicted in Fig. 4(a–d). The images confirm the presence of a porous structure with elliptical cavities suitable for bone tissue engineering. The porosity, created spontaneously without the addition of porosity, resulted from the material dissolution in water. As the Ti-NP concentration increased, the porosity percentage rose to approximately 76.5 %, marginally greater than that of hyaluronic acid/HA scaffolds. The interconnected porosity facilitates the exchange of nutrients, waste, water, and oxygen, essential for cell growth, angiogenesis, spatial organization, and ECM production. Scaffolds with a mean porosity diameter of 400 μm exhibit more bone growth, proliferation, angiogenesis, and are more conducive to nutrient transfer. CaP particles and Ti-NP are homogeneously dispersed throughout the scaffold, denoting their availability to create a bioactive surface. Porosity and pore interconnectivity are crucial factors in designing bone scaffolds for tissue engineering applications. Adequate porosity and interconnected pores can enhance nutrient and oxygen distribution within the scaffold, facilitating the growth and proliferation of stem cells. In studies that have used fillers in bone scaffolds, porosity and pore interconnectivity have played important roles. Several studies showed that porosity and pore interconnectivity in bone scaffolds with filler led to increased mechanical strength and biocompatibility of the material. Therefore, when designing bone scaffolds with fillers, porosity and pore interconnectivity should be considered as two critical factors (Rahman et al., 2009; Lison et al., 2008; Yang et al., 2023; Sanapalli et al., 2022; Bian and Xing, 2022). Fig. 5 shows as the amount of Ti-NP increases, the porosity percentage increases and the strength of the first two samples decreases while the sample with the highest percentage of Ti-NP may exhibit a significant increase in strength. Nevertheless, the porosity difference between the last two samples is relatively small and can be verified by the SEM outcomes. Furthermore, the augmentation of Ti-NP volume possessing catalytic features results in the enhancement of tensile strength in the scaffold network structure, and hence, the likelihood of calcium ion presence and its bonding with Ti-NP on the surface of the sample increases.

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Fig. 4

SEM images of hyaluronic acid-Alginate/HA scaffold specimens containing (a) 0 wt% (b) 5 wt%, (c) 10 wt% Ti-NP, and (d) 15 wt% Ti-NP.

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Fig. 5

Results of porosity measurement in samples with different amounts of Ti-NP.

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3.3 Mechanical properties results

The natural extracellular matrix (ECM) plays a crucial role in bone repair as it possesses conductive and inductive signals that can expedite bone repair and remodeling. In scaffold design for tissue engineering, mechanical properties are of paramount importance. The scaffold must be capable of bearing loads until new bone tissue forms, and the mechanical properties should be biomechanically compatible with the environment and capable of load transfer. Incompatibility can lead to tension shield or scaffold collapse. HA is a bioceramic that is chemically similar to the primary bone minerals and is frequently employed in bone and tooth compounds to improve bioactivity. The utilization of HA can improve the mechanical properties and biocompatibility of scaffolds (Al-Taweel and Saud, 2016; Smith et al., 2012; Mishra et al., 2019; Zhang et al., 2020; Wiedmer et al., 2018). Although porous scaffolds are favorable for osteogenic environment conducive to bone tissue growth and formation, excessively large pore size and porosity percentage can significantly impact mechanical properties, leading to the loss of mechanical integrity (Zhang et al., 2020; Wiedmer et al., 2018; Hasan and Fatalla, 2023; Alnajm and Shukri, 2023; Kim et al., 2017). Ti-NP ceramics have higher compressive strength than other bone conductor scaffolds, with pure Ti-NP scaffolds exhibiting a compressive strength of approximately 2.5 MPa. After the implantation of the scaffold in the body, the high strength is maintained. Scaffolds composed of CaP ceramics and polymer/CaP scaffolds such as HA, with a similar porosity percentage, have a strength value of approximately 2 MPa (Kopec et al., 2007; Li et al., 2020; Gao et al., 2017; Liu et al., 2004). This indicates that the presence of Ti-NP is more effective than bioceramics in enhancing the mechanical properties. Additionally, the compressive strength of normal spongy and dense bone has been reported to be about 2–12 MPa and 100–230 MPa, respectively (Rahman et al., 2009). While the addition of HA nanoparticles can improve the mechanical properties, it may also increase the brittleness of the scaffold. A single HA may not provide the desired mechanical properties for bone (Lison et al., 2008). Since Ti-NP alone lacks good toughness, it is usually combined with other materials to form bone scaffolds that provide a structure for bone conduction with adequate mechanical strength and load-bearing capacity necessary for bone repair. The addition of Ti-NP to polymers and hydrogels improves strength, mechanical properties, and bioactivity (Hasan et al., 2023; Shuai et al., 2017; Cui et al., 2020; Kopec et al., 2007; Li et al., 2020; Gao et al., 2017; Liu et al., 2004; AL-Anezi et al., 2023; Rahman et al., 2009). Therefore, the application of Ti-NP, HLA-Alginate, and n-HA together seems to enhance the mechanical properties and compensate for their respective weaknesses. In this study, the mechanical properties of scaffolds were investigated, and the results are depicted in Fig. 6(a–c). As shown in Fig. 5(a), the addition of 15 % Ti-NP increased the tensile strength of the specimens. The low tensile strength of ceramics such as HA is one of their challenges, and the addition of 5 % Ti-NP and 10 % Ti-NP does not have a significant effect on tensile strength. However, 15 % Ti-NP can compensate for this weakness and increase the tensile strength to about 100 kPa. A similar trend was observed in the elastic modulus, and the addition of different amounts of Ti-NP may increase the elastic modulus. The elastic modulus of HA-Alginate/HAp/15Ti was less than 1 MPa compared to other samples, making it more suitable for bone applications as show in Fig. 6(b). The results of fracture toughness evaluation are also presented in Fig. 6(c). The findings indicate that the inclusion of Ti-NP enhances fracture toughness, with the best results observed in the 15 % Ti-NP sample. HA exhibits bioactivity, non-inflammatory and non-toxic properties, and promotes ossification and differentiation of osteoblastic cells.

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Fig. 6

Results of a) Tensile strength, b) elastic modulus and c) fracture toughness of samples containing hyaluronic acid-Alginate/HA of different amounts of Ti-NP.

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Various strategies have been considered, including adding different reinforcing agents to the structure of calcium phosphate ceramics and improving manufacturing and processing methods, to enhance their mechanical properties while maintaining biocompatibility. Reinforcing agents like metal particles, ceramic whiskers, biocompatible glass, and neutral ceramic phases such as alumina, zirconia, and Ti-NP have been added to improve the mechanical properties of HA. Ti-NP is naturally found in minerals like safety (FeTiO₃), roulette (TiO₂), and aspher (CaSiTiO₅), and its density varies depending on the polymorph. Different techniques have been developed to create biodegradable three-dimensional porous scaffolds, each with unique porosity characteristics. In this study, the freeze-drying method was used, where the polymer was dissolved in a solvent, frozen at a low temperature, and then the solvent was sublimated under vacuum pressure to create a highly porous sponge. Traditional drying methods, such as heating, are not appropriate for drying heat-sensitive materials, such as polymers, biological and inorganic compounds, due to the risk of damage or degradation. Solvent extraction and water removal are critical processes in various industries, including pharmaceuticals, food, medical, petrochemicals, biochemicals, and nanotechnology. Freeze-drying is a widely used method for drying sensitive and valuable substances, such as drugs, vaccines, antibiotics, blood components, biological samples, microorganisms, sensitive inorganic substances, trace metal powders, and ceramics, particularly in the nanotechnology industry. The freeze-drying process offers several advantages over other drying methods, including no oxidation, no formation of new compounds, reduced adhesion and agglomeration of materials, uniform particle size distribution, extended shelf life, and low cost. This method also causes minimal damage to heat-sensitive products and preserves their sensory and nutritional attributes.

3.4 Contact angle of three-component scaffold

The wettability of the samples was evaluated by analyzing the contact angle as shown in Fig. 7. The findings from a single test on the samples in Fig. 7 indicate that the scaffold lacking pure nanoparticles is hydrophobic, resulting in poor absorption of the culture medium. After introducing Ti-NP, the contact angle decreased, except in sample four where there was a slight increase. However, compared to the scaffold without nanoparticles, there was a significant decrease in contact angle, indicating an enhancement in the hydrophilicity of the samples. Improved hydrophilicity leads to better penetration of fluids, nutrients, and removal of cellular wastes. Moreover, cells tend to adhere to hydrophilic surfaces and spread more readily.

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Fig. 7

Contact angle of samples A: 1, B: 2, C: 3 and D: 4 with different amounts of Ti-NP.

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3.5 Weight loss analysis

The porous scaffold used to encourage tissue growth and replacement within the body gradually degrades, allowing the inserted cells to multiply and eventually replace the damaged tissue. To facilitate bone formation, the scaffold or bone-replacement composite material should be able to ossify, either through containing growth factors and extracellular proteins or being made of bio-nanocomposite n-HA. Biodegradable polymers such as HLA-Alginate and gelatin are commonly used in bone replacement surgeries to avoid the need for additional surgical procedures. Hyaluronic acid, a molecule found in connective tissue, plays diverse roles in the body, including regulating tissue regeneration and cell movement, and it promotes composite material adhesion when in a solid state. Porous Ti-NP scaffolds are also biocompatible and have demonstrated promising results in promoting osteoblast cell growth and providing a suitable substrate for human seminal vesicles to thrive and multiply. Conversely, the HLA-Alginate/HAp/5Ti sample showed an increased slope of degradation owing to its improved hydrophilic properties, low crystallinity, and Ti-NP content. Furthermore, it should be noted that environmental changes and material release could greatly affect implant performance in vivo, with pH being one of the critical factors. The pH levels of different samples were assessed, and the results are presented in Fig. 8(a). The present study investigate the degradability of porous Ti-NP scaffolds in different compositions. To this end, the scaffolds were immersed in PBS and the weight loss was measured after a specific time, as illustrated in Fig. 8(b). The results demonstrated that adding small amounts of Ti-NP augmented the degradation process, whereas increasing the Ti-NP content decreased the process and reached a minimum in the HLA-Alginate/HAp/15Ti sample. This decrease in degradation could be ascribed to a decrease in pH acidity, an increase in the crystalline phase, and the low degradation rate of Ti-NP. During the calcium release test, it was observed that samples containing higher amounts of Ti-NP exhibit more intense calcium ion release, leading to the formation of a layer of HA on the samples, and reducing the degradation in the HLA-Alginate/HAP/15 TiNP, as depicted in Fig. 8(b). In this study, we evaluated the amount of ion release from different samples, as shown in Fig. 8(c). The findings indicate that the HLA-alginate/HAp/5Ti sample does not significantly differ in calcium ion release from the titanium-free sample. However, the release rate increases with increasing titanium content in the HLA-alginate /HAp/10 TiNP sample, and the HLA-alginate/HAp/15TiNP sample exhibits the highest release rate. Despite the positive effects of calcium ions, it should be noted that the HLA-alginate/HAp/15Ti sample shows better results than the other samples, while the amount of release does not exceed the allowable range to avoid forming a calcium carbonate layer. The maximum adsorption percentage is for the second sample, and the lowest attractive rate occurs in the sample containing the highest amount of Ti-NP. We observe an increase in the adsorption percentage of PBS solution by increasing the Ti-NP. Our results demonstrate that the HLA/HA samples create a somewhat acidic environment, which could cause cell damage after degradation (Rahman et al., 2009; Lison et al., 2008). On the other hand, the addition of Ti-NP increases the pH, and the HA/HAp/15Ti sample, in particular, exhibits the highest pH due to Ti-NP's somewhat alkaline property. This has been able to neutralize the relatively acidic environment, resulting in a pH close to 7, which is a more suitable neutral pH for the treatment of bone lesions. In this study, the bioactivity of the samples was evaluated by immersing them in SBF and analyzing the deposited layer using SEM imaging, as shown in Fig. 9(a–d). Results indicate that an increased percentage of Ti-NP led to an increase in the deposited HA layer, which can be attributed to the increase in sample bioactivity. Furthermore, the calcium release test demonstrated that an increased percentage of Ti-NP resulted in an increased amount of calcium released into the environment. The release of calcium ions can stimulate the deposition of calcium phosphates (CaPs) and accelerate the growth of HA, ultimately leading to the formation of a Ca-OH layer on the sample.

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Fig. 8

a) pH concentration b) weight loss, and c) Ca release of sample containing various amount of Ti-NP.

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Fig. 9

SEM images of the HA layer deposited on a hyaluronic acid-Alginate/HA sample containing (a) 0 wt%, (b) 5 wt%, and (c) 10 wt%, and d) 15 wt% Ti-NP.

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Maji et al. (Maji et al., 2018) introduced a more straightforward method to create a three-dimensional microporous scaffold for bone tissue engineering. They used a foaming combination approach that involved intense agitation of gelatin/carboxymethyl chitosan (CMC)/nanoHA followed by freezing to create a scaffold with interconnected networks of macropores. The resulting built-in macro porous (SGC) scaffold had higher porosity, water holding capacity, slower enzyme degradation, and greater compressive strength than the non-porous (NGC) scaffold. Histopathology and radiographic examinations were performed on bone biopsy samples in the eighth week to assess fusion rate, ossification activity, and remodeling. Radiographic examination showed a significant difference between the groups, with the n-HA group exhibiting higher ossification activity in the sixth week.

Dennis et al. (Dennis et al., 2017) investigated the use of colloidal HLA-Alginate/HAp derivative gels in bone tissue engineering and found them to be non-cytotoxic and supportive of cell viability. Subramaniam et al. (Subramaniam et al., 2016) proposed an alternative approach to bone tissue engineering by combining HA/calcium sulfate/HLA with collagenase to enhance bone regeneration. The mechanical tests and WST-1 cell assays revealed high biocompatibility and mechanical strength of 6.69 MPa. The release of collagenase from the samples was found to accelerate the process of bone regeneration.

Fig. 10(a–d) displays the cell survival against different samples over 24 h, 48 h and 72 h. As the mineral phase of bone consists primarily of calcium and phosphorus, the measurement of calcium ions is a critical parameter in designing bone and knee fillers. The apatite layer is a calcium phosphate bioceramic that resembles the ECM of bone and functions as a bone conductor. The physicochemical properties of a synthesized scaffold are essential in determining its behavior and suitability for various applications, including molecular weight, solubility, chemical composition, morphology, and surface charge. The critical-sized bone defects are a significant challenge in bone damage, and bone scaffolds can provide mechanical support and act as a template for bone tissue formation. Additionally, stress shielding is an important phenomenon to consider when designing bone scaffolds, as it can cause bone resorption and eventual failure of the scaffold (Dennis et al., 2017). The intricate nature of bone tissue and its unique characteristics necessitate the use of an ex vivo model to incorporate all its properties. This is only achievable due to the significant resemblance between the external environment and the body's biological environment (Liu et al., 2004; AL-Anezi et al., 2023; Rahman et al., 2009; Lison et al., 2008). Current research is actively exploring this area, highlighting the importance of closely mimicking the biological environment when designing tissue engineering scaffolds to achieve favorable outcomes. Studies have shown that composite scaffolds hold the potential to stimulate and guide bone cell growth and proliferation.

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Fig. 10

Cell Viability Analysis (a-d) The viability of cells is assessed at 24 h, 48 h, and 72 h for various samples containing Ti-NP.

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In addition, the similarity in structure between calcium phosphate in scaffold structures and natural bone, along with the release of similar substances and ions in the body, promotes bone cell growth. To assess the capacity of samples to support cell growth and viability, a cell viability test was conducted on scaffolds with and without 15 % Ti-NP. The results demonstrated that both scaffolds exhibited high percentages of survival at all timepoints, with an increase in cell growth after 48 h. This outcome is attributed to the scaffolds' porous and biocompatible fabrication, critical for cell growth and nutrient exchange. The inclusion of 15 % Ti-NP further improved cell viability, confirming its positive effect on cell growth and survival. Fig. 11 highlights the specialized use of bio-nanocomposite hydrogels in repairing tissue damage resulting from sports injuries, particularly fractures and cartilage damage. These types of injuries are common in sports and can have a significant impact on an athlete's performance and overall quality of life. Bio-nanocomposite hydrogels, made up of a hydrogel matrix infused with nanoparticles, are increasingly being studied for their potential in tissue engineering and regenerative medicine. These hydrogels can provide additional structural support and enhance the material's bioactivity. The use of these hydrogels in repairing tissue damage caused by sports injuries is a promising area of research with the potential to revolutionize the treatment of these injuries. Additionally, there are several other treatment options available for sports-related joint injuries. Nonsteroidal anti-inflammatory drugs (NSAIDs) can effectively alleviate joint pain and inflammation. However, due to potential negative side effects, prolonged use of NSAIDs should only occur under the supervision of a healthcare professional. Corticosteroid injections can be highly effective in reducing joint inflammation and pain, but their use should be limited and only under the guidance of a healthcare professional, as overuse may result in joint damage. Platelet-rich plasma (PRP) therapy involves the injection of a concentrated solution of the patient's own platelets into the joint, containing growth factors that can promote joint healing and reduce inflammation. Metal and metal oxide nanoparticles (NPs) have advantages such as modulating ROS and RNS, disrupting cell membranes, and targeted delivery, but their excessive production of ROS and RNS can be cytotoxic, and the potential toxicity of released metal ions requires evaluation for long-term effects on the environment and human health (Martínez-Vázquez et al., 2010; Alavi and Yarani, 2023). Titanium dioxide nanoparticles have shown promising potential in promoting healing mechanisms related to bone injuries. These nanoparticles possess unique properties that make them suitable for bone tissue engineering applications, interacting with surrounding cells and tissues to trigger a cascade of biological responses that contribute to the healing process. One of their key healing mechanisms is the enhancement of osteogenesis, stimulating the differentiation and proliferation of osteoblasts responsible for bone formation. TiO₂ NPs also upregulate the expression of genes and proteins involved in osteogenic differentiation, leading to increased bone matrix deposition and mineralization. Additionally, TiO₂ NPs exhibit antibacterial properties, inhibiting the growth and activity of bacteria, thereby reducing the risk of infection and creating a more favorable environment for bone healing. Furthermore, these nanoparticles modulate the inflammatory response in bone injuries by suppressing the release of pro-inflammatory cytokines and promoting the secretion of anti-inflammatory factors. This balanced and controlled inflammatory reaction helps to reduce tissue damage and expedite the healing process. Through these mechanisms, TiO₂ NPs hold promise for improving bone injury outcomes and advancing the field of bone tissue engineering.

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Fig. 11

Specialized use of bio-nanocomposite hydrogels in the repair of tissue damage resulting from sports injuries, including fracture or cartilage damage.

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