Tocotrienol-Incorporated Gelatin Hydrogel Crosslinked with Genipin for Future Bone Tissue Engineering Applications: Physiochemical Characterization and Biocompatibility

Abstract

Oral administration of tocotrienol has poor systemic distribution due to poor selectivity by the α-tocopherol transfer protein at the liver. Local injection of tocotrienols with appropriate drug delivery systems is significant to ensure that the drug is delivered directly to the site of injury or fracture. This paper presents a tocotrienol-loaded gelatin hydrogel crosslinked with genipin for bone regeneration. This innovative method improves the incorporation and sustained delivery of tocotrienol while overcoming its incompatibility with hydrophilic biomaterials. It establishes a novel platform for targeted therapeutic applications in bone treatment. The cytotoxicity and physicochemical properties of tocotrienol were examined using the genipin-crosslinked gelatin hydrogel. A 10% tocotrienol nanoemulsion (TTE) was prepared using a sonicator and characterized with a zeta sizer and FTIR. A dose–response analysis was conducted to determine the appropriate tocotrienol concentration for hydrogel integration with gelatin (7% or 10% w/v) and crosslinked with genipin (0.1% or 0.3% w/v). The dose–response study’s tocotrienol nanoemulsion was added to gelatin before polymerization. With 141.9 nm particles and 0.150 PDI, the nanoemulsion was homogeneous and stable. The 1% tocotrienol nanoemulsion was chosen due to its viability. Formulations 1% TTE_0.1% GNP_7% GEL and 1%TTE_0.3% GNP_7% GEL had superior physicochemical properties compared to other groups. The 1% TTE_0.3% GNP_7% GEL had outstanding hydrophilicity, low weight loss, and a suitable swelling ratio for bone application. SEM scans of the surface and cross-section showed that 1% TTE_0. 3% GNP_7% GEL had interconnected pores with an optimal average pore size of 292 ± 37 μm. Adding tocotrienol to the gelatin hydrogel matrix did not affect FTIR, XRD, or EDX. In vitro cytotoxicity studies indicated >90% cell viability of hFOB 1.19 cells cultured on 1% TTE_0.1% GNP_7% GEL and 1% TTE_0.3% GNP_7% GEL (105 ± 4.36% and 95.36 ± 9.78%). Combining tocotrienol with a genipin-crosslinked gelatin hydrogel demonstrated superior physicochemical properties and no in vitro toxicity.

1. Introduction

Bone regeneration is a complicated process that involves multifaceted interactions between the matrix and cells, as well as various biological pathways related to ion channels and their signaling mechanisms [1]. The dysfunction between bone formation and resorption is the leading cause of impaired bone healing, which is common in situations of infections, tumors, osteoporosis, and trauma [2]. Manufactured therapies are the primary approach for treating bone defects, sustaining bone mass, and reducing consequences such as fractures [3]. The regulation between bone formation and resorption is crucial in preventing osteoporosis. These medications are designed based on this concept, which is divided into two categories: anabolic agents and antiresorptive agents. Denosumab and bisphosphonates (BPs) are anti-resorptive medications that regulate osteoclast activity. Their efficacy in managing bone diseases has been demonstrated, and they frequently serve as the initial line of defense [4]. On the other hand, anabolic agents, including teriparatide and abaloparatide, are specifically designed to target osteoblasts and facilitate bone formation. The use of anabolic medications is still restricted, typically due to their high costs, even though they have demonstrated their efficacy through their superior outcomes in comparison to antiresorptive agents [5]. Regardless of the efficacy of synthetic medications, their prolonged use can result in substantial adverse effects. The prolonged use of anabolic agents can result in an elevated risk of cardiovascular disorders and osteosarcoma, as well as an extensive cost [6]. The most common adverse effects of anti-resorptive agents after prolonged use are gastrointestinal events, osteonecrosis of the jaw, typical femur fractures, and esophageal cancer [7].

Vitamin E, an important agent for bone remodeling, protects against excessive bone resorption by regulating the osteoclast and increases bone formation by enhancing the activity of the osteoblasts. Vitamin E, also known as tocochromanols, is categorized into tocopherols and tocotrienols. Tocopherols are fat-soluble antioxidants with a chromanol ring and a saturated phytyl side chain; meanwhile, tocotrienol contains an unsaturated isoprenoid side chain [8]. In a study by Renò. et al. (2005), they demonstrated the efficacy of tocopherol as a stimulating factor for osteoblasts when incorporated with poly(d,l)-lactic acid [9]. Different studies demonstrated the efficacy of the tocotrienols regarding superior antioxidant properties compared to tocopherol [10,11]. Oral administration of tocotrienol has poor systemic distribution due to poor selectivity by the α-tocopherol transfer protein at the liver. Local injection of tocotrienols with an appropriate drug delivery system is significant to ensure that the drug is delivered directly to the bone defect site. Natural carriers such as hydrogels have been developed to control the release of vitamin E and protect it from degradation due to their biodegradability and biocompatibility [12,13]. However, no previous research used gelatin hydrogel crosslinked with genipin as a drug delivery system for tocotrienol.

Gelatin is a denatured macromolecular form of the protein collagen, used for tissue healing and to control the delivery of drugs. It begins with the triple-helix structure of collagen, which disassembles into individual strands upon partial hydrolysis. This natural biomaterial can be classified into two types of gelatin (A and B) based on its extraction method. Gelatin offers high quality, adaptability, biocompatibility, degradability, low viscosity, and excellent water retention and rheological properties, making it a preferred medium for drug delivery applications [14]. Genipin, a natural crosslinking agent sourced from Gardenia jasminoides and Genipa americana, is extensively reported for its superior biocompatibility, biodegradability, and durability as a crosslinker in biological materials. Cytotoxicity testing of genipin on preosteoblasts revealed that genipin enhanced the proliferation, adhesion, and differentiation of the preosteoblast cell line [15]. As compared with other natural hydrogels, such as Chitosan, it is a natural hydrogel that provides benefits, including mucoadhesion, despite the need for acidic conditions and other chemical modifications in physiological conditions to degrade. Chitosan/gelatin hydrogels are frequently used to solve these obstacles, as gelatin enhances biodegradability and cellular compatibility within these systems. This emphasizes the pivotal function of gelatin in biopolymeric hydrogels and confirms its selection as the principal hydrogel matrix in the current study [16]. Gelatin hydrogel crosslinked with genipin is generally associated with a strong and stable 3D network due to the chemical interaction between genipin and gelatin amino acids, which is reflected in the improved mechanical strength of the gelatin hydrogel. Genipin has been demonstrated to improve the physicochemical properties of the gelatin scaffold [17]. Gelatin crosslinking with genipin significantly improves the mechanical strength and resistance to enzymatic degradation compared with physically crosslinked or uncrosslinked gelatin matrices, which liquefy at body temperature and degrade too quickly for effective sustained delivery [18]. Furthermore, genipin demonstrated its ability compared with other crosslinking agents, such as carbodiimide, and with different hydrogel systems, such as the extracellular matrix (ECM). Genipin achieved a higher degree of crosslinking and superior resistance to degradation [19]. Genipin-crosslinked gelatin hydrogel has effectively demonstrated the ability to encapsulate and release various compounds [20]; however, most studies on genipin-crosslinked gelatin hydrogel do not provide specific insights into its use as a drug delivery system for tocotrienols.

Bone tissue engineering has proven effective in treating critical-sized bone damage, including fractures and initial tumor resections. Its main goal is to enhance and accelerate the physiological process of bone healing, which does not occur in cases of critical-sized damage. The biocompatibility and physicochemical properties of bone scaffolds make them ideal for tissue regeneration and the delivery of bioactive compounds and medications [21]. The present study focuses on the development of a tocotrienol-fortified gelatin hydrogel crosslinked with genipin for potential use in bone regeneration. The innovative concept of this hydrogel is to serve as a drug delivery system for tocotrienol. Its gradual degradation helps sustain tocotrienol release, thereby exposing the site of injury to tocotrienol for a longer period. The sustained release achieved by the hydrogel system is expected to facilitate and accelerate healing, while also supporting the repair and regeneration of bone tissue. In this study, gelatin hydrogels crosslinked with genipin and incorporated with tocotrienol in various formulations were demonstrated to have potential as an effective drug delivery method for tocotrienols in bone repair. Tocotrienol, however, is a hydrophobic compound with poor water solubility, which contributes to its low bioavailability and limited absorption. Additionally, oral administration of tocotrienol exhibits poor systemic distribution due to the limited selectivity by the α-tocopherol transfer protein in the liver [22]. Therefore, in this study, we hypothesized that in situ injection of tocotrienols with a suitable macromolecular carrier as a drug delivery system is critical for delivering the drug directly to the injury or fracture site.

Based on these considerations, this study provides a novel integration of tocotrienol and gelatin hydrogel crosslinked with genipin for targeted bone drug delivery, which enhances the efficacy of the tocotrienol and reduces its limitations. This study aimed to evaluate the cytotoxicity of the tocotrienol in combination with the genipin-crosslinked gelatin polymeric matrix. Additionally, the physicochemical and mechanical properties of the hydrogel were examined.

2. Results and Discussion

2.1. Morphology and Characteristics of Tocotrienol Nanoemulsion

This study developed a tocotrienol nanoemulsion through simple mixing followed by ultrasonication. Varying surfactant concentrations, sonication time, and amplitude optimized the nanoemulsion. It showed no phase separation for one month at 4 °C in a refrigerator, formulated with 10% tocotrienol, 3% Tween 20 as a surfactant, and 7% ethanol as a cosolvent (Figure 1a,b). The nanoemulsion demonstrated good homogeneity and stability, with a particle size of 141.9 nm and a polydispersity index (PDI) of 0.150 (Figure 1c). The tocotrienol nanoemulsion appeared milky; this observation aligns with nanoemulsions less than 200 nm (100–200 nm) because the droplets scatter light strongly. On the other hand, nanoemulsions less than 50 nm may appear translucent or transparent due to weak droplet-light scatter [23]. The FTIR spectra (Figure 1d) indicated chemical changes between tocotrienol and the tocotrienol nanoemulsion, with the prominent peaks remaining intact. The peaks in the range 2852–3922 cm⁻¹ reflect the asymmetric and symmetric stretching vibrations of the alkane (CH) bonds in tocotrienol. The interaction between the alkane chain stretches of Tween 20 and tocotrienol resulted in a slight shift from 2852 to 2857 cm⁻¹, attributed to weak intermolecular interactions or alterations in the molecular packing. The stability can be attributed to the sonication technique used during preparation, which induces cavitation forces that fragment droplets into smaller particles [24]. FTIR analysis confirmed the preservation of the principal tocotrienol peaks, verifying the stability of the active compounds, with a slight shift in the alkane chain attributed to the interactions between the hydrophobic tail of Tween 20 and the hydrophobic region of tocotrienol.

2.2. Dose Response of Tocotrienol Nanoemulsion Study

A study was conducted using the MTT assay to investigate the effect of varying tocotrienol nanoemulsion concentrations (0.2%, 0.3%, 1%, 5%, and 9%) on the viability of HFOb cells. The results demonstrated a dose-dependent reduction in cell viability. Figure 2 shows the viability after 1, 3, and 5 days. On all days, concentrations up to 1% preserved considerable cell viability: 100.3%, 96.5%, and 115.4%, respectively. However, significant declines occurred at 5% and 9%, with viability falling below 50%, indicating cytotoxicity at these elevated dosages. Based on these findings, we carefully selected the 1% concentration, which had a significant yet non-lethal impact on cell viability, for all subsequent experiments to examine its effects without producing excessive cellular toxicity and with substantial pharmacological implications. Although in vitro osteogenic activity has been demonstrated at 0–25 µg/mL of δ-tocotrienol in MC3T3-E1 cells [25] and at low micromolar levels of γ-tocotrienol has been shown to protect against oxidative stress [26], the concentrations used in this study were comparatively higher. Higher tocotrienol concentrations were chosen as emulsification significantly reduces cytotoxicity while preserving bioactivity. This strategy allows for increased doses that enhance the therapeutic efficacy without causing cellular toxicity, significantly improving local availability compared to non-emulsified tocotrienols, which are limited by cytotoxic effects, whereby this observation aligns with previous studies involving both pure oils and emulsified formulations [27,28]. Notably, the MTT assay results indicate that the selected concentration yielded the highest viability for hFOB 1.19 cells on days 1, 3, and 5, making it suitable for use in bone regeneration scaffolds.

2.3. Phytochemical and Mechanical Characterization of Tocotrienol Nanoemulsion Incorporated Genipin-Crosslinked Gelatin Hydrogel

2.3.1. Swelling Ratio, Biodegradation, and Porosity

The swelling ratio is an essential parameter for evaluating hydrogels for bone applications, as it reflects the material’s capacity to absorb fluids and maintain structural integrity in physiological environments [17]. These factors are crucial for bone regeneration, as they establish an appropriate hydrated microenvironment and deliver sufficient rigidity that promotes cellular activity and osteogenesis [21]. This study evaluated swelling behavior to optimize water absorption, thereby enhancing nutrient transport, cellular penetration, and mechanical strength. Based on the results (Figure 3a), 0.1% GNP showed a significantly higher swelling capacity (p < 0.0001) compared to 0.3% GNP. Therefore, 0.3% was suitable for bone application due to the moderate swelling ratio, which maintained the mechanical strength. The hydrogel’s mechanical strength is essential for the bone application to withstand physiological loads and preserve sufficient space for the growth of new bone tissue [15]. All hydrogels exhibited an acceptable swelling ratio (more than 500%) compared to established bone scaffold materials such as Polycaprolactone (PCL) and β-tricalcium phosphate (β-TCP) scaffolds [29]. The high swelling ratio is related to the low mechanical strength of the biomaterial. The 0.3% GNP groups exhibited a moderate swelling ratio (≈600) compared to the 0.1% GNP groups (≈800–1100) (p < 0.0001), which is ideal for bone applications to promote water uptake and maintain stable structural strength [15]. The reason for these findings is that as the genipin concentration increases, the biomaterial becomes denser and exhibits lower water absorption capacity [18].

The degradation of the biomaterial was evaluated using collagenase type-1 to mimic the enzymatic environment. The findings indicated that gelatin hydrogel loaded with tocotrienol was degraded in the absence of genipin after one hour. The 0.3% GNP groups are more suitable than 0.1% GNP groups for the bone application (Figure 3b) (p < 0.0001). These results revealed that polymers containing 0.3% GNP can resist the environment around the fracture site and release tocotrienol for a long time. The in vitro degradation of hydrogels was a significant factor, as a primary drawback of hydrogels is their rapid degradation after injection at the injured site. The 0.3% GNP groups show significantly greater durability than the 0.1% GNP groups. Meanwhile, tocotrienol showed a slightly higher weight loss than the groups without tocotrienol; however, the difference was not significant. Allowing prolonged release profiles suitable for bone regeneration implants or targeted drug delivery in orthopedic applications [18], the 0.3% GNP groups have demonstrated their ability in this regard. In addition to its effective crosslinking ability, genipin also promotes micro-stability in hydrogels, thereby enhancing the sustained release of the drug at injured sites [30]. Overall, the inhibition of enzymatic degradation and prolonged drug release occurred when an increase in the genipin concentration led to a denser hydrogel network and reduced water absorption.

To facilitate osteoblast attachment and proliferation, an optimal scaffold should possess an interconnected porous structure. Figure 3c indicates that the 0.1% and 0.3% GNP groups exhibited good porosity (50–60%), especially using 7% GEL. This is suitable for bone applications and tissue engineering designs, as the pores facilitate cell migration and nutrient transfer [31]. In all groups, 10% GEL showed significantly lower porosity. Gelatin was the primary factor influencing the porosity of the hydrogels, as it acts as a stabilizer; thus, an increase in the gelatin content results in a reduction in pore size and, consequently, a decrease in porosity [32]. At the end of the preliminary study, 0.1GNP_7% GEL, 1%TTE_0.1% GNP_7% GEL, 0.3 GNP_7% GEL, and 1% TTE_0.3 GNP_7% GEL were selected for subsequent studies.

Compared with established bone scaffold materials, the gelatin hydrogel crosslinked with genipin demonstrated its ability to serve as a drug delivery system after the incorporation of tocotrienol. In the study conducted by Bruyas et al., 2018, Polycaprolactone (PCL) and β-tricalcium phosphate (β-TCP) scaffolds showed a lower swelling ratio and the same range of porosity (50–70%) [33]. The low water uptake leads to the formation of a more rigid scaffold and extremely high mechanical strength, which is unsuitable as a drug delivery system due to the low drug loading ability and controlled release. Our findings were aligned with the Kirchmajer et al., 2013, study that enhanced the stable water uptake with moderate porosity, which is necessary for bioactive diffusion, along with maintaining the structural stability [34].

The 0.1% and 0.3% GNP groups exhibited good porosity (50–60%), especially using 7% GEL compared to 10% GEL (** p < 0.01; **** p < 0.0001, respectively). Therefore, the 10% GEL was excluded from subsequent studies.

2.3.2. Gross Appearance and 3D-Microporous Architecture Hydrogel

The tocotrienol nanoemulsion hydrogel crosslinked with genipin was successfully developed. The crosslinking process was chemically initiated, as indicated by the color change from milky yellow to dark blue during fabrication (Figure 4a). The gross appearance of the selected hydrogels is attributed to the reaction of genipin with oxygen radicals, accompanied by the hydrogen loss and ring-opening facilitated by the primary amino groups in gelatin. Figure 4b shows the microstructure of the groups 0.1% GNP_7% GEL, 0.3% GNP_7% GEL, 1% TTE_0.1% GNP _7% GEL, and 1% TTE_0.3% GNP_7% GEL, observed using field emission scanning electron microscopy (FESEM). All groups exhibited a porous structure with high interconnectivity after freeze-drying. The results suggest that increasing the GNP concentration decreased the pore size (Figure 4c), which may affect the hydrogel’s microstructure. Additionally, incorporating oil could lead to a denser hydrogel network with smaller pores. The incorporation of tocotrienol into the formulations resulted in denser internal morphology, as shown by SEM analysis. This could be explained by the formation of an emulsion phase during polymerisation, in which distinct oil droplets, upon drying, either break or hinder pore formation, ultimately leading to a smaller average pore size. These findings are aligned with the existing research on porous materials made using emulsion templating, where a higher oil content produces a denser, less porous network [35].

2.3.3. FTIR Analysis of Functional Groups

Figure 5a shows FTIR spectra for the tocotrienol nanoemulsion, gelatin, and tocotrienol nanoemulsion (1%) incorporated with gelatin (7%) hydrogel crosslinked with genipin (0.1% and 0.3%). The non-crosslinked gelatin showed distinct peaks for amide I and amide II, as seen in Figure 5a. These peaks are attributed to N-H stretching vibrations (amide I) at band 1628 cm⁻¹, aliphatic C-H stretching vibrations (amide II) at band 1628 cm⁻¹, and CH₂ wagging vibrations (amide III) at 1233 cm⁻¹. A shift in the amide II peak following crosslinking suggested that gelatin’s triple-helix structure was preserved. The presence of genipin was indicated by sharper amide peaks in the FTIR spectra of the 1%TTE_0.1% GNP_7% GEL and the 1%TTE_0.3% GNP_7% GEL. Both the asymmetric and symmetric stretching vibrations of the alkane bond (CH) in tocotrienol, between 2852 and 3922 cm⁻¹, remained intact. Additionally, the amino groups of the gelatin were preserved. As the concentration of genipin increased, all prominent peaks remained and became sharper, as indicated by the results.

2.3.4. X-Ray Diffraction Analysis (XRD)

Figure 5b illustrates the XRD profiles of tocotrienol-fortified gelatin hydrogels. XRD analysis can provide essential information about the crystalline and amorphous phases in the hydrogel matrix, offering insights into the average crystalline size, orientation, and diffraction patterns of gelatin, genipin, and tocotrienol inside the structure. A prominent peak at 20° in the XRD profile corresponds to the polymer network structure of the hydrogels. The investigation indicates that all hydrogels possess an amorphous structure; nevertheless, the results were not statistically significant across all groups.

Table 1. The crystalline and amorphous content of the selected hydrogels.

Hydrogels	Crystallinity	Amorphous
0.1%GNP_7% GEL	19.5	80.5
0.3%GNP_7% GEL	14.9	85.1
1%TTE_0.1% GNP_7% GEL	17.9	82.1
1% TTE_0.3% GNP_7% GEL	16.2	83.7

summarizes the crystalline and amorphous contents of hydrogels. This amorphous structure enhances the biodegradability and mechanical flexibility and supports cell growth [18]. The absence of additional peaks in the XRD patterns of the composite hydrogels indicated that no new phase was formed within the polymeric matrix [36]. The findings indicate that the essential properties of the hybrid biomaterials were preserved despite the incorporation of the natural crosslinking agents genipin and tocotrienol. Maintaining the intrinsic features of these materials is vital to facilitating appropriate interactions with specific cells, enhancing their efficacy while minimizing cell mortality.

2.3.5. Elemental Analysis Using EDX

Energy dispersive X-ray (EDX) spectroscopy was used to examine the elemental compositions of the hydrogels, as presented in

Table 2. The elemental compositions of the hydrogels were observed by energy dispersive X-ray (EDX).

Hydrogels	C (%)	O (%)	N (%)
0.1%GNP_7% GEL	60.6 ± 0.50	26.8 ± 0.7	13.7 ± 1.2
0.3%GNP + 7% GEL	61.6 ± 0.6	28.1 ± 0.3	14.3 ± 0.7
1%TTE_0.1%GNP_7% GEL	65 ± 0.4	21.8 ± 0.8	11.1 ± 1.7
1%TTE_0.3%GNP_7% GEL	62.9 ± 0.1	20.8 ± 0.5	9.2 ± 0.1

. The presence of carbon (C) was attributed to the gelatin and tocotrienol components. EDX measurements showed that integrating tocotrienol into the gelatin hydrogel matrix increased the carbon content of the hydrogels. The 0.1% GNP_7% GEL and 0.3% GNP_7% GEL hydrogels demonstrated a slight reduction in carbon content. Nonetheless, these alterations were not statistically significant. This observation was critical to make sure that the primary properties of the gelatin hydrogel were maintained, which is necessary for the hydrogels as biomaterials in bone applications.

2.3.6. Mechanical Strength and Resilience Properties

Mechanical strength is a crucial characteristic for biomaterials used in bone applications, as the scaffold must withstand physiological pressures while facilitating tissue regeneration. This study evaluated the compressive strength to determine the material’s ability to preserve its structural integrity under stress. As shown in Figure 6b, the 1% TTE_0.3% GNP_7% GEL group demonstrated the highest mechanical strength (85.4%) among the groups, especially compared to 0.3% GNP_7% GEL (56.4%) (p < 0.0001). The enhanced compressive strength of tocotrienol-fortified gelatin hydrogels is ascribed to the amphiphilic characteristics of tocotrienols, which are thought to engage with the gelatin polymer chains. The hydrophobic tail of the tocotrienol molecule can integrate into the hydrophobic areas of the gelatin triple helix, whilst the chromanol head may engage in hydrogen bonding with the polypeptide backbone. This dual interaction functions as a physical crosslinker, forming a more complex and resilient polymer network. The increasing density and strength of these intermolecular bonds hinder deformation under compressive stress, therefore improving the overall compressive modulus. Resilience is measured by its capacity to absorb elastic energy during deformation and thereafter return to its original shape upon the removal of the load. In this study, all the groups exhibited the capacity to return to their former shape after being squeezed (compressed); 1% TTE_0.1% GNP_7% GEL, 1% TTE_0.3% GNP_7% GEL, and 0.1GNP_7% GEL (181.2%, 106.3%, 121.4% respectively) exhibited the highest resilience compared to 0.3% GNP_7% GEL (89.6%) (Figure 6a). Tocotrienols enhance the resilience of the hydrogel by improving its elasticity, which is the capacity of a substance to absorb energy during elastic deformation and revert to its original form. The non-covalent connections between tocotrienols and gelatin chains are sufficiently robust to strengthen the network, yet they remain dynamic. These contacts may fracture and restore under stress, enabling the polymer network to release energy instead of experiencing plasticization. Upon the removal of the load, the network can return to its previous structure due to the entropic elasticity of the gelatin chains, which have been maintained in a more ordered yet elastic state by the tocotrienols.

Scaffolds fabricated for bone applications exhibited a high compressive modulus and rigid structure; most of them had decreased hydration, which made them less effective in delivering and encapsulating the bioactive compounds [29]. In this study, 0.3% GNP maintains suitable mechanical strength and regulates hydration, reducing the high swelling ratio.

2.3.7. Thermal Stability Assessment

Thermal stress testing was performed via thermogravimetric analysis (TGA) to assess the stability of tocotrienol gelatin hydrogels at elevated temperatures. The TGA curves of the tocotrienol hydrogels are shown in Figure 7a, and the onset temperatures were recorded. The degradation pattern of the hydrogels shows a consistent trend, with degradation starting at an initial temperature of about 120 °C and ending around 400 °C. However, there are differences in the peak around 300 °C for tocotrienol hydrogels (Figure 7b). The 1% TTE_0.3% GNP_7% GEL hydrogel, which has the highest GNP content, shows three small peaks near 300 °C, while the 1% TTE_0.1% GNP_7% GEL has two peaks, reflecting different stages of polymer degradation. The appearance of these small peaks at approximately 300 °C suggests that adding tocotrienol improves the thermal stability of the hydrogels, likely due to the specific interactions between gelatin and tocotrienol. These findings align with TG (stage 2). All samples experienced a major mass loss between 25 °C and 800 °C. The thermal stability of the 1% TTE_0.1% GNP_7% GEL was lower at stage 3 compared to the 1% TTE_ 0.3% GNP_7% GEL and to hydrogels without tocotrienol.

2.3.8. Surface Wettability

Surface wettability is a crucial factor in hydrogel–liquid interactions, which can impact drug release, degradation, and swelling behavior. In the present study, all hydrogel formulations exhibited contact angles below 90°, indicating a hydrophilic surface (Figure 8). Hydrogels containing tocotrienol showed significantly lower contact angles compared with hydrogels without tocotrienol (p < 0.0001), suggesting enhanced surface wettability. Among the tocotrienol-loaded formulations, the 1% TTE_0.3% GNP_7% GEL group exhibited the lowest contact angle (22.86 ± 2.87), which was significantly lower than that of the 1% TTE_0.1% GNP_7% GEL group (37.42 ± 1.87). This rise in wettability is consistent with previous reports involving nonionic surfactants such as Tween 20, where the abundance of hydrophilic and hydroxyl groups promotes surface hydration through hydrogen bonding interactions. As a result, the presence of Tween 20 promotes the migration of hydrophilic moieties toward the hydrogel surface, thereby reducing the contact angle. Enhanced wettability is advantageous for drug delivery applications, as it can improve hydrogel–cell interactions and promote more predictable swelling and degradation behavior [37,38], supporting the selection of the 1% TTE_0.3% GNP_7% GEL formulation as the most favorable among the tested groups.

2.4. Cytotoxicity Evaluation

The tocotrienol-incorporated gelatin hydrogels grown with hFOB 1.19 were evaluated using a live/dead assay, revealing no harmful effects after 24 h of incubation, as illustrated in Figure 9. Green staining indicated healthy cells, whereas red staining indicated dead cells, thus confirming the biocompatibility and applicability of the hydrogels for cell growth. Among the hydrogel treatment groups, 1% TTE_0.1% GNP_7% GEL had the highest cell viability percentage (105 ± 4.36%), followed by 1% TTE_0.3% GNP_7% GEL (95.36 ± 9.78%), 0.1% GNP_7% GEL (77.6 ± 4%), and 0.3% GNP_7% GEL (74 ± 8.62%). Tocotrienol serves as a shield against the stress of the biomaterials on the cells due to its high antioxidant and anti-inflammatory properties. The incorporation of tocotrienol with gelatin hydrogel crosslinked with genipin increases the adhesion properties (more hydrophilicity) of the hydrogel matrix, thereby enhancing cell attachment and proliferation [39].

3. Materials and Methods

3.1. Materials and Reagents

Gelatin powder (12% moisture, 2% minerals, and 86% protein) was purchased from Nia-Gelatin Ltd. (Osaka, Japan). A natural full-spectrum palm tocotrienol/tocopherol complex (50%) was purchased from PhytoGaia Sdn. Bhd. (Kuala Lumpur, Malaysia). This complex contains alpha-tocotrienol (11–19%), beta-tocotrienol (1%), gamma-tocotrienol (15–25%), delta-tocotrienol (4–10%), and alpha-tocopherol (9.5–17%). Genipin (molecular weight: 226.23, purity: 98%) was supplied by FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and dissolved in ethanol, which was obtained from Merck KGaA (Darmstadt, Germany). Phosphate-buffered saline (PBS) (pH 7.4) was from NACALAI TESQUE, INC. (Kyoto, Japan). Collagenase type I (lyophilized powder) was purchased from Worthington (Lakewood, NJ, USA). Polyoxyethylene (20) sorbitan monolaurate (Tween 20) was purchased from Sigma-Aldrich (Kansas, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the LIVE/DEAD viability and cytotoxicity kit for mammalian cells, supplied by Thermo Fisher Scientific (Waltham, MA, USA), were used with the human fetal osteoblast cell line (hFOB 1.19). The remaining chemicals used were of analytical grade.

3.2. Preparation of Tocotrienol Nanoemulsion (TTE)

The tocotrienol-rich fraction of palm oil (TRF) was dissolved in ethanol at various ratios. Tween 20 was incorporated as a surfactant and stabilizer. All components were combined using a vortex for 3 min, then distilled water (dH₂O) was added gradually until the total volume reached 10 mL. The produced emulsion was homogenized for 15 min at 3000 rpm using a homogenizer (JOANLAB, Huzhou, China). The micro-emulsion was sonicated at 70% amplitude (under cooling) using SONICS VCX 130 PB and CV 188 probe sonicator (Sonics & Materials, Inc., Newtown, CT, USA). The optimal tocotrienol and Tween–ethanol ratios were selected based on phase separation. All samples were refrigerated at 4 °C, and phase separation was monitored. For one month of control, 10% (v/v) TRF was used, given the lack of separation between phases in subsequent studies.

3.3. Characterization of Tocotrienol Nanoemulsion

The uniformity of the TTE was validated using droplet size and PDI measurements with a zeta sizer (Malvern Instruments, Worcestershire, UK) under a voltage level of 3.00 kV. FTIR measurements were conducted to verify the chemical stability of tocotrienol using FTIR (PE, Waltham, MD, USA).

3.4. Dose Response of Tocotrienol Emulsion Study

The optimal tocotrienol emulsion concentration to incorporate into the fabricated hydrogel was determined using a cell viability test with the MTT assay. The osteoblast cell line was used for this purpose. The human fetal osteoblast cell line (hFOB 1.19) was provided by Dr. Sok Kuan Wong, Department of Pharmacology, Faculty of Medicine, National University of Malaysia, and grown in α-modified essential medium (α-MEM) supplemented with 10% fetal bovine serum and 10% antibiotic–antifungal agent in a humidified condition at 37 °C and 5% carbon dioxide. In all experiments, the cells were subcultured at a density of 1 × 10⁴ cells/mL in growth media. Different tocotrienol emulsion concentrations were prepared in a complete medium (α-MEM supplemented with 10% fetal bovine serum and 10% antibiotic–antifungal agent) using serial dilution. The concentrations were 0.2%, 0.3%, 1%, 5%, and 9% (v/v). Cytotoxicity of tocotrienol was assessed in four osteoblast samples. Osteoblasts were seeded in a 48-well plate for 24 h. Then, osteoblasts were treated with different concentrations of tocotrienol for days 1, 3, and 5. After that, the MTT assay was performed to evaluate cell viability following tocotrienol treatment. Cell viability was calculated using the following formula:

$$Cell viability=\left({\displaystyle \frac{At}{Af}}\right)\times 100$$

where At is the absorbance of cells treated with tocotrienol and Af is the absorbance of cells in complete medium.

3.5. Fabrication of Tocotrienol Emulsion Hydrogel

A gelatin hydrogel was fabricated as previously described by Kirchmajer et al. (2013) with some modifications [34]. Two different concentrations of gelatin solution (7% and 10% w/v) were prepared by dissolving and stirring gelatin powder (Nia-Gelatin Ltd., Osaka, Japan) in distilled water (dH₂O) at 40 °C for 1 h at 400 rpm using a hotplate stirrer. Genipin (GNP) solution (0.1%, 0.3%, and 0.5% w/v) was made by mixing crystalline GNP powder (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in 70% ethanol (EtOH) (MERCK, Darmstadt, Germany) at room temperature (22–24 °C). The GNP solution was added to the prepared gelatin solution to obtain different formulations of GNP-crosslinked GH (GNP_GH). The selected tocotrienol emulsion from the dose–response study is incorporated into the gelatin solution before the polymerisation step. Polymerisation time for each formulation was determined via the inverted tube test method at room temperature (22–24 °C), as performed elsewhere [17]. The polymerisation time was recorded by observing the polymerisation in a tilted tube.

Table 3. Experimental groups: tocotrienol, genipin, and gelatin hydrogel formulations.

Code	Tocotrienol Emulsion	Genipin %	Gelatin %
1	1%	0.1%	7%
2	1%	0.1%	10%
3	1%	0.3%	7%
4	1%	0.3%	10%
7	_____	0.1%	7%
8	_____	0.1%	10%
9	_____	0.3%	7%
10	_____	0.3%	10%

Note: The values listed in the table indicate the final concentrations of each component in the hydrogel formulations after mixing. “_____” indicates that the tocotrienol nanoemulsion was not included in that specific group.

summarizes the composition of the experimental hydrogel formulations. All values represent the final concentrations of each component after preparation. For groups 7–10, the tocotrienol nanoemulsion was omitted to serve as controls.

3.6. Phytochemical and Mechanical Characterization of Tocotrienol Nanoemulsion Incorporated Genipin-Crosslinked Gelatin Hydrogel

3.6.1. Gross Appearance

A digital camera (Nikon, Tokyo, Japan) was used to record the overall appearance of the synthesized hydrogels, including both top and side views.

3.6.2. Fourier Transform Infrared Spectroscopy

The FTIR spectrum of tocotrienol emulsion, gelatin, gelatin crosslinked with genipin, and tocotrienol emulsion-loaded gelatin hydrogel crosslinked with genipin was analyzed using FTIR (PE, Waltham, MD, USA) within the wavenumber range of 500–4000 cm⁻¹ to investigate the interaction between the chemical bonds of the tocotrienol emulsion and hydrogel components.

3.6.3. X-Ray Diffraction

The crystallinity of the freeze-dried control hydrogels and drug-loaded hydrogels was examined using an X-ray diffractometer (Bruker, D8 Advance, Coventry, UK). The XRD diffraction angle was recorded at 2θ, and 0–80 °C, and the crystalline and amorphous structures were evaluated using the integrated software (Diffrac. Suite EVA, V4.0, Bruker, Coventry, UK).

3.6.4. Field Emission Scanning Electron Microscopy

The surface and cross-sectional morphology of the freeze-dried hydrogels were evaluated by FESEM (Supra 55VP model, Jena, Germany) after coating with gold using an ion sputter coater. Then, the pore size was examined using ImageJ software (V1.5, Bethesda, MD, USA).

3.6.5. Energy Dispersive X-Ray Spectrum (EDX)

The element composition was examined by FESEM and provided with an energy-dispersive X-ray spectrum (EDX), which can directly analyze the composition of the hydrogels in both qualitative and quantitative terms.

3.6.6. Mechanical Properties

The simple compression test was applied to assess the strength and applicability of the selected hydrogels [40]. The fabricated hydrogels, approximately 2 cm in diameter and 4 mm in height, were tested for compression at ambient temperature. The compression modulus (E) was determined by the formula shown below [41]:

$$E=\sigma /\epsilon$$

where ε indicates changes in volume per unit volume (strain) and σ indicates compressive force per unit area (stress).

3.6.7. Resilience

Resilience is the ability of scaffolds to recover and return to their original form after being subjected to pressure. A measurement of 300 g of metal was used and placed on the scaffolds for 5 min. Subsequently, images of the scaffolds were obtained before and after pressure using a digital camera. The thickness of the scaffolds before pressure, during pressure (when 300 g of metal was placed on the scaffolds for 5 min, and by the end of the 5 min, the thickness was measured), and after pressure was analyzed using ImageJ software version 1.54k (NIH, Bethesda, MD, USA) [42]. The equation below was used to measure the resilience (R):

$$R\left(\%\right)={\displaystyle \frac{Ai-Ac}{Af}}\times 100$$

Ai indicates the thickness area before compression, Ac indicates the thickness area during compression, and Af indicates the thickness area post-compression.

3.6.8. Thermal Stress Testing

The variations in the mass of the hydrogels against temperature were evaluated via thermogravimetric analysis (TGA) using the simultaneous thermal analyzer STA 449 F3 Jupiter (NETZSCH, Bavaria, Germany). The hydrogels’ weight loss was recorded as a function of the temperature within 30–800 °C. The readings obtained were analyzed using OriginPro 2024 software.

3.6.9. Swelling Properties

The swelling ratio of the hydrogels was examined to evaluate their capacity to absorb exudates. The weights of the freeze-dried hydrogels were measured before immersion in phosphate-buffered saline (PBS) for 24 h. The following formula was used to measure the swelling ratio:

$$Swelling Ratio (\%)={\displaystyle \frac{W2-W1}{W1}}\times 100$$

W1 is the weight of the hydrogels before submersion.

W2 is the weight of the hydrogels after submersion

3.6.10. Degradation Analysis

The in vitro enzymatic biodegradation analysis was conducted to assess the biodegradability of the hydrogels subjected to the enzymatic reaction. In brief, the initial weight of the hydrogels was recorded prior to immersion in collagenase type I (0.0006% (w/v)) (Worthington, Lakewood, NJ, USA) for 24 h at 37 °C. The final weight of the hydrogels was recorded after the enzyme was removed and the hydrogels were rinsed with distilled water. The following equation was used to measure the weight loss %:

$$Weight Loss \left(\%\right)={\displaystyle \frac{Wf-Wi}{Wi}}\times 100$$

Wf indicates the final weight and Wi indicates the initial weight.

3.6.11. Surface Wettability

The surface wettability was evaluated via a contact angle test to determine the hydrophilicity (water-loving) of the hydrogels. A measurement of 10 μL of distilled water was applied to the hydrogel surface, and images were captured using a digital camera. The captured pictures were examined via ImageJ software version 1.54k (NIH, Bethesda, MD, USA).

3.6.12. Porosity

The porosity of the freeze-dried hydrogels was assessed by the liquid displacement method. Ethanol was selected as the displacement liquid because it can pass through the porous hydrogels without causing the scaffolds to shrink or swell. After collecting the initial weight of the freeze-dried hydrogels, the hydrogels were immersed in ethanol. The hydrogels’ final weight was recorded following immersion. The porosity of the hydrogels was measured using the following equation:

$$Porosity(\%)={\displaystyle \frac{Wb-Wa}{\rho V}}\times 100$$

Wb indicates the final weight of the hydrogel.

Wa indicates the initial weight of the hydrogel.

V indicates the volume of the hydrogel.

ρ indicates the density of ethanol (0.789 g/m³).

3.7. In Vitro Viability Analysis—Live/Dead Cell Fluorescence Microscopy

The human fetal osteoblast cell line (hFOB 1.19) was seeded on top of the sterile hydrogels placed on a 48-well tissue culture plate at a density of 2 × 10⁷ cells/mL. The cells were cultured using FBS-free DMEM in the incubator at 5% CO₂ and 37 °C for 1 and 5 days. To determine the viability of the Hfob 1.19 cells, live and dead assay was performed. In brief, the staining solution (Thermo Fisher Scientific, Waltham, MA, USA), containing 4 mM ethidium homodimer-1 (EthD-1) for dead cells and 2 mM acetomethoxy calcein derivative (calcein-AM) for live cells, was added to the wells (300 μL) for 30 min. The stained hydrogels were assessed utilizing a fluorescence microscope (Nikon A1R-A1, Tokyo, Japan).

3.8. Statistical Analysis

GraphPad Prism version 10.0 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis. The mean values from the group comparisons were analyzed using one- and two-way analysis of variance (ANOVA). Statistical significance was set at a p-value of less than 0.05, and all data are provided as mean ± standard deviation (SD).

4. Conclusions

In conclusion, the genipin-crosslinked gelatin hydrogel serves as a platform on which various physicochemical interactions with incorporated drugs sustain drug release and prevent drug degradation. In this study, we successfully evaluated a biodegradable gelatin hydrogel crosslinked with genipin for tocotrienol delivery directly at the bone-injured site. The emulsion showed good homogeneity, stability, and viability for hydrogel incorporation. The formulation 1% TTE_0.3% GNP_7% GEL exhibited a suitable swelling ratio for bone application, optimal weight loss, and excellent hydrophilicity. FESEM images of the cross-section revealed that 1% TTE_0.3% GNP_7% GEL had an ideal average pore size of 292 ± 37 μm with interconnected pores. No significant changes in the chemical analysis were observed after incorporating tocotrienol into the gelatin hydrogel matrix. The contact angle of the hydrogels containing tocotrienol decreased (wettability increased) compared to the hydrogels without tocotrienol, thereby increasing the hydrogel’s adhesion properties (more hydrophilicity) and enhancing cell attachment. The live/dead assay showed >90% cell viability of hFOB 1.19 cells cultured on 1% TTE_0.1% GNP_7% GEL and 1% TTE_0.3% GNP_7% GEL. A tocotrienol-fortified gelatin hydrogel crosslinked with genipin showed excellent physicochemical properties. It exhibited no detectable in vitro toxicity, which can specifically maintain long-term delivery at the desired site in bones. Further NMR studies are required to better understand the chemical interactions within the tocotrienol emulsion. Osteogenic activity studies, such as alkaline phosphatase activity, mineralization, and osteogenic gene expression, are also required to demonstrate the ability of the tocotrienol-incorporated gelatin hydrogels crosslinked with genipin to activate bone healing and support the osteogenic differentiation of cells.