Biofabricated genistein incorporated collagen scaffold for enhanced mineralization with refined osteogenic efficacy

Abstract

Bone remodeling is a natural process that involves osteoblasts and osteoclasts. However, this balance can be disrupted due to aging, accidents, and bone defects from conditions like arthritis. Traditionally, these issues have been addressed through metal implantation, tissue grafting, and surgical interventions. However, these methods have limitations, including the risks of inflammation, infection, and delays in donor availability. Bone tissue engineering has become a modern approach to overcoming these challenges. This field utilizes biocompatible and biodegradable biomaterials with osteogenic properties, which reduce the risk of post-surgery infections and inflammation. The development of natural composite scaffolds provides a synergistic effect for osteogenic regeneration. In this study, a collagen-fibrin scaffold incorporated with genistein (Gn) and crosslinked with genipin (Gp), (C-F-Gp-Gn) has been developed to repair osteogenic defects. The binding of genistein to collagen and fibrin was evaluated and verified using Fourier Transform Infrared Spectroscopy. Structural analysis demonstrated that the scaffold’s interconnected pore structure, with a porosity of 60%, is ideal for bone regeneration. Biochemical assays indicated a good swelling potential and an optimal degradation rate of 82% in 14 days. In vitro assays were carried out in MG-63 cells, exhibiting the scaffold’s biocompatibility, while Live/Dead cell staining displayed enhanced cell proliferation and adherence properties. Alizarin Red staining and alkaline phosphatase assays indicated an increased mineralization capacity in the C-F-Gp-Gn scaffold compared to the scaffold without genistein. Gene expression studies showed that the C-F-Gp-Gn complex enhances osteogenic gene expression and improves bone matrix formation. The results suggest that the genistein-incorporated collagen-fibrin scaffold possesses improved osteogenic properties and superior mineralization, making it a promising biomaterial for bone tissue engineering applications.

Keywords

Collagen genistein bone tissue engineering osteogenic repair mineralization

Introduction

Bone remodeling is a physiological process that rejuvenates skeletal tissue by replacing old and newly formed bone.¹ This process helps in providing mechanical stability by remodeling osteoblast, osteoclast, osteocytes, and bone lining cells, and it maintains the balance of calcium and phosphorus.^2,3 However, individuals who are aged, dealing with arthritis, injured by accidents, or have undergone bone trauma face challenges in replacing the injured bone and bone remodeling, resulting in delays in achieving complete bone regeneration. It results in intense pain, infection, and inflammation throughout the regeneration process.^3,4 Various treatments have been developed, including surgery, bone grafting, and metal implantation. However, these approaches have complications of postsurgical problems, immunogenic response, the risk of infection, limited availability of donor tissue, and poor integration in metal implantation.⁵ A contemporary approach has been carried out to avoid this limitation by introducing a biomaterial-based scaffold to regenerate repaired bone, known as bone tissue engineering. This process involves using bone substitute biomaterial to promote regeneration and enhance the functionality of bone tissue in the damaged region.^5,6 Biomaterials are components that mimic the internal environment and induce the formation of new tissue.⁷ There are two categories of biomaterials involved: natural biomaterials and synthetic biomaterials. Natural biomaterials possess high biocompatibility and biodegradability, resembling the biological extracellular matrix components within the body, and synthetic biomaterials are mechanically robust and possess thermal stability.^8,9 Biomaterials can be further classified as either single or composite. Single compound biomaterials typically focus on one or two specific properties, which may not provide a complete range of characteristics necessary for effective application. In contrast, composite biomaterial scaffolds combine different biomaterials with varying properties to provide a synergistic effect on improving bone tissue engineering.¹⁰ An ideal biomaterial should possess biocompatibility, biodegradability, mechanical stability, and non-immunogenicity, enhance osteogenic differentiation, and support cell growth and attachment.³ A natural composite scaffold can encapsulate all these properties within a single material. In this study, composite material is synthesized by combining natural biomaterial collagen and fibrin with the incorporation of genistein for improved bone tissue regeneration. Firstly, collagen has been chosen as the most preferable material; it is a crucial protein constituting 30% of the total protein in mammals, playing a vital role in forming both hard and soft tissue.⁹ Among the 29 types of collagen, type 1 is predominantly present in tissues such as skin, bone, cornea, tendon, and ligament.^10,11 Collagen exhibits favorable characteristics such as excellent dimensional stability, regenerating osteoblast, low immunogenicity, effective hemostatic capacity, and biodegradability.¹² However, collagen alone exhibits suboptimal structural characteristics in the scaffolds. To overcome this limitation, fibrin has been chosen to bolster structural resilience. Fibrin contributes to structural reinforcement and other properties, like biocompatibility and biodegradability, and has good pro-angiogenic effects, along with tunable physiochemical features.^13,14 Additionally, fibrin is crucial in maintaining homeostasis and possesses properties that aid in bone healing and osteoblast differentiation.¹⁵ It provides a temporary matrix during tissue regeneration, eventually replaced by more permanent extracellular matrix components as the tissue heals and matures.¹⁶ To improve osteoblast activity and regulate signaling pathways related to bone regeneration, genistein has gained preference. Genistein, a phytoestrogen, has garnered significant attention in the medical and scientific community.¹⁷ It is a major isoflavone found in soybeans and structurally similar to estrogen. Two subtypes of estrogen receptors, α, and β, are expressed in bone cells after estrogen binding; it triggers the osteogenic-related genes, leading to osteoblast activity and the facilitation of new bone regeneration. Genistein has the ability to bind to the estrogen receptor due to structural similarity and activate estrogen-related gene expression.¹⁸ It also enhances osteoblastogenesis by triggering osteoblast-based expression like alkaline phosphatase, osteocalcin, and mineral matrix deposition.¹⁹ It decreases the osteoclast resorption by inhibiting the signaling activity of NF-κB. These attributes contribute to its substantial biocompatibility, which is desirable for bone regeneration.²⁰ Genistein is typically administered orally for osteoporosis and postmenopausal bone loss, systemic delivery faces challenges such as low bioavailability, first-pass metabolism, and non-targeted distribution.²¹ This study uniquely addresses these limitations by locally delivering genistein through a biodegradable scaffold, allowing for site-specific and sustained release directly at the bone defect site. The use of collagen and fibrin, both natural, biocompatible polymers, provides a physiologically relevant matrix that supports osteoblast proliferation, differentiation, and extracellular matrix formation in delayed bone regeneration.²² Integration of genistein into a collagen–fibrin scaffold with a focus on bone regeneration, thus offering both material and therapeutic innovation.

The synthesized scaffolds were subjected to physiochemical characterization like Fourier transform infrared spectroscopy (FTIR) to analyze the interaction between the scaffold and scanning electron microscopy (SEM) to evaluate the morphology and structural integrity of the scaffold. Further analysis included a ninhydrin assay to assess the crosslinking percentage of the genipin crosslinked scaffold. Additionally, biochemical characterization was performed to evaluate biodegradation, swelling potential, and porosity. The cytocompatibility of the scaffold was analyzed through 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide assay (MTT), and cell morphology and proliferation were viewed by using Live/dead cell staining. To assess the mineralization properties of the scaffold, Alizarin red staining and alkaline phosphatase assays were conducted. Finally, the osteogenic-related protein marker expression level of RunX2, Osteocalcin (OCN), Collagen type I (Col type 1), BMP-2, and TGF-β1 was evaluated using an enzyme-linked immunosorbent assay.

Experimental methodology

Experimental materials

Genistein was procured from TCI Development Co Ltd, Genipin, Dulbecco’s Modified Eagle Medium, DMSO (Dimethyl Sulfoxide), Calcein AM, Propidium Iodide, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Alizarin red was bought from Sigma Co Ltd, Ninhydrin, Fetal bovine serum (FBS) from Biowest Co Ltd, Antibiotic (penicillin/streptomycin), 4% paraformaldehyde purchased from Himedia, ALP assay Kit was purchased from Thermofisher Scientific Inc, ELISA Kit from R & D Systems and Bone protein markers- RunX2, Osteocalcin, Collagen type I, BMP-2, and TGF-β1 were purchased from Santa Cruz.

Collagen extraction and characterization

Collagen was isolated from rat tail tendons using the following isolation procedure. The tendons were gently separated and thoroughly washed with a 0.9% saline solution. The cleaned tendons were then dissolved in a 0.5M acetic acid solution and left overnight. Afterward, the dissolved tendons were filtered through muslin cloth to eliminate any undissolved material. A 5% NaCl was added to the supernatant to induce collagen precipitation, and the mixture underwent centrifugation. In a 0.5M acetic acid solution, the obtained pellet was dissolved again and stirred overnight. The collected solution was treated with a 0.02M dibasic sodium phosphate solution using a dialysis process until a dense precipitate formed. The precipitated collagen was subsequently re-dissolved in acetic acid and dialyzed again against a 0.05M acetic acid solution. All steps were conducted at 4°C. Following dialysis, the collagen is freeze-dried and stored for further use.²² The purified collagen was characterized using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis to examine the molecular size, purity, and structural characteristics. Additionally, Circular Dichroism (CD) analysis was performed with a Jasco Circular Dichroism Spectropolarimeter to evaluate the triple helix structure. Fourier Transform Infrared Spectroscopy (FTIR) was also conducted using a spectrometer model JASCO FT/IR-4700 type A; it detects the chemical bonds within a molecule by producing a spectrum of infrared absorption. Molecules absorb IR radiation ranging between 4000 and 600 cm⁻¹, detecting the functional groups of collagen.

Fibrin isolation and characterization

From the slaughterhouse, bovine blood was collected, and the collected blood was stirred continuously by homogenizer. The clotted crude fibrin was thoroughly cleansed using sterile water to eliminate any remaining traces of blood. Fibrin was subsequently treated with 0.5M sodium acetate for 2 days, and the solution was changed thrice during this interval. After treatment, the fibrin was filtered and further purified by immersing it in 30% hydrogen peroxide, followed by an extensive rinsing with distilled water. The purified fibrin was stored in the deep freezer at −80°C and lyophilized for future work.⁷ To confirm its chemical composition, the isolated fibrin was characterized using Fourier Transform Infrared spectroscopy (FTIR).

Scaffold preparation and characterization

Scaffold was fabricated by dissolving 50 mg of collagen in 10 ml of 0.5 molar acetic acid, followed by overnight stirring to ensure complete dissolution. Concurrently, 5% fibrin was dissolved in 1 N NaOH and continuously stirred for 48 h.²² Once both solutions were well-dissolved, they were mixed in a 1:1 volume ratio. To induce crosslinking, 0.5 mM of genipin was added from a 10 mM stock solution. Subsequently, genistein was incorporated into the mixture at a concentration of 50 µM. The resultant solution was then aliquoted into well plates, freeze-dried, and prepared for subsequent characterization.

Physiochemical characterization

The fabricated scaffold was characterized using Fourier Transform Infrared Spectroscopy (FTIR-4700 JASCO, Japan) to verify the presence of constituent materials and evaluate the bond formation between the biomaterial. Differential Scanning Calorimetry (DSC) was also conducted using DSC2A-00837, Type DSC25, to assess thermal stability. Analysis began at a starting temperature of 30°C, with the temperature increasing at a rate of 10°C/min until reaching 300°C. Scanning Electron Microscopy (SEM) analysis was conducted to evaluate the surface and cross-sectional morphology, pore size, and structural integrity of the scaffold.

Mechanical strength

The compressive strength of the scaffold was evaluated to assess its mechanical integrity under load-bearing conditions. Testing was conducted using a Universal Testing Machine (UTM; INSTRON 3369, equipped with load cell J 7257), fitted with a 10 N capacity load cell. Scaffolds with dimensions of (30 mm height × 20 mm width × 15 mm thickness) were subjected to uniaxial compression at a crosshead speed of 0.5 mm/min.²³ The force displacement data were recorded, and compressive strength was calculated based on the maximum load and the cross-sectional area of the scaffold.

Ninhydrin-based crosslinking analysis

The extent of crosslinking within the scaffolds was determined through a chemical reaction using ninhydrin. Equal amounts of each scaffold sample were prepared, and approximately 4 ml of ninhydrin reagent was introduced. The mixtures were subjected to heating at 100°C for 20 min, followed by cooling to room temperature. Then, 50% of 5 ml isopropanol was introduced to the solution.²² The absorbance was then recorded at 570 nm using a UV-Vis spectrometer (JASCO). A graph was plotted, and the degree of crosslinking was assessed using the following formula

Crosslinking % = \frac{\begin{array}{l} Absorbance of Non - \\ crosslinked sample - \\ Absorbance of Cross \\ linked sample \end{array}}{\begin{array}{l} Absorbance of Non - \\ Crosslinked sample \end{array}}

Kinetic study of drug delivery

Kinetic experiments were performed to evaluate the drug delivery efficacy of the genistein-loaded scaffold. Equal weights of the scaffold were prepared in triplicate, 1× phosphate buffer saline solution (PBS. pH 7.4), and soaked at room temperature for predetermined time intervals. At the end of each interval, a defined volume incubation solution was obtained and substituted with an equivalent amount of unused PBS.²⁴ The amount of genistein release was quantified using a UV-visible absorption spectrometer (JASCO) at a specific wavelength of 260 nm. The cumulative release profile was quantified by referencing the obtained data with a predetermined standard graph. For accuracy, the mean values derived from the triplicate measurements were utilized for subsequent analysis.²⁵

Biochemical characterization

Swelling analysis

Investigation of swelling properties of the scaffold was estimated by recording its initial dry weight of scaffold, denoted as Wi. The scaffold was subsequently submerged in 1× PBS and kept at 37°C for the designated time periods. The scaffold was collected, rinsed, and gently blot-dried at the termination of each interval.⁷ The final wet weight of the scaffold was recorded as Wt and then measured. The following formula calculated to determine the swelling ratio:

Swelling Ratio = \frac{W i - W t}{W t}

Porosity

To assess the scaffold’s porosity, the initial volume of ethanol (V1) was recorded. The weight of the scaffold was measured before immersing it in ethanol. After immersion, the wet weight of the scaffold was recorded, and the absorbed ethanol volume was recorded as V2. Subsequently, the remaining ethanol volume in the tube, following the removal of the scaffold, was recorded as V3.⁷ The scaffold’s porosity was subsequently determined using the provided formula:

Porosity % = \frac{V 1 - V 3}{V 2 - V 3} \times 100

Biodegradation

To measure the degradation rate of the scaffold, it was immersed in 1× PBS enriched with lysozyme at a concentration of 10,000 U/L. Before immersion, the scaffold’s initial weight (Wi) was measured. Following this, it was maintained at room temperature with different intervals of time between 7 and 14 days. Following incubation, the scaffold underwent lyophilization, and its post-lyophilization weight (Wt) was recorded.⁷ Analyses were undertaken were conducted in three replicates. The scaffold’s degradation rate was subsequently calculated using the following formula:

Biodegradation % = \frac{W i - W t}{W t} \times 100

Biocompatibility assay

MTT assay

In vitro experiments were carried out in MG-63 cells. The assay was performed in three groups: Group 1 (Control): Media only, no biomaterial was added (DMEM + 10% FBS + 1% Antibiotics), Group 2 (C-F-Gp): Media incubated with collagen and fibrin, crosslinked with genipin, Group 3 (C-F-Gp-Gn): Media incubated with collagen, fibrin, and genistein, crosslinked with genipin. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS) and antibiotics to support growth and maintain culture conditions. Cultures were maintained in a CO₂ environment with 5% at 37°C. Conditioned media were prepared by sterilizing the scaffolds with ethanol, exposing them to UV light for decontamination, soaking them in prepared media, and kept for incubation. After incubation, the collected media was used for downstream experiments.²²

The biocompatibility of the material was evaluated using the 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Here, the cell viability evaluation was performed to analyze the scaffold’s biocompatibility. Initially, MG-63 cells (10⁴ cells/wells) were seeded and left for adherence. The experiments were carried out in triplicates. After the adherence of cells, the existing media was discarded, and the conditioned media was introduced. The assay for biocompatibility was carried out at 24-, 48-, and 72-h intervals. At the completion of each time period, the conditioned media were taken out, and MTT solution (5 mg/ml) was introduced in every well, then incubated for 4 h. Discard the MTT, add 10% DMSO solution to dissolve the formazan crystal, and incubate for 30 min. After incubation, the solution was thoroughly mixed, and the absorbance value was estimated at 570 nm with a microplate assay reader⁷ (Jasco Spectrometer V-750).

Cellular viability by fluorescence staining

The viability of cells, morphology, and distribution were evaluated by dual staining with Calcein AM/Propidium Iodide (PI) fluorescence staining. A 96-well plate was seeded with 10⁴ cells, and after cell adherence, the conditioned media were replaced. The cells were incubated for varying durations of 24, 48, and 72 h. Upon reaching each time interval, the media were discarded, and each well was gently rinsed with 1x ice-cold PBS. Following this, 3 µM of Calcein AM and 1 µg/ml of Propidium Iodide were added to the well.²⁶ The samples were visualized using a fluorescence microscope (Leica, DMI8), where green fluorescence was observed in viable cells, and red fluorescence was detected in non-viable cells, indicating live and dead populations, respectively.

Analyzing the mineralization property

Biomineralization

The evaluation of mineral deposition on the scaffold was conducted using biomineralization techniques. The scaffold was weighed equally and immersed in Simulated Body Fluid (SBF) at room temperature for the specified duration of 7 and 14 days. Following incubation, the materials were washed with sterile distilled water and subsequently subjected to lyophilization.²⁷ The dried sample underwent Scanning Electron Microscopy (SEM) analysis to visualize mineral deposition on the scaffold, which was further validated through Energy Dispersive X-ray Spectroscopy (EDAX) analysis.

Alkaline phosphatase assay

The activity of alkaline phosphatase (ALP) was quantified to assess early osteogenic differentiation, a key marker indicating phosphate ion release necessary for hydroxyapatite formation and bone matrix maturation. This analysis provides insights into the scaffold’s osteoinductive capabilities and its potential to facilitate mineralization.⁷ The procedure followed is based on the manuscript protocol of the product. A total of 10⁴ cells were plated into a 96-well plate, and after cell adherence, the media were replaced with conditioned media. The cells were incubated for specified durations of 7, 14, and 21 days. After each incubation time interval, an ALP assay was performed on the collected media. Initially, 980 µl of assay buffer solution was introduced to each well, and subsequently, a substrate solution (167 mM) was added. The sample was kept for incubation for 30 min at 37°C. A standard positive control was established by introducing an ALP enzyme, and conditioned media were introduced into each well in triplicates. After a further 30 min of incubation, the optical density was recorded at 410 nm to quantify ALP activity.

Assessment of mineralization using alizarin red

Alizarin Red staining Assay was employed to evaluate calcium deposition on the cells monolayer, serving as a critical indicator of mineralization and osteogenic differentiation. It confirms the scaffold’s ability to support bone-like matrix formation. Cells were grown in 12-well plates with conditioned media for specified durations of 7 and 14 days. After incubation, ice-cold 1xPBS was used to rinse the cells, treated with 10% formalin for fixation, and restored to hydration using 1 ml of distilled water for a duration of 5 min. To stabilize the monolayer, the cells were stained with alizarin red (1%) in ethanol (2%) for fixation.⁷ Following 30 min of incubation at 37°C, the stain was removed and rinsed with 1xPBS. Images of alizarin-stained cells were viewed and captured under a light microscope. For quantitative evaluation, stained cells were incubated with 10% acetic acid, detached using a cell scraper, and transferred into microcentrifuge tubes. The samples were then heated at 85°C for 10 min, followed by centrifugation to separate the supernatant, which was subsequently analyzed for absorbance at 405 nm using a JASCO UV-Vis spectrophotometer.²⁸

Quantitative analysis of bone protein marker expression using ELISA

ELISA-Enzyme-linked immunosorbent Assay was conducted to assess the osteogenic marker protein expression following scaffold treatment and conditioned media preparation. The procedure was performed as per the manufacturer’s instructions. For this analysis, 10⁴ cells were seeded into plates and cultured with conditioned media for 7, 14, and 21 days. After each incubation period, the media were collected, and the Bradford assay was carried out for quantification of protein to ensure equal protein loading for ELISA. An indirect ELISA method was employed, where the first layer was coated with the antigen (collected media). A primary antibody was then added to specifically bind to the target antigen, followed by introducing the secondary antibody designed to bind to the primary antibody. Adding a substrate resulted in a color change, which was quantified by measuring optical density (OD). This process confirmed the level of osteogenic protein expression.

The ELISA procedure started with plate preparation involving antigen coating. Antibodies targeting RunX2, BMP-2, Collagen type I (Col type I), Osteocalcin, and TGF-β1 were utilized. To prepare the plate, 50 µl of a plate coating solution was made by mixing a quantified sample with the coating buffer. The well plate was kept for overnight incubation at 37°C to ensure proper antigen adherence. After the specified time interval, the coating solution was discarded, and the wells were rinsed 2–3 times with 1x washing buffer, ensuring gentle tapping to eliminate residual liquid. Subsequently, 50 µl of blocking buffer was introduced to each well to avoid nonspecific binding, and the sample was incubated at room temperature for 30 min. Following another wash cycle, 100 µl of primary antibody, diluted at a 1:1000 ratio, was introduced and incubated for 2 h. The wells were then washed again, and the secondary antibody was added, followed by 1 h of incubation. After the final washing step, the substrate was added. Once incubated for 1 h, add the stop solution and read the absorbance at 570 nm.

Statistical analysis

All assays were undertaken in triplicates. Data were analyzed using one-way analysis of variance (ANOVA) to compare differences among independent groups, and pairwise comparisons were carried through using the Student T-test. Statistical significance was considered for p-values of below 0.05.

Results and discussion

Collagen and fibrin characterization

The collagen extracted from rat tail tendons was confirmed through several methods: SDS-PAGE, circular dichroism (CD) analysis, and Fourier-transform infrared spectroscopy (FTIR). SDS-PAGE revealed α1 and α2 bands at 120 and 110 kDa, respectively, along with a β band at 200 kDa, confirming the presence of Type I collagen, the result is enclosed in Supplemental Figure S1. FTIR analysis shown in Figure 1, has a peak at 1631 cm−¹ for Amide I, which corresponds to the C=O vibration of the stretching peptide bond, confirming the secondary structure of the collagen. The peak at 1548 cm⁻¹ for Amide II is primarily due to N-H flexural vibration and C-N vibrational elongation, while the peak reached maximum at 1238 cm⁻¹ for Amide III reflects a fusion of N-H deformation and C-N vibration of expansion, associated with the three-dimensional helix configuration of collagen.²⁹ The CD analysis supported the triple helix structure of the collagen, displaying a maximum rotatory value at 221 nm and a minimum at 205 nm.³⁰ The results are enclosed in Supplemental Figure S1. Similarly, in Figure 1, fibrin displayed a maximum at 1655 cm⁻¹, indicating the C=O vibration of the stretching peptide bond, and a maximized at 1518 cm⁻¹, representing N-H bending deformation and C-N stretching vibrations. Additionally, peaks at 1239 and 1402 cm⁻¹ correspond to Amide III of fibrin.²⁰ A peak at 3423 cm⁻¹ represents the hydroxyl group (OH) in genistein.³¹

Physiochemical analysis of the Scaffold

Infrared spectroscopic characterization

In the characterization of genistein-incorporated collagen fibrin scaffold, the FTIR results in Figure 1 show the disappearance of peak at 3276 cm⁻¹, indicating that the hydroxyl group in genistein forms a bond with collagen and fibrin.³² The disappearance of the peak at 1243 cm⁻¹ signifies the interaction between the collagen’s primary amine functional groups and genipin. Furthermore, peaks at 1402, 1541, and 1636 cm⁻¹ confirm the presence of both collagen and fibrin.^7,29

Thermal analysis by DSC

Differential thermal analysis is performed to analyze the thermal transitions of the scaffold with increasing temperature. Their thermal stability was evaluated based on weight loss as the temperature increased. The results in Figure 1 show that C-F-Gp analysis initially reveals endothermic peaks, indicating a phase transition with melting occurring at 50°C for the scaffold. Following these endothermic peaks, exothermic peaks emerge, signifying the degradation of the scaffold.²² Initial degradation begins at 100°C, and by 300°C, the scaffold shows significant weight loss, suggesting that it has poor thermal stability. In the genistein-incorporated scaffold, the first endothermic peak also occurs around 50°C, indicating the phase transition.⁷ A notable peak is observed at 150°C, which indicates degradation and the curves begin to flatten after reaching 200°C, suggesting a reduction in activity at that temperature.³³ Among the samples, C-F-Gp exhibits the least thermal stability due to its early onset of degradation and considerable weight loss. In contrast, C-F-Gn-Gp displays smoother peaks, indicating a slower transition.

Structural property

The scaffold’s structural characteristics were analyzed using SEM analysis. Key factors like structural integrity and pore size were crucial for enhancing osteogenic properties. The optimal pore size and porosity of the scaffold facilitate interconnectivity, which was essential for nutrient and waste exchange, as well as for cell migration and integration.⁷ If the pores are too small, they impede cell infiltration and nutrient flow. Conversely, if the pores are too large, they may weaken the scaffold and reduce its adhesion properties.³⁴ Both surface and cross-sectional images of the scaffold were examined. In Figure 2 the SEM images show that the C-F-Gn-Gp has well-interconnected pores comparatively, which enhances osteoconductivity during bone regeneration. The pore sizes were optimal, which supports cell adherence and penetration.

Mechanical strength

The mechanical stability of the scaffold was assessed through compressive strength testing. As shown in Table 1 and Figure 3 the C-F-Gp scaffold exhibited a compressive strength of 0.073 MPa, whereas the genistein-incorporated scaffold (C-F-Gp-Gn) demonstrated a significantly higher value of 0.191 MPa. This value falls within the typical range for cancellous bone (0.1–12 MPa),²³ suggesting that the genistein-incorporated scaffolds possess sufficient mechanical integrity to support osteoblast regeneration and the surrounding microenvironment. The incorporation of genistein into collagen–fibrin scaffolds notably enhanced their compressive strength. This improvement can be attributed to increased intermolecular interactions, such as hydrogen bonding. These interactions facilitate a tighter formation of the polymeric network, thereby enhancing the structural integrity and load-bearing capacity of the scaffold.³⁵ The improved mechanical properties observed in the genistein group imply greater structural stability, likely contributing to enhanced osteogenic support.

Table 1.

Compressive strength of scaffold without genistein (C-F-Gp) and the scaffold with genistein (C-F-Gp-Gn).

Sample	Compressive strength (MPa)
C-F-Gp	0.07 ± 0.01
C-F-Gp-Gn	0.19 ± 0.02
Cancellous bone	0.1–12

Ninhydrin-based crosslinking analysis

The ninhydrin assay was employed to evaluate the extent of crosslinking in a collagen-fibrin matrix modified with genipin by measuring the reduction of unreacted amino groups. A lower concentration of free amino groups indicates a higher degree of crosslinking.²² This reaction can be quantitatively analyzed to estimate the level of unbound amino groups in the sample. The incorporation of genipin is assessed at concentrations of 0.3, 0.5, and 1 mM. The ninhydrin assay results in Figure 4 indicate crosslinking levels of 37%, 60%, and 53% for the respective concentrations. The findings suggest that 0.5 mM results in the maximum crosslinking capacity; therefore, this concentration was selected for further scaffold fabrication.

Kinetic study of drug delivery

Drug release studies are crucial for evaluating the kinetics and mechanisms by which a drug is released from its delivery system. Understanding the release profile is essential for predicting the drug’s behavior in the body.³⁶ In our study, we assessed the release kinetics of two different concentrations of genistein (30 and 50 µM) loaded into a collagen-fibrin scaffold. The results in Supplemental Figure S2 indicated that the scaffold containing 30 µM of genistein released 90% of the drug within 48 h and 98% within 72 h. In contrast, Figure 4 shows the scaffold with a 50 µM concentration released 74% of the drug in 48 h and 80% in 72 h, taking a total of 7 days to achieve complete release. The interactions between genistein and the scaffold material can significantly influence the release rate. These interactions are more pronounced at the 50 µM concentration compared to 30 µM, which restricts the mobility of the genistein molecules, resulting in a slower and more sustained release.³⁷ This slower release profile could be particularly beneficial for bone regeneration. Further MTT results of genistein, tested at different concentrations, are presented in Supplemental Figure S2. The results indicate that up to a concentration of 50 μM, genistein demonstrates good biocompatibility and a sustainable increase in cell proliferation. By combining the outcomes of drug release kinetics and the MTT assay for genistein, concluded that the 50 μM concentration was the most suitable for scaffold incorporation. Given these findings, the scaffold with the 50 µM concentration is preferred for further experiments due to its sustainable release characteristics.

Biochemical characterization

Swelling analysis

The swelling potential of a scaffold is essential for absorbing body fluids and delivering nutrients to the regenerating area. An increased swelling potential enhances fluid adsorption and retention, thereby expanding the material’s surface area, which promotes better cell infusion and adherence.³⁸ In Figure 5 Scaffolds loaded with genistein exhibit a greater swelling capacity compared to those without genistein. This is attributed to the hydroxyl group in genistein, which binds with water molecules in body fluids.³⁹ Additionally, the hydroxyl group forms hydrogen bonds with the functional groups of collagen and fibrin, resulting in increased water uptake and swelling potential. This heightened swelling potential directly correlates with increased porosity, providing a larger surface area for cell attachment and growth.

Porosity

The porosity of a scaffold is crucial for bone regeneration, as it facilitates the exchange of nutrients and gases while also supporting cell migration and vascularization.⁴⁰ A minimum pore supports cell adhesion, whereas a maximum pore increases the surface area available for cell proliferation and enhances cell migration.⁴¹ Therefore, it is essential to maintain a balanced porosity to promote both cell adherence and proliferation. The optimal range for porosity is between 40% and 65%.⁷ In Figure 5, the scaffold’s porosity was determined through the ethanol displacement technique. The scaffold without genistein illustrated a porosity of 43%, while the scaffold containing genistein had an increased porosity of 60%. The enhancement results from hydrogen bonds forming between the genistein hydroxyl group and the scaffold material, which increases pore size and overall porosity.³⁷ This porosity level provides the best architectural balance and pore interconnectivity for cell proliferation and osteogenic differentiation.⁴¹

Biodegradation

To facilitate effective bone regeneration, a scaffold must exhibit precisely regulated degradability, ensuring that its breakdown aligns with the progression of new tissue formation. This synchronization is pivotal for maintaining structural stability and adequately supporting the healing tissue throughout the regenerative process. An ideal scaffold should degrade in a controlled manner, gradually releasing its components to foster osteogenesis.⁷ The findings from this study in Figure 5 indicate that the scaffold incorporated with genistein and incubated in a 1× PBS solution enriched with lysozyme exhibited a degradation rate of 82% over 14 days. This demonstrates a slower and more controlled degradation than scaffolds without genistein, which showed a higher degradation rate of 97% during the same period. These results underscore the role of genistein in modulating scaffold degradation, making it more suitable for meeting the requirements of effective bone regeneration.^42,43

Biocompatibility assay

MTT assay

The MTT assay was performed using various concentrations of genistein, showing a steady increase in cell proliferation up to 50 µM, without exhibiting toxicity. This concentration was then utilized for scaffold preparation. The biocompatibility assay was performed to determine the compatibility of the scaffold. The results in Figure 6 indicate that the scaffold containing genistein promotes greater cell proliferation than the scaffold without genistein. Analysis with one-way ANOVA highlighted significant differences (p < 0.01 for 24, 48, and 72 h). These findings suggest that the incorporation of genistein does not negatively impact cell viability and, in fact, enhances proliferation.⁴⁵ This was further confirmed by a Student’s t-test comparing the scaffolds with and without genistein, which showed significance at (p < 0.05) in the 24-h, 48-h and 72-h groups.

Cellular viability by fluorescence staining

Live/dead cell staining was carried out to examine the cell viability and bioactivity of the scaffold and the liquid media extracted from the scaffold soaked in media. The assay was performed at hours 24, 48, and 72. The results in Figure 6 show a high proportion of live cells (indicated by green fluorescence) at 24, 48, and 72 h for both scaffolds C-F-Gp (without genistein) and C-F-Gp-Gn (with genistein). In comparison, cells exposed to the conditioned media from scaffolds incorporated with genistein demonstrated slightly higher viability than those from scaffolds without genistein and the control group. This finding suggests that the presence of genistein enhances the bioactive potential of the scaffold by promoting cell proliferation and survival. The accompanying image in Supplemental Figure S3 indicates that the cells maintain good structural integrity, as evidenced by unaltered morphology, intact cellular structure, and good adherence properties.⁴⁴ Genistein incorporation increases the cell proliferative capacity by enhancing the ERα and ERβ.⁴⁵

Mineralization property

Biomineralization

Biomineralization of the scaffold was conducted to evaluate its capacity for mineral deposition and to assess its interaction with a simulated body fluid (SBF) environment.⁷ In Figure 7, both scaffolds, with and without genistein, demonstrated calcium phosphate deposition on their surfaces. Scanning Electron Microscopy (SEM) analysis revealed distinct rod-like and flower-like structures dispersed across the scaffold layers, indicative of mineral formation. The mineral deposition was further confirmed by EDAX Table 2, which validated calcium and phosphate concentrations. While both scaffolds exhibited mineralizing capabilities, the genistein-incorporated scaffold displayed a significantly higher percentage of phosphorus content, whereas calcium levels were relatively comparable between the two groups. This increase in phosphorus may be attributed to the formation of calcium phosphate compounds, such as hydroxyapatite, facilitated by the genistein incorporation. Genistein is known to promote osteogenic differentiation, which can lead to increased deposition of mineralized matrix components.⁴⁶ The higher phosphorus levels, in conjunction with calcium, indicate the potential formation of these mineral phases.⁴⁷ This indicates that including genistein enhances the scaffold’s mineralization properties, contributing to the formation of the mineralized layer. This layer not only improves the scaffold’s osteoinductivity but also provides an ideal surface for cell adhesion and bone tissue integration, ultimately accelerating the process.⁴⁸

Table 2.

EDAX analysis of Scaffolds with and without genistein.

C-F-Gp
Element	Weight	Atomic	Net Int.
P K	85.29	88.24	41.4
Ca K	14.71	11.76	2.64
C-F-Gp-Gn
Element	Weight	Atomic	Net Int.
P K	90.55	92.54	53.6
Ca K	9.45	7.46	2.05

Alkaline phosphatase assay

Alkaline phosphatase assay was conducted using MG-63 cells treated with conditioned scaffold media, both with and without genistein. Alkaline phosphatase is a critical enzyme involved in the mineralization process, as it catalyzes the hydrolysis of phosphate ester substrates, typically p-nitrophenyl phosphate (pNPP), releasing free phosphate ions that contribute to hydroxyapatite formation. It serves as an early marker of osteogenesis, usually peaking during the initial phase of bone formation.⁷ The results in Figure 8 demonstrated increased alkaline phosphatase activity in cells treated with scaffold media containing genistein. In particular, the C-F-Gp-Gn (with genistein) group showed significant activity (p < 0.01) relative to the control group during the first and third week of the incubation period. This finding highlights the enhanced osteogenic properties of the scaffold when genistein is incorporated. The ALP activity peaked during the first week and subsequently gradually declined in the following weeks. The results suggest that the upregulation of alkaline phosphatase (ALP) is attributable to the incorporation of genistein, which may up-regulate estrogen receptor alpha (ERα). It results in an increase in hydroxyapatite deposition and the formation of bone matrix.¹⁹

Assessment of mineralization using alizarin red

The mineralization process was evaluated through alizarin red staining to assess the mineralization properties of the scaffold. The alizarin red stains bind directly to the calcium salts present in the extracellular matrix, resulting in a red color deposition. The intensity of the color defines the level of mineralization. In Figure 9, the cells treated with the C-F-Gp-Gn group exhibited greater mineral deposition compared to the C-F-Gp group. The incorporation of genistein activated the estrogen pathway, promoting osteogenic differentiation and enhancing bone matrix formation. Additionally, genistein stimulates mineralization, which induces calcium deposition in the extracellular matrix. These observations highlight that incorporating genistein elevates the osteogenic properties and supports the formation of mineral matrix formation.⁴⁸ Quantitative analysis showed higher absorbance values in the genistein-loaded scaffold on both days 7 and 14. Additionally, mineral deposition significantly increased by day 14 compared to day 7, and these results were consistent with the staining images.

Quantitative analysis of bone protein marker expression using ELISA

ELISA is performed to determine the expression of bone protein markers related to osteogenesis. The Col 1A1 marker is essential for extracellular matrix (ECM) production and the deposition of collagen, which is the initial step in bone regeneration.⁴³ The results in Figure 10 indicate that the expression of Col Type 1 increases in the C-F-Gp-Gn group at days 7, 14, and 21. T-test analysis demonstrates a significance level of p > 0.01 when comparing C-F-Gp-Gn to both the Control and C-F-Gp groups. TGF-β1 plays a vital role in recruiting osteoblast progenitor cells and is vital for maintaining the balance between the formation of osteoblasts and osteoclasts. This factor is particularly important during the early stages of bone matrix formation. The expression of TGF-β1 from scaffolds incorporating genistein shows significant differences relative to the control group, with p > 0.01 on days 7, 14, and 21. Estrogen activates the TGF-β1 pathway, and the introduction of genistein, which mimics estrogen, also triggers this pathway, leading to enhanced osteogenesis and increased bone formation.⁴⁹ Additionally, in Figure 10, there is a substantial difference with p > 0.05 on days 14 and 21 for scaffolds without genistein. RunX2 plays a role in the differentiation of osteoprogenitor and osteoblast cells.⁴⁸ RunX2, a master regulator of osteoblast differentiation, is continuously expressed throughout the osteogenic timeline, specifically at days 7, 14, and 21. It continues to promote the expression of downstream osteogenic genes.^50,51 The results indicate an increased expression of RunX2 in the C-F-Gp-Gn group at days 7, 14, and 21 when compared to the control and C-F-Gp groups. The t-test shows a significant value below 0.05 when comparing C-F-Gp-Gn to both control groups. Osteocalcin expression detected in Figure 10 illustrates the early regeneration phase, with the osteoblastic bone formation marker protein showing an increase on the seventh day of incubation.^52,53 This increase indicates the initiation of the scaffold’s mineralization process. Although osteocalcin (OCN) is traditionally recognized as a late-stage marker of osteoblasts, several studies indicate that it can also be expressed in the early phases of osteogenic differentiation. During these initial stages, OCN plays a role in activating key transcription factors such as RUNX2, which supports the onset of other osteogenic markers.^54,55 BMP-2 activity is predominant in the bone matrix and is important in the bone resorption process, playing a significant role in activating osteogenic genes like RunX2. The results in Figure 10 show a consistent increase in BMP-2 expression from day 7 to day 14, which stimulates osteoblast cells and induces bone matrix formation.^4,56 Overall, the expression of bone protein markers increased in the genistein-incorporated scaffold due to the activation of estrogen receptors by genistein. This activation induces mechanisms that promote the differentiation of osteoprogenitor cells, bone matrix formation, and mineral deposition.

Conclusion

In summary, a natural composite scaffold was fabricated using collagen fibrin incorporated with genistein (an isoflavone) and crosslinked with genipin. This scaffold was analyzed for its binding properties with genistein, as well as its thermal properties, structural characteristics, biochemical properties, mineralization, and osteogenic potential. The scaffold exhibited well-interconnected pores with optimal porosity and structural integrity, along with good adhesion properties. It demonstrated an increase in bone matrix formation and hydroxyapatite deposition. Additionally, the scaffold degraded in a controlled manner, showcasing enhanced biocompatibility. Incorporating genistein facilitated the binding of hydroxyl groups to functional groups in collagen and fibrin, resulting in increased swelling potential and controlled porosity. Genistein also enhanced alkaline phosphatase (ALP) activation by binding to estrogen receptors, which improved the mineral deposition properties of the scaffold. Genistein played a crucial role in activating estrogen receptors, leading to the recruitment of progenitor cells and fostering increased bone matrix formation and mineralization. This was confirmed through ELISA, which showed higher expression levels of TGF-β1, BMP-2, type I collagen, Runx2, and osteocalcin in the scaffold containing genistein compared to the scaffold without it, demonstrating significant statistical differences. Overall, the genistein-incorporated scaffold supports cell adherence and proliferation while exhibiting osteoconductivity. With refined osteogenic properties and the ability to recruit osteogenic cells, it promotes bone matrix formation with good osteoinductivity. Thus, this scaffold shows enhanced osteogenic and mineralization capabilities, making it a prominent candidate for bone tissue engineering applications.

Supplemental Material

sj-docx-1-jbc-10.1177_08839115251359662 – Supplemental material for Biofabricated genistein incorporated collagen scaffold for enhanced mineralization with refined osteogenic efficacy

Supplemental material, sj-docx-1-jbc-10.1177_08839115251359662 for Biofabricated genistein incorporated collagen scaffold for enhanced mineralization with refined osteogenic efficacy by Kannadasan Pranathy, Srinivetha Pathmanapan, Ponesakki Ganesan and Suresh Kumar Anandasadagopan in Journal of Bioactive and Compatible Polymers

Footnotes

Acknowledgements

The author would like to thank the Director of the CSIR-Central Leather Research Institute in Chennai for their support of the research study and the publication of this paper (Communication No: 2075). We also express our gratitude to CATERS and the CSIR-Central Leather Research Institute for their invaluable assistance in the characterization and execution of the research work. We are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, for providing financial support to Ms. Pranathy K (sanction letter no.31/0006(16490)/2023-EMR-I)

Authorship contribution statement

Kannadasan Pranathy: Study design, Experimental analysis, data analysis, and writing—original draft. Srinivetha Pathmanapan: Data visualization, Refinement of methodology, Review analysis. Ponesakki Ganesan: Data validation and review analysis. Suresh Kumar Anandasadagopan: Conceptualization, Project administration, ensured project integrity, supervision, and validation.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research received funding from CLRI-OLP-2403 (Collagen Biomaterial Theme).

ORCID iD

Suresh Kumar Anandasadagopan

Supplemental material

Supplemental material for this article is available online.

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