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. 2023 May 16;17(4):376–386. doi: 10.1049/nbt2.12136

Osteogenesis promotion by injectable methacryloylated gelatin containing psoralen and its bacteriostatic properties

Qi Zhang 1, Fuhang Chu 2, Yingjie Xu 2, Xiaonan Wu 2, Jie Yu 1, Beibei Cong 2,✉, Yingtao Wu 2,✉
PMCID: PMC10288355  PMID: 37191270

Abstract

The treatment of periodontitis focuses on controlling the progression of inflammation, reducing plaque accumulation, and promoting bone tissue reconstruction. Among them, the reconstruction of irregular bone resorption caused by periodontitis is a long‐standing challenge. At present, the local drug treatment of periodontitis is mainly anti‐inflammatory and antibacterial drugs. In this study, psoralen (Pso), a Chinese herbal medicine with anti‐inflammatory, antibacterial, and osteogenic effects, was selected for the local treatment of periodontitis. Meanwhile, an injectable methacrylate gelatin (GelMA) platform loading with Pso was constructed. Pso‐GelMA had the properties of fluidity, light cohesion, self‐healing, and slow release, which could be better used in the deep and narrow structure of the periodontal pocket, and greatly increased the effectiveness of local drug delivery. The pore size of Gelma hydrogel did not change after loading Pso by SEM. In vitro, Pso‐GelMA effectively upregulated the expression of osteogenic genes and proteins, increased alkaline phosphatase activity, promoted the mineralisation of rat bone marrow mesenchymal stem cells (BMSCs) extracellular matrix, and had significant antibacterial effects on Staphylococcus aureus and Fusobacterium nucleatum. Therefore, Pso‐GelMA has immense promise in the adjuvant treatment of periodontitis.

Keywords: antibacterial activity, biomedical materials, dentistry, drugs, hydrogels

The treatment of periodontitis focuses on controlling the progression of inflammation, reducing plaque accumulation, and promoting bone tissue reconstruction. Among them, the reconstruction of irregular bone resorption caused by periodontitis is a long‐standing challenge. At present, the local drug treatment of periodontitis is mainly anti‐inflammatory and antibacterial drugs. In this study, psoralen (Pso), a Chinese herbal medicine with anti‐inflammatory, antibacterial, and osteogenic effects, was selected for the local treatment of periodontitis. Meanwhile, an injectable methacrylate gelatin (GelMA) platform loading with Pso was constructed. Pso‐GelMA had the properties of fluidity, light cohesion, self‐healing, and slow release, which could be better used in the deep and narrow structure of the periodontal pocket, and greatly increased the effectiveness of local drug delivery. The pore size of Gelma hydrogel did not change after loading Pso by SEM. In vitro, Pso‐GelMA effectively upregulated the expression of osteogenic genes and proteins, increased alkaline phosphatase activity, promoted the mineralisation of rat bone marrow mesenchymal stem cells (BMSCs) extracellular matrix, and had significant antibacterial effects on Staphylococcus aureus and Fusobacterium nucleatum. Therefore, Pso‐GelMA has immense promise in the adjuvant treatment of periodontitis.

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1. INTRODUCTION

Periodontitis is a chronic progressive inflammatory disease with plaque as the initiating factor [1]. As the disease progresses, periodontitis causes destruction and loss of periodontal supporting tissues, including gingiva, periodontal membrane, alveolar bone, and dental bone, ultimately causing irreversible damage, such as loosening and loss of teeth. These pathological changes significantly impact the quality of life and psychological health of patients [2]. In addition, periodontal disease is closely related to systemic health or disease. Periodontal infection and bacterial‐induced organism response are important risk factors for respiratory infections, diabetes mellitus, and cardiovascular diseases [3]. Therefore, the treatment of periodontitis should not be neglected. The effectiveness of periodontal disease treatment depends on the standardisation and effectiveness of the primary treatment. The current periodontal basic treatment is aimed at plaque control, including mechanical clinical scaling, scraping, and effective brushing and flossing by the patient. However, due to the complexity of periodontal lesions and dental anatomy [4], such as narrow and deep subosseous pockets, bacterial invasion of periodontal tissues, posterior root bifurcation areas, and cervical enamel protrusions, it is difficult for periodontal instruments to reach the depths of infection and completely remove plaque bacteria [5, 6]. The residual plaque causes periodontal inflammation and alveolar bone resorption to continue to develop. Therefore, in some cases, pharmacological treatment of periodontitis can be used as a complementary treatment to scaling and scraping [7]. Currently, the drugs used to treat chronic periodontitis are antibacterial drugs, host immunomodulatory drugs, and traditional Chinese medicine. Antibacterial drugs are commonly used in clinical practice, including nitroimidazoles (metronidazole, tinidazole, and ornidazole), tetracyclines, and macrolides [8]. Adjunctive use of antibacterial drugs can reduce the number of bleeding sites and deep periodontal pockets [9, 10, 11]. However, systemic antibiotics are not recommended for the vast majority of patients with periodontal disease [12]. It was found that resistant strains of bacteria could be identified from subgingival plaque and saliva samples of patients with chronic periodontitis after subgingival scraping and root planing (SRP) and oral antibiotic treatment [13]. Oral antibiotics may also cause gastrointestinal problems such as diarrhoea and nausea, and even serious adverse events such as allergic reactions and pseudomembranous colitis [9, 14]. Whether used locally or systemically, antibiotics mainly act as anti‐inflammatory and antibacterial agents but cannot restore the regeneration of defective alveolar bone.

Psoralen (Pso) belongs to the furanocoumarins group and has multiple pharmacological effects such as oestrogen‐like, anti‐osteoporotic, antibacterial, anti‐cancer, anti‐inflammatory, and immunomodulatory [15]. Psoralen can act on osteogenesis‐related pathways through direct or indirect oestrogenic effects to regulate cellular osteogenic differentiation, reduce bone loss, and promote bone regeneration. A previous study by our group [16] demonstrated that the expression of serum anti‐inflammatory factor IL‐10 could be up‐regulated, the expression of inflammatory factor TNF‐α was down‐regulated, and alveolar bone resorption was significantly reduced in rats with periodontitis model after 4 weeks of gavage with Pso. Although oral administration is a classical route of administration, it has disadvantages, such as first‐pass effects of liver and intestinal enzymes, one‐time metabolism, and slow onset of action [17]. Whereas oral administration often requires increased dosage to achieve therapeutic efficacy, topical administration can avoid these problems. It was found [18] that topical application of Pso, compared to oral osteopontin, ensures that a higher concentration of Pso is achieved in the epidermis and a lower concentration of Pso in the blood. This suggests that topical Pso is safer for humans at a lower dose to perform a specific therapeutic effect.

GelMA is a double‐bond modified gelatin that can be crosslinked into a gel under the action of a photoinitiator with a 405 nm light source. GelMA facilitates cell adhesion, proliferation, and differentiation and has the advantage of good solubility and low antigenicity [19]. Studies have demonstrated that GelMA loaded with nanoparticles can be injected into the bone defect area at room temperature [20] or treated locally, and can effectively fill irregular bone defects caused by periodontitis and provide space for cell growth. This injectable property is significant for the repair of bone defects. In this study, Pso and GelMA were co‐crosslinked into a new material Pso‐GelMA, which is injectable, easy to handle, and can be injected into close contact with irregular bone defects or periodontal pockets by injection, and can be rapidly crosslinked with stable properties after irradiation with specific light sources. Bone marrow mesenchymal stem cells (BMSCs) can grow and multiply well in Pso‐GelMA with good biocompatibility. In vitro, we have demonstrated that Pso‐GelMA material can promote osteogenic differentiation of BMSCs, and inhibit the growth of Staphylococcus aureus and Fusobacterium nucleatum. Therefore, it has the potential to be used as an adjunctive therapy for periodontitis.

2. MATERIAL AND METHODS

2.1. Preparation of Pso‐GelMA

Firstly, 0.01 g of lithium phenyl (2,4,6‐trimethyl benzoyl)phosphate (LAP) was mixed with 4mLPBS and shaken for 15 min at 55°C in a water bath to obtain 0.25% LAP solution. Secondly, 50 mg of GelMA was placed in a centrifuge tube and mixed with 1 mL of the LAP solution. The mixture was heated in a 65°C water bath for 25 min to obtain a 5% GelMA solution, which was then filtered and sterilised. Thirdly, 1 mg of Pso was mixed with 20 μL of DMSO to obtain a Pso solution with a concentration of 0.269 moL/L. Then, 10 μL of the Pso solution (with a concentration of 0.269 moL/L) was added to 2.68 mL of the LAP solution to obtain a Pso solution with a concentration of 1mmoL/L. Finally, 5% GelMA solution 900 μL, mixed with 1mmoL/L Pso solution 100 μL to get 1 mL 100 μmoL/L Pso‐GelMA solution (Pso‐GelMA solution contains 100 μmoL/L Pso and 5% GelMA solution), filtered and de‐bacterised, irradiated under 405 nm light source for the 20 s to make it crosslinked into the glue (Figure 1).

FIGURE 1.

FIGURE 1

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Synthesis process of Pso‐GelMA.

2.2. Characterisation of Pso‐GelMA

The injectability of the hydrogel was assessed by extruding the Pso‐GelMA solution through a 1 mL syringe and observing the continuity and gelling state of the hydrogel. The self‐healing properties of the hydrogels were evaluated by placing the stained and unstained hydrogels together and observing their adhesion status. Pso‐GelMA and GelMA were lyophilised and sprayed with gold, and then the hydrogel images were taken by SEM.

2.3. Extraction and identification of BMSCs

Healthy male SD rats at 4 weeks of age were executed, the femur of the rats was stripped, the epiphyses were cut at both ends, and the fluid from the flushed bone marrow cavity was collected. After centrifugation, the cell sediment was resuspended, the medium was changed every 3 days, and the 3rd generation cells were taken for subsequent experiments. The cell surface markers CD90 and CD45 were detected by flow cytometry. Rat BMSCs were cultured for 21 days after osteogenic and lipogenic induction medium and stained with alizarin red and oil red O to observe the osteogenic and lipogenic conditions. (This experiment was approved by the Experimental Animal Ethics Committee of Qingdao Stomatological Hospital, Approval No. 2021KQYX034).

2.4. Observation of cell‐loaded Pso‐GelMA and live/dead cell staining

BMSCs were inoculated into the material containing 100 μmoL/L Pso‐GelMA, and cell proliferation within the material was evaluated after 1, 3, and 7 days by microscopic observation and staining with a live/dead cell staining kit, with live cells showing green fluorescence and dead cells showing red fluorescence.

2.5. Pso release assay

One millilitre of 100 μmoL/L Pso‐GelMA solution and 1 mL of GelMA solution were separately added to a 10 mL centrifuge tube, and cross‐linked into a gel by irradiation at 405 nm for the 20 s. 8 mL of PBS solution was added to the centrifuge tube and at predetermined times (1, 2, 3, 4, 5, 6, 7, 8 and 9 days), the PBS solution was removed in its entirety (8 mL of fresh PBS solution was added to the tube again), centrifuged, and set aside. Under a UV spectrophotometer, the sample solution is measured at the corresponding concentration.

Releaserate=sampleconcentration/knownconcentration×100%

2.6. CCK‐8 experiment

One millilitre of 100 μmoL/L Pso‐GelMA solution was added to a 5 mL centrifuge tube and cross‐linked into a gel by irradiation at 405 nm for the 20 s. 1 mL of complete medium solution was added to the centrifuge tube for 5 days. 1 mL of the extract was diluted into different concentrations and the experiment was divided into 6 groups: 100 μmoL/L Pso‐GelMA extract group, 25 μmoL/L Pso‐GelMA extract group, 12.5 μmoL/L Pso‐GelMA extract group, 10 μmoL/L Pso‐GelMA extract group, 5 μmoL/L Pso GelMA extract group, and complete medium group (control group). BMSCs were inoculated in 96‐well plates at 2 × 103 cells/well, and culture in each of the six groups, and the culture medium was changed every 3 days. The cell proliferation was detected by the CCK8 method on d 1, 3, and 7 to determine the optimal concentration for subsequent experiments.

2.7. ALP staining

The experiment was divided into 4 groups: 10 μmoL/L Pso‐GelMA extract group, 10 μmoL/L Pso group (positive control group), GelMA scaffold extract group, and complete medium group. BMSCs were inoculated in 6‐well plates at 5 × 103 cells/well, and the 4 groups were cultured for BMSCs cells separately. After 12 days of culture, each group was stained with an ALP staining kit, and the staining results were observed under the microscope.

2.8. ALP activity assay

The experimental grouping was the same as the ALP staining experimental grouping described above, and BMSCs were inoculated in 6‐well plates at 1 × 104 cells/well, and the culture medium was changed every 3 days. After 12 days of cell culture, the cells of each group were lysed, and the lysate was collected for protein quantification and ALP activity assay.

2.9. Alizarin red staining

The experimental grouping was the same as the ALP staining experimental grouping described above, and BMSCs were inoculated in 6‐well plates at 1 × 104 cells/well, and the culture medium was changed every 3 days. After culturing the cells for 21 days, each group added 4% paraformaldehyde to fix the cells for 15 min, and the fixative was discarded before adding alizarin red staining solution for 30 min. The staining solution was discarded, and each well was washed with double‐distilled water and observed under a microscope for photographs.

2.10. Real‐time quantitative PCR

The experimental grouping was the same as the ALP staining experimental grouping described above, and BMSCs were inoculated in 6‐well plates at 1 × 104 cells/well, and the culture medium was changed every 3 days. After 12 days of incubation, RNA was extracted from each group and the RNA concentration was measured. cDNA was synthesised using a reverse transcription kit, and Real‐time RCR detected the expression levels of Smad4 and TGF‐β1 genes, and the results were quantified.

2.11. Western blot

The experimental grouping was the same as the ALP staining experimental grouping described above, and BMSCs were inoculated in 6‐well plates at 1 × 104 cells/well, and the culture medium was changed every 3 days. After 12 days of incubation, the pre‐treatment was the same as the ALP activity assay experiment described above. The protein concentration was measured in the supernatant, and the supernatant was added to 1 × loading buffer and boiled at 100°C for 15 min. Protein samples were electrophoresed and electrotransferred to the PVDF membrane by SDS‐PAGE. The PVDF membrane was confined with confinement solution, incubated with primary antibody against the membrane at 4°C and secondary antibody at room temperature, exposed to exposure solution, and the gel image analysis system picked up the image for analysis.

2.12. Bacteriostatic activity

The sterilised filter papers with a diameter of 6 mm were placed in Pso‐GelMA extract and GelMA extracts for 1 day. After 1 day, the filter papers were separately on the surface of a TSB solid medium inoculated with 1 × 106 CFU/mL of Staphylococcus aureus (SA) and with 1 × 106 CFU/mL of Fusobacterium nucleatum (FN) and incubated in a bacterial incubator for 24 h. For 24 h, the size of the inhibition circle of each filter paper was observed and recorded, and the strength of the inhibition activity of the drug was evaluated by the size of the inhibition circle.

2.13. Bacterial inhibition rate

Pso‐GelMA extract 1 mL and GelMA extract 1 mL, were co‐cultured with Staphylococcus aureus suspension (1 × 106CFU/mL) and Fusobacterium nucleatum suspension (1 × 106CFU/mL), respectively, for 1 day. The bacterial solution was diluted (1 × 106CFU/mL), and 10 μL of the diluted bacterial solution was added to the solid medium for 1 day, and the colony The bacterial inhibition rate (BR) of the material was determined by the colony counting method.

The bacterial inhibition rate was calculated by the formula: BR = (number of colonies in the control group ‐ number of colonies in the experimental group)/number of colonies in control group × 100%

2.14. Statistical analysis

GraphPad Prism 8.0 software was used for data statistics, and data were expressed as mean ± standard deviation (SD). Data were compared between two groups by t‐test and multiple groups by one‐way ANOVA and LSD‐t‐test two groups, and p < 0.05 was considered statistically significant.

3. RESULTS

3.1. Characterisation of Pso‐GelMA

The 5% GelMA and 1 mmoL/L Pso solution were mixed in proportion and irradiated to obtain a material containing 100 μmoL/L of Pso‐GelMA (Figure 2a,b). Two separate pieces of hydrogel adhere well together within 20 min (Figure 2c). The Pso‐GelMA solution passed freely through a 1 mL syringe needle with continuity and cross‐linked into a gel under light (Figure 2d). SEM images showed that the GelMA scaffold and Pso‐GelMA scaffold had a loose porous structure with different pore sizes and uniform distribution, the pore size of the GelMA scaffold was about 149.17 ± 34.24 μm for hydrogel pore size, and the pore size of Pso‐GelMA was about 146.33 ± 43.77 μm. The pore size did not change much before and after drug loading (Figure 2e,f).

FIGURE 2.

FIGURE 2

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Characterisation of Pso‐GelMA. (a) Liquid‐like GelMA hydrogel. (b) Pso‐GelMA. (c) Self‐repairability of Pso‐GelMA. (d) Injectability of Pso‐GelMA. (e) SEM image of GelMA hydrogel scaffold. (f) SEM image of Pso‐GelMA.

3.2. In vitro culture and identification of BMSCs cells

The primary cells were irregularly arranged and showed various morphologies, such as shuttle, round, and polygonal. The second‐generation cells were mainly small round, and shuttle‐shaped, mostly growing in parallel, and some swirled. The third‐generation cells were mainly shuttle‐shaped (Figure 3a). After 21 days of lipogenesis and osteogenesis induction, round orange lipid droplets and reddish‐brown calcium nodules were seen under the microscope, with the orange arrow pointing to the lipid droplets and reddish‐brown calcium nodules (Figure 3b). The expression rate of positive marker CD90 on the cell surface of BMSCs was more than 99%, and negative marker CD45 on the cell surface was about 0.05% (Figure 3c).

FIGURE 3.

FIGURE 3

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Identification and culture of BMSCs. (a) Primary BMSCs, secondary BMSCs, and tertiary BMSCs. (b) BMSCs were identified as lipogenic and osteogenic. (c) BMSCs were expressed with positive marker CD90 and negative marker CD45 on the cell surface.

3.3. Biocompatibility of Pso‐GelMA with BMSCs

Most cells are round on day 1, and a few are polygonal. On day 3, most cells were shuttle‐shaped and started to join. On day 7, BMSCs could stretch well and grow in Pso‐GelMA (Figure 4). The live/dead cell staining results showed that after putting into Pso‐GelMA, most of the cells survived, and only a few dead cells were visible. On day 3, the number of cells increased significantly, and after 7 days, the cells proliferated rapidly with a few dead cells (Figure 4).

FIGURE 4.

FIGURE 4

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Growth of BMSCs in Pso‐GelMA observed by microscopy and Live/Dead staining on days 1, 3, and 7.

3.4. Effect of Pso‐GelMA on the proliferation of BMSCs cells

The results of the drug release assay of Pso showed that the cumulative release of Pso from Pso‐GelMA reached 91.1% at 5 days. Pso was released stably at 1, 2, 3, 4, and 5 days, indicating that Pso‐GelMA has an excellent sustained release (Figure 5a).

FIGURE 5.

FIGURE 5

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Effect of Pso‐GelMA extracts on the proliferation of BMSCs. (a) Pso‐GelMA in vitro release profile. (b) Proliferative effect of Pso‐GelMA on BMSCs.

The effect of each group of materials on cell proliferation was not statistically significant on day 1 compared to the control group (p > 0.05). On day 3, there was a statistically significant difference (p < 0.001) in the promotion of BMSCs by the groups diluted to 25 μmoL/L, 12.5 μmoL/L, and 10 μmoL/L concentrations of Pso‐GelMA material extracts. On day 7, there was a statistically significant difference (p < 0.05) between the groups diluted to 12.5 μmoL/L and 10 μmoL/L concentrations of Pso‐GelMA material infusion on the promotion of BMSCs, with a statistically significant difference (p < 0.001) between the groups with 10 μmoL/L concentration of Pso‐GelMA infusion (Figure 5b).

3.5. Effect of Pso‐GelMA on osteogenic differentiation of BMSCs

The alizarin red staining site was reddish‐brown, observed by alizarin red staining after 21 days of cell culture: compared to the control group, no obvious reddish‐brown was seen in the GelMA group; the Pso‐GelMA group, Pso group had obvious reddish‐brown, which was relatively more extensive and more numerous (Figure 6a). The ALP active site was blue after staining, located in the cytoplasm, after 12 days of cell culture ALP staining was observed: compared to the control group, no obvious blue colour was seen in the GelMA group; the Pso‐GelMA group and Pso group had deep blue staining (Figure 6b). The quantitative assay of ALP viability in each group after 12 days of cell culture showed that there was no statistical difference between the control and GelMA groups (p > 0.05), a statistically significant difference between the control and Pso groups (p < 0.05), and a statistically significant difference between the control and Pso‐GelMA groups (p < 0.001), while there was no statistically significant difference between the Pso and Pso‐GelMA groups (p > 0.05) (Figure 6c).

FIGURE 6.

FIGURE 6

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Effect of Pso‐GelMA on promoting cellular osteogenesis. (a) alizarin red staining. (b) ALP staining. (c) ALP viability test.

3.6. Pso‐GelMA promotes the expression of genes and proteins related to osteogenic differentiation of BMSCs

RT‐PCR results showed that the expression of Smad4 and TGF‐β1 mRNA was significantly higher in the Pso‐GelMA and Pso groups compared with the control group, and the difference was significant (p < 0.01), while there was no statistical difference between the Pso‐GelMA and Pso groups (p > 0.05) (Figure 7AB). Smad4 and TGF‐β1 protein expression in the Pso‐GelMA group was significant (p < 0.01) compared with the control group, and there was no statistical difference (p > 0.05) between the Pso‐GelMA group and the Pso group (Figure 7c).

FIGURE 7.

FIGURE 7

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Effect of Pso‐GelMA on promoting cellular osteogenic genes and proteins. (a) TGF‐β1 gene expression (b) Smad4 gene expression. (c) Smad4 and TGF‐β1 protein expression.

3.7. Inhibition of Staphylococcus aureus and Fusobacterium nucleatum by Pso‐GelMA

The effect of inhibition was verified by measuring the diameter of the inhibition circle between groups. No significant inhibition circle was seen in the GelMA group (control group), and the Pso‐GelMA material group had significantly larger round inhibition circles; the diameter of the inhibition circle of the Pso‐GelMA material group against Staphylococcus aureus was 23.67 ± 0.0897 mm (Figure 8a). The inhibition rate of Pso‐GelMA material against Staphylococcus aureus was 86.4% (Figure 8b). The inhibition circle diameter of the Pso‐GelMA material group was 23.93 ± 0.0374 mm against Fusobacterium nucleatum (Figure 8c), and the inhibition rate of Pso‐GelMA material against Fusobacterium nucleatum was 72.15% (Figure 8d).

FIGURE 8.

FIGURE 8

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Antibacterial activity of Pso‐GelMA material. (A‐B) Antibacterial activity of Pso‐GelMA material against Staphylococcus aureus. (C‐D) Antibacterial activity of Pso‐GelMA material against Fusobacterium nucleatum.

4. DISCUSSION

The resorption of alveolar bone due to chronic periodontitis results from an infection caused by an inflammatory reaction, accompanied by an imbalance in the bone immune system. Bone and immune cells have a close interaction and perform together the functions of the ‘bone immune system’ [21]. The current gold standard of periodontal non‐surgical treatment is still subgingival scaling and root surface planing (SRP), but there are still some limitations, such as the presence of deep periodontal pockets and root bifurcation lesions, that make mechanical debridement more difficult. It was found [22, 23] that SRP supplemented with pharmacological treatment could reduce recurrence and improve efficacy to some extent. Currently, the main drugs commonly used clinically to treat chronic periodontitis are antibacterial drugs, non‐steroidal drugs, rinse‐containing drugs, and herbal medicines. Antibacterial drugs are mainly antibacterial. NSAIDs are mainly used to inhibit infection, relieve symptoms, and inhibit alveolar bone resorption, but excessive intake of NSAIDs may cause increased sodium retention and decreased renal blood flow, leading to renal failure [24]. In recent years statins and herbal preparations have played a positive role in the oral cavity, but studies have confirmed that the administration of high doses (27 mg/kg) of atorvastatin prevented alveolar bone loss, but low doses (below 3 mg/kg) of atorvastatin increased bone resorption [25]. Chinese medicine has unique advantages in bone regeneration, and numerous studies have confirmed [26, 27] that Chinese medicine can promote bone regeneration through multiple mechanisms or targets. Chinese herbal medicine avoids the drug resistance associated with antibiotics and chemically synthesised drugs, has less toxic side effects [28], and is suitable for the long‐term treatment of periodontal disease.

Pso is an effective extracted component of the legume Psoralea, and numerous studies have confirmed that Pso induces miR‐488 to participate in the osteogenic differentiation of BMSCs by targeting Runx2 [29]. Pso inhibits osteoclast formation and reduces osteolytic activity by enhancing the expression of oestrogen receptor ERα and inhibiting IL‐17 signalling by decreasing IL‐17 expression [30]. It is the anti‐inflammatory, anti‐osteoporotic, and antibacterial pharmacological effects of Pso that make it a promising drug for the treatment of periodontitis. The study found that [31] that after 4 weeks of gavage of Pso to Wistar rats after incisal to molar force, the periodontal fibres of molar teeth in the osteopontin group were more neatly and densely aligned relative to the control group. There was a new bone formation on the alveolar bone surface, a large amount of cellular dental bone present on the apical surface and the root surface is flat and free of residue. However, it was found [32] that prolonged low‐dose oral administration of osteopontin caused abnormal metabolism of amino acids in the serum of female rats. Based on the above factors, in this study, we chose Pso, which is traditionally used for bone repair, for the topical treatment of periodontal, but a better carrier is needed.

GelMA is a gelatin derivative [33] that contains many amino acid sequences that promote cell adhesion in different biomedical applications, such as bone regenerative medicine, drug delivery, and tissue adhesives. One study [34] showed that the combination of riboflavin with GelMA could promote osteogenic differentiation of osteoblasts. It was shown [35] that 5% GelMA hydrogels were better at promoting osteogenic differentiation in BMSCs compared to 10% GelMA hydrogels. This may be related to the fact that the pore size of GelMA hydrogels is inversely proportional to the concentration [36], with lower concentrations of GelMA gels having larger pore sizes, facilitating the entry of cells, growth factors, and nutrients Therefore, in this study, 5% GelMA was chosen as the experiment and the Psoralen‐laden injectable GelMA hydrogel material was prepared by the photo‐crosslinking method. In vitro experiments showed that Pso‐GelMA could continuously pass through a 1 mL needle and the two gels with different colours could be well bonded together, which suggested that Pso‐GelMA could effectively fill irregular bone defects or periodontal pockets and maintain a stable state. The structure of GelMA hydrogel did not change before and after the loading of psoralen, and the pore size was about 146.33 ± 43.77 μm, and the live‐dead staining of BMSCs on days 1, 3, and 7 after inoculation into Pso‐GelMA showed that the majority of cells survived on the first day, and only a small number of dead cells were visible. The number of cells increased significantly at 3 days, and cell proliferation was seen after 7 days. The results show that the porous structure of hydrogel provides space for cell growth, and the cells can adhere, grow and proliferate well in Pso‐GelMA material with good biocompatibility.

In order to verify the osteogenicity of Pso‐GelMA, ALP staining and alizarin red staining were performed, and it was found that the Pso‐GelMA extract group and the Pso group (positive control group) had a darker colour and a stronger ability to osteogenic differentiation relative to the control group. ALP activity can be used as a measure of the differentiation of BMSCs towards osteoblasts. ALP viability assay showed a statistically significant increase in the level of ALP viability in the experimental group relative to the control group. Smad4 is an important intracytoplasmic signalling cascade molecule that transduces TGF‐β signals and functions as a transcription factor [37]. TGF‐β exerts its function through Smad4, especially by stimulating the expression of downstream genes, such as Runx2, Dlx5, and Msx2, downstream genes in turn can promote skeletal development [37, 38]. TGF‐β/Smad4 plays an important role in the osteoclast and endochondral ossification of MSCs. The RT‐PCR and Western blot experiments further demonstrated that Pso‐GelMA may promote osteogenic differentiation by upregulating Smad4, TGF‐β gene, and protein expression. This study confirmed the inhibitory effect of Pso‐GelMA on Staphylococcus aureus and Fusobacterium nucleatum. Staphylococcus aureus is a gram‐positive bacterium that is closely associated with periodontitis and peri‐implantitis [39, 40, 41]. Staphylococcus aureus not only causes infections in the oral cavity but also affects the osteogenic process [42, 43]. Fusobacterium nucleatum is a gram‐negative bacterium that is not only an important periodontal pathogen in chronic periodontitis but also plays a protective role against Porphyromonas gingivalis, another important periodontal pathogen [44, 45]. Pso‐GelMA materials have osteogenic and antibacterial effects, and Pso‐GelMA materials are of great interest for the treatment of periodontitis.

Studies have confirmed that Pso‐GelMA promotes the proliferation and osteogenic differentiation of BMSCs and has an anti‐osteoporotic effect [46]. By using a minimally invasive injection method, Pso‐GelMA was delivered into the organism and remains in a stable state, avoiding the trauma caused by larger surgical procedures. At the same time, Pso‐GelMA effectively inhibited the proliferation of Staphylococcus aureus and Fusobacterium nucleatum, which can reduce infections caused by bacteria in the oral environment. Pso‐GelMA has good biocompatibility and can promote the differentiation of BMSCs, as well as influence the osteogenic differentiation of adult stem cells (periodontal stem cells) with differentiation potential in periodontal tissues. This suggests that Pso‐GelMA has the potential to achieve periodontal tissue regeneration by promoting the proliferation and osteogenic differentiation of some cells with stem cell properties in periodontal tissue.

5. CONCLUSION

In this study, a novel Pso‐GelMA scaffold was prepared by combining psoralen with GelMA. Pso‐GelMA can effectively promote the growth, propagation, and osteogenic differentiation of BMSCs, and promote osteogenic differentiation of BMSCs through TGF‐β1/Smad4 pathway. Pso‐GelMA effectively inhibited the growth of Staphylococcus aureus and Fusobacterium nucleatum. As a good injectable material, Pso‐GelMA has a good potential for periodontal adjunctive drug therapy and defective alveolar bone repair. Its application prospects are to be further investigated by in vivo experiments.

AUTHOR CONTRIBUTIONS

Qi Zhang: Investigation; Formal analysis; Methodology; Writing – original draft; Writing – review & editing. Fuhang Chu: Investigation; Formal analysis; Investigation; Methodology. Yingjie Xu: Investigation; Writing – review & editing; Software; Resources. Xiaonan Wu: Investigation; Formal analysis; Methodology; Visualisation. Jie Yu: Investigation; Analysis; Data management; Software. Beibei Cong: Supervision; validation; conceptualisation; Writing – review & editing. Yingtao Wu: Supervision; conceptualisation; funding acquisition; Writing – review & editing; Project administration.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

ACKNOWLEDGEMENTS

The study was supported by Qingdao Key Health Discipline Development Fund (2022–2024), Qingdao Clinical Research Center for Oral Diseases (22‐3‐7‐1czx‐7‐nsh), and Shandong Province Chinese Medicine Science and Technology Project (2021Z030), Qingdao Chinese Medicine Science and Technology Project (2022‐zyym26).

Zhang, Q. , et al.: Osteogenesis promotion by injectable methacryloylated gelatin containing psoralen and its bacteriostatic properties. IET Nanobiotechnol. 17(4), 376–386 (2023). 10.1049/nbt2.12136

Qi Zhang and Fuhang Chu contributed equally to this work.

Contributor Information

Beibei Cong, Email: xinruo1986@163.com.

Yingtao Wu, Email: 347107303@qq.com.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding authors.

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