Degradable hydrogel fibers encapsulate and deliver metformin and periodontal ligament stem cells for dental and periodontal regeneration

Abstract Human periodontal ligament stem cells (hPDLSCs) are promising cells for dental and periodontal regeneration. Objective This study aimed to develop novel alginate-fibrin fibers that encapsulates hPDLSCs and metformin, to investigate the effect of metformin on the osteogenic differentiation of hPDLSCs, and to determine the regulatory role of the Shh/Gli1 signaling pathway in the metformin-induced osteogenic differentiation of hPDLSCs for the first time. Methodology CCK8 assay was used to evaluate hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red S staining, and the expression of osteogenic genes were evaluated. Metformin and hPDLSCs were encapsulated in alginate-fibrinogen solutions, which were injected to form alginate-fibrin fibers. The activation of Shh/Gli1 signaling pathway was examined using qRT-PCR and western blot. A mechanistic study was conducted by inhibiting the Shh/Gli1 pathway using GANT61. Results The administration of 50 μM metformin resulted in a significant upregulation of osteogenic gene expression in hPDLSCs by 1.4-fold compared to the osteogenic induction group (P < 0.01), including ALP and runt-related transcription factor-2 (RUNX2). Furthermore, metformin increased ALP activity by 1.7-fold and bone mineral nodule formation by 2.6-fold (P<0.001). We observed that hPDLSCs proliferated with the degradation of alginate-fibrin fibers, and metformin induced their differentiation into the osteogenic lineage. Metformin also promoted the osteogenic differentiation of hPDLSCs by upregulating the Shh/Gli1 signaling pathway by 3- to 6- fold compared to the osteogenic induction group (P<0.001). The osteogenic differentiation ability of hPDLSCs were decreased 1.3- to 1.6-fold when the Shh/Gli1 pathway was inhibited, according to ALP staining and alizarin red S staining (P<0.01). Conclusions Metformin enhanced the osteogenic differentiation of hPDLSCs via the Shh/Gli1 signaling pathway. Degradable alginate-fibrin hydrogel fibers encapsulating hPDLSCs and metformin have significant potential for use in dental and periodontal tissue engineering applications. Clinical Significance Alginate-fibrin fibers encapsulating hPDLSCs and metformin have a great potential for use in the treatment of maxillofacial bone defects caused by trauma, tumors, and tooth extraction. Additionally, they may facilitate the regeneration of periodontal tissue in patients with periodontitis.


Introduction
Periodontitis is an inflammatory illness of the periodontium that includes the gingiva, alveolar bone, periodontal ligament (PDL), and cementum. It is characterized by inflammation and alveolar bone loss, and it may lead to tooth loss if left untreated. Ideally, regenerative periodontology aims to regenerate these lost tissues to their original architecture and function, which is a challenging task. Several approaches for periodontal regeneration have been explored, including the use of gingival margin-derived stem/progenitor cells combined with IL-1ra short term releasing HA hydrogel synthetic extracellular matrix, which has shown periodontal regenerative potential, 1  In recent years, an increasing number of studies have confirmed that metformin is an antihyperglycemic biguanide compound, and has many biochemical activities, such as anti-aging, anti-tumor, antiinflammatory, and anti-cardiovascular diseases. [3][4][5] Furthermore, metformin stimulates the osteogenic/ dentinogenic differentiation of various mesenchymal stem cells (MSCs), such as adipose stromal cells, 6 dental pulp stem cells (DPSCs), 7 human periodontal ligament stem cells (hPDLSCs), 8 and induced pluripotent stem cell-derived MSCs. 9 However, few studies have reported the combined application of metformin and hPDLSCs for the regeneration of alveolar bone defects.
Biomaterials loaded with metformin have been shown to promote cellular osteogenesis and dentinogenesis, such as nanosphere-laden photocrosslinkable gelatin hydrogels, tricalcium silicate-based cements, polydopamine-templated hydroxyapatite, and resin. [10][11][12][13] Some of these materials, however, induced the production of reactive oxygen species. As a result, cells are damaged and apoptosis occurs. These systems are unable to carry cells and drugs at the same time, which is not conducive to drug and stem cell delivery to bone defects.
As a highly hydrated natural material with good biocompatibility, alginate hydrogels are expected to be able to carry drugs and cells at the same time.
Therefore, we developed degradable alginate-fibrin hydrogel fibers to encapsulate hPDLSCs and metformin simultaneously. We hypothesized that the degradation process of the hydrogel fibers would lead to the sustained release of metformin, which, in combination with the progressive proliferation and osteogenic differentiation of hPDLSCs, could effectively promote the regeneration of alveolar bone.
Hedgehog is a secreted signaling molecule that regulates all stages of embryonic development and the production of many tissues and organs, including tooth development. 14  was used to perform qRT-PCR with SYBR Premix DimerEraser TM (Takara, Shiga, Japan). Figure 1 lists the primer sequences utilized in the tests. Three separate experiments were performed using human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene to normalize the mRNA levels.
The 2 -∆∆CT approach was used to calculate the relative expression of target genes. All the experiments were performed in triplicate.

Western blot analysis
Cell lysates were produced and western blot analysis was performed as previously described. 17

Encapsulation of cells and metformin in alginate-fibrin fibers
Alginate (64% guluronic acid, MW = 75,000-220,000 g/mol, ProNova, Oslo, Norway) was oxidized to 7.5% using reported procedures to increase the The alginate-fibrin fibers incubated with 0.4% fibrinogen lost their integrity and released most of the encapsulated cells on day four, according to our previous study. 23 Therefore, three groups were tested by performing live/dead staining and ALP activity assays, and alizarin red S staining of the second and third groups was performed: 1) alginate-fibrin fiber-encapsulated hPDLSCs cultured in growth medium; 2) alginate-fibrin fiber-encapsulated hPDLSCs cultured in osteogenic medium; and 3) alginate-fibrin fiber-encapsulated hPDLSCs with 50 μM metformin cultured in osteogenic medium.

Statistical analysis
The area fractions of ALP staining, alizarin red S staining, and xylenol orange staining were calculated using Image-Pro Plus software. The western blot results were calculated using ImageJ software. The data were statistically evaluated utilizing GraphPad Prism software (GraphPad, USA). An unpaired t test was used to assess significant differences. All data are presented as the means ± SEM, and the significance level was set to P<0.05 based on results from at least three independent samples.

Results
Metformin was not toxic to hPDLSCs hPDLSCs were successfully isolated from ALP, alizarin red S, and xylenol orange ( Figure 3A).
The area of stained cells (%) in the 50 μM metforminosteogenic group was 3.1-fold higher than that of the osteogenic group ( Figure 3B). The mineralized areas (%) of alizarin red S staining and xylenol orange staining in the 50 μM metformin-osteogenic group were 1.7-fold and 2.6-fold higher than those of the osteogenic induction group, respectively (P<0.01) ( Figure 3C and D).
Western blot analyses showed higher levels of the RUNX2 (2.0-fold) and ALP (1.4-fold) proteins in the 50 μM metformin-osteogenic group than in the osteogenic    Metformin was nontoxic to the cells, but it didn't stimulate cell proliferation as effectively. Zhao,et al. 18 (2019) showed that metformin and hPDLSCs seeded on calcium phosphate cement scaffolds presented no effect on the proliferation of hPDLSCs. However, metformin combined with photobiomodulation therapy exerted a synergistic effect on promoting cell proliferation and reducing the production of reactive oxygen species in hPDLSCs pretreated with high glucose to simulate diabetes. 25 Therefore, the authors speculated that metformin presents no effect on the   activity might also participate in this mechanism. 36,37 However, the related mechanism of the osteogenic differentiation of hPDLSCs by metformin was unclear.
Metformin promoted the osteogenesis of hPDLSCs by upregulating the Akt/Nrf2 signaling pathway and protecting cells from oxidative stress, according to Jia, et al. 8 (2020). The osteogenic effect of metformin was inhibited by administering LY294002, an inhibitor of Akt phosphorylation. 8  However, researchers have not reported whether Degradable hydrogel fibers encapsulate and deliver metformin and periodontal ligament stem cells for dental and periodontal regeneration metformin promotes osteogenic differentiation via the Shh/Gli1 signaling pathway.
We further explored the relationship between the increased expression of Shh/Gli1 and osteogenesis in hPDLSCs. The capacity of metformin to promote the osteogenic differentiation of hPDLSCs was decreased by 1.2-to 1.7-fold compared to the osteogenic induction group when the Shh/Gli1 signaling pathway was downregulated by 1.6-fold, as evidenced by the administration of GANT61, a selective transcriptional inhibitor of Gli1. Based on these findings, we suggest that Shh/Gli1 signaling is involved in the metforminmediated enhancement of osteogenesis in hPDLSCs.
Previous studies by our group also revealed that the Shh signaling pathway played an important role in regulating the osteogenic differentiation of DPSCs.
In addition, many studies have shown that the