Effect of overexpression of GRN on the proliferation and osteogenic capacity of human periodontal cells
- Authors:
- Published online on: December 17, 2024 https://doi.org/10.3892/etm.2024.12783
- Article Number: 33
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Copyright: © Yao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Periodontitis is a chronic and progressive inflammatory disease that affects the supporting tissues of the periodontium and primarily results from the invasion of specific microorganisms. This microbial intrusion into the periodontal tissues ultimately leads to tooth loosening and loss (1,2). Periodontitis is a risk factor for various systemic diseases, including diabetes mellitus and coronary heart disease (3), which has a prevalence of 45-50% overall, with the severe form affecting 11.2% of the general population (4). Consequently, there has been a growing focus on the prevention and treatment of periodontitis.
The advent of tissue engineering techniques has opened new avenues for addressing periodontitis (5,6). Tissue engineering involves three crucial elements: Seed cells, growth factors and scaffolding materials, with seed cells as the core components (7). In 2004, Seo et al (8) first isolated multipotent stem cells from the periodontal ligament, confirming the multipotential differentiation, robust self-renewal and self-repair capacity of human periodontal ligament cells (hPDLCs). These cells can differentiate into various cell types under specific conditions, thereby facilitating true periodontal tissue regeneration. Additionally, hPDLCs exhibit low immunogenicity, post-implantation stability, adaptability to the implant environment and minimal harm to the body (9), making them the most promising stem cell populations for regenerative periodontal therapy. Currently, hPDLCs are the most widely used seed cells for periodontal tissue engineering. However, the inflammatory microenvironment caused by cytokines, such as TNF-α and IL-1, limits the proliferation and osteogenic capacity of hPDLCs (10). Therefore, targeted antagonism of TNF-α or its receptors is crucial for reducing inflammatory factors and enhancing the proliferation and osteogenesis of hPDLCs.
Progranulin (PGRN), also known as pro-epithelin, 88 kDa glycoprotein (GP88) or PC cell-derived growth factor and granulin-epithelin precursor (11), is a growth factor encoded by the granulin precursor (GRN) gene with anti-inflammatory and osteogenic effects. It plays pivotal roles in various physiological and pathological processes such as early embryonic development, inflammation (12,13), wound healing (14), tumorigenesis and neurological disorders. In inflammatory response, PGRN inhibits neutrophil activation and proteolytic enzyme secretion by antagonizing TNF-α. In the context of anti-inflammatory mechanisms, the macrophage-derived factor secretory leukocyte protease inhibitor interacts with the inner domain of PGRN, providing protection against cleaving enzymes, such as proteinase 3 and elastase (15-17). Furthermore, studies have revealed that recombinant PGRN proteins and TNF-α competitively bind to tumor necrosis factor receptor (TNFR), leading to the antagonism of TNF-α and inhibition of the inflammatory response (17,18).
PGRN can induce mesenchymal stem cells to differentiate into cartilage. As a major downstream molecule of bone morphogenetic protein 2 (BMP2) (19), PGRN activates ERK1/2 signaling and the transcription factor JunB, which play significant roles in cartilage formation. Additionally, PGRN knockout mice exhibit dwarfism and severe skeletal defects, emphasizing the essential role of PGRN in skeletal development (20-22).
Periodontal regeneration involves recruiting endogenous stem cells to the defect site and using bioactive factors with anti-inflammatory and tissue-repair properties to enhance stem cell proliferation and differentiation. In contrast to TNF-α inhibitors that directly stimulate osteogenic differentiation (23), PGRN acts by antagonizing TNF-α and serving as a downstream protein of BMP2, promoting the osteogenic differentiation of cells. However, the direct use of exogenous growth factors presents challenges, such as high cost, complex protein extraction, short in vivo half-life, susceptibility to degradation by proteases and limited functionality (24,25). This often necessitates the use of large quantities of recombinant proteins, leading to increased economic costs and potentially excessive dosages as a side effect.
To address these challenges, researchers have proposed gene therapy, which relies on effective gene transfection and expression methods, as an innovative approach for periodontal regeneration (26). Viral vectors, particularly lentiviral or adenoviral vectors, are highly efficient and safe delivery vehicles for transferring exogenous genes to target cells (27,28). By transfecting target cells with these vectors, specific genes can be overexpressed or knocked down, enabling cells to proliferate and undergo osteogenic differentiation at the genetic level. Consequently, the present study hypothesized that lentivirus-mediated GRN could promote the proliferation and osteogenesis of periodontal ligament cells and aimed to construct a human periodontal cell line stably overexpressing GRN using the lentiviral method. Subsequently, it focused on assessing the effect of GRN overexpression on the proliferation and osteogenic capacity of hPDLCs, offering a theoretical foundation for advancing the understanding of periodontal regeneration.
Materials and methods
Isolation, culture and identification of hPDLCs
The study protocol was approved by the Ethics Committee of Lanzhou University School of Stomatology (approval no. LZUKQ-2019-045). A 28-years-old volunteer with good oral health donated four intact caries-free premolars that were extracted due to orthodontic treatment and used for PDLCs isolation; written informed consent was obtained from the patient. Extracted teeth were placed in α-MEM (Gibco; Thermo Fisher Scientific, Inc.) containing 2% penicillin-streptomycin solution (PS; Gibco; Thermo Fisher Scientific, Inc.) and hPDLCs extraction was completed within 2 h. The teeth were rinsed from root to crown with PBS containing 10% double antibody and the periodontal membrane tissue from the middle 1/3 of the isolated root was scraped and collected in a tube, which was added to 1 ml of α-MEM and then centrifuged at 100 x g for 5 min at 4˚C. After discarding the supernatant, collagenase type I (Gibco; Thermo Fisher Scientific, Inc.) was added and the tissue was digested for 15 min in an incubator at 37˚C. Following centrifugation at 100 x g for 5 min at 4˚C, trypsin was aspirated and 5 ml of complete medium [1% PS; 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 89% α-MEM] was added. The cells were cultured for 3-5 days in a cell culture incubator at 37˚C and the cell culture flask was removed to observe cell growth under an optical electron microscope (magnification, x40). The medium was changed every 2 days after cell adhesion to the flask and these cells were recorded as primary cells (P0) (19).
Finally, the third hPDLCs passage (P3) exhibiting well-grown were seeded into six-well plates and cultured in an incubator for 1 day. After fixation with 4% paraformaldehyde for 10 min at 37˚C, 0.1% Triton X-100 (Millipore Sigma) was added and incubated for 20 min at room temperature. Cells were washed three times with PBS and each well was treated with 300 µl of 5% BSA blocking solution and allowed to stand for 1 h at room temperature. After aspirating the liquid, anti-vimentin (1:1,000 dilution; rabbit; cat. no. ab137321; Abcam) and anti-keratin (1:1,000 dilution; cat. no. ab8068 mouse; Abcam) antibodies were added and incubated overnight at 4˚C. Following antibody removal, the cells were rinsed three times with TBST (0.1% Tween-20) and the mouse anti-rabbit IgG (HRP) secondary antibody (1:5,000 dilution; cat. no. ab99697; Abcam) was added and incubated at room temperature for 1 h in the dark. Following TBST rinsing, cell nuclei were stained with DAPI at room temperature for 10 min. Subsequently, the DAPI solution was discarded, the cells were washed three times with PBS, the slides were mounted and the entire cell area was observed under an inverted fluorescence microscope (magnification, x20). Based on immunofluorescence staining, protein staining characteristics of hPDLCs were therefore observed in 15 random fields of view at x20 magnification.
Lentiviral construction of a stable periodontal cell line overexpressing pLV-GRN
Primers were designed according to the gene sequence of GRN in NCBI (NM_002087.4), as shown in Table I. The Xho1 restriction site and protective bases were added to the forward primer and the Nhe1 restriction site, HA tags and protective bases were added to the reverse primer. All components were added following the manufacturer's instructions for the PrimerSTAR Max kit (Invitrogen; Thermo Fisher Scientific, Inc.). Specific PCR amplification of the coding sequence of the GRN gene was conducted, followed by nucleic acid electrophoresis detection and the target bands were excised and recovered. The PCR products of GRN and pLV-puro plasmid (Addgene, Inc.) underwent double digestion with the restriction enzymes Xho1 (New England BioLabs, Inc.) and Nhe1 (New England BioLabs, Inc.) The gel mixture was incubated at 50-60˚C for 10 min, according to the instructions for the GeneJET gel extraction kit (Thermo Fisher Scientific, Inc.). The gel was recovered, ligated with T4 ligase (New England BioLabs, Inc.) and the ligated product was combined with DH5α (Invitrogen; Thermo Fisher Scientific, Inc.) receptor cells for transformation. The plasmid was extracted using the TIANprep Mini Plasmid Kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The plasmid was identified by double digestion with Xho1 and Nhe1 and then sent to Tsingke Biotechnology Co., Ltd. for sequencing.
293T cells (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China) were cultured in T25 culture flasks. Upon reaching 80% confluence, cell transfection was performed using the jetPRIME Transfection Reagent Kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. A total of 300 µl of jetPRIME buffer was added to a sterile EP tube and combined with 6 µg of pLV-puro or pLV-GRN plasmid, 4 µg of psPAX2 plasmid (Addgene, Inc.) and 2 µg of pMD2.G plasmid (Addgene, Inc.). To this mixture, 12 µl of jetPRIME reagent was added, thoroughly mixed and left at room temperature for 15 min. The resulting solution was added dropwise to a 293T cell culture flask, followed by the addition of 5 ml of complete medium. The medium was changed after 12 and 36 h later, the supernatant was aspirated, centrifuged at 100 x g for 5 min at 4˚C, filtered through a 0.22 µm filter, collected into a centrifuge tube, aliquoted into 1 ml EP tubes and stored at -80˚C (29).
Finally, P3 hPDLCs were divided into hPDLCs, pLV-puro and pLV-GRN groups and inoculated into T25 culture flasks (n=3). The medium was changed when the cell confluence reached 70-80% and then 1 ml of the collected pLV-puro or pLV-GRN lentiviral plasmids was added dropwise to the corresponding flasks, mixed and cultured for 24 h at 37˚C. Based on the resistance screening concentration determined by the previous research, the final concentration of 5 µg/ml polybrene (Gibco; Thermo Fisher Scientific, Inc.) was added to the culture medium. After 3-4 days, when, upon microscopic investigation, most cells in the blank group appeared to have disintegrated and died, the polybrene concentration was halved and screening continued. After ~7 days, when all cells in the blank group had died, the cells that remained adherent to the wall in the PLV-puro and pLV-GRN groups were successfully infected and continued to be cultured and passaged. The experiment for constructing an hPDLCs line overexpressing GRN using lentivirus was repeated at least three times.
Reverse transcription-quantitative (RT-q) PCR of the mRNA expression level of GRN
After the confluence of cells reached 90%, total RNA was extracted from P5 cells in the hPDLCs group, pLV-puro group and pLV-GRN group, followed by reverse transcription into cDNA using the PrimerScript RT Master Mix Reverse Transcription Kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Gene primers were designed with β-actin serving as an internal reference (Table I). qPCR amplification was performed using 2X SYBR Green QPCR Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. PCR conditions were as follows: 95˚C for 5 min, followed by 30 cycles at 95˚C for 30 sec, 58˚C for 20 sec and 72˚C for 20 sec. Each experiment was repeated in three sets (n=3). Following the completion of the reaction, melting curves were analyzed and the data were processed based on 2-ΔΔCq and the experiment was repeated three times (30,31).
Western blot analysis of GRN protein expression
Total protein from P5 cells in the hPDLCs, pLV-puro and pLV-GRN groups was extracted (Laemmli buffer 2x; cat. no. S3401; MilliporeSigma) and protein quantification was performed using a BCA kit (n=3) following the manufacturer's instructions. The cells were evenly scraped with a cell scraper and the mixed solution was transferred to a 2 ml EP tube and ultrasonically lysed for 2 min and centrifuged at 16,000 x g for 10 min at 4˚C. The supernatant was added to a new 2 ml EP tube. Meanwhile, 5x loading Buffer (Thermo Fisher Scientific, Inc.) was added in a ratio of 1:4 and was denatured at 95˚C for 10 min, and finally the protein was stored at -20˚C. The pre-thawed protein was added to the electrophoresis tank (10% SDS-PAGE) at 12 µl per well and electrophoresed for 2.5 h (80 V 30 min, 120 V 120 min; n=3). Afterward, the membrane was transferred in the order of sponge-filter paper gel-PVDF membrane-filter paper-sponge, and the program was set to 2,000 mA for 120 min. Then, the PVDF membrane was placed in 15 ml of 5% skimmed milk powder and blocked on a shaker for 3 h. After blocking, the desired gene bands were cut into diluted anti-GRN (1:1,000 dilution; rabbit; cat. no. ab191211; Abcam) and β-actin (1:1,000 dilution; rabbit; cat. no. ab8227; Abcam) at 4˚C overnight. The next day, the strip was removed and washed three times with 15 ml TBST (0.1% Tween-20) for 10 min each time. After washing, the strip was placed in the goat anti-rabbit (IgG) secondary antibody HRP (1:5,000 dilution; cat. no. ab6721; Abcam) and incubated for 1 h at room temperature on a shaker. Then, the strip was washed three times with 15 ml TBST for 10 min each time. Finally, after adding the developer solution to the stripes, the image could be exposed and saved by the exposure instrument. The whole process of western blot experiment was replicated for three times. ImageJ 2.2.0-beta6 software (National Institutes of Health) was used to determine the protein grayscale values and the experiment was repeated three times (32).
MTT assay of cell proliferative capacity
P2 hPDLCs were digested with 0.25% trypsin, counted and inoculated into 96-well plates at a density of 1x104 cells/ml and 100 µl per well with three replicates per group (n=3). Culturing was conducted for 1-7 days at a constant temperature of 37˚C with 5% CO2. After aspirating the medium, 10 µl of MTT labeling reagent (Abcam) was added to each well. Subsequently, 100 µl of lysis buffer was added to each well after continuous culture for 4 h and then mixed thoroughly on a shaker and the absorbance (OD) value at 450 nm was determined with a spectrophotometer. Each experiment was performed in triplicate (33).
Alizarin Red staining
P6 cells from the hPDLCs, pLV-puro and pLV-GRN groups were inoculated into 35 mm2 dishes, with three replicates per group (n=3). After the cells adhered to the surface, 2 ml of osteogenic induction solution was added and continuous culturing was performed for 21 days with the solution changed every 2 days. On the 7th, 14th and 21st day, the medium was aspirated and washed with PBS for three times and 300 µl of 4% paraformaldehyde was added to each group of cells for 30 min at room temperature. After aspirating the paraformaldehyde, the cells were washed three times with PBS and 1 ml of alizarin red solution (Procell Life Science & Technology Co., Ltd.) was added to each group of cells, allowed to stand for 20 min and observed the entire cell area under an optical electron microscope (magnification, x4) (19).
Determination of alkaline phosphatase (ALP) activity
Cells from the hPDLCs, pLV-puro and pLV-GRN groups were seeded into 96-well plates after 7, 14 and 21 days of induction and ALP activity was assessed using an ALP activity assay kit (Procell Life Science & Technology Co., Ltd.) according to the manufacturer's instructions. The absorbance (OD) of each well was measured at 405 nm using a spectrophotometer and a standard curve was constructed. The ALP activity of each cell group was calculated using an enzyme activity assay. Each experiment was performed in triplicate (34).
RT-qPCR determination of mRNA expression levels of osteogenesis-related genes
Total After the confluence of cells reached 90%, RNA was extracted from cells in the hPDLCs group, pLV-puro group and pLV-GRN group after induction for 7, 14 and 21 days, with three replicates per group (n=3) and reverse transcribed into cDNA using the PrimerScript RT Master Mix Reverse Transcription Kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. PCR conditions were as follows: 95˚C for 5 min, followed by 30 cycles at 95˚C for 30 sec, 58˚C for 20 sec and 72˚C for 20 sec. Primers for osteogenesis-related genes, including ALP, runt-related transcription factor 2 (Runx2) and osteopontin (OPN), are listed in Table I. qPCR amplification was performed according to the instructions for 2X SYBR Green QPCR Master Mix. Each experiment for each gene was conducted in triplicate, with β-actin serving as an internal reference. The relative mRNA expression of each gene was calculated according to 2-ΔΔCq (30,31).
Western blot determination of osteogenesis-related protein expression levels
Total protein was extracted from cells in each group following osteogenic induction for 7, 14 and 21 d, with three replicates per group (n=3). Protein quantification was conducted using the BCA kit according to the manufacturer's instructions. Western blot analysis was performed using GADPH as an internal reference to assess the expression of osteogenesis-related proteins, including OPN (1:400 dilution; rabbit; cat. no. ab8448; Abcam), Runx-2 (1:200 dilution; rabbit; cat. no. ab114133; Abcam) and GADPH (1:1,000 dilution; rabbit; cat. no. ab263962; Abcam). The specific method was the same as that in Western blot analysis of GRN protein expression. ImageJ 2.2.0-beta6 software (National Institutes of Health) was used to determine the grayscale values of proteins (32).
Statistical analysis
SPSS Statistics for Windows (version 26.0; IBM Corp.) was used for the statistical analysis. One-way ANOVA and two-way ANOVA followed by Tukey's post hoc test were used to analyze the differences. P<0.05 was considered to indicate a statistically significant difference.
Results
Culture and identification of primary hPDLCs
After 5 days of culture, black clusters of periodontal ligament tissue were observed under an inverted microscope. Cells could be observed expanding around the tissue mass, which were spindle or spindle-shaped, uniform in shape and full of cytoplasm (Fig. 1A). The immunofluorescence staining of P3 hPDLCs was negative, indicating that the extracted cells were not contaminated with epithelial cells (Fig. 1B). Positive vimentin staining indicated that the cells were derived from the mesenchyme (Fig. 1B). The cells were characterized as hPDLCs based on immunofluorescence staining.
Identification of pLV-GRN recombinant plasmid by double digestion
The electrophoresis results showed a distinct band at ~1,866 bp (Fig. 2A). Following double digestion of the homologous recombinant plasmid pLV-GRN with XhoI and NheI, electrophoresis displayed clear bands at ~1,866 and 7,800 bp (Fig. 2B), which were consistent with the size of the 1,866 bp GRN gene along with its HA tag. The sequencing results were aligned with the cDNA sequence of the GRN gene (NM_002087.4) in the NCBI database (Fig. 2C). The sequence demonstrated 100% homology with the target sequence, confirming the successful construction of the recombinant plasmid.
Screening results of hPDLCs overexpressing GRN
After periodontal ligament cells were infected with the lentivirus for 24 h (Fig. 3A), the cell morphology in the hPDLCs, pLV-puro and pLV-GRN groups remained unchanged, maintaining a radial or spiral arrangement. After 7 days of puromycin resistance screening, microscopic examination revealed numerous cell fragments in the hPDLCs group, indicating the loss of intact cell morphology. Varying degrees of cell disintegration and death were observed in the pLV-puro and pLV-GRN groups. However, a small number of hPDLCs with surface adherence persisted, exhibiting an unaltered long spindle or fusiform shape. At this stage, cells with normal morphology, clear edges and continued adherence observed under the microscope were identified as hPDLCs that were successfully infected with the lentiviral vector.
RT-qPCR and western blot analysis of GRN expression
RT-qPCR results revealed robust expression of the GRN gene in the pLV-GRN group, which was 80-90 times higher than that in the hPDLCs and pLV-puro groups (P<0.01; Fig. 3B).
With β-actin as the internal reference, western blotting results demonstrated the expression of HA-tagged protein, indicating strong PGRN expression in periodontal cells transfected with GRN. Protein expression assays showed a significant enhancement in PGRN protein expression in the pLV-GRN group, which was ~1.8 times higher than that in the hPDLCs and pLV-puro groups (P<0.05; Fig. 3C).
Comparison of cell proliferation capacity
The MTT assay results indicated a similar increase in the cell proliferation rate for all groups cultured for 12-72 h. The hPDLCs group did not differ significantly from the pLV-puro group; however, the cell proliferation capacity of the pLV-GRN group was significantly higher (Fig. 4A; P<0.05).
ALP activity assay
The ALP activity assay results revealed that the ALP activity of the hPDLCs, pLV-puro and pLV-GRN groups peaked at 14 days. The ALP activity of the pLV-GRN group was significantly higher than that of the other two groups (P<0.01) and subsequently declined but remained higher than that of the hPDLCs and pLV-puro groups (P<0.05). No significant differences were observed between the hPDLCs and pLV-puro groups (Fig. 4B).
Alizarin red staining
At 7 days after osteogenic induction, no obvious orange-red mineralized nodules were observed in the hPDLCs (Fig. 4Ca), pLV-puro (Fig. 4Cb), or pLV-GRN (Fig. 4Cc) groups. After osteogenic induction for 14 days, orange-red mineralized nodules were observed in the hPDLCs (Fig. 4Cd), pLV-puro (Fig. 4Ce) and pLV-GRN (Fig. 4Cf) groups. The pLV-GRN group exhibited more mineralized nodules than the other two groups. After 21 days of osteogenic induction, the pLV-GRN group (Fig. 4Ci) produced more orange-red mineralized nodules than the hPDLCs (Fig. 4Cg) and pLV-puro groups (Fig. 4Ch) and there was no significant difference between the hPDLCs (Fig. 4Cg) and pLV-puro groups (Fig. 4Ch).
RT-qPCR analysis of osteogenesis-related genes
RT-qPCR results demonstrated that after 7, 14 and 21 days of osteogenic induction, the mRNA expression levels of osteogenesis-related genes, including ALP and Runx2, in the pLV-GRN group were 2-3 times higher than those in the hPDLCs and pLV-puro groups (P<0.05). The mRNA expression level of the osteogenesis-related gene OPN was ~1.5 times higher in the pLV-GRN group than in the hPDLCs and pLV-puro groups (P<0.05; Fig. 5A).
Western blot analysis of osteogenesis-related protein expression
Western blotting results indicated that the expression level of the osteogenic protein Runx-2 in the pLV-GRN group was ~1.5-fold higher than that in the hPDLCs and pLV-puro groups at 7, 14 and 21 days of osteogenic induction (P<0.05). The expression level of the osteogenic protein OPN in the pLV-GRN group did not differ significantly from that in the hPDLCs and pLV-puro groups on days 7 and 14 of osteogenic induction. However, on day 21 of osteogenic induction, the expression level of OPN in the pLV-GRN group was ~2-fold higher than that in the hPDLCs and pLV-puro groups (P<0.05; Fig. 5B).
Discussion
Periodontitis, characterized by chronic inflammation affecting the supporting periodontal tissues, poses a significant threat, leading to the progressive destruction of the alveolar bone and potential tooth loss in severe cases. Current clinical treatments primarily focus on addressing etiological factors and controlling inflammation through scaling and root planning. However, these methods failed to achieve comprehensive periodontal regeneration. Therefore, exploring effective strategies for complete periodontal regeneration is imperative for managing periodontitis. Tissue engineering techniques, which are gaining prominence in domestic and international research, offer a promising avenue for advancing periodontal regeneration (10).
hPDLCs, which are multipotent cells that can differentiate into fibroblasts, osteoblasts and cementoblasts, have emerged as the ideal seed cells for periodontal tissue engineering (10). In the present study, primary hPDLCs were successfully isolated and cultured from teeth extracted during orthodontic procedures and from third molars. Immunofluorescent staining for keratin and vimentin confirmed the mesenchymal origin of the cells, ensuring stable biological characteristics following passaging. Microscopic examination revealed a consistent fusiform or long-spindle morphology, indicating a robust growth status suitable for subsequent investigation (35).
The dual protective and regenerative attributes of PGRN make it a promising target for novel therapies to treat diseases associated with tissue defects (36). Periodontal regeneration promotes the proliferation and differentiation of stem cells by recruiting endogenous stem cells to the defect site and using bioactive factors with anti-inflammatory and tissue repair effects. Therefore, the effect of PGRN on hPDLCs is key to promoting periodontal tissue regeneration (37). While local application of exogenous PGRN has shown promise in promoting periodontal regeneration (37), direct administration of recombinant proteins presents challenges, such as frequent dosing and high quantities. To address these problems, a stable hPDLCs cell line overexpressing GRN was successfully constructed using a lentiviral vector. Western blotting results unequivocally indicated that the GRN gene was successfully constructed in the present study using a lentiviral vector and the protein expression of GRN protein in transfected cells of the pLV-GRN group was compared with that in the hPDLCs and pLV-puro groups. The RT-qPCR results demonstrated robust expression of the GRN gene in the pLV-GRN group, exhibiting a substantial difference from that in the hPDLCs and pLV-puro groups. Moreover, following passaging, the morphological consistency of hPDLCs transfected with the GRN gene, resembling primary cells with a fusiform or long spindle shape, confirmed the stable effect of GRN on hPDLCs. This stable research model provides a foundation for exploring the effects of GRN on the proliferation and osteogenic capacity of hPDLCs.
Exogenous PGRN has been shown to induce cell proliferation and increase ALP activity in hPDLCs (37,38). In the present study, the proliferative capacity of hPDLCs successfully transfected with pLV-GRN was assessed using MTT and ALP activity assays. The results revealed that the proliferative capacity of cells in the pLV-GRN group significantly surpassed that of cells in both the hPDLCs and pLV-puro groups, underscoring the role of GRN in promoting hPDLCs proliferation.
Studies have shown that the intricate process of osteogenic differentiation in hPDLCs involves the orchestration of various factors, including BMPs, Wnt family proteins and transcription factors (Runx-2, β-catenin), with BMP2 being a pivotal inducer in bone formation (5,6,10,39). PGRN, a downstream target of BMP2, plays a crucial role in inducing bone formation (19). In the present study, osteogenic induction spanning 21 days was conducted on cells from the hPDLCs, pLV-puro and pLV-GRN groups, followed by Alizarin Red staining on days 7, 14 and 21. Microscopic observations revealed a higher occurrence of orange-red mineralized nodules in the pLV-GRN group than in the other two groups. This observation supported the robust osteogenic-promoting capability of GRN in hPDLCs at the cellular level.
Runx-2 regulates osteoblast differentiation and maturation primarily through the Wnt and BMP signaling pathways (40,41). PGRN promotes the expression of Runx-2, exerting a pivotal role in the proliferation, maturation and differentiation of mesenchymal stem cells (19). Additionally, OPN plays a crucial role in bone metabolism, not only as a vital factor in the neuron-mediated and endocrine regulation of bone mass but also in various biological activities (42-44). ALP, recognized as the principal mineralizing enzyme in osteogenesis, metabolism and regeneration (45,46), serves as an early differentiation marker for osteoblasts and a characteristic indicator for evaluating differentiation toward osteogenic lineages. In the present study, RT-qPCR results revealed that the mRNA expression of osteogenesis-related genes, including OPN, Runx-2 and ALP, in the pLV-GRN group significantly surpassed that in both the hPDLCs and pLV-puro groups. No significant differences were observed between the hPDLCs and pLV-puro groups. Consistent with these findings, western blotting demonstrated higher protein expression levels of OPN and Runx-2 in the pLV-GRN group than in the hPDLCs and pLV-puro groups during osteogenic induction. Moreover, OPN expression in the pLV-GRN group was notably higher than that in the hPDLCs and pLV-puro group on the 21st day. These results further substantiated that GRN effectively enhanced the ability of hPDLCs to differentiate into an osteogenic lineage at both the molecular and protein levels.
The present study unequivocally demonstrated that GRN possessed a pronounced ability to enhance both the proliferation and osteogenic differentiation of hPDLCs. Importantly, it addressed the drawbacks associated with the frequent administration and high dosage of exogenous GRN observed in previous studies. Using lentiviral-mediated methods, hPDLCs cell lines that overexpressed GRN and exhibited stable and noteworthy osteogenic effects were successfully constructed. The present study provided a solid theoretical foundation for future investigations on periodontal regeneration.
To summarize, the present study successfully established a stable hPDLCs cell line overexpressing the GRN gene through the use of a lentiviral vector. The ability of GRN to promote proliferation and osteogenic differentiation of hPDLCs was confirmed. Further exploration of this signaling pathway is needed to comprehensively verify the role of GRN in promoting the proliferation and osteogenesis of hPDLCs. The present study not only served as a robust experimental basis for advancing the understanding of periodontal tissue regeneration but also charted a novel direction for preventing and treating periodontal disease.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by the Research Fund of Lanzhou University (grant no. 20JR10RA653-ZDKF20210103) and the Key Research Fund of Gansu Province (grant no. 21YF5GA100).
Availability of data and materials
The data generated in the present study are included in the figures and/or tables of this article.
Authors' contributions
XY and RQ conducted the synthesis of recombinant plasmids and osteogenic differentiation experiments and were major contributors to writing the manuscript. ZC, DH and XS collected the clinical samples and experimental data. YS and XH designed the experiments and reviewed and edited the manuscript. XY and XH confirm the authenticity of all the raw data. All the authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
Human periodontal ligament cells were isolated and used in accordance with the ethical standards established in the Declaration of Helsinki. The present study was approved by the ethics committee of the School of Stomatology, Lanzhou University (approval no. LZUKQ-2019-045) and informed consent form was signed by the patient prior to participation in the study.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Authors' information
Professor Xiangyi He ORCID: 0000-0002-5687-0991.
References
|
Guzik TJ and Czesnikiewicz-Guzik M: Mounting pressure of periodontitis. Hypertension. 78:552–554. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Sirisereephap K, Maekawa T, Tamura H, Hiyoshi T, Domon H, Isono T, Terao Y, Maeda T and Tabeta K: Osteoimmunology in periodontitis: Local proteins and compounds to alleviate periodontitis. Int J Mol Sci. 23(5540)2022.PubMed/NCBI View Article : Google Scholar | |
|
Michalowicz BS, Hodges JS, DiAngelis AJ, Lupo VR, Novak MJ, Ferguson JE, Buchanan W, Bofill J, Papapanou PN, Mitchell DA, et al: Treatment of periodontal disease and the risk of preterm birth. N Engl J Med. 335:1885–1894. 2006.PubMed/NCBI View Article : Google Scholar | |
|
Chatzaki N, Zekeridou A, Paroz E, Gastaldi G and Giannopoulou C: Knowledge and practice attitudes regarding the relationship between diabetes and periodontitis: A survey among Swiss endocrinologists and general physicians. BMC Prim Care. 24(238)2023.PubMed/NCBI View Article : Google Scholar | |
|
Lee HS, Byun SH, Cho SW and Yang BE: Past, present, and future of regeneration therapy in oral and periodontal tissue: A review. Appl Sci. 9(1046)2019. | |
|
Raveau S and Jordana F: Tissue engineering and three-dimensional printing in periodontal regeneration: A literature review. J Clin Med. 9(4008)2020.PubMed/NCBI View Article : Google Scholar | |
|
Tsuchida S and Nakayama T: Periodontal tissue regeneration therapy using stem cells. Stem Cell Rev Rep. 19:825–826. 2023.PubMed/NCBI View Article : Google Scholar | |
|
Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY and Shi S: Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 364:149–155. 2004.PubMed/NCBI View Article : Google Scholar | |
|
Tomokiyo A, Wada N and Maeda H: Periodontal ligament stem cells: Regenerative potency in periodontium. Stem Cells Dev. 28:974–985. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Zhao B, Zhang W, Xiong Y, Zhang Y, Zhang D and Xu X: Effects of rutin on the oxidative stress, proliferation and osteogenic differentiation of periodontal ligament stem cells in LPS-induced inflammatory environment and the underlying mechanism. J Mol Histol. 51:161–171. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Mendsaikhan A, Tooyama I and Walker DG: Microglial progranulin: Involvement in Alzheimer's disease and neurodegenerative diseases. Cells. 8(230)2019.PubMed/NCBI View Article : Google Scholar | |
|
Yin FF, Banerjee R, Thomas B, Zhou P, Qian LP, Jia T, Ma XJ, Ma Y, Iadecola C, Beal MF, et al: Exaggerated inflammation, impaired host defense, and neuropathology in progranulin-deficient mice. J Exp Med. 207:117–128. 2010.PubMed/NCBI View Article : Google Scholar | |
|
Saeedi-Boroujeni A, Purrahman D, Shojaeian A, Poniatowski ŁA, Rafiee F and Mahmoudian-Sani MR: Progranulin (PGRN) as a regulator of inflammation and a critical factor in the immunopathogenesis of cardiovascular diseases. J Inflamm (Lond). 20(1)2023.PubMed/NCBI View Article : Google Scholar | |
|
He Z, Ong CH, Halper J and Bateman A: Progranulin is a mediator of the wound response. Nat Med. 9:225–229. 2003.PubMed/NCBI View Article : Google Scholar | |
|
Gulluoglu S, Tuysuz EC, Sahin M, Yaltirik CK, Kuskucu A, Ozkan F, Dalan AB, Sahin F, Ture U and Bayrak OF: The role of TNF-α in chordoma progression and inflammatory pathways. Cell Oncol (Dordr). 42:663–677. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Noguchi T, Ebina K, Hirao M, Kawase R, Ohama T, Yamashita S, Morimoto T, Koizumi K, Kitaguchi K, Matsuoka H, et al: Progranulin plays crucial roles in preserving bone mass by inhibiting TNF-α-induced osteoclastogenesis and promoting osteoblastic differentiation in mice. Biochem Biophys Res Commun. 465:638–643. 2015.PubMed/NCBI View Article : Google Scholar | |
|
Pogonowska M, Poniatowski ŁA, Wawrzyniak A, Królikowska K and Kalicki B: The role of progranulin (PGRN) in the modulation of anti-inflammatory response in asthma. Cent Eur J Immunol. 44:97–101. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Wei F, Zhang Y, Jian J, Mundra JJ, Tian Q, Lin J, Lafaille JJ, Tang W, Zhao W, Yu X and Liu CJ: PGRN protects against colitis progression in mice in an IL-10 and TNFR2 dependent manner. Sci Rep. 4(7023)2014.PubMed/NCBI View Article : Google Scholar | |
|
Qin R, Cui Z, Zhou H, Guo R, Yao X, Wang T, Qin X and He X: Effect of lentivirus-mediated BMP2 from autologous tooth on the proliferative and osteogenic capacity of human periodontal ligament cells. J Periodont Res. 57:869–879. 2022.PubMed/NCBI View Article : Google Scholar | |
|
Ding Y, Wei J, Hettinghouse A, Li G, Li X, Einhorn TA and Liu CJ: Progranulin promotes bone fracture healing via TNFR pathways in mice with type 2 diabetes mellitus. Ann N Y Acad Sci. 1490:77–89. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Yang Y, Feng N, Liang L, Jiang R, Pan Y, Geng N, Fan M, Li X and Guo F: Progranulin, a moderator of estrogen/estrogen receptor α binding, regulates bone homeostasis through PERK/p-eIF2 signaling pathway. J Mol Med (Berl). 100:1191–1207. 2022.PubMed/NCBI View Article : Google Scholar | |
|
Zhao Z, Li E, Luo L, Zhao S, Liu L, Wang J, Kang R and Luo J: A PSCA/PGRN-NF-κB-Integrin-α4 axis promotes prostate cancer cell adhesion to bone marrow endothelium and enhances metastatic potential. Mol Cancer Res. 18:501–513. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Sfikakis PP and Tsokos GC: Towards the next generation of anti-TNF drugs. Clin Immunol. 141:231–235. 2011.PubMed/NCBI View Article : Google Scholar | |
|
Han CM, Cheng B and Wu P: Writing group of growth factor guideline on behalf of Chinese Burn Association. Clinical guideline on topical growth factors for skin wounds. Burns Trauma. 8(tkaa035)2020.PubMed/NCBI View Article : Google Scholar | |
|
Hefka Blahnova V, Dankova J, Rampichova M and Filova E: Combinations of growth factors for human mesenchymal stem cell proliferation and osteogenic differentiation. Bone Joint Res. 9:412–420. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Ridet JL and Privat A: Gene therapy in the central nervous system direct versus indirect gene delivery. J Neurosci Res. 42:287–293. 1995.PubMed/NCBI View Article : Google Scholar | |
|
Milone MC and O'Doherty U: Clinical use of lentiviral vectors. Leukemia. 32:1529–1541. 2018.PubMed/NCBI View Article : Google Scholar | |
|
Perry C and Rayat ACME: Lentiviral vector bioprocessing. Viruses. 13(268)2021.PubMed/NCBI View Article : Google Scholar | |
|
Cui Z, Qin R, Feng J, Liu Y, Zhou X, Qin X, Li Y, Zhang Z and He X: XBP1s gene of endoplasmic reticulum stress enhances proliferation and osteogenesis of human periodontal ligament cells. Tissue Cell. 83(102139)2023.PubMed/NCBI View Article : Google Scholar | |
|
Fathi E, Farahzadi R and Charoudeh HN: L-carnitine contributes to enhancement of neurogenesis from mesenchymal stem cells through Wnt/β-catenin and PKA pathway. Exp Biol Med (Maywood). 242:482–486. 2017.PubMed/NCBI View Article : Google Scholar | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar | |
|
Bagheri Y, Barati A, Nouraei S, Jalili Namini N, Bakhshi M, Fathi E and Montazersaheb S: Comparative study of gavage and intraperitoneal administration of gamma-oryzanol in alleviation/attenuation in a rat animal model of renal ischemia/reperfusion-induced injury. Iran J Basic Med Sci. 24:175–183. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Kumar P, Nagarajan A and Uchil PD: Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc. 2018(pdb.prot095505)2018.PubMed/NCBI View Article : Google Scholar | |
|
Suga T, Usui M, Onizuka S, Sano K, Sato T, Nakazawa K, Ariyoshi W, Nishihara T and Nakashima K: Characterization and study of gene expression profiles of human periodontal mesenchymal stem cells in spheroid cultures by transcriptome analysis. Stem Cells Int. 2021(5592804)2021.PubMed/NCBI View Article : Google Scholar | |
|
Fathi E, Azarbad S, Farahzadi R, Javanmardi S and Vietor I: Effect of rat bone marrow derived-mesenchymal stem cells on granulocyte differentiation of mononuclear cells as preclinical agent in cell based therapy. Curr Gene Ther. 22:152–161. 2022.PubMed/NCBI View Article : Google Scholar | |
|
Li L, Jiang H, Chen R, Zhou J, Xiao Y, Zhang Y and Yan F: Human β-defensin 3 gene modification promotes the osteogenic differentiation of human periodontal ligament cells and bone repair in periodontitis. Int J Oral Sci. 12(13)2020.PubMed/NCBI View Article : Google Scholar | |
|
Zheng C, Chen J, Liu S and Jin Y: Stem cell-based bone and dental regeneration: A view of microenvironmental modulation. Int J Oral Sci. 11(23)2019.PubMed/NCBI View Article : Google Scholar | |
|
Chen Q, Wu Z and Xie L: Progranulin is essential for bone homeostasis and immunology. Ann N Y Acad Sci. 1518:58–68. 2022.PubMed/NCBI View Article : Google Scholar | |
|
Chen Q, Cai J, Li X, Song A, Guo H, Sun Q, Yang C and Yang P: Progranulin promotes regeneration of inflammatory periodontal bone defect in rats via anti-inflammation, osteoclastogenic inhibition, and osteogenic promotion. Inflammation. 42:221–234. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Sun R, Wang D, Song Y, Li Q, Su P and Pang Y: Correction: Granulin as an important immune molecule involved in lamprey tissue repair and regeneration by promoting cell proliferation and migration. Cell Mol Biol Lett. 27(96)2022.PubMed/NCBI View Article : Google Scholar | |
|
Gomathi K, Akshaya N, Srinaath N, Moorthi A and Selvamurugan N: Regulation of Runx2 by post-translational modifications in osteoblast differentiation. Life Sci. 245(117389)2020.PubMed/NCBI View Article : Google Scholar | |
|
Komori T: Regulation of proliferation, differentiation and functions of osteoblasts by Runx2. Int J Mol Sci. 20(1694)2019.PubMed/NCBI View Article : Google Scholar | |
|
Yang J, Ye L, Hui TQ, Yang DM, Huang DM, Zhou XD, Mao JJ and Wang CL: Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway. Int J Oral Sci. 7:95–102. 2015.PubMed/NCBI View Article : Google Scholar | |
|
Min KK, Neupane S, Adhikari N, Sohn WJ, An SY, Kim JY, An CH, Lee Y, Kim YG, Park JW, et al: Effects of resveratrol on bone-healing capacity in the mouse tooth extraction socket. J Periodont Res. 55:247–257. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Zernik J, Twarog K and Upholt WB: Regulation of alkaline phosphatase and alpha 2(I) procollagen synthesis during early intramembranous bone formation in the rat mandible. Differentiation. 44:207–215. 1990.PubMed/NCBI View Article : Google Scholar | |
|
Wang Y, Li X, Zhou X, Wang T, Liu Y, Feng J, Qin X, Zhang Z, Li Y and He X: Regulation of proliferation and apoptosis of aging periodontal ligament cells by autophagy-related gene 7. Mol Biol Rep. 50:6361–6372. 2023.PubMed/NCBI View Article : Google Scholar |
