Periodontal ligament fibroblasts utilize isoprenoid intermediate farnesyl diphosphate for maintaining osteo/cementogenic differentiation abilities
Fibroblastic cells in periodontal ligament tissue (periodontal ligament fibroblasts, PDLFs) possess stem cell–like properties and, thus, can differentiate into osteo/cementogenic cells. , , , , Inhibition of Wingless, the Drosophila melanogaster segment-polarity gene, and integrase-1, the vertebrate homologue (Wnt) secretion, such as Wnt3a, a key inducer of osteogenesis and cementogenesis, in murine PDLFs leads to a higher width of periodontal ligament tissue and suppresses the expression of osteogenic markers in periodontal ligament tissue. Therefore, osteo/cementogenic abilities of PDLFs are indispensable for maintaining periodontal tissue homeostasis. During periodontal regeneration, PDLFs proliferate and migrate from the remaining periodontal ligament tissue to the surgically disinfected space and reconstruct the physiological periodontal tissue composed of cementum, alveolar bone, and periodontal ligament tissue. PDLFs utilize energy metabolic processes to maintain osteo/cementogenic differentiation abilities. Knockdown of peroxisome proliferator-activated receptor γ (PPARγ), a key transcription factor for genes involved in energy metabolic pathways, in PDLFs results in a marked reduction in the expression of osteo/cementogenic-related and extracellular matrix (ECM)-related genes. Moreover, thiazolidinedione compounds (TZDs), chemical agonists of PPARγ, such as rosiglitazone and troglitazone, enhance the osteo/cementogenic abilities in vitro . Periodontal tissue/cells express three isoforms of PPARγ: a full-length PPARγ and two partially deleted isoforms, ubiquitous isoform of PPARγ (PPARγ-UBI) and periodontal isoform of PPARγ (PPARγ-PDL). PPARγ-UBI was previously identified in adipocytes, and PPARγ-PDL was newly identified as a PDL-specific isoform. , Local application of procyanidin B2, a PPARγ ligand, into the periodontal tissue of ligature-induced periodontitis mice prevents alveolar bone loss. Mechanistically, PDLFs treated with procyanidin B2 exhibit suppressed expression of PPARγ-UBI, which stabilizes the nuclear factor kB (NF-kB) p65 and the expression of genes encoding pro-inflammatory cytokines. PPARγ isoform-dependent functions have been identified; however, the distinct functions of PPARγ isoforms in osteo/cementogenic differentiation remain unclear. PPARγ is a key modulator of energy metabolism, such as lipid and glucose metabolism; acetyl coenzyme A (acetyl-CoA) is a central coordinator for lipid and glucose metabolism. However, which metabolic pathways and intermediates contribute to maintaining the osteo/cementogenic abilities of PDLFs remain unclear. In the present study, we assessed the effects of TZDs on periodontal tissue regeneration. Next, the osteo/cementogenic abilities of the three PPARγ isoforms in PDLFs were explored. Subsequently, the isoprenoid metabolic pathway was identified as the most severely downregulated metabolic pathway in PPARγ-knockdown PDLFs. Isoprenoid synthesis originates from the mevalonate pathway, which starts with the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, and then synthesizes intermediates such as farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) by the condensation reaction of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Therefore, FPP and GGPP were used to identify the key intermediates for osteo/cementogenic abilities. 

Reagents Rosiglitazone (R2408), FPP ammonium salt (F6892), and GGPP ammonium salt (G6025) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Troglitazone (209–19481) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Experimental animals All experimental procedures conformed to the “Regulations for Animal Experiments and Related Activities at Tohoku University” and were reviewed by the Institutional Laboratory Animal Care and Use Committee of Tohoku University and approved by the President of the University (Permit No. 2020DnA-044-06). Eleven-week-old male C57BL6/J mice (specific pathogen-free grade) were purchased from CLEA Japan, Inc. The mice were allowed to adapt to the new environment for one week before the experiments. The mice were anesthetized, and silk ligatures (Elp Sterile Blade Silk, Black, 5–0, Akiyama Medical MFG, Tokyo, Japan) were tied around the left second maxillary molar for 14 days. After the 14-day periodontal inflammation period, rosiglitazone (1.2 mg/kg) dissolved in solvent (50% DPBS, 40% PEG300, 5% Tween 80, 5% DMSO) was locally applied into the mesial and distal sides of the left second maxillary molars every other day for 14 days. The mice in the control group were administered the same amount of solvent at the same frequency. The right maxillae of the rosiglitazone and solvent-treated control groups were not treated throughout the experimental period. Micro-computed tomography Micro-computed tomography (μCT) was conducted as previously described. , The vertical distances from the cemento-enamel junction to the alveolar bone crest at the mesial and distal roots of the second maxillary molars were measured and added. This sum was used to quantitatively compare bone regeneration levels in the periodontal regeneration stage. Histology The maxilla samples used for μCT analysis were decalcified using ethylenediaminetetraacetic acid at 4 °C for 2 weeks. Masson's trichrome staining was performed on 5 μm-thick paraffin sections as previously described. , , Cell culture and stable cell line generation Human PDLFs were purchased from Lonza Inc. (Walkersville, MD, USA) and maintained in low-glucose Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine system. PDLFs were cultivated at 37 °C under humidified atmospheric conditions (5% CO 2 and 95% air). PDLFs stably expressing the full-length (PPARγ), PPARγ-UBI, and PPARγ-PDL were generated as previously described. For inducing osteo/cementogenic differentiation, PDLFs were cultured in an induction medium (low-glucose DMEM with 10% FBS, ascorbic acid (100 μg/mL), and β-glycerophosphate (10 mM)) in the presence or absence of troglitazone or rosiglitazone for 12 days. Alkaline phosphatase activity Alkaline phosphatase (ALP) activity was measured as previously described and normalized to the cell numbers obtained from a parallel cell culture. Alizarin red S staining Alizarin red S staining and the average intensity calculations were performed as previously described. RNA-seq data processing The RNA-seq dataset deposited in NCBI's Gene Expression Omnibus (GEO, accession number: GSE178607 ) was processed as previously described. , Subsequently, gene ontology analyses of enhanced biological processes of the PDLFs transfected with control siRNA relative to the cells transfected with siRNA for PPARγ were conducted to identify the pathways suppressed by PPARγ knockdown. Quantitative reverse transcription polymerase chain reaction Total RNA purification, cDNA preparation, and quantitative reverse transcription polymerase chain reaction (qPCR) were conducted as previously described. , , Osteocalcin ( OCN ) and collagen type I alpha 1 chain ( COL1A1 ) expression analyses were conducted previously. Hypoxanthine phosphoribosyltransferase 1 ( HPRT ) was used as the internal reference control. The qPCR primer sequences used for the target genes are listed in . Immunohistochemical analyses Immunostaining was performed on 5-μm thick paraffin sections. The fixed maxillae of 1.5-month-old male mice were decalcified and embedded in paraffin as described above. The sections were stained with Alexa Fluor™ 488 Tyramide SuperBoost™ Kit (B40922, Thermo Fisher Scientific) according to the manufacturer's instructions. The sections were incubated with rabbit IgG (DA1E, Cell Signaling Technologies, Danvers, MA, final concentration 10 μg/mL), rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1) (13128-1-AP, Proteintech, Rosemont, IL, final concentration 10 μg/mL), rabbit anti-farnesyl pyrophosphate synthetase (FDPS) (16129-1-AP, Proteintech, final concentration 10 μg/mL), and rabbit anti-geranylgeranyl diphosphate synthase 1 (GGPS1) (14944-1-AP, Proteintech, final concentration 10 μg/mL) at 4 °C overnight. Immunofluorescent signals were observed using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan). Clinical sample preparation Clinical samples were prepared as previously described with the approval of the Ethics Committee of Tohoku University Graduate School of Dentistry (approval number: 2020-3-045). Statistical analysis Statistical analysis was performed using a one-way analysis of variance, followed by Tukey's test ( ) and Dunnett's test ( ). 

Rosiglitazone (R2408), FPP ammonium salt (F6892), and GGPP ammonium salt (G6025) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Troglitazone (209–19481) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 

All experimental procedures conformed to the “Regulations for Animal Experiments and Related Activities at Tohoku University” and were reviewed by the Institutional Laboratory Animal Care and Use Committee of Tohoku University and approved by the President of the University (Permit No. 2020DnA-044-06). Eleven-week-old male C57BL6/J mice (specific pathogen-free grade) were purchased from CLEA Japan, Inc. The mice were allowed to adapt to the new environment for one week before the experiments. The mice were anesthetized, and silk ligatures (Elp Sterile Blade Silk, Black, 5–0, Akiyama Medical MFG, Tokyo, Japan) were tied around the left second maxillary molar for 14 days. After the 14-day periodontal inflammation period, rosiglitazone (1.2 mg/kg) dissolved in solvent (50% DPBS, 40% PEG300, 5% Tween 80, 5% DMSO) was locally applied into the mesial and distal sides of the left second maxillary molars every other day for 14 days. The mice in the control group were administered the same amount of solvent at the same frequency. The right maxillae of the rosiglitazone and solvent-treated control groups were not treated throughout the experimental period. 

Micro-computed tomography (μCT) was conducted as previously described. , The vertical distances from the cemento-enamel junction to the alveolar bone crest at the mesial and distal roots of the second maxillary molars were measured and added. This sum was used to quantitatively compare bone regeneration levels in the periodontal regeneration stage. 

The maxilla samples used for μCT analysis were decalcified using ethylenediaminetetraacetic acid at 4 °C for 2 weeks. Masson's trichrome staining was performed on 5 μm-thick paraffin sections as previously described. , , 

Human PDLFs were purchased from Lonza Inc. (Walkersville, MD, USA) and maintained in low-glucose Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine system. PDLFs were cultivated at 37 °C under humidified atmospheric conditions (5% CO 2 and 95% air). PDLFs stably expressing the full-length (PPARγ), PPARγ-UBI, and PPARγ-PDL were generated as previously described. For inducing osteo/cementogenic differentiation, PDLFs were cultured in an induction medium (low-glucose DMEM with 10% FBS, ascorbic acid (100 μg/mL), and β-glycerophosphate (10 mM)) in the presence or absence of troglitazone or rosiglitazone for 12 days. 

Alkaline phosphatase (ALP) activity was measured as previously described and normalized to the cell numbers obtained from a parallel cell culture. 

Alizarin red S staining and the average intensity calculations were performed as previously described. 

The RNA-seq dataset deposited in NCBI's Gene Expression Omnibus (GEO, accession number: GSE178607 ) was processed as previously described. , Subsequently, gene ontology analyses of enhanced biological processes of the PDLFs transfected with control siRNA relative to the cells transfected with siRNA for PPARγ were conducted to identify the pathways suppressed by PPARγ knockdown. 

Total RNA purification, cDNA preparation, and quantitative reverse transcription polymerase chain reaction (qPCR) were conducted as previously described. , , Osteocalcin ( OCN ) and collagen type I alpha 1 chain ( COL1A1 ) expression analyses were conducted previously. Hypoxanthine phosphoribosyltransferase 1 ( HPRT ) was used as the internal reference control. The qPCR primer sequences used for the target genes are listed in . 

Immunostaining was performed on 5-μm thick paraffin sections. The fixed maxillae of 1.5-month-old male mice were decalcified and embedded in paraffin as described above. The sections were stained with Alexa Fluor™ 488 Tyramide SuperBoost™ Kit (B40922, Thermo Fisher Scientific) according to the manufacturer's instructions. The sections were incubated with rabbit IgG (DA1E, Cell Signaling Technologies, Danvers, MA, final concentration 10 μg/mL), rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1) (13128-1-AP, Proteintech, Rosemont, IL, final concentration 10 μg/mL), rabbit anti-farnesyl pyrophosphate synthetase (FDPS) (16129-1-AP, Proteintech, final concentration 10 μg/mL), and rabbit anti-geranylgeranyl diphosphate synthase 1 (GGPS1) (14944-1-AP, Proteintech, final concentration 10 μg/mL) at 4 °C overnight. Immunofluorescent signals were observed using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan). 

Clinical samples were prepared as previously described with the approval of the Ethics Committee of Tohoku University Graduate School of Dentistry (approval number: 2020-3-045). 

Statistical analysis was performed using a one-way analysis of variance, followed by Tukey's test ( ) and Dunnett's test ( ). 

Rosiglitazone accelerates periodontal tissue regeneration after ligature removal To investigate whether TZDs such as rosiglitazone and troglitazone can induce periodontal tissue regeneration in vivo , ligature ties surrounding the second molar were removed after 14 days. Subsequently, rosiglitazone was administered locally every alternate day for 14 days. Quantitative analysis of the distance between the cemento-enamel junction and the alveolar bone crest showed that the distance was significantly narrower in the rosiglitazone-treated group than in the solvent-treated group ( P = 0.0496), indicating that rosiglitazone administration accelerated alveolar bone regeneration ( A). No apparent effect was observed on the non-ligature side ( A). Masson's trichrome staining of ligated second molars that exhibited median values of the distance between the cemento-enamel junction and the alveolar bone crest in the rosiglitazone and solvent-treated control groups showed that rosiglitazone treatment induced alveolar bone regeneration ( B). Overexpression of full-length peroxisome proliferator-activated receptor γ specifically enhances troglitazone-induced osteo/cementogenic differentiation of periodontal ligament fibroblasts PDLF-PPARγ, PDLF-PPARγ-UBI, PDLF-PPARγ-PDL, and PDLF-empty, which stably expressed the empty vector, were cultured in osteo/cementogenic induction medium in the presence or absence of troglitazone (10 μM) for 12 days. ALP activity and calcium nodule formation, which are specific markers of osteo/cementogenic differentiation, were evaluated every three days. PDLF-PPARγ caused a marked increase in ALP activity upon troglitazone treatment on day 6 ( A). Concomitantly, troglitazone treatment of PDLF-PPARγ showed strong calcium nodule formation at days 9 and 12 ( B) Therefore, analyses of the distinct isoforms revealed that the agonistic actions of troglitazone for osteo/cementogenic differentiation were caused by the full-length PPARγ isoform. Intermediates of the isoprenoid metabolic process regulate the osteo/cementogenic abilities of periodontal ligament fibroblasts To explore the key energy metabolic pathways associated with the osteo/cementogenic abilities of PDLFs, differentially downregulated genes of PDLFs transfected with siRNA for PPARG by comparing with PDLFs transfected with control siRNA were extracted from publicly available RNA-seq datasets. Subsequently, gene ontology analysis was conducted to rank the pathways suppressed by PPARG knockdown ( ). Pathways associated with osteo/cementogenic differentiation and bone matrix mineralization, and with metabolic pathways are highlighted in blue and red, respectively ( A). The isoprenoid metabolic process ranked first among the metabolic pathways. To assess whether TZD treatment upregulated the expression of isoprenoid metabolic process-related genes with high values of fragments per kilobase of exon per million mapped reads and significantly suppressed the expression level by PPARG knockdown, PDLFs were stimulated with TZDs for 3 and 6 days under osteo/cementogenic induction. We found that three of the four examined genes, aldo-keto reductase family 1 member C3 ( AKR1C3 ), aldo-keto reductase family 1 member C1 ( AKR1C1 ), and syndecan 3 ( SDC3 ), were upregulated by TZDs ( B). These results indicate that TZDs were at least partially able to trigger an isoprenoid pathway in an osteogenic environment. Next, PDLFs were stimulated with the key isoprenoid intermediates FPP (20 μM) or GGPP (20 μM) for 6 days, and ALP activity was assessed ( C). FPP increased ALP activity. However, GGPP synthesized from FPP by GGPS1 did not enhance ALP activity. TZD treatment enhanced the ALP activity of PDLFs compared to DMSO treatment, as expected, and FPP, but not GGPP, increased ALP activity. Alizarin Red S staining showed that FPP treatment accelerated calcified nodule formation ( D). Subsequently, the expression of three catalytic enzymes in mouse periodontal ligament tissues was analyzed: FDPS, which catalyzes the production of FPP from IPP and DMAPP; GGPS1; and FDFT1, which enzymatically converts FPP to squalene. Immunohistochemical analysis of the one-third apex of the periodontal tissue of the first and second molars showed that FDPS, GGPS1, and FDFT1 were expressed in the periodontal ligament tissue, indicating that the isoprenoid metabolic process was active ( E). Farnesyl pyrophosphate synthetase expression is correlated with osteocalcin expression, and farnesyl diphosphate farnesyltransferase 1 expression is strongly correlated with collagen type I alpha 1 chain expression in human clinical periodontal tissues Human clinical periodontal tissue samples obtained during surgical or nonsurgical periodontal treatment, as described previously, were used to examine whether a correlation existed between the expression levels of OCN , an osteo/cementogenic-related gene; COL1A1 , an ECM-related gene; and FDPS , GGPS1 , or FDFT1 . A weak correlation between OCN and FDPS was identified (r = 0.221); however, no apparent correlation between OCN and GGPS 1 (r = 0.173) or between OCN and FDFT1 (r = −0.036) was observed ( ). COL1A1 expression was strongly correlated with FDFT1 (r = 0.947) but not with FDPS (r = −0.014) or GGPS1 (r = −0.032). 

To investigate whether TZDs such as rosiglitazone and troglitazone can induce periodontal tissue regeneration in vivo , ligature ties surrounding the second molar were removed after 14 days. Subsequently, rosiglitazone was administered locally every alternate day for 14 days. Quantitative analysis of the distance between the cemento-enamel junction and the alveolar bone crest showed that the distance was significantly narrower in the rosiglitazone-treated group than in the solvent-treated group ( P = 0.0496), indicating that rosiglitazone administration accelerated alveolar bone regeneration ( A). No apparent effect was observed on the non-ligature side ( A). Masson's trichrome staining of ligated second molars that exhibited median values of the distance between the cemento-enamel junction and the alveolar bone crest in the rosiglitazone and solvent-treated control groups showed that rosiglitazone treatment induced alveolar bone regeneration ( B). 

PDLF-PPARγ, PDLF-PPARγ-UBI, PDLF-PPARγ-PDL, and PDLF-empty, which stably expressed the empty vector, were cultured in osteo/cementogenic induction medium in the presence or absence of troglitazone (10 μM) for 12 days. ALP activity and calcium nodule formation, which are specific markers of osteo/cementogenic differentiation, were evaluated every three days. PDLF-PPARγ caused a marked increase in ALP activity upon troglitazone treatment on day 6 ( A). Concomitantly, troglitazone treatment of PDLF-PPARγ showed strong calcium nodule formation at days 9 and 12 ( B) Therefore, analyses of the distinct isoforms revealed that the agonistic actions of troglitazone for osteo/cementogenic differentiation were caused by the full-length PPARγ isoform. 

To explore the key energy metabolic pathways associated with the osteo/cementogenic abilities of PDLFs, differentially downregulated genes of PDLFs transfected with siRNA for PPARG by comparing with PDLFs transfected with control siRNA were extracted from publicly available RNA-seq datasets. Subsequently, gene ontology analysis was conducted to rank the pathways suppressed by PPARG knockdown ( ). Pathways associated with osteo/cementogenic differentiation and bone matrix mineralization, and with metabolic pathways are highlighted in blue and red, respectively ( A). The isoprenoid metabolic process ranked first among the metabolic pathways. To assess whether TZD treatment upregulated the expression of isoprenoid metabolic process-related genes with high values of fragments per kilobase of exon per million mapped reads and significantly suppressed the expression level by PPARG knockdown, PDLFs were stimulated with TZDs for 3 and 6 days under osteo/cementogenic induction. We found that three of the four examined genes, aldo-keto reductase family 1 member C3 ( AKR1C3 ), aldo-keto reductase family 1 member C1 ( AKR1C1 ), and syndecan 3 ( SDC3 ), were upregulated by TZDs ( B). These results indicate that TZDs were at least partially able to trigger an isoprenoid pathway in an osteogenic environment. Next, PDLFs were stimulated with the key isoprenoid intermediates FPP (20 μM) or GGPP (20 μM) for 6 days, and ALP activity was assessed ( C). FPP increased ALP activity. However, GGPP synthesized from FPP by GGPS1 did not enhance ALP activity. TZD treatment enhanced the ALP activity of PDLFs compared to DMSO treatment, as expected, and FPP, but not GGPP, increased ALP activity. Alizarin Red S staining showed that FPP treatment accelerated calcified nodule formation ( D). Subsequently, the expression of three catalytic enzymes in mouse periodontal ligament tissues was analyzed: FDPS, which catalyzes the production of FPP from IPP and DMAPP; GGPS1; and FDFT1, which enzymatically converts FPP to squalene. Immunohistochemical analysis of the one-third apex of the periodontal tissue of the first and second molars showed that FDPS, GGPS1, and FDFT1 were expressed in the periodontal ligament tissue, indicating that the isoprenoid metabolic process was active ( E). 

expression is correlated with osteocalcin expression, and farnesyl diphosphate farnesyltransferase 1 expression is strongly correlated with collagen type I alpha 1 chain expression in human clinical periodontal tissues Human clinical periodontal tissue samples obtained during surgical or nonsurgical periodontal treatment, as described previously, were used to examine whether a correlation existed between the expression levels of OCN , an osteo/cementogenic-related gene; COL1A1 , an ECM-related gene; and FDPS , GGPS1 , or FDFT1 . A weak correlation between OCN and FDPS was identified (r = 0.221); however, no apparent correlation between OCN and GGPS 1 (r = 0.173) or between OCN and FDFT1 (r = −0.036) was observed ( ). COL1A1 expression was strongly correlated with FDFT1 (r = 0.947) but not with FDPS (r = −0.014) or GGPS1 (r = −0.032). 

PPARγ agonists, such as rosiglitazone and procyanidin B2, can prevent periodontal tissue breakdown in experimental periodontitis models. , The present study demonstrated for the first time that local administration of TZDs into regenerating periodontal tissue accelerated periodontal tissue regeneration in an animal model ( ). Mechanistically, the TZD-induced differentiation abilities of PDLFs relied on full-length PPARγ rather than the other two deleted isoforms ( ). Furthermore, both FPP and GGPP showed potent but inverse effects on the osteo/cementogenic abilities of PDLFs, with FPP showing inducible abilities ( ). Among the PPARγ agonists, troglitazone showed the most drastic inducible effects on osteo/cementogenic differentiation and matrix mineralization of PDLFs in vitro , we conducted in vitro experiments primarily with troglitazone as the PPARγ agonist ( ). However, troglitazone was replaced with rosiglitazone for clinical use as a drug for type 2 diabetes mellitus because of its hepatotoxicity. , , Therefore, rosiglitazone was used for the in vivo study ( ). As the original ALP activities (day 0) and the ALP activities at the early differentiation stage (day 3) of PDLF-PPARγ, PDLF-PPARγ-UBI, and PDLF-PPARγ-PDL were higher than those of PDLF-empty ( A). Not only full-length PPARγ, but PPARγ-UBI and PPARγ-PDL possessed osteo/cementogenic inducible abilities independent of TZDs. In particular, PPARγ-PDL expression in clinical periodontal tissue was highly correlated with COL1A1 expression. The deduced amino acid sequence indicates that PPARγ-UBI and PPARγ-PDL lack the ligand binding domain. Therefore, PPARγ-UBI and PPARγ-PDL may not act as a nuclear receptor. Some portions of the overexpressed PPARγ-UBI and PPARγ-PDL localized in the cytoplasm. Further studies are required to determine how PPARγ-UBI and PPARγ-PDL enhance osteo/cementogenic abilities by modulating protein-protein interactions in PDLFs. Acetyl-CoA participates in lipid, glucose, and amino acid metabolism and is the starting material for the mevalonate pathway. Amino-bisphosphonate, an analog of inorganic pyrophosphate, which inhibits FDPS enzymatic ability in osteoclasts, protects against osteoporosis. , , Thus, inhibition of the mevalonate pathway can retain bone volume and bone mineral density in vivo . In contrast, the in vitro finding that FPP accelerated the osteo/cementogenic differentiation and matrix mineralization abilities of PDLFs suggested a preferable role of FPP in PDLFs ( ). In line with the present study, PDLFs stimulated with alendronate, an amino-bisphosphonate, at physiological concentrations (2–25 μM) showed lower ALP activity and suppressed calcified nodule formation. As FPP showed preferential but GGPP showed adverse effects ( ), FDPS expression was more correlated with OCN than GGPS1 and FDFT1 ( ), and the expression levels of COL1A1 and FDFT1 were highly correlated ( ), selective conversion of FPP into squalene rather than GGPP might be key for FPP-induced matrix mineralization and differentiation of PDLFs ( D). Further studies are necessary to investigate the precise molecular mechanisms by which exogenous FPP and its derived intermediates participate in the osteo/cementogenic differentiation and matrix mineralization of PDLFs. In conclusion, by applying a PPARγ agonist to a murine periodontal regeneration model and utilizing PDLFs expressing various isoforms of PPARγ, we found that PPARγ agonistic actions facilitated periodontal tissue regeneration by specifically activating the most abundant isoform, full-length PPARγ. FPP, but not GGPP, induced osteo/cementogenic differentiation of PDLFs. Thus, in addition to the local application of TZDs and FPP, precise control of the activities of FDPS, GGPS1, and FDFT1 in periodontal ligament tissue could be a novel therapeutic method for periodontal regeneration. 

The authors have no conflicts of interest relevant to this article. 

