Osteogenic capacity and cytotherapeutic potential of periodontal ligament cells for periodontal regeneration in vitro and in vivo.
ABSTRACT: Background:The periodontal ligament cells (PDLCs) contain heterogeneous cell populations and possess stem-cell-like properties. PDLCs have attracted considerable attention as an option for periodontal regeneration. However, the osteogenic differentiation of PDLCs remains obscure owing to variable osteo-inductive methods and whether PDLCs could be directly used for periodontal regeneration without stem cell enrichment is uncertain. The aim of the present study was to clarify the osteogenic differentiation capacity of PDLCs and test PDLCs as an alternative to stem cells for periodontal regeneration. Methods:We tested the performance of human PDLCs in osteo-inductive culture and transplantation in vivo while taking human bone marrow derived mesenchymal stem cells (hMSCs) as positive control. Proliferation of PDLCs and hMSCs in osteo-inductive condition were examined by MTT assay and colony formation assay. The osteogenic differentiations of PDLCs and hMSCs were assessed by Alkaline phosphatase (ALP) activity measurement, von Kossa staining, Alizarin red S staining and quantitative RT-PCR of osteogenic marker gene including RUNX2, ALP, OCN, Col I, BSP, OPN. We transplanted osteo-inductive PDLCs and hMSCs with hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds to immunodeficient mice to explore their biological behaviors in vivo by histological staining and immunohistochemical evaluation. Results:After 14 days of osteo-induction, PDLCs exhibited significantly higher proliferation rate but lower colony-forming ability comparing with hMSCs. PDLCs demonstrated lower ALP activity and generated fewer mineralized nodules than hMSCs. PDLCs showed overall up-regulated expression of RUNX2, ALP, OCN, Col I, BSP, OPN after osteo-induction. Col I level of PDLCs in osteo-inductive group was significantly higher while RUNX2, ALP, OCN were lower than that of hMSCs. Massive fiber bundles were produced linking or circling the scaffold while the bone-like structures were limited in the PDLCs-loaded HA/TCP samples. The fiber bundles displayed strong positive Col I, but weak OCN and OPN staining. The in vivo results were consistent with the in vitro data, which confirmed strong collagen forming ability and considerable osteogenic potential of PDLCs. Conclusion:It is encouraging to find that PDLCs exhibit higher proliferation, stronger collagen fiber formation capacity, but lower osteogenic differentiation ability in comparison with hMSCs. This characteristic is essential for the successful periodontal reconstruction which is based on the synchronization of fiber formation and bone deposition. Moreover, PDLCs have advantages such as good accessibility, abundant source, vigorous proliferation and evident osteogenic differentiation capacity when triggered properly. They can independently form PDL-like structure in vivo without specific stem cell enrichment procedure. The application of PDLCs may offer a novel cytotherapeutic option for future clinical periodontal reconstruction.
Project description:BACKGROUND:Patients with diabetes mellitus (DM) often suffered with many musculoskeletal disorders, such as tendon rupture and tendinopathy. However, the understanding of the pathogenesis of these alternations is limited. This study was designed to investigate the role of tendon-derived stem cells (TDSCs) in histopathological alterations of DM tendons. METHODS:Forty-two SD rats were randomly and equally divided into a diabetes group (DG) and control group (CG). DM was induced by streptozotocin (65?mg/kg). The patellar tendons were isolated at weeks 1, 2, and 4 for histological analysis. TDSCs were isolated at week 2 for osteo-chondrogenic differentiation analysis. Mann-Whitney U test was used with SPSS. p?<?0.050 was statistically significant. RESULTS:Micro-tears of collagen fibers and altered appearance of tendon cells were observed in DG tendons. DG tendons exhibited significantly higher expression of OPN, OCN, SOX9, and Col II and decreased expression of Col I and tenomodulin (TNMD) at week 2. Diabetic TDSCs (dTDSCs) demonstrated significantly decreased proliferation ability and increased osteogenic and chondrogenic differentiation ability. Osteo-chondrogenic markers BMP2, ALP, OPN, OCN, Col II, and SOX9 were also significantly increased while tenogenic markers Col I and TNMD were decreased in dTDSCs. CONCLUSION:These results suggested the erroneous differentiation of dTDSCs might account for the structural and non-tenogenic alternations in DM tendons, which provided new cues for the pathogenesis of tendon disorders in DM.
Project description:Periodontal ligament cells (PDLCs), which have potential for multilineage differentiation, are candidates for use in regeneration of periodontal tissue defects; however, our understanding of the mechanisms underlying the lineage commitment of PDLCs remains limited. C4orf7, which is specifically expressed in the periodontal ligament (PDL) tissue, may be crucial in deciding the fate of PDLCs and regulating the periodontal bone balance. In this study, we examined the expression of C4orf7 in PDL tissue, using immunohistochemical staining. We transfected PDLCs with lentiviral vectors expressing C4orf7 and examined the effect of C4orf7 on the balance of PDLC osteogenic and osteoclastogenic differentiation. Osteogenic induction resulted in the downregulation of mRNA and protein expression levels of the osteogenic/cementoblastic markers: ALP, RUNX2, COL1, OPN, OPG, OSX, IBSP, CAP, and CEMP1. Transfected cells also exhibited an increased RANKL/OPG ratio, which is an indicator of osteoclastogenic differentiation. ALP activity assays and Alizarin red staining confirmed the negative effect of C4orf7 on PDLC osteogenic differentiation. Finally, we investigated the effect of C4orf7 on the lineage commitment of PDLCs to adipocytes. We observed increased expression levels of PPAR?2, GLUT4, ZFP423, FABP4, and LPL mRNAs, as well as a gradual accumulation of lipid droplets in the C4orf7-overexpressing group compared with controls. In summary, our data confirm that C4orf7 has an important role in the regulation of periodontal bone remodeling through promotion of the adipogenic/osteoclastogenic, and inhibition of the osteogenic/cementoblastic, differentiation of PDLCs. Therefore, C4orf7 is a potential therapeutic target for the treatment of periodontal disease and other bone metabolic disorders.
Project description:This study inspected whether calcitriol could exert a mineralization-inductive effect comparable to that of vitamin C in cultured human periodontium cells (hPDCs). The mRNA expression of the mineralization-related biomarkers core-binding factor subunit alpha-1 (Cbfa1), collagen 1 α1 (Col-I), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), vitamin D receptor (VDR), cementum protein 1 (CEMP-1), cementum attachment protein (CAP), interleukin 6 (IL-6), transforming growth factor-β1 (TGF-β1) and osteoprotegerin (OPG) was surveyed after incubation of hPDCs with vitamin C and calcitriol for 2 weeks. Translational expression information from ALP activity and CEMP-1 and CAP immunofluorescence assays was acquired from hPDCs at the second and third weeks. Extracellular calcifications were confirmed by von Kossa staining, Alizarin Red staining and synchrotron transmission X-ray microscopy (TXM) at the fourth and fifth weeks. It was found that both vitamin C and calcitriol not only increased mineralization-related mRNA fold-changes but also enhanced ALP activity, CEMP-1 immunofluorescence, von Kossa and Alizarin Red staining and TXM-associated calcifications. Generally, 10-8 M calcitriol displayed greater mineralization significance than 10-7 M calcitriol in the assays tested. However, vitamin C stimulated lower Cbfa1, Col-1, ALP, OPN, BSP, OCN, VDR, CEMP-1 and IL-6 mRNA fold-changes than 10-8 M calcitriol. Finally, TXM analysis indicated that a 10-8 M calcitriol treatment stimulated greater calcifications than vitamin C treatment. Therefore, the analytical results confirmed the osteo-inductive potential of vitamin C in cultured hPDCs. In contrast, 10-8 M calcitriol could potentially function as a substitute because it stimulates a greater mineralization effect than vitamin C or 10-7 M calcitriol.
Project description:OBJECTIVES:The present study aimed to investigate overall effect of quercetin on proliferation and osteogenic differentiation of mouse adipose stem cells (mASCs) in vitro. MATERIALS AND METHODS:Mouse adipose stem cells were isolated from subcutaneous fat pads and induced into the osteogenic lineage. Effects of quercetin on cell proliferation were assessed using MTT assay. Then they were treated by quercetin for 3, 7 and 11 days in a range of concentrations. Finally, effects of quercetin on osteogenic differentiation of mASCs were analysed by real-time PCR. RESULTS:Data of MTT assay showed that quercetin did not enhance mASC proliferation in a dose-dependent or time-dependent manner. Results of qPCR indicated that quercetin promoted expressions of Osx, Runx2, BMP-2, Col-1, OPN and OCN at the mRNA level in the presence of osteo-induction medium. CONCLUSIONS:Our data demonstrated that quercetin was not active in terms of enhancing mASCs proliferation; however, it increased osteogenesis of mASCs by up-regulation of genes including Osx, Runx2, BMP-2, Col-1, OPN and OCN.
Project description:The aim of the present study was to investigate the effects of advanced glycation end products (AGEs) and berberine hydrochloride (BBR) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs) in vitro, and their underlying mechanisms. hPDLSCs were subjected to osteogenic induction and were treated with AGEs or AGEs + BBR. Following varying numbers of days in culture, alkaline phosphatase (ALP) activity assays, ALP staining, alizarin red staining, ELISAs, and reverse transcription?quantitative polymerase chain reaction (RT?qPCR) and western blot analyses were performed to determine the osteogenic differentiation ability of hPDLSCs; RT?qPCR, western blot analysis, and immunofluorescence staining were conducted to investigate the underlying mechanisms. The canonical Wnt/??catenin pathway inhibitor XAV?939 and agonist CHIR?99021 were used to determine the contribution of the canonical Wnt/??catenin pathway to differentiation. Treatment with AGEs resulted in reduced ALP activity and Collagen I protein levels, decreased ALP staining, fewer mineralized nodules, and downregulated expression of osteogenic?specific genes [Runt?related transcription factor 2 (Runx2), Osterix, ALP, osteopontin (OPN), Collagen I and osteocalcin (OCN)] and proteins (Runx2, OPN, BSP and OCN); however, BBR partially rescued the AGE?induced decrease in the osteogenic potential of hPDLSCs. Furthermore, AGEs activated the canonical Wnt/??catenin signaling pathway and promoted the nuclear translocation of ??catenin; BBR partially attenuated this effect. In addition, XAV?939 partially rescued the AGE?induced reduction in the osteogenic potential of hPDLSCs, whereas CHIR?99021 suppressed the BBR?induced increase in the osteogenic potential of hPDLSCs. The present study indicated that AGEs attenuated the osteogenic differentiation ability of hPDLSCs, in part by activating the canonical Wnt/??catenin pathway; however, BBR attenuated these effects by inhibiting the canonical Wnt/??catenin pathway. These findings suggest a role for BBR in periodontal regeneration induced by hPDLSCs in patients with diabetes mellitus.
Project description:OBJECTIVES:Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered to be a promising method for periodontitis treatment. The molecular mechanism of functional regulation by MSCs remains unclear, thus limiting their application. Our previous study discovered that Periostin (POSTN) promoted the migration and osteogenic differentiation of periodontal ligament mesenchymal stem cells (PDLSCs), but it is still unclear whether POSTN is able to restore the regenerative potential of PDLSCs under inflammatory conditions. In this study, we investigated the effect of POSTN on PDLSCs under inflammatory conditions and its mechanism. MATERIALS AND METHODS:PDLSCs were isolated from periodontal ligament tissue. TNF-? was used at 10 ng/mL to mimic inflammatory conditions. Lentivirus POSTN shRNA was used to knock down POSTN. Recombinant human POSTN (rhPOSTN) was used to stimulate PDLSCs. A scratch assay was used to analyse cell migration. Alkaline phosphatase (ALP) activity, Alizarin Red staining and expression of osteogenesis-related genes were used to investigate the osteogenic differentiation potential. Western blot analysis was used to detect the mitogen-activated protein kinases (MAPK) and AKT signalling pathways. RESULTS:After a 10 ng/mL TNF-? treatment, knockdown of POSTN impeded scratch closure, inhibited ALP activity and mineralization in vitro, and decreased expression of RUNX2, OSX, OPN and OCN in PDLSCs, while 75 ng/mL rhPOSTN significantly accelerated scratch closure, enhanced ALP activity and mineralization in vitro, and increased expression of RUNX2, OSX, OPN and OCN. In addition, knockdown of POSTN inhibited expression of phosphorylated c-Jun N-terminal kinase (p-JNK), while 75 ng/mL rhPOSTN increased expression of p-JNK in PDLSCs with TNF-? treatment. Furthermore, inhibition of JNK by its inhibitor SP600125 dramatically blocked POSTN-enhanced scratch closure, ALP activity and mineralization in PDLSCs. CONCLUSIONS:Our results revealed that POSTN might promote the migration and osteogenic differentiation potential of PDLSCs via the JNK pathway, providing insight into the mechanism underlying MSC biology under inflammatory conditions and identifying a potential target for improving periodontal tissue regeneration.
Project description:Objective:The need for orthodontic treatment continues to increase. Strategies that shorten the treatment course and reduce discomfort are most welcome in clinic. Circadian rhythm plays important role in various physiological processes, including bone formation. This study intended to depict a possible circadian releasing property of the osteogenic factors within the periodontal tissue during orthodontic treatment, which may direct a more efficient and satisfactory orthodontic treatment to the patient. Methods:Primary periodontal ligament cells (PDLCs) were obtained from the Sprague-Dawley (SD) rats. An equibiaxial strain value of 12% was applied on rat PDLCs (rPDLCs). After 2 h stimuli of 10-7 M dexamethasone (DX), the osteogenic genes' expressions were detected by real-time polymerase chain reaction (RT-PCR) at Zeitgeber times 0, 4, 8, 12, 16, 20, and 24. An orthodontic appliance was placed on 45 SD rats. Animals were maintained under 12-h light/dark periods and euthanized at 9 time points over the diurnal cycle. The orthodontic sensitive tissues of the mesial root of the maxillary first molar were collected for RT-PCR and immunohistological assay. Results:The rPDLCs displayed typical fibroblastic spindle shape, and subcultured steadily in vitro. Induced by DX, the mRNA expression of Col-1, OPN, and IBSP within the loaded/unloaded rPDLCs oscillated as that of the main clock gene Per-1. The osteogenic genes' expressions as well as the protein releases sustained a circadian oscillation trend in vivo. Conclusions:This study indicates the existence of a circadian rhythm of the osteogenic factors within the orthodontic sensitive tissues, which highlights the importance of precise timing of force loading in further orthodontic treatment. Thus, a periodicity pattern of orthodontic traction at night may prove a more efficient tooth movement while minimizing the treatment window and discomfort complains.
Project description:As a biocompatible and low cytotoxic nanomaterial, graphene oxide (GO) has captured tremendous interests in tissue engineering. However, little is known about the behavior of dental stem cells on GO. This study was to evaluate the bioactivity of human periodontal ligament stem cells (PDLSCs) on GO coated titanium (GO-Ti) substrate in vitro as compared to sodium titanate (Na-Ti) substrate. By scanning electron microscope (SEM), confocal laser scanning microscope (CLSM), methylthiazol tetrazolium (MTT) assay, alkaline phosphatase (ALP) activity, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, we investigated the attachment, morphology, proliferation and osteogenic differentiation of PDLSCs on these two substrates. When seeded on GO-Ti substrate, PDLSCs exhibited significantly higher proliferation rate, ALP activity and up-regulated gene expression level of osteogenesis-related markers of collagen type I (COL-I), ALP, bone sialoprotein (BSP), runt related transcription factor 2 (Runx2) and osteocalcin (OCN) compared with those on Na-Ti substrate. Moreover, GO promoted the protein expression of BSP, Runx2 and OCN. These findings suggest that the combination of GO and PDLSCs provides a promising construct for regenerative dentistry.
Project description:Bone morphogenetic protein-9 (BMP9) shows great osteoinductive potential in bone regeneration. Periodontal ligament stem cells (PDLSCs) with multi-differentiation capability and low immunogenicity are increasingly used as seed cells for periodontal regenerative therapies. In the present study, we investigated the potent osteogenic activity of BMP9 on human PDLSCs (hPDLSCs), in which the c-Jun N-terminal kinase (JNK) pathway is possibly involved. Our results showed that JNK inhibition by the specific inhibitor SP600125 or adenovirus expressing small interfering RNA (siRNA) targeting JNK (AdR-si-JNK) significantly decreased BMP9-induced gene and protein expression of early and late osteogenic markers, such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN), in hPDLSCs. We also confirmed the in-vivo positive effect of JNKs on ectopic bone formation induced by hPDLSCs injected into the musculature of athymic nude mice and BMP9 ex vivo gene delivery. For the cellular mechanism, we found that BMP9 activated the phosphorylation of JNKs and Smad2/3, and that JNKs may engage in cross-talk with the Smad2/3 pathway in BMP9-mediated osteogenesis.
Project description:HPDLSCs derived from periodontal ligament tissues contribute to tooth development and tissue regeneration. Exploring the effects of long noncoding RNAs (lncRNAs) in the process of osteogenic differentiation of periodontal ligament stem cells would provide novel therapeutic strategies for tissue regeneration. The expression levels of lncRNA, which significantly changed during osteogenic differentiation, were observed by real-time quantitative PCR (q-PCR). Then, we screened for osteogenic-related lncRNA, which was initially named lncRNA-TWIST1. Moreover, we detected the mRNA expression levels of TWIST1 and osteogenesis-related genes after upregulating and downregulating lncRNA-TWIST1 in PPDLSCs (periodontal mesenchymal stem cells from periodontitis patients) and HPDLSCs (periodontal mesenchymal stem cells from healthy microenvironment), respectively. The osteogenic degree was verified by detecting ALP activity and alizarin red staining. LncRNA-TWIST1 decreased the mRNA levels of TWIST1 and promoted osteogenic differentiation in PPDLSCs, which was confirmed by the increase in osteogenesis-related gene levels (Runx2, ALP, and OCN), the increase in ALP activity, and the formation of more osteogenic nodules. In contrast, downregulating lncRNA-TWIST1 decreased the expression of osteogenesis-related genes, ALP activity, and osteogenic nodules both in PPDLSCs and in HPDLSCs. LncRNA-TWIST1 promoted osteogenic differentiation both in PPDLSCs and in HPDLSCs by inhibiting the TWIST1 expression. LncRNA-TWIST1 may be a novel therapeutic strategy to regenerate dental tissues.