Cell Tissue Res (2010) 340:323–333 DOI 10.1007/s00441-010-0953-0
REGULAR ARTICLE
Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth Kiranmai Chadipiralla & Ji Min Yochim & Bindu Bahuleyan & Chun-Yuh Charles Huang & Franklin Garcia-Godoy & Peter E. Murray & Eric J. Stelnicki
Received: 19 October 2009 / Accepted: 23 February 2010 / Published online: 23 March 2010 # Springer-Verlag 2010
Abstract Multipotent stem cells derived from periodontal ligaments (PDLSC) and pulp of human exfoliated deciduous teeth (SHED) represent promising cell sources for bone regeneration. Recent studies have demonstrated that retinoic acid (RA) and dexamethasone (Dex) induce osteogenesis of postnatal stem cells. The objective of this study was to examine the effects of RA and Dex on the proliferation and osteogenic differentiation of SHED and PDLSC and to
This work was supported by the President’s Faculty Research and Development Grant from Nova Southeastern University and the Craniofacial Research Grant from Joe DiMaggio Children’s Hospital. P. E. Murray Department of Endodontics, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Fla., USA C.-Y. C. Huang (*) Department of Biomedical Engineering, University of Miami, 219A McArthur Annex, 1251 Memorial Drive, Coral Gables FL 33146, USA e-mail:
[email protected] F. Garcia-Godoy Bioscience Research Center, College of Dentistry, University of Tennessee Health Science Center, Memphis, Tenn., USA K. Chadipiralla : J. M. Yochim : B. Bahuleyan : C.-Y. C. Huang : E. J. Stelnicki Craniofacial Research Laboratory, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Fla., USA E. J. Stelnicki Cleft and Craniofacial Center, Joe DiMaggio Children’s Hospital, Hollywood, Fla., USA
compare the osteogenic characteristics of SHED and PDLSC under RA treatment. SHED and PDLSC were treated with serum-free medium either alone or supplemented with RA or Dex for 21 days. The proliferation of SHED and PDLSC was significantly inhibited by both RA and Dex. RA significantly upregulated gene expression and the activity of alkaline phosphatase in SHED and PDLSC. Positive Alizarin red and von Kossa staining of calcium deposition was seen on the RA-treated SHED and PDLSC after 21 days of culture. The influences of RA on the osteogenic differentiation of SHED and PDLSC were significantly stronger than with Dex. Supplemention with insulin enhanced RA-induced osteogenic differentiation of SHED. Thus, RA is an effective inducer of osteogenic differentiation of SHED and PDLSC, whereas RA treatment in combination with insulin supplementation might be a better option for inducing osteogenic differentiation. Significantly higher cell proliferation of PDLSC results in greater calcium deposition after 3-week culture, suggesting that PDLSC is a better osteogenic stem cell source. This study provides valuable information for efficiently producing osteogenically differentiated SHED or PDLSC for in vivo bone regeneration. Keywords Adult stem cells . Osteogenesis . Dental pulp . Periodontal ligament . Retinoic acid . Human
Introduction Multipotent postnatal stem cells have been isolated from various types of tissues, e.g., bone marrow, adipose tissue, muscle, periosteum, synovial tissue, dental pulp, and periodontal ligaments (Campagnoli et al. 2001;
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Fickert et al. 2003; Gronthos et al. 2000; Jaiswal et al. 1997; Miura et al. 2003; Mizuno et al. 2002; Pittenger et al. 1999; Seo et al. 2004; Young et al. 2001; Zuk et al. 2001). Since such stem cells are capable of differentiating into diverse mesenchymal lineages including bone, the transplantation of replacement cells or tissues generated by the patient’s own stem cells has been considered as a potential treatment for repairing bone defects (Krampera et al 2006). In particular, stem cells derived from the pulp of human exfoliated deciduous teeth (SHED; Miura et al. 2003) and periodontal ligaments (PDLSC; Seo et al. 2004) might be an ideal cell source for repairing bone defects in pediatric patients. For instance, the exfoliation of human deciduous teeth usually occurs between age 6 and 12 and lost bony segments in the alveolar ridge of cleft palate patients are often restored by autologous bone grafting after age 7–8 (Collins et al. 1998). Since bone grafts are harvested by surgical procedures with high risk of donor site morbidity, stem cells derived from the dental tissues may offer a possible potential treatment of bone defect for such a population of patients. Few studies have investigated the osteogenic potential of stem cells derived from dental pulp and periodontal ligaments (Gronthos et al. 2000; Miura et al. 2003; Seo et al. 2004, 2007). Mineral deposition of stem cells derived from such dental tissues can be induced in monolayer cultures by treatment with dexamethasone (Dex; Gronthos et al. 2000; Miura et al. 2003; Seo et al. 2004), which has traditionally been used to promote osteogenic differentiation of postnatal stem cells (Pittenger et al. 1999; Young et al. 2001; Zuk et al. 2001). Retinoic acid (RA) has been shown to influence the differentiation and proliferation of osteoblasts (Choong et al. 1993; Kawaguchi et al. 2005; Skillington et al. 2002; Song et al. 2005), and a recent study has demonstrated that RA is able to induce osteogenic differentiation of stem cells derived from adipose tissues (Malladi et al. 2006). Previous studies have also shown that the activities of alkaline phosphatase (ALP), an osteogenic marker, are upregulated in cells of human dental pulp and periodontal ligaments by shortterm treatment with RA (San Miguel et al. 1998, 1999). These previous findings indicate that RA is a potential inducer promoting the osteogenesis of stem cells derived from dental pulp and periodontal ligament. Therefore, the objectives of this study have been to investigate (1) the effects of RA on the proliferation and osteogenic differentiation of SHED and PDLSC and (2) differences in the osteogenic potential between SHED and PDLSC under RA treatment. This study has also compared the effects of Dex treatment on the osteogenic differentiation of SHED and PDLSC side-by-side with those of RA treatment.
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Materials and methods In vitro osteogenic differentiation of SHED and PDLSC SHED and PDLSC were made available through a material transfer agreement with the National Institute of Dental and Craniofacial Research (NIDCR, Bethsda, Md., USA). The cells were isolated based on the protocol reported in previous studies (Miura et al. 2003; Seo et al. 2004). Bone-marrowderived mesenchymal stem cells (MSCs; Sciencell Research Laboratories, Carlsbad, Calif, USA) were used for comparison. The frozen cells were thawed and expanded in monolayer with Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, Calif., USA) containing 10% fetal calf serum (FBS; Invitrogen) and 1% antibioticantimycotic (Invitrogen) at 37°C in a 5% CO2 incubator. For experiments on osteogenic differentiation, the cells were plated in 12-well culture dishes at a density of 40,000 cells/ well and treated with a basic serum-free medium either alone as control or supplemented with RA (0.5, 1, or 2 μM; Sigma, St. Louis, Mo., USA), or Dex (1, 10, or 100 nM; Sigma). The basic serum-free medium used in all experiments was DMEM containing 50 μg/ml ascorbic acid (Sigma), 10 mM β-glycerophosphate (Sigma), and 1% antibiotic-antimycotic. Since insulin-transferrin-selenous acid (ITS) supplement was often previously used in the defined serum-free medium for chondrogenesis of adult stem cells (Fickert et al. 2003; Pittenger et al. 1999; Zuk et al. 2001), the basic serum-free medium (defined above) supplemented with ITS+ Premix (final concentrations: 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenous acid, 1.25 mg/ml bovine serum albumin, 5.35 μg/ml linoleic acid; BD Biosciences, Bedford, Mass., USA) was examined in control and RA (2 μM)-treated groups of SHED. The medium was changed every 3 days. Osteogenic gene expression [ALP, RUNX2, osteonectin (ONN), osteopontin (OPN), and collagen type I (ColI)] and ALP activity of SHED and PDLSC were examined at 7 and 14 days. Gene expression of fibroblastic (fibroblast-specific protein-1; FSP-1), chondrogenic (collagen type II; ColII), and adipogenic (peroxisome proliferatoractivated receptor gamma isoform 2; PPARγ2) was also examined after 7 days of RA treatment. After 3 and 4 weeks of treatment, calcium deposition was detected by calcium assay and Alizarin red and von Kossa staining. Western blot analysis was performed to examine the protein levels of RUNX2 and OPN in SHED at 7 days. Immunochemical analysis of RUNX2 and OPN in SHED was conducted at 21 days. Proliferation assay Five thousand cells were seeded in 96-well microtiter plates and cultured in 200 μl basic serum-free medium alone (as a
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control) or basic serum-free medium supplemented with various concentrations of RA or Dex (as described above). After 5 days of culture, cell proliferation was examined by using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega Biosciences, San Luis Obispo, Calif. USA) according to the manufacturer’s instructions. Briefly, a reagent solution containing a tetrazolium compound [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H tetrazolium, inner salt; MTS] and an electron-coupling reagent (phenazine methosulfate; PMS) was added to each well of the 96-well microtiter plates. MTS can be bioreduced by cells into the aqueous soluble formazan whose quantity is directly proportional to the number of living cells in culture and can be measured by absorbance at 490 nm. After a 4-h incubation at 37°C under 5% CO2 in an incubator, the 96-well microtiter plates were read on a Wallac VICTOR3™ plate-reader (PerkinElmer Life and Analytical Sciences, Waltham, Mass., USA). The cell proliferation of SHED and PDLSC was also examined after 1 and 2 weeks of RA or Dex treatment.
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Analysis by reverse transcription and real-time polymerase chain reaction
After removal of culture medium, the cells were rinsed with phosphate-buffered saline (PBS) and lysed in an NP40 buffer (50 mM TRIS-HCl at pH 8.0, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40 [NP40]; Sigma). Following centrifugation at 12,000g for 10 min, the ALP activity in the supernatant of the cell lysate was assayed by using an enzyme assay. A volume (15 μl) of 150 mM p-nitrophenyl phosphate solution (Sigma) was added to a 1-ml tube containing 1 M diethanolamine buffer (pH 9.8 at 37°C; Sigma) and 0.5 mM MgCl2 (Sigma), followed by the addition of 10 μl supernatant. The samples were incubated at 37°C for 30 min. Absorbance was measured at 405 nm with a Wallac VICTOR3™ plate-reader (PerkinElmer Life and Analytical Sciences). The quantity of ALP activity was normalized against the total protein quantity as measured by the Bradford protein assay.
Gene expression of osteogenic (ALP, RUNX2, ONN, OPN, and ColI), fibroblastic, (FSP-1), chondrogenic (ColII), and adipogenic (PPARγ2) markers was analyzed by using realtime polymerase chain reaction (PCR). The total RNA of cells was extracted by using the reagent Trizol (Invitrogen). The cDNA synthesis and real-time PCR were performed with the iScript cDNA synthesis kit and iQ Supermix (Bio-Rad Laboratories, Hercules, Calif., USA), respectively, by using a real-time PCR detection system (Bio-Rad Laboratories). For the relative comparison of each target gene, the threshold cycle (CT) obtained from the real-time PCR was analyzed by using the 2 ΔΔCT method (Livak and Schmittgen 2001). Target gene transcript levels were normalized against the endogenous control of β-actin gene expression. PCR products were examined by agarose gel electrophoresis and stained by ethidium bromide. Real-time PCR was performed in triplicate with the following sequences of sense and antisense primers: ColII, 5′-GAACCACTCTCACCCTT CACA-3′ and 5′-GCCTCAAGGATTTCAAGGCAA-3′, size 285 bp; FSP-1, 5′-AGCTTCTTGGGGAAAAGGAC-3′ and 5′-CCCCAACCACATCAGAGG-3′, size 200 bp; PPARγ2, 5′-CTCCTATTGACCCAGAAAGC-3′ and 5′-GTAGAGCT GAGTCTTCTCAG-3′, size 351 bp; ColI, 5′-CTGACCTTC CTGCGCCTGATGTCC-3′ and 5′-GTCTGGGGCACCAAC GTCCAAGGG-3′, size: 300 bp; RUNX2, 5′-TTCATCCCT CACTGAGAG-3′ and 5′-TCAGCGTCAACACCATCA-3′, size: 354 bp; ALP, 5′-CCACGTCTTCACATTTGGTG-3′ and 5′-AGACTGCGCCTGGTAGTTGT-3′, size: 196 bp; OPN, 5′-TGAAACGAGTCAGCTGGATG-3′ and 5′-TGAA ATTCATGGCTGTGGAA-3′, size: 162 bp; ONN, 5′-GTGC AGAGGAAACCGAAGAG-3′ and 5′-TCATTGCTGCACA CCTTCTC-3′, size: 172 bp; β-actin, 5′-CATGTACGTTGCT ATCCAGGC-3′ and 5′-CTCCTTAATGTCACGCACGAT3′, size: 250 bp. The optimum annealing temperature of each primer was determined by traditional thermal gradient PCR.
Staining of mineral deposition
Western blot analysis
The cells were rinsed twice with PBS and fixed in 10% buffered formalin for 10 min at room temperature. The fixative was carefully removed, and the cells were gently rinsed three times with distilled water, followed by staining with 1% Alizarin red S (Sigma) solution for 20 min. In addition, mineralized matrix was also detected by von Kossa staining. After being fixed in 10% buffered formalin, the cells were rinsed with deionized water and incubated in 1% silver nitrate (Sigma) under UV light for 45 min, followed by the addition of 3% sodium thiosulfate (Sigma) to fix the positive dark staining.
Cells were lysed in buffer consisting of 50 mM TRIS, 150 mM NaCl, 2% sodium dodecyl sulfate (SDS), and a protease inhibitor mixture. After centrifugation at 12,000g for 12 min, protein levels in the supernatant were measured by the bicinchoninic acid method (Thermo Scientific, Rockford, Ill., USA). Cell lysates containing equal amounts of protein (40 mg) were subjected to SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes. Following a blocking step with 3% TRIS-buffered saline containing 5% milk and 0.1% Tween-20 for 2 h at room temperature, the membranes were
Measurement of ALP activity
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incubated with primary antibodies (1:300) at 4°C overnight and subsequently with horseradish-peroxidase-conjugated secondary antibodies (1:2000) for 2 h at room temperature. Membranes were visualized by using an enhanced chemiluminescence system (ECL) according to the manufacturer’s instruction (Pierce Biotechnology, Rockford, Ill., USA). Concomitently, β-actin was run as a reference protein. The following primary antibodies were used: rabbit polyclonal antibody against human β-actin, rabbit polyclonal antibody against human OPN, and goat polyclonal antibody against human RUNX2 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA).
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One-way analysis of variance was performed to investigate the effects of RA and Dex treatments on osteogenic gene expression, ALP activity, and proliferation of SHED and PDLSC and to compare MSC with SHED and PDLSC (significance assumed for P
