Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Summary

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)₂D₃]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Abstract

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.

Vitamin D₃ and 1,25-(OH)₂D₃ have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D₃ and Vitamin C on MSCs.

Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)₂D₃ may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)₂D₃ (10⁻¹⁰, 10⁻⁹, 10⁻⁸, and 10⁻⁷ M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

Introduction

Although numerous relevant techniques have been developed in recent years, bone reconstruction remains limited by multiple constraints, and estimating the extent of necessary reconstruction is often impossible. Hard-tissue augmentation is required to achieve both esthetic and functional goals in addition to a favorable long-term success rate. Methods commonly used for such procedures include autogenous and allogenic bone grafting, xenografting, and alloplastic bone grafting. Among the various types of bone graft, autogenous bone grafts are considered the most effective. However, donor site morbidity, compromised vascularity, and limited tissue availability¹ have been major drawbacks for autogenous bone grafting. In addition, allogenic bone grafts and xenografts have been associated with disease transmission. Currently, synthetic bone grafts are widely used to resolve these problems. However, with their lack of osteogenic potential, clinical outcomes have varied widely. Materials such as cellulose are associated with volume fluctuation, infection, and a lack of strength.

Bone augmentation using tissue engineering has generated considerable interest. In this technique, mesenchymal stem cells (MSCs) are initially used to promote osteoblast differentiation, which are then transplanted to the site of bone loss to achieve bone repair. This procedure is currently applied in cell therapy. Achieving bone reconstruction by extracting a limited amount of tissue is simpler and less invasive compared with other methods.

The potential role of MSCs as a tool for cell-based therapies aimed at dental regeneration is an emerging interest among various research groups. Studies have confirmed that MSCs can be differentiated from the following types of tissue: bone marrow, adipose, synovial membrane, pericyte, trabecular bone, human umbilical cord, and dental tissues^2,3. Common sources of MSCs include bone marrow, adipose tissue, and dental tissues. Compared with MSCs derived from adipose tissue and bone marrow, the advantages of dental stem cells are easy accessibility and less morbidity after harvesting. Compared with embryonic stem cells, MSCs derived from dental tissue appear nonimmunogenic and are not associated with complex ethical concerns³.

In 2006, the International Society for Cellular Therapy recommended using the following standards to identify MSCs: First, MSCs must be capable of attaching to plastic. Second, MSCs must be positive for the surface antigens CD105, CD73, and CD90 and negative for the markers for monocytes, macrophages, and B cells in addition to the hematopoietic antigens CD45 and CD34⁴. As a final criterion, MSCs must be able to differentiate into the following three types of cells under standard conditions of in vitro differentiation: osteoblasts, adipocytes, and chondrocytes⁴. To date, six types of human dental stem cell have been isolated and characterized. The first type was isolated from human pulp tissue and termed postnatal dental pulp stem cells⁵. Subsequently, three additional types of dental MSCs have been isolated and characterized: stem cells from exfoliated deciduous teeth⁶, the periodontal ligament⁷, and the apical papilla⁸. More recently, dental follicle-derived⁹, gingival tissue-derived¹⁰, dental bud stem cells(DBSCs)¹¹, and periapical cyst MSCs (hPCy-MSCs)¹² have also been identified.

Friedenstein was the first to define MSCs¹³. MSCs exhibit a high proliferation potential and can be manipulated to differentiate before being transplanted, which suggests that they are ideal candidates for regenerative procedures¹⁰.

Although most studies have used bone marrow as a source of stem cells, periosteum-derived cells (PDCs) have also been used recently¹⁴. The periosteum is more easily accessible than is the bone marrow. Therefore, in this technique, we use alveolar periosteum to eliminate the need for additional incisions during surgery and to reduce postsurgical morbidity in patients. The periosteum is the connective tissue that forms the outer lining of long bones and comprises two distinct layers: the outer fibrous layer composed of fibroblasts, collagen, and elastic fibers¹⁵, and the inner cell-rich cambium layer in direct contact with the bone surface. The cambium layer contains a mixed cell population, primarily fibroblasts¹⁶, osteoblasts¹⁷, pericytes¹⁸, and a critical subpopulation identified as MSCs^19,20,21. Most studies have reported that PDCs are comparable, if not superior, to bone marrow-derived stem cells (bMSCs) in bone healing and regeneration^22,23,24. The periosteum is easily accessible and exhibits excellent regenerative effectiveness. However, few studies have focused on the periosteum^25,26,27.

Regarding bone repair, the current clinical practice involves the transplantation of periosteal progenitor cells amplified within supportive scaffolds. Recent studies have focused on acquiring stem cells in defective regions and employing progenitor cells for tissue regeneration²⁰. Dentists also anticipate future application of periodontal bone regeneration in periodontal treatments and dental implants. Regarding the donor site, the periosteum can be easily harvested by general dental surgeons. This compares favorably against marrow stromal cells, as the periosteum can be accessed during routine oral surgery. Thus, the objective of this study is to establish a protocol for harvesting PDCs and to assess the morphology, attachment, viability, and proliferation of human periosteum stem cells.

Vitamin D metabolites affect the in vivo bone-mineral dynamic equilibrium. One study reported that the 24R,25-(OH)₂D₃ active form of Vitamin D is essential for the osteoblastic differentiation of human MSCs (hMSCs)²⁸. Bone homeostasis and repair are regulated by a network of Vitamin D₃ metabolites, of which 1,25-(OH)₂D₃ (calcitriol) is the most biologically active and relevant in the regulation of bone health. Vitamin D₃ is essential for calcification²⁹. In one study using 2-d-old Kunming white mice, the embryoid bodies in the mice indicated that Vitamin C and Vitamin D supplements effectively promoted the differentiation of ESC-derived osteoblasts³⁰. Among its other biological activities, 1,25-(OH)₂D₃ stimulates the in vitro differentiation of hMSCs to osteoblasts, which can be monitored based on the increase in alkaline phosphatase (ALP) enzyme activity or OCN gene expression.

Few studies have detected a dose-response relationship of combined treatments with Vitamin C and 1,25-(OH)₂D₃ in human PDCs with a particular focus on bone tissue engineering. Therefore, in this study, we examine the optimal concentrations for single or combined treatment of 1,25-(OH)₂D₃ and Vitamin C for inducing osteogenic differentiation of human PDCs. The goal of this protocol is to determine whether a cell population isolated from the dental alveolar periosteum contains cells with an MSC phenotype and whether these cells can be expanded in culture (in vitro) and differentiated to form the desired tissue. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes. The second part of the study evaluates the effects of Vitamin C and 10⁻¹⁰, 10⁻⁹, 10⁻⁸, and 10⁻⁷ M 1,25-(OH)₂D₃ on the osteogenic activity of PDCs. The primary objective of this study is to assess the functions of Vitamin C and 1,25-(OH)₂D₃ during the osteoblastic differentiation of PDCs by ALP activity, and pro-osteogenic genes, such as ALP, collagen-1, OCN, BSP, and CBFA1. In addition, this study determines the optimal osteoinductive conditions for human PDCs based on these findings.

Protocol

The study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital. All participants provided written informed consent.

1. Tissue Preparation

1. Harvest the periosteal tissues from patients during dental surgery (Figure 1). After flap reflection under local anesthesia, take a piece of periosteum tissue from the alveolar bone using a periosteal separator³¹.
2. After harvesting, store the periosteal tissues slices of approximately 5 mm × 2 mm in Dulbecco’s phosphate buffered saline (DPBS) with 300 U/mL of penicillin and 300 mg/mL of streptomycin. Transfer the tissues to the laboratory within 24 h.
3. Mince the alveolar periosteal tissue fragments with scalpels until well minced and maintain the samples in passage 0 medium (composed of 300 U/mL of penicillin and 300 mg/mL of streptomycin, α-MEM, and 5% fetal bovine serum [FBS]) cultured in an incubator at 37 °C with humidified air (5% carbon dioxide). In the incubator, prepare a water basin to maintain humidity.
4. Change the medium after 3 days and twice per week thereafter.
5. When the cells have reached subconfluence (80%), release the adherent cells with 200 µL of 0.25% trypsin in the incubator (37 °C) for 3 min, and then use 4.5 mL of medium to terminate the reaction.
6. Plate the cells again in fresh passage 1 medium (α-MEM, 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin). Plate is 5 × 10³ Cells (as counted by a hemocytometer).
7. Perform each subsequent passage after the cells achieve 80% confluence³¹.
   NOTE: In the beginning, the medium will be orange red. After roughly three days, the medium color should change to a yellowish shade. The doubling time of the cells is roughly 30–40 h, needing 7 days for 3–5 generations.
8. Culture the isolated PDCs in α-MEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin in an incubator (37 °C, 5% CO₂).
9. Observe cell growth daily and replace the growth medium twice per week. Use third- to fifth-generation cells for all further experiments.

2. Flow Cytometry

1. Harvest 1 × 10⁶ cells through trypsin digestion:
   1. Add 200 µL of 0.25% trypsin. After 3 min, use 4.5 mL of culture medium containing FBS to terminate the reaction of trypsin and collect through centrifugation (1500 rpm, 5 min, 22 °C).
   2. Wash the cell pellets three times using 1x DPBS. Resuspend the cells in 200 µL 1x permeabilization buffer. Count the cells with a hemocytometer.
2. Examine the expressed surface markers expressed of the PDCs through flow cytometry (fluorescence-activated cell sorting)³². Use monoclonal antibodies (MAb) against CD19 (fluorescein isothiocyanate (FITC)), CD34 (FITC), CD44 (Phycoerythrin [PE]), CD45 (FITC), CD73 (PE), CD90 (allophycocyanin [APC]), CD146 (PE), STRO-1 (Alex), and HLA-DR (FITC)³².
   1. Add the antibodies to the samples and culture in the dark at 4 °C for 30 min.
   2. Wash the cells with 1x DPBS three times.
   3. Fix the cells using 2% formaldehyde and proceed with flow cytometry analysis³².

3. Cell Attachment and Viability with Osteogenic, Adipogenic, and Chondrogenic Differentiation

Induce differentiation in the cells into osteogenic, adipogenic, and chondrogenic lineages by culturing the periosteal cells in all three passages on six-well plates with specific differentiation media.

1. For osteogenic differentiation, culture the cells in α-MEM at a density of 5000 cells per well on six-well culture plates.
   1. On achieving 80% confluence, culture the cells in media containing α-MEM, 5% FBS, β-glycerophosphate (10 mM), 10⁻⁷ M dexamethasone (0.1 mM), and 5 mL of ascorbic acid (100 uM). Culture the negative control in media consisting of α-MEM and 5% FBS.
   2. Change the media twice per week.
   3. After 4 weeks, assess the potential of the cells to differentiate into an osteogenic lineage by staining the cells using a von Kossa assay, which distinguishes the presence of calcified deposits in a culture³³.
      1. Add 10% formalin for fixing. Within 30 min, add 5% silver nitrate and treat under ultraviolet (UV) light for 1 h at room temperature.
      2. Add 5% Na₂SO₄ four times, allowing 3 min for each reaction. Finally, wash the cells twice with distilled water.
         NOTE: Von Kossa staining is a precipitation reaction where silver ions and phosphate react in the presence of acidic material; this staining technique is not specific to calcium. In this study, when the investigated cells were treated with silver nitrate solution, calcium—which is reduced by strong UV light—was replaced by silver, which became visible as metallic silver.
2. For adipogenic differentiation, culture the cells at a density of 5000 cells per well on six-well culture plates containing α-MEM.
   1. On achieving 80% confluence, culture the cells in media containing α-MEM, 5% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (100 mg/mL), 1% penicillin, 10⁻⁶ M dexamethasone (1 mM), insulin (5 mg/mL), and indomethacin (60 mM).
   2. Culture the negative control in media consisting of α-MEM and 5% FBS.
   3. After 6–9 weeks, use oil red O to stain the cells and highlight lipid drops, thereby determining the presence of adipogenic differentiation³³:
      1. Add 10% formalin for fixing. Within 30 min, stain the cells with 0.5% oil red O at room temperature for 10 min, and then wash the cells three times with 60% isopropanol for 3 min each time; finally, wash the cells with distilled water.
         NOTE: Oil red O is a lysochrome (fat-soluble) diazo dye used for the staining of neutral lipids, primarily triacylglycerol, lipoprotein, and cholesterol esters. The dye dissolves in the lipid droplets in the cell, turning the lipid droplets red.
3. For chondrocyte differentiation, culture cells on six-well culture plates with each well containing 5000 cells.
   1. Add a differentiation medium containing α-MEM, 5% FBS, 10⁻⁷ M dexamethasone (0.1 mM), sodium pyruvate (100 µg/mL), insulin-transferrin-selenium-A (1 × ITS), transforming growth factor-β (10 ng/mL), and 5 mL of ascorbic acid (100 µM).
   2. Culture the negative control in media consisting of α-MEM and 5% FBS.
   3. After 4–5 weeks, use Alcian blue staining to highlight the acidic polysaccharides, such as glycosaminoglycans or some types of mucopolysaccharides, in the cartilage of the cells to determine the presence of chondrocyte differentiation³³.
      1. Add 10% formalin for fixing. Within 30 min, add 3% acetic acid and allow 2 min for the reaction. Subsequently, wash with distilled water three times, add 1% Alcian blue 8GX incubated for 30 min, and then wash with distilled water. Finally, add 3% acetic acid and wash for 3 min, and then wash with distilled water to finish.
         NOTE: The parts of the cell that specifically stain by this dye become blue to blue-green after staining and are called “alcianophilic.”

4. Effects of 25-Dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on Osteogenesis

1. Split the P3 to P5 PDCs into six groups and culture on 6-well plates with various culture media:
2. negative group (α-MEM and 5% FBS);
3. Vitamin C group (α-MEM, 5% FBS, 10 mM β-glycerophosphate, and 10⁻⁷ M dexamethasone + 100 uM Vitamin C);
4. and 10⁻⁷ M 1,25-(OH)₂D₃ group (α-MEM, 5% FBS, 10 mM β-glycerophosphate, and 10⁻⁷ M dexamethasone + 10⁻⁷ M 1,25-(OH)₂D₃).
5. Add 2 mL of the medium to an incubator (37 °C) in each well and change the medium twice every week.

5. Reverse Transcription/Quantitative Real-Time Polymerase Chain Reaction

1. After 7 d of osteoblast differentiation, isolate the total RNA on ice by using a commercial reagent (see Table of Materials):
   1. Add 1 mL of isolation reagent to each well. Add 200 µL of bromochloropropane and leave for 10 min to generate layered RNA, and then centrifuge at 10,000 rpm for 15 min at 4 °C.
   2. After centrifugation, add 500 µL of 2-propanol to 500 µL of the supernatant containing RNA. Precipitate the RNA on the ice and allow 15 min for the reaction.
   3. After the procedure, centrifuge at 12,000 rpm for 15 min at 4 °C, after which the RNA is located at the bottom of the tube. Subsequently, remove the supernatant by using 1 mL of 75% alcohol for washing and centrifuge at 7,500 rpm for 8 min in 4 °C.
   4. Remove the supernatant and use a vacuum concentrator to produce RNA from the bottom of the liquid. Recover with water.
2. Reverse transcribe the RNA (1 μg) using avian myeloblastosis virus reverse transcriptase. Set the polymerase chain reaction (PCR) to run at 25 °C for 10 min followed by 50 °C for 60 min and then 85 °C for 5 min, before finally maintaining at 4 °C.
3. Synthesize first-strand complementary DNA (cDNA). Perform quantitative PCR (qPCR) using 5 μL of 1:10 diluted cDNA. Conduct quantitative RT-PCR (qRT-PCR) using primers for ALP, BSP, OCN, CBFA1, and collagen-1.
   NOTE: To avoid DNA contamination by the signals, the forward and reverse sequences of each primer were designed on distinct exons.
4. Perform qPCR by using a commercial PCR Master Mix (see Table of Materials) in accordance with the manufacturer’s instructions. Set the thermal cycler at 50 °C for 2 min followed by 95 °C for 10 min and then 40 cycles each at 95 °C for 15 s followed by 60 °C for 60 s.
   NOTE: The SYBR protocol also requires a melt curve stage, which is run at 95 °C for 15 s, 60 °C for 60 s, and then 95 °C for 15 s.
5. Normalize the cycle threshold values for ALP, BSP, CBFA1, collagen-1, and OCN to that of the housekeeping gene GAPDH.³⁴
6. Primer pairs are listed in the Table of Materials.

6. Alkaline Phosphatase Activity

1. Assess the ALP enzyme activity of the cells using the previously described technique³⁵, which converts p-nitrophenyl phosphate to p-nitrophenol; p-nitrophenyl phosphate is a phosphatase substrate that turns yellow when dephosphorylated by ALP, as determined at a 405-nm wavelength. Normalize the total p-nitrophenol formed based on the total protein as determined by the Bradford assay.
2. Compare the ALP activities of the control (α-MEM, 5% FBS), Vitamin C (α-MEM, 5% FBS, 10 mM β-glycerophosphate, 10⁻⁷ M dexamethasone + 100 uM Vitamin C), 1,25-(OH)₂D₃ (α-MEM, 5% FBS, 10 mM β-glycerophosphate, and 10⁻⁷ M dexamethasone + 10⁻⁸ M 1,25-(OH)₂D₃), and Vitamin C + 1,25-(OH)₂D₃ (α-MEM, 5% FBS, 10 mM β-glycerophosphate, 10⁻⁷ M dexamethasone + 100 uM Vitamin C +1,25-(OH)₂D₃) groups after 1, 2, 3, and 4 week of culture.
3. Perform a protein extraction procedure on ice:
   1. Scrape the cells from the 6-well plates and add 50 µL of lysis buffer. Subsequently, place the cells on the ice for 30 min to destroy the cells and free the protein.
   2. After 30 min, centrifuge at 13000 rpm at 4 °C for 15 min. After centrifugation, extract the supernatant for storage and use.

Results

For all quantitative assays, the data are presented as mean ± standard deviation (SD). All statistical analyses were performed using Student's t-test. In total, 34 samples were obtained with a mean participant age of 48.1 ± 12.3 y. Eleven of these samples were obtained from male patients and 23 from female patients. Twenty-eight samples were obtained from the molar regions and six from the anterior regions; 26 were obtained from the maxilla and 8 from the mandible. The mean durat...

Discussion

A recently developed therapeutic modality, namely tissue engineering entailing MSCs, has numerous advantages. MSCs, which are present in several tissue types, are multipotent cells that can differentiate into a variety of functional mesodermal tissue cells³⁷ and other cells such as osteoblasts.

The periosteum serves as a niche for progenitor cells and as a rich vasculature supply for the bone it envelops³⁸. In our study, of the 34 investigated sa...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco	25200-056
2-phospho-L-ascorbic acidtrisodium salt	Sigma	49752
35-mm culture dishes	Corning	430165
3-isobutyl-1-methylxanthine	Sigma	I5879
6  well plate	Corning	3516
Alkaline phosphatase	ABI	Hs01029144_m1
Alkaline Phosphatase Activity Colorimetric Assay Kit	BioVision	K412-500
avian myeloblastosis virus reverse transcriptase	Roche	10109118001
CD146	BD	561013
CD19	BD	560994
CD34	BD	560942
CD44	BD	561858
CD45	BD	561088
CD73	BD	561014
CD90	BD	561974
Cell banker1	ZEAOAQ	11888
core binding factor alpha-1	ABI	Hs00231692_m1
dexamethasone	Sigma	D4902
DPBS	Gibco	14190250
FBS	Gibco	26140-079
GAPDH	ABI	Hs99999905_m1
HLA-DR	BD	562008
indomethacin	Sigma	I7378
insulin	sigma	91077C
insulin–transferrin–selenium-A	Sigma	I1884
MicroAmp Fast 96 well reaction plate(0.1ml)	Life	4346907
MicroAmp optical adhesive film	Life	4311971
Minimum Essential Medium 1X Alpha Modification	HyClone	SH30265.02
Penicillin/Streptomycin	Gibco	15140-122
Permeabilization buffer	eBioscience	00-8333-56
Sodium pyruvate	Gibco	11360070
STRO-1	BioLegend	340103
SYBER Green PCR Master Mix	AppliedBiosystems	4309155
TaqMan Master Mix	Life	4304437
transforming growth factor-β	Sigma	T7039
Trizol reagent (for RNA isolation)	Life	15596018
β-glycerophosphate	Sigma	G9422
collagen-1	Invitrogen	forward primer 5' CCTCAAGGGCTCCAACGAG-3 reverse primer 5'-TCAATCACTGTCTTGCCCCA-3'
OCN	Invitrogen	forward primer 5'-GTGCAGCCTTTGTGTCCAAG-3' reverse primer 5'-GTCAGCCAACTCGTCACAGT-3'
BSP	Invitrogen	forward primer 5' AAAGTGAGAACGGGGAACCT-3' reverse primer 5'-GATGCAAAGCCAGAATGGAT-3'
Commercial ALP primers
Commercial CBFA1 primers