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 availability

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....

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