The study protocol was approved by the Institutional Review Board for Clinical Research of Chang Gung Memorial Hospital (IRB99-1828B, 100-3019C, 99-3814B, 102-1619C, 101-4728B, and 103-4223C). This study was supported by Chang Gung Memorial Hospital (CMRPG392071, CMRPG3A1141, CMRPG3A1142, and NMRPG3C0151). This manuscript was edited by Wallace Academic Editing.

The study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital. All participants provided written informed consent.
1. Tissue Preparation
<ol>
	<li>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.</li>
	<li>After harvesting, store the periost...

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...

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 tissues2,3. 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 MSCs19,20,21. Most studies have reported that PDCs are comparable, if not superior, to bone marrow-derived stem cells (bMSCs) in bone healing and regeneration22,23,24. The periosteum is easily accessible and exhibits excellent regenerative effectiveness. However, few studies have focused on the periosteum25,26,27.
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&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;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.

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>2-phospho-L-ascorbic acidtrisodium salt</td>
			<td>Sigma</td>
			<td>49752</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#160; well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>ABI</td>
			<td>Hs01029144_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Activity Colorimetric Assay Kit</td>
			<td>BioVision</td>
			<td>K412-500</td>
			<td></td>
		</tr>
		<tr>
			<td>avian myeloblastosis virus reverse transcriptase</td>
			<td>Roche</td>
			<td>10109118001</td>
			<td></td>
		</tr>
		<tr>
			<td>CD146</td>
			<td>BD</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD</td>
			<td>560994</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34</td>
			<td>BD</td>
			<td>560942</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44</td>
			<td>BD</td>
			<td>561858</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>BD</td>
			<td>561088</td>
			<td></td>
		</tr>
		<tr>
			<td>CD73</td>
			<td>BD</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90</td>
			<td>BD</td>
			<td>561974</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell banker1</td>
			<td>ZEAOAQ</td>
			<td>11888</td>
			<td></td>
		</tr>
		<tr>
			<td>core binding factor alpha-1</td>
			<td>ABI</td>
			<td>Hs00231692_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>ABI</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>BD</td>
			<td>562008</td>
			<td></td>
		</tr>
		<tr>
			<td>indomethacin</td>
			<td>Sigma</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>sigma</td>
			<td>91077C</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin&#8211;transferrin&#8211;selenium-A</td>
			<td>Sigma</td>
			<td>I1884</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast 96 well reaction plate(0.1ml)</td>
			<td>Life</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp optical adhesive film</td>
			<td>Life</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium 1X Alpha Modification</td>
			<td>HyClone</td>
			<td>SH30265.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>STRO-1</td>
			<td>BioLegend</td>
			<td>340103</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBER Green PCR Master Mix</td>
			<td>AppliedBiosystems</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Master Mix</td>
			<td>Life</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>transforming growth factor-&#946;</td>
			<td>Sigma</td>
			<td>T7039&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol reagent (for RNA isolation)</td>
			<td>Life</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">collagen-1</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39; CCTCAAGGGCTCCAACGAG-3</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-TCAATCACTGTCTTGCCCCA-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">OCN</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39;-GTGCAGCCTTTGTGTCCAAG-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-GTCAGCCAACTCGTCACAGT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">BSP</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39; AAAGTGAGAACGGGGAACCT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-GATGCAAAGCCAGAATGGAT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial ALP primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial CBFA1 primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mesenchymal stem cells from human alveolar periosteum and effects of vitamin d on osteogenic activity of periosteum-derived cells

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&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;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).

For all quantitative assays, the data are presented as mean &#177; standard deviation (SD). All statistical analyses were performed using Student&#39;s t-test. In total, 34 samples were obtained with a mean participant age of 48.1 &#177; 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. ...

Watch this Scientific Journal Video about Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells at JoVE.com

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

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.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

The overall goal of this study is to investigate the osteoinductive effects of Vitamin D on cultured mesenchymal stem cells that have been harvested from the dental alveolar periosteum. The use of mesenchymal stem cells is a great advance in dentistry for the past decade. They have been successfully isolated and cultured from many part of dental tissue.The objective though is to isolate and culture mesenchymal stem cells from alveolar periosteum and to investigate the osteoinductive potential of Vitamin D compound. Generally speaking, individuals new to this matter will struggle because of the new idea of bone formation in mesenchymal stem cells derived from alveolar periosteum. The laboratory staff demonstration of procedure will be Miss Hsin-Wen Chi.She is a research assistant from my laboratory. To begin, harvest the periosteal tissues from patients during dental surgery using a periosteal separator. Collect slices that are about two by five millimeters.Store the tissue slices in DPBS with antibiotics. At the lab, use a scalpel to thoroughly mince the alveolar periosteal tissue fragments within 24 hours of harvesting. Next, culture the tissue on 35 millimeter petri dishes in the appropriate culturing medium.Use a humidified 37 degree celsius incubator with 5%carbon dioxide gas. Three days later, discard the collected periosteal tissues and change the medium. When the cells have proliferated to about 80%confluence, detach the cultured cells by adding Trypsin.And incubating the culture for three minutes. After this, add 0.8 milliliters of medium to terminate the detachment. After collecting and counting, distribute 5, 000 cells in one milliliter per plate.Mark the plated cells as passage one at this stage. Culture these cells and separate them again after they have proliferated to 80%confluence. The medium with passage one is initially an orange/red color.When the medium begins to turn orange three days later, change it. When the passage one cells reach 80%confluence, create the second to the fifth generation cells by repeating the same procedures used to prepare the zero generation to the first generation. Change the medium twice per week.Four weeks later, assess the potential of the cells to differentiate into an osteogenic lineage by observing the morphology of the cells under a microscope. Stem the cells using a von kossa assay. This assay distinguishes the presence of calcified deposits in your culture.Wash the cells with PBS and the fix them for 30 minutes in one milliliter of 10%Formalin. After this, remove the Formalin and then wash the cells and treat them with 5%silver nitrate. Expose the cells to UV light for one hour.Next, remove the silver nitrate and treat the cells with two milliliters of 5%sodium sulfate four times for three minutes each time. Replace the solution between treatments. Finally, wash the cells twice using distilled water.20 of 34 periosteal tissue samples successfully yielded marrow-derived stem cell colonies. No factors with relation to the donors were found to significantly affect the culturing success. Initially, PDCs formed colonies and spherical clusters with a mix of round and spindle-shaped cells.After the first passage, the cells were homogeneously fibroblast-like with a spindle shape. At the end of the osteogenic differentiation, von kossa assay indicated the possible presence of calcium deposits and osteogenic differentiation. These results strongly indicate that among periosteal cell populations, progenitor cells possess the potential to differentiate into osteogenic cells.The osteogenic precursor cells were also treated with Calcitriol and examined using RTPCR to assess the expression level of alkaline phosphatase which is a strong indicator for early osteogenic differentiation of cells. Both treatment of vitamin C and Calcitriol, an active metabolite of vitamin D, increased alkaline phosphatase expression. Also, collagen one mRNA levels expression increased in response to the same treatments.The expression of bone sialoprotein mRNA was found to increase for the cells treated with a higher concentration of Calcitriol. However, CBFA one, and ostocalcium mRNA expression did not respond to the treatments. After watching this video, the audience should have a good understanding of the mineralization and culture protocol of human alveolar periosteum.Once mastered, this pursuit can be done in four to five weeks if performed properly. When attempting this experiment, it is important to use a careful and sterile technique to avoid contamination of cell.

isolation-mesenchymal-stem-cells-from-human-alveolar-periosteum

Research

JoVE Journal

Bioengineering

9.2K visualizações.  Chang Gung University. The overall goal of this study is to investigate the osteoinductive effects of Vitamin D on cultured mesenchymal stem cells that have been harvested from the dental alveolar periosteum. The use of mesenchymal stem cells is a great advance in dentistry for the past decade. They have been successfully isolated and cultured from many part of dental tissue.The objective though is to isolate and culture mesenchymal stem cells from alveolar periosteum and to investigate the osteoinductive po...