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 periosteal tissues slices of approximately 5 mm &#215; 2 mm in Dulbecco&#8217;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.</li>
	<li>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, &#945;-MEM, and 5% fetal bovine serum [FBS]) cultured in an incubator at 37 &#176;C with humidified air (5% carbon dioxide). In the incubator, prepare a water basin to maintain humidity.</li>
	<li>Change the medium after 3 days and twice per week thereafter.</li>
	<li>When the cells have reached subconfluence (80%), release the adherent cells with 200 &#181;L of 0.25% trypsin in the incubator (37 &#176;C) for 3 min, and then use 4.5 mL of medium to terminate the reaction.</li>
	<li>Plate the cells again in fresh passage 1 medium (&#945;-MEM, 10% FBS, 100 units/mL of penicillin, and 100 &#956;g/mL of streptomycin). Plate is 5 &#215; 103 Cells (as counted by a hemocytometer).</li>
	<li>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&#8211;40 h, needing 7 days for 3&#8211;5 generations.</li>
	<li>Culture the isolated PDCs in &#945;-MEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin in an incubator (37 &#176;C, 5% CO2).</li>
	<li>Observe cell growth daily and replace the growth medium twice per week. Use third- to fifth-generation cells for all further experiments.</li>
</ol>
2. Flow Cytometry
<ol>
	<li>Harvest 1 &#215; 106 cells through trypsin digestion:
	<ol>
		<li>Add 200 &#181;L 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 &#176;C).</li>
		<li>Wash the cell pellets three times using 1x DPBS. Resuspend the cells in 200 &#181;L 1x permeabilization buffer. Count the cells with a hemocytometer.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Add the antibodies to the samples and culture in the dark at 4 &#176;C for 30 min.</li>
		<li>Wash the cells with 1x DPBS three times.</li>
		<li>Fix the cells using 2% formaldehyde and proceed with flow cytometry analysis32.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>For osteogenic differentiation, culture the cells in &#945;-MEM at a density of 5000 cells per well on six-well culture plates.
	<ol>
		<li>On achieving 80% confluence, culture the cells in media containing &#945;-MEM, 5% FBS, &#946;-glycerophosphate (10 mM), 10&#8722;7 M dexamethasone (0.1 mM), and 5 mL of ascorbic acid (100 uM). Culture the negative control in media consisting of &#945;-MEM and 5% FBS.</li>
		<li>Change the media twice per week.</li>
		<li>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.
		<ol>
			<li>Add 10% formalin for fixing. Within 30 min, add 5% silver nitrate and treat under ultraviolet (UV) light for 1 h at room temperature.</li>
			<li>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&#8212;which is reduced by strong UV light&#8212;was replaced by silver, which became visible as metallic silver.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For adipogenic differentiation, culture the cells at a density of 5000 cells per well on six-well culture plates containing &#945;-MEM.
	<ol>
		<li>On achieving 80% confluence, culture the cells in media containing &#945;-MEM, 5% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (100 mg/mL), 1% penicillin, 10&#8722;6 M dexamethasone (1 mM), insulin (5 mg/mL), and indomethacin (60 mM).</li>
		<li>Culture the negative control in media consisting of &#945;-MEM and 5% FBS.</li>
		<li>After 6&#8211;9 weeks, use oil red O to stain the cells and highlight lipid drops, thereby determining the presence of adipogenic differentiation33:
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For chondrocyte differentiation, culture cells on six-well culture plates with each well containing 5000 cells.
	<ol>
		<li>Add a differentiation medium containing &#945;-MEM, 5% FBS, 10&#8722;7 M dexamethasone (0.1 mM), sodium pyruvate (100 &#181;g/mL), insulin-transferrin-selenium-A (1 &#215; ITS), transforming growth factor-&#946; (10 ng/mL), and 5 mL of ascorbic acid (100 &#181;M).</li>
		<li>Culture the negative control in media consisting of &#945;-MEM and 5% FBS.</li>
		<li>After 4&#8211;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.
		<ol>
			<li>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 &#8220;alcianophilic.&#8221;</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Effects of 25-Dihydroxyvitamin D3 (1,25-(OH)2D3) on Osteogenesis
<ol>
	<li>Split the P3 to P5 PDCs into six groups and culture on 6-well plates with various culture media:</li>
	<li>negative group (&#945;-MEM and 5% FBS);</li>
	<li>Vitamin C group (&#945;-MEM, 5% FBS, 10 mM &#946;-glycerophosphate, and 10&#8722;7 M dexamethasone + 100 uM Vitamin C);</li>
	<li>and 10&#8722;7 M 1,25-(OH)2D3 group (&#945;-MEM, 5% FBS, 10 mM &#946;-glycerophosphate, and 10&#8722;7 M dexamethasone + 10&#8722;7 M 1,25-(OH)2D3).</li>
	<li>Add 2 mL of the medium to an incubator (37 &#176;C) in each well and change the medium twice every week.</li>
</ol>
5. Reverse Transcription/Quantitative Real-Time Polymerase Chain Reaction
<ol>
	<li>After 7 d of osteoblast differentiation, isolate the total RNA on ice by using a commercial reagent (see Table of Materials):
	<ol>
		<li>Add 1 mL of isolation reagent to each well. Add 200 &#181;L of bromochloropropane and leave for 10 min to generate layered RNA, and then centrifuge at 10,000 rpm for 15 min at 4 &#176;C.</li>
		<li>After centrifugation, add 500 &#181;L of 2-propanol to 500 &#181;L of the supernatant containing RNA. Precipitate the RNA on the ice and allow 15 min for the reaction.</li>
		<li>After the procedure, centrifuge at 12,000 rpm for 15 min at 4 &#176;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 &#176;C.</li>
		<li>Remove the supernatant and use a vacuum concentrator to produce RNA from the bottom of the liquid. Recover with water.</li>
	</ol>
	</li>
	<li>Reverse transcribe the RNA (1 &#956;g) using avian myeloblastosis virus reverse transcriptase. Set the polymerase chain reaction (PCR) to run at 25 &#176;C for 10 min followed by 50 &#176;C for 60 min and then 85 &#176;C for 5 min, before finally maintaining at 4 &#176;C.</li>
	<li>Synthesize first-strand complementary DNA (cDNA). Perform quantitative PCR (qPCR) using 5 &#956;L 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.</li>
	<li>Perform qPCR by using a commercial PCR Master Mix (see Table of Materials) in accordance with the manufacturer&#8217;s instructions. Set the thermal cycler at 50 &#176;C for 2 min followed by 95 &#176;C for 10 min and then 40 cycles each at 95 &#176;C for 15 s followed by 60 &#176;C for 60 s. 
	NOTE: The SYBR protocol also requires a melt curve stage, which is run at 95 &#176;C for 15 s, 60 &#176;C for 60 s, and then 95 &#176;C for 15 s.</li>
	<li>Normalize the cycle threshold values for ALP, BSP, CBFA1, collagen-1, and OCN to that of the housekeeping gene GAPDH.34</li>
	<li>Primer pairs are listed in the Table of Materials.</li>
</ol>
6. Alkaline Phosphatase Activity
<ol>
	<li>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.</li>
	<li>Compare the ALP activities of the control (&#945;-MEM, 5% FBS), Vitamin C (&#945;-MEM, 5% FBS, 10 mM &#946;-glycerophosphate, 10&#8722;7 M dexamethasone + 100 uM Vitamin C), 1,25-(OH)2D3 (&#945;-MEM, 5% FBS, 10 mM &#946;-glycerophosphate, and 10&#8722;7 M dexamethasone + 10&#8722;8 M 1,25-(OH)2D3), and Vitamin C + 1,25-(OH)2D3 (&#945;-MEM, 5% FBS, 10 mM &#946;-glycerophosphate, 10&#8722;7 M dexamethasone + 100 uM Vitamin C +1,25-(OH)2D3) groups after 1, 2, 3, and 4 week of culture.</li>
	<li>Perform a protein extraction procedure on ice:
	<ol>
		<li>Scrape the cells from the 6-well plates and add 50 &#181;L of lysis buffer. Subsequently, place the cells on the ice for 30 min to destroy the cells and free the protein.</li>
		<li>After 30 min, centrifuge at 13000 rpm at 4 &#176;C for 15 min. After centrifugation, extract the supernatant for storage and use.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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 samples, 20 successfully yielded MSC colonies, with a successful isolation rate of 58.8%. MSCs derived from the periosteum were selected in this study based on their proliferation and osteogenic differentiation abilities. The proliferation rate of the periosteal cells was much higher than that of the marrow stromal cells22. Regarding the osteogenic ability of the periosteum, Ousual24 contended that the osteogenic ability of periosteum-derived stem cells (PMSCs) was inferior to that of BMSCs. However, Yoshimura attested that the osteogenic ability of PMSCs outperformed that of BMSCs39. Hayashi et al. demonstrated periosteal adult MSCs as ideal candidates for bone tissue regeneration24. In addition, periosteal adult MSCs are considered useful in shortening the cell culture period, thus reducing both cost and the risk of contamination22. Reportedly, aging also affects the mitogenic activity of the osteoprogenitor cells40. A study reported 1,25-(OH)2D3 induced a higher degree of stimulation of in vitro osteoblast differentiation in hMSCs from younger participants (aged &#60; 65 y)41. The present study involved the use of periosteal cells obtained from young adults (aged &#60; 65 y). Further studies are required to compare the bone regeneration ability of periosteal cells between old and young donors.
In this study, a cell population was isolated based on its plastic-adherent property, as described by Friedenstein et al.13. The PDCs harvested here adhered to the cell culture plastic. The esenchymal markers (CD 73, CD 90, STRO-1, and CD 44) of the surface antigens of these primary cultures cells exhibited significant expressions. Furthermore, hematopoietic markers (CD 45, CD 34, CD 19, and HLA-DR) were absent, indicating that the cultured cells were of a mesenchymal origin. The osteogenic, chondrogenic, and adipogenic differentiation of the PDCs could be induced using specific differentiation media, as described in other studies evaluating samples obtained from gingival tissue and bone marrow42. The results of the present study comply with the minimal standards for the phenotypic and functional definition of human MSCs accepted by the International Society for Cellular Therapy4. In the present study, the MSCs from human periosteum were isolated and expanded, which revealed characteristics similar to those typically described of BMSCs.
Studies have proposed that dexamethasone, ascorbate, and &#946;-glycerophosphate cause BMSCs to undergo osteogenic differentiation43. Another study demonstrated that 1&#945;, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) in combination with ascorbic acid promoted stem cell differentiation44. Therefore, dexamethasone, ascorbate, and &#946;-glycerophosphate were added in this study to promote stem cell differentiation. The patterns observed were similar in all three patient cell samples. In this study, MSCs were successfully identified from the alveolar periosteum with an isolation rate of 58.8%. No statistically significant differences were observed in the success rate with respect to age, sex, and location.
The present experimental study investigated two substances, namely 1,25-(OH)2D3 and vitamin C, both of which can stimulate the osteogenic differentiation of stem cells. A 2008 study45 indicated that 1,25-(OH)2D3 is crucial for bone metabolism and calcium homeostasis and affects the cardiovascular system through mechanisms yet to be fully understood. In another previous study46, 1,25-(OH)2D3 stimulated the in vitro differentiation of human MSCs into osteoblasts. The ability of 1,25-(OH)2D3 to induce undifferentiated BMSCs to differentiate into osteoblast-like cells in vitro has been demonstrated in several studies exploring various systems46. Some studies have also demonstrated that, when added to cultures of osteoblast-like cells, 1,25-(OH)2D3 inhibits cellular proliferation but increases the expression of osteoblastic markers such as ALP, osteopontin, OCN, and matrix Gla protein47,48. A previous study reported that 1,25-(OH)2D3 typically inhibits cellular proliferation and induces osteoblast differentiation49. One in vitro study conducted on murine cells indicated that 1,25-(OH)2D3 can promote the early stages of osteoblastogenesis9. Additionally, another in vitro study suggested that, to both early and late MSCs,1,25-(OH)2D3 can stimulate osteogenic differentiation markers, including ALP, osteopontin, BSP, and OCN43.
Both OCN&#8212;considered responsible for the control of matrix synthesis&#8212;and collagen 1A1&#8212;the most abundant matrix protein&#8212;are crucial acidic bone matrix proteins that play vital roles in matrix synthesis. In addition, they are characteristic mature matrix-forming osteoblasts.50 A previous study reported that 1,25-(OH)2D3 treatment stimulated increased collagen 1A1 expression but not CBFA1 or ALP gene expression34. Assessments of genes involved in osteoblastic differentiation have revealed that 1,25-(OH)2D3 treatment increased the expression of OCN51,52,53. In our study, the results showed that 1,25-(OH)2D3 exerts positive effects on osteogenic activity. Compared with the other groups, 10&#8722;8 M 1,25-(OH)2D3 demonstrated increased mRNA expression of ALP, BSP, CBFA1, OCN, and Col1. Among these, the results for ALP and Col1 reached statistical significance. Therefore, optimal conditions were obtained when using 10&#8722;8 M 1,25-(OH)2D3. mRNA expression of BSP in the 10&#8722;10 M 1,25-(OH)2D3 group increased significantly (p &#60; 0.05). However, no statistically significant differences were observed in the fold changes in the mRNA expressions of CBFA1 and OCN. When reviewing the gene mRNA expression, the results of the experiment indicated in vitro osteogenic differentiation. Based on these findings, the osteogenesis observed in the negative, Vitamin C, and 10&#8722;10, 10&#8722;9, 10&#8722;7 M 1,25-(OH)2D3 groups was weaker than that in the 10&#8722;8 M 1,25-(OH)2D3 group. These results led to the speculation that 1,25-(OH)2D3 primes PDCs by upregulating the matrix proteins required for mineralization (collagen 1A1).
A previous study reported OCN as a prime target of 1,25-(OH)2D3, which markedly increased OCN expression52. OCN deposition can be seen as a marker for the osteogenic differentiation of PDCs. In addition, OCN expression has been evaluated as a late osteogenic marker54. OCN is a reliable marker for bone formation and osteogenesis55. Therefore, OCN expression is generally related to the mineralization abilities of cells. OCN is a sensitive biomarker of mature osteoblasts. For an in vitro osteogenic comparison, markedly elevated OCN mRNA expression was observed in the 10&#8722;8 M 1,25-(OH)2D3 group.
One study indicated that ALP activity was inhibited by transforming growth factor beta (0.1-10 ng/mL) and promoted with 1,25-(OH)2D3 (50 nM)46. These results imply that 10&#8722;8 M 1,25-(OH)2D3 promotes stem cell maturation and bone development. ALP activity at the optimal observed concentrations of 10&#8722;8 M 1,25-(OH)2D3 was compared with that at the corresponding concentrations of Vitamin C. The PDCs expressed ALP activity. Moreover, the addition of 1,25-(OH)2D3 increased the ALP activity and promoted the osteoblastic differentiation in the PDCs. In all three settings, Vitamin C, 10&#8722;8 M 1,25-(OH)2D3, and Vitamin C + 10&#8722;8 M 1,25-(OH)2D3 increased ALP activity at Weeks 1 (Figure 4a), 2 (Figure 4b), 3 (Figure 4c), and 4 (Figure 4d) by several fold compared with that of the negative control group. In Weeks 1 and 4, ALP activity increased significantly in the 10&#8722;8 M 1,25-(OH)2D3 group. In Week 2, ALP activity increased significantly in the Vitamin C and 10&#8722;8 M 1,25-(OH)2D3 groups.
Through a comparative study of various human MSCs at passage 3, Sakaguchi et al.56 found that BMSCs and PMSCs possess similar osteogenic abilities. Therefore, the cell culture period can likely be shortened by using periosteal cells, which in turn would reduce the cost and contamination risk. The use of early-passage hMSCs avoids the senescent effects of expansion in the culture. Ousual and Yoshimura used MSCs at passage 3 and subcultured for 2 weeks under osteogenic differentiation conditions35. Therefore, the passage time should be considered when comparing the cellular activities of various cell sources. In this study, third-generation PDCs were used because the osteogenic potential of the stem cells of later generations of PDCs is lower than that of the stem cells of third-generation PDCs.
When cultured in a human serum medium, periosteum stem cells can not only maintain their shape, but can also attach, maintain activity, and achieve cell growth. In summary, based on these results, human PDCs possess the ability to survive, proliferate, and differentiate into the osteogenic lineage in vitro, which is crucial in evaluating efficacy in vivo. The periosteum is rich in MSCs and is thus suitable for tissue engineering. The periosteum obtained from the gingiva poses fewer complications. Therefore, the alveolar bone is considered an ideal donor site. The results clearly demonstrated the possibility of similarly isolating MSCs from the periosteum and achieving their differentiation into osteoblasts, chondrocytes, and adipocytes. In this study, the effects of Vitamin C on the osteogenic potential of PDCs were compared with those of 1,25-(OH)2D3 at various concentrations. The most effective concentration was 10&#8722;8 M 1,25-(OH)2D3. The 10&#8722;8 M 1,25-(OH)2D3 treatment throughout the culture period was an effective and economical method of inducing osteogenic differentiation in PDCs. In conclusion, both Vitamin C and 1,25-(OH)2D3 promote the osteogenic differentiation of PDCs. However, these exhibited no significant synergistic effects in ALP activity results. Considering the small sample size, additional samples are required for further evaluation. Thus, PDCs cultured under osteogenic conditions might be useful for bone tissue engineering and offer the advantage of higher cellular proliferation.
Critical steps of this protocol include steps 1.2 and 1.3 (starting the culturing procedure within 24 hours of harvesting and proper maintenance thereafter). Contamination of the tissue must be avoided and use of aseptic technique is mandatory. One limitation of this protocol is that, compared with sourcing stem cells from bone marrow or adipose tissue, sourcing stem cells from dental tissue is limited in terms of the low number of cells that can be obtained through this technique.
The identification and characterization of periosteal stem cells can provide valuable information regarding the functions and regenerative potential of this tissue as well as its applicability in regenerative therapy. An optimal condition was obtained using 10&#8722;8 M 1,25-(OH)2D3. Therefore, the treatment of PDCs with 10&#8722;8 M 1,25-(OH)2D3 promotes osteogenic differentiation. Future studies should examine the in vivo application of 10&#8722;8 M 1,25-(OH)2D3 for effective and economical bone tissue engineering.

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&amp;nbsp; 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>&lt;em&gt;sigma&lt;/em&gt;</td><td>91077C</td><td></td></tr><tr><td>insulin&amp;ndash;transferrin&amp;ndash;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-&amp;beta;</td><td>Sigma</td><td>T7039&amp;nbsp;</td><td></td></tr><tr><td>Trizol reagent (for RNA isolation)</td><td>Life</td><td>15596018</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma</td><td>G9422</td><td></td></tr><tr><td>collagen-1</td><td>Invitrogen</td><td>forward primer 5&#039; CCTCAAGGGCTCCAACGAG-3&lt;br /&gt;reverse primer 5&#039;-TCAATCACTGTCTTGCCCCA-3&#039;</td><td></td></tr><tr><td>OCN</td><td>Invitrogen</td><td>forward primer 5&#039;-GTGCAGCCTTTGTGTCCAAG-3&#039;&lt;br /&gt;reverse primer 5&#039;-GTCAGCCAACTCGTCACAGT-3&#039;</td><td></td></tr><tr><td>BSP</td><td>Invitrogen</td><td>forward primer 5&#039; AAAGTGAGAACGGGGAACCT-3&#039;&lt;br /&gt;reverse primer 5&#039;-GATGCAAAGCCAGAATGGAT-3&#039;</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).

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.3K Views.  Chang Gung University. We presenteren een protocol om te onderzoeken het mRNA uitdrukking biomarkers voor beenvlies-afgeleide cellen (PDC's) geïnduceerd door vitamine C (vitamine C) en 1,25-dihydroxy vitamine D [1,25-(OH)2D3]. Daarnaast evalueren we het vermogen van PDC's te differentiëren in osteocytes, chondrocyten en adipocytes.

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

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetics

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicine

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Biology

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

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

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