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.
