Mesenchymal stem cell proliferation in carbonate apatite-chitosan scaffold

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The need for bone repair has increased with increasing life expectancy. Bone damage caused by tooth extraction, trauma, and other pathological conditions have limitations in spontaneous bone repair. In this case, strategies to repair bone defects include autografts, allografts, and xenografts. Autografts have become the ideal choice for filling bone damage. This method has several weaknesses that can cause complications in the healing process, additional surgical procedures, pain to the donor and also limited supply of bone donors. On the other hand, allografts and xenografts have a risk of immunogenic reactions and disease transmission from donor fluid and tissue. With these weaknesses, alternative bone grafts providing ideal tissue engineering treatment needs to be improved.

Chitosan powder was dissolved in 5 ml of 2% acetic acid at room temperature, shaken for 15 minutes, then neutralized with 15 ml of 0.1 M NaOH solution After centrifugation at 1500 rpm for 10 minutes, the chitosan gel was put into the mold. To make a mixture of carbonite apatite chitosan scaffolds (CA-ChSs), chitosan scaffolds which contained 200 mg of chitosan powder were chosen. After neutralization, 50, 100, 200 and 300 mg 0.06 M CA were added to the chitosan gel containing 200 mg of chitosan powder.

During the process of making carbonite apatite and chitosan scaffolds, a solution of acetic acid was used to dissolve the chitosan powder and then neutralize it with a solution of NaOH. To remove base salts, ions or some toxic substances in a mixture of carbonite apatite and chitosan scaffolds, desalination was carried out. Furthermore, ultraviolet radiation was done for 2 hours. Samples were then ready to be tested in cell culture.

Scaffolding was chosen randomly and macroscopic photographs of scaffolding were taken using a digital camera. Microscopic structures and porosity scaffolding were observed using scanning electron microscopy.

A compressive strength test was done by using a sample of a mixture of sloping active ingredients whose top and bottom are softened with sandpaper. The sample was placed in the press machine’s compression unit, then the engine was started and set the speed and force to be measured. The load cell was slowly lowered and then stopped and the amount of force obtained was recorded.

Culture mesenchymal stem cells in the form of cell-line were implanted in a dish. After the meeting, the culture was harvested using trypsin versene solution. The media was replaced every 3-4 days and subculture was carried out every 7 days. Then the cells were transferred in small bottles and made with a density of 2 x 104 cells/ml, the cells were ready to be used for sample testing.

This test was carried out according to the recommended protocol standard for MTT essays. Cell proliferation in scaffolds is examined using an MTT essay. A 20 μl cell suspension with a density of 2 x 104 cells added to the active ingredient was placed in a 24-well tissue culture plate. After 2 hours, 980 μl DMEM was added to each well. Then the cells were incubated in an incubator (5% CO2 at 37 ° C).

On day 1, day 3, day 5 and day 7 of observation, chitosan scaffolds and a mixture of carbonate apatite – chitosan scaffolds were washed using PBS, incubated for 2 hours in 500 μl medium culture containing 50 μl MTT reagents, the well was put into an incubator (5% CO2 in 37oC). The media supernatant (110 μl) was transferred to a 96-well culture plate and absorbance was measured using a microplate reader with a wavelength of 450 nm. The results obtained were expressed in optical density (absorbent). The large absorbent from each well showed the amount of cell proliferation in culture media.

From this research it can be concluded that 200 mg of carbonite apatite is the most effective amount to be combined with chitosan scaffolds to produce excellent scaffolding from the perspective of three-dimensional structures with small pores. A mixture of carbonite apatite and chitosan scaffolds developed from 200 mg chitosan plus 50 mg of CA powder showed the highest ability of cell proliferation among groups.

Author: Aqsa Sjuhada Oki, drg, MKes and Maretaningtias Dwi Ariani

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