The Research Of PLA-PEG/HA-g-PLLA Composite Material On The Osteogenesis In Vitro

Objective: To investigate the effect of PLA-PEG/HA-g-PLLAcomposite material with different content of HA-g-PLLA on adhesion,multiplication and osteogensis of osteoblasts, so as to confirm theosteogenic induction effect of PLA-PEG/HA-g-PLLA is well and lay thefoundation of experiment for the repair and healing on bone defect.Methods: Four fiber membrane composite materials with differentcontent of HA-g-PLLA(5%PLA-PEG,5%PLA-PEG/5%HA-g-PLLA,5%PLA-PEG/10%HA-g-PLLA,5%PLA-PEG/15%HA-g-PLLA) weredivided into four experimental groups, while glass is used as the controlgroup. After sterilization, all groups were fixed in24well cell cultureplates. Each group has four holes. Rat osteoblasts were inoculated on thematerials after prewet, put it into incubator with37℃,5%CO2for4hours to make sure the osteoblasts adherent well. Each hole was put into1ml complete medium, then put the plates into incubator and change the culture solution every other day. MTT test was made respectively on1th,4th and7th day after inoculating and then evaluated the multiplicationeffect of cells. We made DAPI staining on4th and7th day afterinoculating and then observed the adhesion of cells in each group by theinverted fluorescence microscope. On7th day after inoculating, accordingto the instructions of alkaline phosphatase (AKP) kit, we measured thealkaline phosphatase activity of osteoblast (AKP) in each group toevaluate the effect of ossification of materials. Results:1. The osteoblastsgrow well on the materials in each group, uniform distribution with manyangle spindle morphological structure.2. In the experiment of cellmultiplication, the OD values of fiber membrane material groups arehigher than that of the control group in all of the three MTT tests. TheOD values from high to low is5%PLA-PEG/10%HA-g-PLLA＞5%PLA-PEG/15%HA-g-PLLA＞5%PLA-PEG/5%HA-g-PLLA＞5%PLA-PEG. The OD value of PLA-PEG/HA-g-PLLA roughly presentsa waveform with the increasing addition of HA-g-PLLA. The OD valueof5%PLA-PEG/10%HA-g-PLLA is most close to the wave crest. Therewas statistical significance in the difference between each two groups ateach time point(P＜0.05).3. In the cell adhesion assay, after DAPIstaining, the experimental groups had more and better growth cells thanthat in the control group. As the same, the result shows that the number ofcells adhered to the materials follows a rule of increasing firstly and then decreasing with the increasing content of HA-g-PLLA in thePLA-PEG/HA-g-PLLA. Especially, the number of cells is the most in thegroup of5%PLA-PEG/10%HA-g-PLLA.4. In the test of alkalinephosphatase (AKP) activity, the result indicated that the activity ofalkaline phosphatase of PLA-PEG/HA-g-PLLA presents a waveform ofincreasing firstly and then decreasing as well with rising addition ofHA-g-PLLA. There also was statistical significance in the differencebetween each two group（sP＜0.05）. In the three tests, the results of themagreed well with each other and authenticated mutually. The accuracy ofthe results is improved. Conclusion:1. The effect ofPLA-PEG/HA-g-PLLA composite material on adhesion, multiplicationand osteogensis of osteoblasts is better than that of PLA-PEG and glass.PLA-PEG/HA-g-PLLA is a good material for repair and healing on bonedefect.2. The effect of PLA-PEG/HA-g-PLLA composite material onadhesion, multiplication and osteogensis of osteoblasts increases withrising addition of HA-g-PLLA in a certain range, just5%PLA-PEG/10%HA-g-PLLA approximately reaches the maximum.