Effects of Two Amino Acids in Porous Hydroxyaptatite Scaffolds

Objectives: For tooth regeneration, a porous hydroxyapatite (HA) component is necessary, because a tooth has three-dimensional construction. The structure of an HA scaffold and any factors for differentiation of undifferentiated mesenchymal cells have been examined for hard-tissue formation by (a smaller number of) undifferentiated mesenchymal cells. It has been reported that L-leucine (leu) and L-lysine (lys) play roles in the formation of osseous tissue by bone marrow cells. The purpose of this study was to compare the effects of leu and lys for osteogenesis in the pores of a porous HA scaffold with a smaller number of bone marrow cells.

Methods: Porous cylindrical HA scaffolds were soaked in 100 mM concentrations of leu and lys solutions and dried. They were kept for 2 hrs in bone marrow cell suspension obtained from bone shafts of the femora of 6-week-old male Fischer 344 rats. The suspension was at a concentration of 1 x 10⁶ cells/mL. The scaffolds were implanted into the dorsal subcutis of the syngeneic rats. Four weeks post-operatively, they were removed from the subcutis. The paraffin-embedded specimens were cut into 6-µm serial sections and stained with hematoxylin-eosin. Scaffolds immersed in MEM were used as controls. To compare osseous formation in each amino acid prepond scaffold, we measured osteocalcin and alkaline phosphatase (ALP) activity.

Results: The quantity of osteocalcin in the scaffold immersed in leu was 6.48 ng/mL. In the scaffold immersed in lys, it was 9.88 ng/mL. ALP activity in the scaffold immersed in leu was 75.15 µM, and in the scaffold immersed in lys, 686.16 µM. There was a statistically significant difference in the quantities of osteocalcin and ALP activity between the scaffolds immersed in leu and lys.

Conclusion: Bone formation in the pores of the HA scaffolds was enhanced more clearly by lys than by leu.