Malicev E, Marolt D, Kregar Velikonja N, Kreft M E, Drobnic M, Rode M
Educell d.o.o., Letaliska c. 33, 1000 Ljubljana, Slovenia.
Biotechnol Bioeng. 2008 Jul 1;100(4):773-81. doi: 10.1002/bit.21815.
The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.
通过植入自体成骨细胞并结合合适的支架来增强骨再生的能力在临床上具有重大意义。我们研究的目的是比较组织工程构建物和单层培养物中牙槽骨细胞的生长和分化情况，作为开发常规制备类骨组织构建物程序的基础。从四名人类供体获取牙槽骨组织，并建立细胞外植体培养。将扩增后的细胞接种到大孔羟基磷灰石颗粒上，并在添加有成骨分化因子的培养基中培养长达3周。同时建立平行的对照单层培养物，并在添加或不添加成骨补充剂的培养基中培养。每周在不同培养条件下测定细胞增殖、碱性磷酸酶（AP）活性以及AP、骨桥蛋白和骨钙素的基因表达。组织构建物中的细胞呈现出与对照单层培养物相似的生长模式：在培养的前2周观察到增殖增强，随后细胞数量减少。在成骨培养基中培养3周时，所有培养物中的AP活性均高于对照培养基。在两种类型的培养基中，单层培养物中的基因表达水平稳定，而在组织构建物中，它们呈现出成骨分化模式。对接种细胞的构建物进行光镜和扫描电镜检查显示细胞分布均匀，以及细胞附着并生长到羟基磷灰石颗粒的内部区域。我们的结果表明，通过在测试的羟基磷灰石颗粒上培养牙槽骨细胞，可以制备出具有呈现分化表型的活细胞的类骨构建物。
