Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering

As THE FIELD OF DENTAL IMPLANTS continues to grow at a rapid rate so does o ur quest to find new techniques to enhance bone grafting. Tissue engineerin g is an exciting new technique in bone grafting. Therefore, the purposes of this study were to develop a simple, reproducible method to isolate human osteoblast-like cells (HOBs) and to evaluate in vitro cell proliferation wi thin 2 different 3-dimensional (3-D) constructs targeted for tissue enginee ring applications. Ultimately, HOBs that have been amplified within 3-D con structs may be employed for bone regeneration techniques, such as onlay and sinus grafting prior to implant placement. Our cell isolation protocol emp loyed human fetal calvaria tissue sequentially digested with trypsin and co llagenase. The HOB cells from only the third and fourth digests were obtain ed, cultured and evaluated within the constructs. An osteoblast-like phenot ype was in part verified for these HOB cells by demonstrating a significant ly higher alkaline phosphatase activity than for human gingival fibroblasts , and a comparable level to the osteoblast cell line MG-63. The HOB cells w ere cultured within either poly (D,L-lactide) (PLA) or a fused fiber cerami c and evaluated for the ability to support in vitro HOB amplification. HOB proliferation was validated by scanning electron microscopy, identifying ce lls throughout the 3-D constructs. Continuous cell viability was demonstrat ed for the duration of the 33-day evaluation period and the extent of cell amplification reached approximately 20 times the seeding density. The in vi tro amplification results further indicate that tissue engineering strategi es with either the PLA or fused fiber construct may be suitable for bone re generation therapy for dental implants.