Isolation of Human Osteoblast‐Like Cells and In Vitro Amplification for Tissue Engineering

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Abstract
As the field of dental implants continues to grow at a rapid rate so does our quest to find new techniques to enhance bone grafting. Tissue engineering 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 within 2 different 3‐dimensional (3‐D) constructs targeted for tissue engineering applications. Ultimately, HOBs that have been amplified within 3‐D constructs may be employed for bone regeneration techniques, such as onlay and sinus grafting prior to implant placement. Our cell isolation protocol employed human fetal calvaria tissue sequentially digested with trypsin and collagenase. The HOB cells from only the third and fourth digests were obtained, cultured and evaluated within the constructs. An osteoblast‐like phenotype was in part verified for these HOB cells by demonstrating a significantly higher alkaline Phosphatase activity than for human gingival fibroblasts, and a comparable level to the Osteoblast cell line MG‐63. The HOB cells were cultured within either poly (D,L‐lactide) (PLA) or a fused fiber ceramic and evaluated for the ability to support in vitro HOB amplification. HOB proliferation was validated by scanning electron microscopy, identifying cells throughout the 3‐D constructs. Continuous cell viability was demonstrated for the duration of the 33‐day evaluation period and the extent of cell amplification reached approximately 20 times the seeding density. The in vitro amplification results further indicate that tissue engineering strategies with either the PLA or fused fiber construct may be suitable for bone regeneration therapy for dental implants. J Periodontol 1998;69:1256–1262.