Use of 3-Dimensional Culture Chambers for Osseous Tissue Engineering

We have previously demonstrated enhanced aggregate formation and accelerated mineralization of human preosteoblastic cells when cultured in 3D-rotary wall vessel chambers. Objectives: The hypothesis of this study was rotary wall vessel culture chambers can be used to engineer osseous tissue. Methods: Human preosteoblastic cells (HEPM, ATCC 1486) were cultured in rotary wall vessels for up to 7 days. Aggregate size was measured; mineralization and collagen expression was detected using Von Kossa and Masson Trichrome stain; and immunohistochemistry was used to detect BSPII and osteopontin. Scanning electron microscopy, elemental analysis, and micro-CT was used for structural comparison, 3D reconstruction, and evaluation of relative mineral density. PCR was used to detect BMP2. Results: Calcium expression correlated to cell number (P<0.01). Calcium, collagen, BSPII, and osteopontin were detected throughout the aggregate. SEM and micro-CT analysis showed similar microscopic structural patterns within the aggregates. Progression of mineralization in 3D cultures was evident by 3 days as shown by micro-CT. A uniform ring of dense mineral on the outermost surface with an amorphous pattern of mineral within the aggregate was noted starting at 3 days. Interestingly, this expression of mineral correlated to the expression of BMP2. Indeed, BMP2 expression peaked at 3 days when mineralization was occurring as seen in micro-CT. By 7 days when mineralization appeared complete, BMP2 expression was significantly reduced. No BMP2 expression was noted in the control 2D tissue cultures. This demonstrates that 3D-rotary wall vessel cultures can be used to enhance osseous tissue engineering methods. Conclusions: These results suggest that we are engineering small volumes of bone from preosteoblasts cultured 3-dimensionally in rotary wall vessels. These methods may someday translate clinically into use for repair of small intraoral osseous defects with engineered osseous tissue. This work was supported by NIH/NIDCR R21-DE016677-01 (GS) and a 2007 AADR Student Research Fellowship(JB).