Analysis of the attachment and differentiation of three-dimensional rotary wall vessel cultured human preosteoblasts on dental implant surfaces.

PURPOSE: The purpose of this study was to determine whether osseous tissues engineered in three-dimensional (3D) environments preserved their mineralizing capacity and retained biologic characteristics when cultured on dental implant surfaces. MATERIALS AND METHODS: Human preosteoblast cells were cultured in both 3D rotary wall vessels and on 2D tissue culture plastic plates for 3 days. Aggregates from the 3D chambers and cells from the 2D plates were collected and transferred to commercially pure titanium disks with either 600-grit polished or sandblasted surfaces. These were cultured for an additional 7 days. The aggregates and cells from the disks were collected and prepared for scanning electron microscopy for microscopic evaluation and atomic adsorption assays for mineral content analysis. Additionally, staining with Alizarin red S was performed to compare the mineralization amount and pattern in each group. Polymerase chain reaction analysis was performed to evaluate expression of osteogenic genes, including Runx2, FAK, bone morphogenetic protein 2, and osteocalcin. RESULTS: Cells from 3D rotary wall vessel cultures attached to implant surfaces and presented cell attachment and growth patterns similar to those of standard 2D cultured cells, showing evidence of radial and random growth, yet they formed multiple focal niches on implant surfaces out of which cells proliferated. The 3D cultured cells and osseous tissues retained higher amounts of mineral formed during the initial culture and showed a higher tendency toward mineralization on implant surfaces compared to standard cultured cells. The 3D cultured cells and osseous tissues on implant surfaces at 1 week showed higher key gene protein expression. RNA expression at 1 week was equivalent to that of standard cultured cells. CONCLUSION: Culture of human osteogenic cells and tissues in 3D rotary wall vessels may expedite the osseointegration process on dental implant surfaces, thus reducing the overall treatment time.

Three-dimensional culture environments enhance osteoblast differentiation.

PURPOSE: In previous work from our laboratory, we demonstrated that the three-dimensional (3D) cell cultures developed in simulated microgravity environments enhanced osseous-like aggregate formation and accelerated preosteoblast cell differentiation. Thus, as described here, we hypothesize that aggregate formation and mineralization would occur with fewer than 10 x 10(6) cells as previously described. MATERIALS AND METHODS: Human preosteoblastic cells were cultured at different concentrations in a rotary wall vessel to simulate microgravity for 7 days. Aggregate size was assessed, and mineralization and collagen expression detected using Von Kossa and Masson Trichrome staining. Scanning electron microscopy was used for structural and elemental analysis. Immunohistochemistry was used to detect expression of the osteogenic markers BSPII and osteopontin (OP). RESULTS: Size and calcium expression were dependent upon cultured cell number (p < 0.01). Calcium and collagen expression were detected throughout the aggregate, but organization was independent of cell number. Aggregates had similar microscopic structural patterns demonstrating organized development. Presence of BSPII and OP showed that the aggregates share common differentiation proteins with in vivo bone formation. CONCLUSIONS: These results may lead to novel bone engineering techniques associated with dental treatment.

Osteoblast differentiation is enhanced in rotary cell culture simulated microgravity environments.

PURPOSE: As the aging population increases, more people will become reliant on regenerative dental medicine for implant therapy. The objective of this study was to test the hypothesis that 3D rotary cell culture (RCC) environments created by simulated microgravity would enhance osteogenic gene expression using integrin mediated pathways. MATERIALS AND METHODS: Human embryonic palatal mesenchymal (HEPM, ATCC 1486) pre-osteoblasts were cultured in either RCC to create 3D environments or in 2D monolayers for 72 hours. Gross phenotypic analysis was performed using Alizarin Red S staining for calcium and microscopy. Real-time PCR analysis was used to detect differences in osteoblast gene expression. Aggregates developed in 3D RCC environments were treated with or without antibody to the collagen-I integrin receptor alpha2beta1 to determine whether this molecular pathway might contribute to the development of a mineralized matrix. RESULTS: Microscopic analysis demonstrated that RCC environments promoted 3D aggregate formation by 72 hours without any scaffold. The mass appeared osseous-like with a white, shiny, translucent surface. The center was amorphous with areas of vacuolization, tubule-like structures, and fibrous-like extensions. Real-time PCR data showed that 3D environments enhanced osteogenic gene expression as compared with 2D monolayer culturing conditions. At 72 hours, changes in levels of osteogenic gene expression were noted. Cbfa1, a necessary transcription factor for osteoblast differentiation, was expressed 33% higher (p= 0.26); Collagen 1, 69% higher (p= 0.05); Osterix, 49% higher (p= 0.001); and BSPII, 54% higher (p= 0.001) than osteoblasts cultured for 72 hours in standard 2D monolayer conditions. When cultured in the presence of collagen alpha2beta1 integrin receptor antibody, 3D aggregates had decreased levels of mineralization as compared with non-treated aggregates. CONCLUSION: RCC enhances osteoblast differentiation using integrin mediated pathways.