Bone tissue engineering with human stem cells

Treatment of extensive bone defects requires autologous bone grafting or implantation of bone substitute materials. An attractive alternative has been to engineer fully viable, biological bone grafts in vitro by culturing osteogenic cells within three-dimensional scaffolds, under conditions supporting bone formation. Such grafts could be used for implantation, but also as physiologically relevant models in basic and translational studies of bone development, disease and drug discovery. A source of human cells that can be derived in large numbers from a small initial harvest and predictably differentiated into bone forming cells is critically important for engineering human bone grafts. We discuss the characteristics and limitations of various types of human embryonic and adult stem cells, and their utility for bone tissue engineering

Primary human alveolar bone cells isolated from tissue samples acquired at periodontal surgeries exhibit sustained proliferation and retain osteogenic phenotype during in vitro expansion.

OBJECTIVES: Bone tissue regeneration requires a source of viable, proliferative cells with osteogenic differentiation capacity. Periodontal surgeries represent an opportunity to procure small amounts of autologous tissues for primary cell isolation. Our objective was to assess the potential of human alveolar bone as a source of autologous osteogenic cells for tissue engineering and biomaterials and drug testing studies. MATERIALS AND METHODS: Alveolar bone tissue was obtained from 37 patients undergoing routine periodontal surgery. Tissue harvesting and cell isolation procedures were optimized to isolate viable cells. Primary cells were subcultured and characterized with respect to their growth characteristics, gene expression of osteogenic markers, alkaline phosphatase activity and matrix mineralization, under osteogenic stimulation. RESULTS: Alveolar bone cells were successfully isolated from 28 of the 30 samples harvested with bone forceps, and from 2 of the 5 samples obtained by bone drilling. The yield of cells in primary cultures was variable between the individual samples, but was not related to the site of tissue harvesting and the patient age. In 80% of samples (n = 5), the primary cells proliferated steadily for eight subsequent passages, reaching cumulative numbers over 10(10) cells. Analyses confirmed stable gene expression of alkaline phosphatase, osteopontin and osteocalcin in early and late cell passages. In osteogenic medium, the cells from late passages increased alkaline phosphatase activity and accumulated mineralized matrix, indicating a mature osteoblastic phenotype. CONCLUSIONS: Primary alveolar bone cells exhibited robust proliferation and retained osteogenic phenotype during in vitro expansion, suggesting that they can be used as an autologous cell source for bone regenerative therapies and various in vitro studies

Growth of alveolar bone cells during the subculturing.

<p>Cells were isolated from 5 samples of alveolar bone in parallel by explant cultures (e-, full symbols) and by collagenase digestion (c-, open symbols) and were continuously subcultured for up to 8 subsequent passages. Increases in total numbers of cells (A) and in number of population doublings (PD) (B) are shown as a function of culture time, for each cell culture sample.</p

Mineralization potential of cultured alveolar bone cells.

<p>*Mineralization was evaluated semi-quantitatively in comparison to control cultures as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092969#pone-0092969-g006" target="_blank"><b>Fig. 6</b></a>, and marked with symbols + (denoting mineralization), +− (denoting minimal mineralization) and – (denoting absence of mineralization). P stands for cell passage that was evaluated. D stands for day of induction that was evaluated. / Denotes that evaluation was not conducted.</p

Relative gene expression levels of alkaline phosphatase (A), osteopontin (B) and osteocalcin (C).

<p>Alveolar bone cells isolated by explant cultures (grey bars) and by collagenase digestion (black bars) from 13 different patient samples were cultured separately and evaluated at the second passage. Data show expression levels of individual samples normalized to the osteoblastic control sample (-fold change). No significant differences in expression levels were found between cells from the two different isolation procedures (p>0.05).</p

Isolation and yield of alveolar bone cells.

<p>Representative images of cells growing in explant cultures (n = 20) (A) and in collagenase digestion cultures (n = 18) (B) are shown. Scale bars: 100 μm. Average cell yields in confluent primary cultures were not significantly different between the two cell isolation techniques (p>0.05) (C). Cell yields varied greatly between the samples obtained from individual patients; no relationship between the cell yield and the site of tissue harvesting was observed (maxilla or mandible, tooth position numbers 1–3 or 4–7) (D). Dotted line indicates the average cell yield in all cultures (3.200 cells/mm³ of tissue).</p