The periosteum as a cellular source for functional tissue engineering

Abstract

The periosteum, a specialized fibrous tissue composed of fibroblast, osteoblast, and progenitor cells, may be an optimal cell source for tissue engineering based on its accessibility, the ability of periosteal cells to proliferate rapidly both in vivo and in vitro, and the observed differentiation potential of these cells. However, the functional use of periosteum-derived cells as a source for tissue engineering requires an understanding of the ability of such cells to elaborate matrix of different tissues. In this study, we subjected a population of adherent primary periosteum-derived cells to both adipogenic and osteogenic culture conditions. The commitment propensity of periosteal cells was contrasted with that of well-characterized phenotypically pure populations of NIH3T3 fibroblast and MC3T3-E1 osteoblast cell lines. Our results demonstrate that the heterogeneous populations of periosteal cells and NIH3T3 fibroblasts have the ability to express both osteoblast-like and adipocyte-like markers with similar potential. This raises the question of whether fibroblasts within the periosteum may, in fact, have the potential to behave like progenitor cells and play a role in the tissue's multilineage potential or whether there are true stem cells within the periosteum. Further, this study suggests that expanded periosteal cultures may be a source for tissue engineering applications without extensive enrichment or sorting by molecular markers. Thus, this study lays the groundwork for future investigations that will more deeply enumerate the cellular sources and molecular events governing periosteal cell differentiation.

FIG. 1.

Magnified (10×) micrographs of alkaline phosphatase (ALP)-stained cell cultures (A, C, D, E) after 1 week in osteoinductive medium and Oil red O–stained cell cultures (B, D, F) after 1 week in adipoinductive medium for all three cell types: MC3T3-E1 osteoblasts (A, B), primary periosteal cells (C, D), and NIH3T3 fibroblasts (E, F). All micrographs are of cell cultures initially seeded at high density; as in all cases, a larger percentage of positive staining was observed (p < 0.01). Panel (D) demonstrates that periosteal cells cultured in adipo-inductive medium for 1 week stain positively for ALP, suggesting that there was a subpopulation within the periosteal culture that expressed ALP in the absence of soluble osteogenic differentiation factors.

FIG. 2.

Osteoblasts, periosteal cells, and fibroblasts stain positively for the osteogenic marker, ALP, when cultured at a high density in osteo-inductive medium. MC3T3-E1 osteoblasts expressed significantly (p < 0.05) higher levels of ALP activity than either periosteal cells of fibroblasts initially seeded at a high density; however, there was no expression of ALP in osteoblast cultures initially seeded at low densities. In both high- and low-density cultures, periosteal cells and NIH3T3 fibroblasts had relatively similar levels of ALP expression with no significant difference.

FIG. 3.

Periosteal cells and fibroblasts have a similar propensity for the adipogenic lineage when cultured for 1 week in adipo-inductive medium. MC3T3-E1 osteoblasts did not stain positively for adipogenic markers, indicating that they are a committed mature cell type. A pure population of fibroblasts has a similar potential to express adipogenic lineage markers.