Isolation and Osteogenic Differentiation of Rat Periosteum-derived Cells

Abstract

Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived osteoprogenitors could be more suitable. Periosteum-derived cells were isolated from the tibiae of adult Wistar rats (n = 12). The osteogenic potential with regard to alkaline phosphatase activity, morphology, nodule formation and mineralization was studied by culturing them in an osteogenic medium for up to 4 months. Seventy-five percent of the cultures (n = 9) did not show any increase in alkaline phosphatase activity nor nodule formation during long-term culture for up to 4 months. Nevertheless, in 25% of the cultures, alkaline phosphatase activity started from negligible (<5 mM pNP/mg protein) and increased towards approximately 50 mM pNP/mg protein. Three-dimensional nodule formation was observed at passages 3-5. In further passages (P5-P7), nodule formation capacity decreased and a diffuse mineralization pattern was observed. Suitable cultures with osteogenic capacity, can be selected at early passages based on the presence of cuboidal cells. These cells have the advantage of retaining their osteogenic potential even after prolonged cultivation (6-7 passages) before final differentiation occurs. Although periosteal cells are suitable for long term in vitro evaluation of biomaterials, the isolation and selection is time consuming. Hence, a more appropriate source to study cell/biomaterial interactions should be more convenient.

Uncovering the periosteum for skeletal regeneration: The stem cell that lies beneath

“…Furthermore, culture expansion of PDC appears to be STAT3 dependent as inhibition of this pathway, either through interference with JAK2 or STAT3 directly, abrogates proliferation induced by serum growth factors [62]. While PDC do not overtly express osteogenic or chondrogenic properties during expansion, they can be induced to differentiate along the osteogenic, chondrogenic and adipogenic lineage in vitro by subjecting them to specific differentiation conditions, confirming their multipotent nature [44,57,60,61,63]. Evidence for the presence of genuine skeletal stem cells in the periosteum has been delivered by single-cell lineage analysis, showing that the periosteum contains cells with mesenchymal multipotency (osteogenic, chondrogenic, adipogenic and myogenic) at the clonal level [61].…”

“…For the isolation of animal PDC, either a similar technique is used or the periosteum is isolated by simply peeling off the periosteal layer from the bone. This method is particularly useful in young animals where the periosteal layer is relatively thick and only loosely attached to the bone [55][56][57][58][59]. In adult mice, the periosteum is only a few cell layers thick and thus difficult to isolate by dissection.…”

Concise Review: The Periosteum: Tapping into a Reservoir of Clinically Useful Progenitor Cells

“…The observed shapes may be elongated, spindle‐shaped, triangular, or cuboidal. Some studies observed uniform morphology within the population, some are able to distinguish subpopulations of cells visually, and others report a more cuboidal or polyhedral morphology during early PDC progression along chondrogenic or osteogenic lineages [69, 86]. The morphology of PDCs also varies from species to species; for instance, rabbit‐derived PDCs are smaller than human PDCs under the same culture conditions [71].…”

Periosteum-Mimicking Tissue-Engineered Composite for Treating Periosteum Damage in Critical-Sized Bone Defects

“…Cell adhesion and proliferation on the scaffold not only define the biocompatibility of the scaffold but also its potential for in vivo applications. To simulate the cell growth characteristics, periosteum cells were isolated from rat tibial periosteum and expanded in culture, as shown in Figure A,B, following an earlier report Figure A shows the explant culture of the rat tibial periosteum, where cells can be observed to migrate from the explant to the tissue culture plate.…”

“…To simulate the cell growth characteristics, periosteum cells were isolated from rat tibial periosteum and expanded in culture, as shown in Figure 3A,B, following an earlier report. 35 Figure 3A shows the explant culture of the rat tibial periosteum, where cells can be observed to migrate from the explant to the tissue culture plate. We first studied the migration of periosteal cells into the PUAOCC membrane.…”