Plenary Poster 33rd ASM of the Australian & New Zealand Bone & Mineral Society 2023

Isolation and comparison of osteoblast lineage cells from periosteal, endocortical and intracortical bone surfaces (#110)

Narelle E McGregor 1 , Patricia WM Ho 1 , Natalie A Sims 1 2 , Ivo Kalaizic 3 , Natalie KY Wee 1 2
  1. Bone Cell Biology and Disease Unit, St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia
  2. Depertment of Medicine at St Vincents Hospital, The University of Melbourne, Melbourne, Victoria, Australia
  3. Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA

Bone houses multiple niche microenvironments with different cellular compositions, including the bone marrow, and the endocortical, intracortical, and periosteal surfaces. This may underpin differences in osteoblast progenitor populations and their responses to therapy. Our aim was to isolate osteoblast-lineage cells from each microenvironment and identify phenotypic and transcriptional differences.

By dissection and serial digestion of long bones, we achieved simultaneous isolation of cells from bone marrow, endocortical, intracortical and periosteal surfaces from 6-12 week old mice, with osteoblast-related gene promoters driving fluorescent reporter expression (OsxCherry.Col1a1GFP). Cells were cultured for 7 days in regular αMEM media.

At days 6-7 in culture, bone marrow, endocortical, and intracortical cells grew sparsely, while periosteal cells grew as colonies. Very few, if any, bone marrow-derived cells expressed OsxCherry+. In contrast, endocortical, intracortical and periosteal derived cells contained >40% OsxCherry+ cells. Very few Col1a1GFP+ cells were observed in any culture in this short time frame. This suggests that isolation of cells close to bone surfaces yields a greater proportion of cells that can commit to the osteoblast lineage.

By qPCR, endocortical, intracortical and periosteal cells exhibited greater mRNA levels for osteoblast commitment (Runx2, Sp7) and differentiation genes (Alpl, Col1a1, Bgalp) than bone marrow stromal cells (BM). Endocortical and intracortical cells had lower adipogenesis-associated mRNAs (PPARγ, AdipoQ) compared to BM. Periosteal cells showed no difference in PPARγ, but greater AdipoQ mRNA transcripts than BM cells. This suggests stromal populations from endocortical and intracortical surfaces have stronger osteogenic potential but less capacity to form adipocytes compared to stromal cells from periosteum and BM.

We conclude that it is feasible to culture stromal cells from different long bone surfaces, and this reveals that stromal cells isolated from close to bone surfaces more readily commit to osteoblast differentiation than marrow cells, and differ in their potential to become adipocytes.