Single-cell RNA sequencing (scRNA-Seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-Seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-Seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-Seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-Seq on freshly recovered long bone endocortical cells from mice that received either vehicle or sclerostin-neutralizing antibody for 1 week. We were unable to detect significant changes in bone anabolism-associated transcripts in immature and mature osteoblasts recovered from mice treated with sclerostin-neutralizing antibody; this might be a consequence of being underpowered to detect modest changes in gene expression, because only 7% of the sequenced endocortical cells were osteoblasts and a limited portion of their transcriptomes were sampled. We conclude that scRNA-Seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single-cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

Metal implants are commonly used in orthopedic surgery. The mechanical stability and longevity of implants depend on adequate bone deposition along the implant surface. The cellular and molecular mechanisms underlying peri‐implant bone formation (ie, osseointegration) are incompletely understood. Herein, our goal was to determine the specific bone marrow stromal cell populations that contribute to bone formation around metal implants. To do this, we utilized a mouse tibial implant model that is clinically representative of human joint replacement procedures. Using a lineage‐tracing approach, we found that both Acta2.creERT2 and Tmem100.creERT2 lineage cells are involved in peri‐implant bone formation, and Pdgfra‐ and Ly6a/Sca1‐expressing stromal cells (PαS cells) are highly enriched in both lineages. Single‐cell RNA‐seq analysis indicated that PαS cells are quiescent in uninjured bone tissue; however, they express markers of proliferation and osteogenic differentiation shortly after implantation surgery. Our findings indicate that PαS cells are mobilized to repair bone tissue and participate in implant osseointegration after surgery. Biologic therapies targeting PαS cells might improve osseointegration in patients undergoing orthopedic procedures. © 2021 American Society for Bone and Mineral Research (ASBMR).

Metal implants are commonly used in orthopaedic surgery. The mechanical stability and longevity of implants depend on adequate bone deposition along the implant surface. The cellular and molecular mechanisms underlying peri-implant bone formation (i.e. osseointegration) are incompletely understood. Herein, our goal was to determine the specific bone marrow stromal cell populations that contribute to bone formation around metal implants. To do this, we utilized a mouse tibial implant model that is clinically representative of human joint replacement procedures. Using a lineage-tracing approach with the Acta2.creERT2 and Tmem100.creERT2 transgenic alleles, we found that Pdgfra- and Ly6a/Sca1-expressing stromal cells (PαS cells) multiply and differentiate in the peri-implant environment to give rise to osteocytes in newly formed bone tissue. Single cell RNA-seq analysis indicated that PαS cells are quiescent in uninjured bone tissue; however, they express markers of proliferation and osteogenic differentiation shortly after implantation surgery. Our findings indicate that PαS cells are mobilized to repair bone tissue and facilitate implant osseointegration following surgery. Biologic therapies targeting PαS cells might improve osseointegration in patients undergoing orthopaedic procedures.

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