Dmp1 Lineage Cells Contribute Significantly to Periosteal Lamellar Bone Formation Induced by Mechanical Loading But Are Depleted from the Bone Surface During Rapid Bone Formation

Abstract

Previous work has shown that osteoprogenitor cells (Prx1+) and pre-osteoblasts (Osx+) contribute to mechanical loadinginduced bone formation. However, the role of mature Dmp1-expressing osteoblasts has not been reported. In this study we assessed the contribution of osteoblast lineage cells to bone formation at an early time point following mechanical loading (day 8 from onset of loading). We labeled Osx-expressing and Dmp1-expressing cells in inducible Osx and Dmp1 reporter mice (iOsx-Ai9, iDmp1-Ai9), respectively, 3 weeks before loading. Mice were then loaded daily for 5 days (days 1-5) and were dosed with 5-ethynyl-2 0 -deoxyuridine (EdU) in their drinking water until euthanasia on day 8. Mice were loaded to lamellar and woven bone inducing stimulation (À7 N/1400 με, À10 N/2000 με) to assess differences in these processes. We found varied responses in males and females to the loading stimuli, inducing modest lamellar (females, À7 N), moderate lamellar (males, À10 N), and robust woven (females, À10 N) bone. Overall, we found that preexisting (ie, lineage positive) Osx-expressing and Dmp1-expressing cells contribute largely to the bone formation response, especially during modest bone formation, while our results stuggest that other (non-lineage-positive) cells support the sustained bone formation response during rapid bone formation. With moderate or robust levels of bone formation, a decrease in preexisting Osx-expressing and Dmp1-expressing cells at the bone surface occurred, with a near depletion of Dmp1-expressing cells from the surface in female mice loaded to À10 N (from 52% to 11%). These cells appeared to be replaced by lineage-negative cells from the periosteum. We also found a dose response in proliferation, with 17% to 18% of bone surface cells arising via proliferation in modest lamellar, 38% to 53% in moderate lamellar, and 59% to 81% in robust woven bone formation. In summary, our results show predominant contributions by preexisting Osx and Dmp1 lineage cells to loading-induced lamellar bone formation, whereas recruitment of earlier osteoprogenitors and increased cell proliferation support robust woven bone formation.

Postnatal Osterix but not DMP1 lineage cells significantly contribute to intramembranous ossification in three preclinical models of bone injury

“…These findings suggest, that pre-existing Osx + lineage osteoprogenitors and their progeny contribute less to total callus area and cellularity in the less traumatic ulnar stress fracture than the femoral fracture model. The overall result that pre-existing Osx + lineage cells contribute more to callus cells than DMP1 + lineage cells with higher degrees of bone damage are consistent with previous reports using anabolic tibial loading at graded force levels (Harris and Silva, 2022), and may potentially reflect the smaller overall cellularity and decreased proliferative processes in stress fracture versus full fracture injuries as previously shown (Coates et al, 2019). However, it may also reflect changes in anatomic location (ulna versus femur) or slight differences in analysis regions between the two fracture models used in the current study (i.e.…”

“…Male and female mice were used as available and in approximately equal numbers among experimental groups. We utilized both males and females in this study since both mouse sexes have been readily utilized in these inducible Cre lines in previous literature ( Buettmann et al, 2019 ; McKenzie et al, 2019 ; Harris and Silva, 2022 ). Mice were group housed under a standard 12-h light/dark cycle and given access to food and water ad libitum .…”

“…However, because cells were labeled before and during healing by continuous tamoxifen, we could not determine the contribution of pre-existing versus newly differentiated Osx + and DMP1 + lineage cells and their progeny (herein labeled Osx + or DMP1 + lineage cells) to fracture callus tissues. More recent work by our lab group used pulse-chase labeling strategies and demonstrated that pre-existing Osx + and DMP + lineage cells and their progeny contribute significantly to early lamellar bone formation following anabolic (non-damaging) skeletal loading ( Zannit and Silva, 2019 ; Harris and Silva, 2022 ). Interestingly, we observed that these lineage-labeled cells, especially DMP1 + lineage cells, are rapidly depleted from the periosteal bone surface when a higher loading stimulus induces woven bone formation ( Zannit and Silva, 2019 ; Harris and Silva, 2022 ).…”

“…More recent work by our lab group used pulse-chase labeling strategies and demonstrated that pre-existing Osx + and DMP + lineage cells and their progeny contribute significantly to early lamellar bone formation following anabolic (non-damaging) skeletal loading ( Zannit and Silva, 2019 ; Harris and Silva, 2022 ). Interestingly, we observed that these lineage-labeled cells, especially DMP1 + lineage cells, are rapidly depleted from the periosteal bone surface when a higher loading stimulus induces woven bone formation ( Zannit and Silva, 2019 ; Harris and Silva, 2022 ). Together these data indicate that Osx + and DMP1 + lineage cells play a role in load-induced bone formation and bone healing, however the relative contributions of pre-existing versus newly-derived Osx + and DMP1 + lineage cells and their progeny across various bone injury types remains poorly defined.…”

A Critical Role of the Bone Marrow Envelope in Human Bone Remodeling

“…5), just as in loading‐induced periosteal bone formation, where preexisting DMP1‐positive cells are supplemented by “lineage‐negative” cells. ( 60 )…”