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Anabolic agents, such as intermittent parathyroid hormone (PTH), exert their treatment efficacy through activation of two distinct bone formation processes, namely, remodeling-based bone formation (RBF, bone formation coupled with prior bone resorption) and modeling-based bone formation (MBF, bone formation without prior activation of bone resorption). However, if not followed by an antiresorptive agent, treatment benefit was quickly lost upon withdrawal from anabolic agents. By using in vivo micro–computed tomography imaging and multiplex cryohistology with sequential immunofluorescence staining, we investigated the temporal response of newly formed bone tissue from MBF and RBF and the preexisting bone tissue to withdrawal from PTH treatment and the associated cellular activity in an ovariectomized (OVX) rat model. We first demonstrated continued mineral apposition at both RBF and MBF sites following PTH discontinuation, resulting in an extended anabolic effect after 1-week withdrawal from PTH. It was further discovered that MBF sites had a greater contribution than RBF sites to the extended anabolic effect upon early withdrawal from PTH, evidenced by a higher percentage of alkaline phosphatase-positive (ALP+) surfaces and far greater bone formation activity at MBF versus RBF sites. Furthermore, significant bone loss occurred after 3 weeks of discontinuation from PTH, resulting from marked loss of newly formed bone tissue from RBF and preexisting bone tissue prior to treatment. In contrast, MBF surfaces had a delayed increase of tartrate-resistant acid phosphatase activity following PTH discontinuation. As a result, newly formed bone tissue from MBF had greater resistance to PTH discontinuation–induced bone loss than those from RBF and preexisting bone. Understanding various responses of two distinct bone formation types and preexisting bone to anabolic treatment discontinuation is critical to inform the design of follow-up treatment or cyclic treatment strategies to maximize treatment benefit of anabolic agents. © 2022 American Society for Bone and Mineral Research (ASBMR).

 

Abstract Mesenchymal stem cells (MSCs) are multipotent cells that can replicate and differentiate to different lineages of mesenchymal tissues, potentiating their use in regenerative medicine. Our previous work and other studies have indicated that mild heat shock enhances osteogenesis. However, the influence of pro-inflammatory cytokines on osteogenic differentiation during mildly elevated temperature conditions remains to be fully explored. In this study, human MSCs (hMSCs) were cultured with Tumor Necrosis Factor-alpha (TNF-a), an important mediator of the acute phase response, and Interleukin-6 (IL-6) which plays a role in damaging chronic inflammation, then heat shocked at 39ºC in varying frequencies - 1 hour per week (low), 1 hour every other day (mild), and 1 hour intervals three times per day every other day (high). DNA data showed that periodic mild heating inhibited suppression of cell growth caused by cytokines and induced maximal proliferation of hMSCs while high heating had the opposite effect. Quantitative osteogenesis assays show significantly higher levels of alkaline phosphatase activity and calcium precipitation in osteogenic cultures following mild heating compared to low heating or non-heated controls. These results demonstrate that periodic mild hyperthermia may be used to facilitate bone regeneration using hMSCs, and therefore may influence the design of heat-based therapies in vivo.