PMID- 34781049
OWN - NLM
STAT- MEDLINE
DCOM- 20220311
LR  - 20230102
IS  - 1873-2763 (Electronic)
IS  - 8756-3282 (Print)
IS  - 1873-2763 (Linking)
VI  - 154
DP  - 2022 Jan
TI  - NAD(P)H autofluorescence lifetime imaging enables single cell analyses of 
      cellular metabolism of osteoblasts in vitro and in vivo via two-photon 
      microscopy.
PG  - 116257
LID - S8756-3282(21)00423-3 [pii]
LID - 10.1016/j.bone.2021.116257 [doi]
AB  - Two-photon fluorescence lifetime microscopy (2P-FLIM) is a non-invasive optical 
      technique that can obtain cellular metabolism information based on the intrinsic 
      autofluorescence lifetimes of free and enzyme-bound NAD(P)H, which reflect the 
      metabolic state of single cells within the native microenvironment of the living 
      tissue. NAD(P)H 2P-FLIM was initially performed in bone marrow stromal cell 
      (BMSC) cultures established from Col (I) 2.3GFP or OSX-mCherry mouse models, in 
      which osteoblastic lineage cells were labelled with green or red fluorescence 
      protein, respectively. Measurement of the mean NAD(P)H lifetime, tau(M), 
      demonstrated that osteoblasts in osteogenic media had a progressively increased 
      tau(M) compared to cells in regular media, suggesting that osteoblasts undergoing 
      mineralization had higher NAD(+)/NAD(P)H ratio and may utilize more oxidative 
      phosphorylation (OxPhos). In vivo NAD(P)H 2P-FLIM was conducted in conjunction 
      with two-photon phosphorescence lifetime microscopy (2P-PLIM) to evaluate 
      cellular metabolism of GFP(+) osteoblasts as well as bone tissue oxygen at 
      different locations of the native cranial bone in Col (I) 2.3GFP mice. Our data 
      showed that osteocytes dwelling within lacunae had higher tau(M) than osteoblasts 
      at the bone edge of suture and marrow space. Measurement of pO(2) showed poor 
      correlation of pO(2) and tau(M) in native bone. However, when NAD(P)H 2P-FLIM was 
      used to examine osteoblast cellular metabolism at the leading edge of the cranial 
      defects during repair in Col (I) 2.3GFP mouse model, a significantly lower tau(M) 
      was recorded, which was associated with lower pO(2) at an early stage of healing, 
      indicating an impact of hypoxia on energy metabolism during bone tissue repair. 
      Taken together, our current study demonstrates the feasibility of using 
      non-invasive optical NAD(P)H 2P-FLIM technique to examine cellular energy 
      metabolism at single cell resolution in living animals. Our data further support 
      that both glycolysis and OxPhos are being used in the osteoblasts, with more 
      mature osteoblasts exhibiting higher ratio of NAD(+)/NAD(P)H, indicating a 
      potential change of energy mode during differentiation. Further experiments 
      utilizing animals with genetic modification of cellular metabolism could enhance 
      our understanding of energy metabolism in various cell types in living bone 
      microenvironment.
CI  - Copyright (c) 2021 Elsevier Inc. All rights reserved.
FAU - Schilling, Kevin
AU  - Schilling K
AD  - Center for Musculoskeletal Research, University of Rochester, School of Medicine 
      and Dentistry, Rochester, NY 14642, USA; Department of Biomedical Engineering, 
      University of Rochester, Rochester, NY 14642, USA.
FAU - Brown, Edward
AU  - Brown E
AD  - Department of Biomedical Engineering, University of Rochester, Rochester, NY 
      14642, USA.
FAU - Zhang, Xinping
AU  - Zhang X
AD  - Center for Musculoskeletal Research, University of Rochester, School of Medicine 
      and Dentistry, Rochester, NY 14642, USA; Department of Biomedical Engineering, 
      University of Rochester, Rochester, NY 14642, USA. Electronic address: 
      Xinping_Zhang@URMC.rochester.edu.
LA  - eng
GR  - P30 AR069655/AR/NIAMS NIH HHS/United States
GR  - R01 DE029790/DE/NIDCR NIH HHS/United States
GR  - R01 DE019902/DE/NIDCR NIH HHS/United States
GR  - R01 AR067859/AR/NIAMS NIH HHS/United States
GR  - R21 AR076056/AR/NIAMS NIH HHS/United States
GR  - R21 DE026256/DE/NIDCR NIH HHS/United States
GR  - R21 DE021513/DE/NIDCR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20211113
PL  - United States
TA  - Bone
JT  - Bone
JID - 8504048
RN  - 0U46U6E8UK (NAD)
SB  - IM
MH  - Animals
MH  - Cells, Cultured
MH  - Energy Metabolism
MH  - Mice
MH  - Microscopy, Fluorescence
MH  - *NAD/metabolism
MH  - *Osteoblasts/metabolism
MH  - Oxidative Phosphorylation
MH  - *Single-Cell Analysis
MH  - Skull
PMC - PMC8671374
MID - NIHMS1757136
OTO - NOTNLM
OT  - 2-photon microscopy
OT  - 2P-FLIM
OT  - 2P-PLIM
OT  - Bone repair
OT  - Energy metabolism
OT  - Glycolysis
OT  - Intravital imaging
OT  - Osteoblasts
OT  - Oxidative phosphorylation
EDAT- 2021/11/16 06:00
MHDA- 2022/03/12 06:00
PMCR- 2023/01/01
CRDT- 2021/11/15 20:16
PHST- 2021/09/05 00:00 [received]
PHST- 2021/10/29 00:00 [revised]
PHST- 2021/11/09 00:00 [accepted]
PHST- 2021/11/16 06:00 [pubmed]
PHST- 2022/03/12 06:00 [medline]
PHST- 2021/11/15 20:16 [entrez]
PHST- 2023/01/01 00:00 [pmc-release]
AID - S8756-3282(21)00423-3 [pii]
AID - 10.1016/j.bone.2021.116257 [doi]
PST - ppublish
SO  - Bone. 2022 Jan;154:116257. doi: 10.1016/j.bone.2021.116257. Epub 2021 Nov 13.