Dysregulated Bone Wound Repair and Marrow Functions in Senescence Accelerated Mice (SAMP6),

The Senescence Accelerated-Prone mouse (SAMP6) shows normal growth followed by rapid aging, development of osteopenia and shortened lifespan, compared with control R1 mice. The bone defect has been attributed to reduced osteoblast potential of marrow stromal cells. We compared in vivo repair of tibial bone wounds. We compared in vitro hematopoiesis, irradiation sensitivity, stromal cell biology, and osteoblastogenesis with SamP6 and R1 marrow. We tested the hypothesis that SAMP6 mice have poor healing of osseous wounds and that in vitro properties of SAMP6 marrow are abnormal.

As a model for spontaneous bone repair, a 2-mm unicortical wound was made in each proximal tibia. At intervals, wound diameter was measured on radiographs. Long term bone marrow cultures (LTBMCs) were established from other SAMP6 and R1 mice to measure hematopoiesis and to establish marrow stromal cell lines (MSCLs) for analysis. MSCL gene expression and sensitivity to irradiation were measured; osteoblastogenic potential was determined by culture in osteoblastogenic medium (1% FBS, 10 nM dexamathasone, 5 mM b-glycerophosphate, 50 mg/mL ascorbate-2-phosphate) and gene expression analysis for osteoblast markers alkaline phosphatase (ALP), PTH-receptor-1 (PTHR1), and Osteocalcin (OC).

There was a faster rate of closure of the tibial wounds in R1 than in SAMP6 mice; at day 21, wounds in R1 mice were 47% the size of those in SAMP6 mice (p = 0.032). For the first 24 weeks, SAMP6 LTBMCs had significantly greater hematopoiesis than R1, shown by more cobblestone islands (sites of stem cell activity) and by more multilineage colonies from nonadherent cells transferred to semisolid medium. After 24 weeks, however, hematopoiesis ceased in SAMP6 cultures but continued in the SAMR1 cultures for 40 weeks. There was constitutive upregulation of TGF-b, an inhibitor of hematopoiesis, in SAMP6 MSCLs, compared with R1 cells. SAMP6 MSCLs were more sensitive to radiation (Do of 1.5 ± 0.1 Gy), compared with R1 (Do of 4.0 ± 0.4 Gy; p = 0.0080). There was unexpected constitutive expression of ALP, PTHR1, and OC in SAMP6 MSCLs, compared with R1. In osteoblastogenic medium, there was greater expression of osteoblast marker genes in the SAMP6 line compared to R1.

SAMP6 showed delayed bone wound repair. Marrow from SAMP6 mice showed shortened in vitro hematopoiesis and greater radiation sensitivity, indicative of greater oxidative stress, but the unexpected finding of constitutive expression of osteoblast genes suggest that in vivo factors are more important than innate cellular defects in marrow to account for impaired bone healing.

This project was supported by NIAID U191A168021-06 and NIA R21AG034254.

No relevant conflicts of interest to declare.