Determination Of Age-Related Changes Of Calvarium And A Critical Size Calcarial Defect In The SAMP6 Osteoporosis Mouse Model

Objectives:1. To compare the age-related changes of calvarial bone in senescence-prone inbred strains (SAMP6) and the normal control senescence-resistant inbred strains (SAMR1); 2. Evaluate differences in the spontaneous healing capacity of these two strains, and determine the critical defect size in a mouse model of senescence-accelerated osteoporosis.Methods:1. In vivo micro-CT scanning was performed for the bone volume density (BV/TV) of cranium in these two strains at their 24-week-old,30-week-old and 36-week-old, respectively.2. Directly after the last scanning of micro-CT, the calvarial specimens were dissected and subjected to H&E staining and tartrate-resistant acid phosphatase (TRAP) staining for histological observation and osteoclasts count.3. Unilateral full-thickness calvarial defects 2 or 4 mm in diameter were made in 6-month-old male SAMP6 and SAMR1 mice. Defects were evaluated in vivo by micro-CT at day 0 and 6 and 12 weeks postoperatively.4. Calvarial specimens were harvested at 12 weeks for hematoxylin and eosin staining, Masson’s trichrome staining, and TRAP staining.Results:1. The BV/TV of cranium in SAMP6 were significantly lower than the age-matched SAMR1 at three time points. BV/TV of cranium in SAMP6 decreased significantly with aging while no significant changes in SAMR1.2. Compared with SAMR1, the internal and external amella of cranium were thinner in SAMP6, and the number of osteoclasts was greater in the diploe of SAMP6 as well.3. Less new bone formation was observed in defects in SAMP6 mice compared to SAMR1 mice at 12 weeks post-surgery (p<0.05), with <5% healing in SAMP6 mice for both 2-and 4-mm defects compared to >5% healing in 2-mm defects in SAMR1 mice (p<0.05).4. Histological analysis revealed a dense connective tissue layer but little bone healing in 2-and 4-mm defects in SAMP6 and 4-mm defects in SAMR1 mice. In contrast, newly formed bone was observed at the periphery of the 2-mm defects in SAMR1 mice. Masson’s trichrome staining also supported these findings. Less osteoblasts were observed at the periphery of any defects in SAMP6 compared to SAMR1 groups (p<0.05). No obvious TRAP-positive cells were observed at the margins of any defects, but more TRAP-positive cells were seen in the diploe of contralateral cranial bone in SAMP6 mice than in that of SAMR1 mice.Conclusions:1. The bone volume density of cranium in SAMP6 decreased with aging and were lower than the age-matched SAMR1, while no significant changes in SAMR1.2. The critical defect size was 2 mm in the cranium in both SAMP6 and SAMR1 mice. Intramembranous ossification of the defect was impaired in SAMP6 mice, likely due to osteoblast insufficiency and excessive osteoclastogenesis.