p21-/- mice exhibit enhanced bone regeneration after injury

Abstract

Background: p21^{(WAF1/CIP1/SDI1)}, a cyclin dependent kinase inhibitor has been shown to influence cell proliferation, differentiation and apoptosis; but more recently, p21 has been implicated in tissue repair. Studies on p21(^-/-) knockout mice have demonstrated results that vary from complete regeneration and healing of tissue to attenuated healing. There have however been no studies that have evaluated the role of p21 inhibition in bone healing and remodeling.

Methods: The current study employs a burr-hole fracture model to investigate bone regeneration subsequent to an injury in a p21^-/- mouse model. p21^-/- and C57BL/6 mice were subjected to a burr-hole fracture on their proximal tibia, and their bony parameters were measured over 4 weeks via in vivo μCT scanning.

Results: p21^-/- mice present with enhanced healing from week 1 through week 4. Differences in bone formation and resorption potential between the two mouse models are assessed via quantitative and functional assays. While the μCT analysis indicates that p21^-/- mice have enhanced bone healing capabilities, it appears that the differences observed may not be due to the function of osteoblasts or osteoclasts. Furthermore, no differences were observed in the differentiation of progenitor cells (mesenchymal or monocytic) into osteoblasts or osteoclasts respectively.

Conclusions: Therefore, it remains unknown how p21 is regulating enhanced fracture repair and further studies are required to determine which cell type(s) are responsible for this regenerative phenotype.

Keywords: Bone healing; Mice; Trabecular bone; p21; μCT.

Fig. 1

Panel of representative μCT images illustrating trabecular morphology at day 0, Week 1, 2 and 4, post burr-hole surgery of p21^−/− (a−d) mice and C57BL/6 (e−h) mice. It is observed that at week 1, both C57BL/6 and p21^−/− mice showed expected regeneration at the ROI. By week 2, the bone has returned to a pre-injury morphology and remains stable through week 4. The red arrows indicate the location of the burr-hole fracture. Each mouse group (n) = 12. Scale bars: 1 mm

Fig. 2

Quantitative analysis of bone histomorphometry properties obtained via μCT scanning of ROI surrounding the burr-hole fractures at day 0, week 1, 2 and 4 for C57BL/6 and p21^−/− mice. *p < 0.05. The error bars indicate standard deviations

Fig. 3

Runx2 (a) and sp7 (b) gene expression in C57BL/6 and p21^−/− bone marrow derived MSCs before and after osteogenic differentiation. Histological (alizarin red) staining of calcium deposits in differentiated MSCs from C57BL/6 (c) and p21^−/− (d) mice respectively. Error bars indicate standard deviation

Fig. 4

No difference was observed in the resorptive capacity of osteoclasts derived from C57BL/6 or p21^−/− mice (A; n = 4). Representative flow cytometry plots of osteoblasts and osteoclasts staining of cells isolated from the bone marrow demonstrating no difference in the number of osteoblasts or osteoclasts between C57BL/6 or p21^−/− mice (B; n = 4). Error bars indicate standard deviation