Background
The natural remodeling process of the bone is effective in maintaining tissue homeostasis, however, a diversity of medical conditions exist that adversely affect the mechanical and biological integrity of the tissue such as physical injuries to the bone, osteoporosis, cancer and inflammatory bone disease [
1]. Current clinical interventions include immobilization with metal rods and pins, implantation of autologous or allogeneic bone grafts and implantation of metals or ceramics. Effective immobilization can be challenging to achieve due to the deteriorated state of the bone [
2,
3]. Although treatment using bone grafts is quite effective, this method is accompanied by serious limitations, including limited supply, donor site morbidity and, in the case of allografts, risk of rejection or disease transmission [
4]. The use of metals or ceramic implants have clear disadvantages in that they exhibit limited integration with the host tissue and can fail as a result of infection [
4]. Gene therapies have been proposed as viable options, both in in vivo and ex vivo models [
5]. Gene therapies provide methods to stimulate molecules or groups of molecules to enhance bone regeneration [
6]. For example, the inhibition of p21 has been observed to increase cell proliferation and subsequently cause regeneration of tissues [
7,
8]. p21
WAF1/CIP1/SDI1, a cyclin dependent kinase inhibitor, is intricately involved in cell proliferation, acting at the G
1 check point during cell cycle. In essence, p21 inhibits the activity of cyclin/cdk2 complexes and inversely affects cell cycle progression [
9].
To evaluate the role of proliferation in tissue repair, a previous study examined fibroblasts obtained from ‘healer’ and ‘non-healer’ mice and tested for their regenerative properties based on a G
1 cell cycle checkpoint deficiency. It was observed that deletion of p21 alone was sufficient to convert non-healer mice into appendage regeneration-competent healers: ear hole closure in 6–7 week old p21
−/− mice was only slightly less than that of MRL (Murphy Roths Large, super healer) mice by 28 days after injury [
7]. In another study, the role of p21 inhibition in liver regeneration was probed, and, continuous hepatocyte proliferation was observed, albeit while also facilitating tumor development [
8]. There have been no studies on specifically cartilage and bone regeneration in p21
−/− mice. Nonetheless, there have been studies on skeletal muscle regeneration and embryonic endochondral ossification, where interestingly enhanced repair was not obtained; specifically, p21
−/− mice display delayed regeneration of muscle after injury compared to wildtype controls [
10]. While several studies assessed p21 inhibition in adult mouse tissue, Chinzei et al. evaluated the role of p21 in embryonic endochondral ossification in vivo, and found that p21 deficiency appears not to influence ossification with other signaling pathways likely compensating [
11]. Despite the lack of studies on the role of p21 in bone and cartilage, other members of the p21 signaling pathway, such as E2F1 have been investigated and could potentially predict the role of p21within bone development/repair. E2F1 like p21 is also involved intricately in proliferation and apoptosis, where its transactivation is governed by pRb (retinoblastoma protein) [
12]. E2F1 and p21 are inversely related, inhibition of E2F1 leads to upregulation of p21, thereby decreasing cell proliferation [
13]. As previously described, the Chinzei et al. study established that p21 inhibition did not have an effect on endochondral ossification, however, Scheijen et al., demonstrated that overexpression of E2F1 delayed endochondral bone formation owing to altered chondrocyte maturation [
14]. In contrast, when E2F1 was inhibited in a Rb-deficient embryo, bone defects caused by Rb deficiency was reversed, demonstrating a role of E2F1 in osteoblast differentiation [
15]. These results, taken together suggest that the p21-E2F1 signaling pathway may play a role in bone development and/or repair after injury, however, to date no study has examined if p21 KO mice demonstrate enhanced fracture repair.
The process of bone healing proceeds with the recruitment and differentiation of mesenchymal stem cells into skeletal and vascular tissues. A cartilaginous callus is formed which is followed by mineralization of the extra cellular matrix. While cartilage is resorbed, bone formation is initiated, and bone remodeling commences where osteoclasts resorb primary bone and secondary bone is formed [
16]. Differentiation of mesenchymal stem cells into osteoblasts is an important factor in determining bone healing and remodeling. The knockdown of p21 in late-passage mesenchymal stem cells (MSCs) exhibited increased proliferation capacity and increased osteogenic potential [
17]. It has been previously shown that there are inherent differences in p21 expression in mesenchymal progenitor cells between normal and osteoarthritic patients; with increased p21 levels correlated with decreased chondrogenic potential [
18].
These studies provide a strong rationale to examine the role of p21 in bone repair; with other kinase inhibitors having being examined in this area of research previously. Drissi et al., demonstrated that osteo-progenitor cells derived from bone marrow of p27
−/− mice proliferated at increased rates compared to wild type mice while retaining differentiation capacity; they also suggested that increased p21 levels were linked to retained differentiation capacity [
19]. These results indicate that kinase inhibitors may play an important role in bone remodeling. While p21 has been implicated in bone remodeling through p27
−/− mice, it has been previously observed that deletion of p21 did not alter embryonic endochondral ossification in mice, as stated earlier [
20]. There have been no studies that have directly examined the role of p21 in bone regeneration, specifically after an injury. Therefore, this study was designed to examine the role of p21 in a non-critical size fracture model [
21].
Discussion
Bone is arguably one of the few tissues in the body that demonstrates true regeneration vs. repair after injury. However, there are still many clinical and pathological conditions that can negatively impact the ability of bone to recover after injury. Over the last decade there have been numerous reports of super-healer mice strains that are able to repair wounds/injuries that normally demonstrate limited repair in mammals. Specifically, both MRL and p21
−/− mice have demonstrated the ability to repair cartilage injuries, while most mice, including C57BL/6 cannot [
7,
26,
27]. The actual mode of healing in these mice is still under debate in regards to if it is true regeneration, therefore, the purpose of this study was to determine if any enhancement in healing could be observed within super-healer mice in a tissue that normally regenerates (e.g. bone). To that end, we have shown that p21
−/− mice demonstrate increased facture repair compared to C57BL/6 mice in a non-critical sized defect model, and while there is no apparent difference in osteogenesis and osteoclastogenesis in p21
−/− vs C57BL/6 mice, increased stem cell retention at the injury site 4 weeks post injury may indicate they play a role in the enhanced healing, potentially at the initial stages during the cartilaginous callus formation. However, since earlier time points were not examined in this study, we cannot conclusively state that p21 is playing a role in the early stages of bone healing, only that p21
−/− mice demonstrate increased fracture repair compared to C57BL/6 controls. Additional experiments examining earlier time points would be required to test this hypothesis.
While the burr-hole model is not widely employed since it is a non-critical defect model, we employed it in this study since it was essential to examine if p21
−/− mice demonstrated increased repair compared to C57BL/6 and while it remains unknown if p21
−/− mice can repair a critical-size defect in bone, it is known that C57BL/6 mice cannot. Furthermore, the healing kinetics of the burr-hole model in the current study demonstrated reproducibility in terms of healing kinetics with previous reports using this model [
28]. Using this model, it was observed that p21
−/− mice demonstrated enhanced bone healing, particularly at week 1, compared to C57BL/6 mice in all bone parameters measured (Fig.
2). Interestingly, we also observed small changes to the uninjured contralateral limb at week 1 (data not shown), which may indicate that changes to the trabecular bone morphology and bone mineral density may be the result of a systemic response to injury [
29], however additional controlled experiments would be required to test this hypothesis. Furthermore, it should be noted that C57BL/6 and p21
−/− mice showed significantly different general bone morphologies, and their physical characteristics such as radial growth of the tibia also varied. Specifically, p21
−/− mice presented with smaller bones and overall lower total body weight, this is in contrast to previously reported work in p27
−/− mice that were larger than age-matched wildtype mice. p27 similar to p21 is a cyclin dependent kinase inhibitor [
30], and both are from the same kinase family but vary in their C terminus and their p53 regulation, where p21 is regulated by p53 [
31]. p53 regulation of p21 could potentially influence size and dimensions of bone by partially overriding effects of p21 inhibition, thereby resulting in smaller animals and could be a factor in the differences in the steady state bone morphology observed.
While previous studies in human MSCs have demonstrated that knocking down p21 leads to an increase in osteogenic potential [
32], a recent study from our lab found that no increase in osteogenic differentiation potential was observed in MSCs derived from MRL super-healer mice [
25]. Correspondingly, no increase in osteogenic potential was observed in this study in p21
−/− MSCs, suggesting that p21 may regulate osteogenesis in human MSCs, but not mouse, and also suggests that the regenerative effect observed in MRL and p21
−/− mice may not be the result of increased differentiation capacity of the MSCs, but instead could be an effect of increased recruitment/retention at the injury site. This hypothesis is supported by our previous study which demonstrated increased numbers of putative MSCs in the defect site of MRL mice while C57BL/6 have very few MSCs the respond to the injury [
25]. In the current study, we observed a retention of MSCs even after the fracture had been healed, suggesting that these cells may also play a role in the long term remodeling of the defect.
While the contribution of other cell types in the enhanced fracture repair cannot be discounted in this study, it was clear that there was no difference in the osteoblast and/or osteoclast function (mineralization vs. resorption), and we also did not observe any change in total numbers of the cells in the bone marrow of the injury limb.
While we have been able to demonstrate that p21
−/− mice display increased fracture repair vs. wild-type C57BL/6 mice, this study is not without limitations. Specifically, we still do not have any direct evidence of how the injury is healing in the p21
−/− mice, and lineage reporter mice may be necessary to track specific cell types (including MSCs) after injury to address this question. Furthermore, since this is a constitutive knockout, we cannot rule out multiple pathways and cell types being differentially affected by the removal of p21. It is quite possible that the cartilaginous callus formed after an injury may also contribute to enhanced healing in p21
−/− mice as we and others have demonstrated p21 also plays a role in chondrogenesis [
18]. Additionally, we cannot discount the role of inflammation in this process. After an injury to the bone, there is an immediate inflammatory response that begins the healing cascade, this inflammatory response is responsible for the recruitment of MSCs that form the initial callus bridging the injury [
33]. Additional controlled experiments will been necessary to dissect out these different pathways to determine how p21 is regulating bone repair after injury and to determine if this mechanism has any potential to be translated into humans.