Numerous in vitro and in vivo studies have investigated the involvement of factors that are crucial for bone formation or differentiation of cells. One of those factors include the cell cycle and its regulators. As discussed in the previous section, cell cycle is a highly complex process that regulates the growth and proliferation of cells, regulation of DNA damage repair, tissue hyperplasia due to an injury, and more [8]. It is a series of events in which the components of the cells are doubled and accurately segregated into daughter cells [33]. The cell cycle regulators are thought to influence the differentiation of cells, as withdrawal from the cell cycle or a temporal arrest in the G1 phase is believed to be a requirement for cell differentiation [34]. Given the constant turnover of cellular components in bone, regulation of the cell cycle is of critical importance in bone development and bone remodeling. Here, we provide a concise summary of the cell cycle regulators that have been reported to control the differentiation of osteoblasts, osteoclasts, chondrocytes, and other types of cells or are currently being studied as a target for bone regeneration or development. Cell cycle regulators that have not been reported to have a role in bone healing or development may not be included here.

The regulation of cell cycle is essential for the survival of the cell, including the detection and repair of genetic damage as well as preventing uncontrolled cell division. The cell cycle is regulated primarily by Cyclin-dependent kinases (CDKs) and Cyclins in complex. CDKs are serine/threonine kinases whose catalytic activities are modulated by interactions with Cyclins and CDK inhibitors (CKIs) [35]. The interaction between CDKs, Cyclin, and CKI is essential for ensuring a systematic progression through the cell cycle. They also play an important role in transcription, metabolism, neural function, and stem cell self-renewal [35]. In vitro experiments as well as genetically engineered mouse models have demonstrated an intricate role for cell cycle regulators in bone and its processes. The following sections will discuss various cell cycle regulators in the context of bone development, healing, and regeneration.

CDKs depend on their association with a noncatalytic regulatory subunit called a cyclin. CDKs drive the major cell cycle transition points (G1, S, G2, M). While CDK protein levels remain stable throughout the cell cycle, cyclin levels fluctuate causing periodic activation of CDKs. Progression through each phase of the cell cycle requires different CDK-cyclin pairs. CDKs exert control of eukaryotic cell division by regulating cell-cycle stages through the phosphorylation of various substrates. The CDKs present in humans include CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8; however, only some cell cycle regulators have been shown to interact with cells that play a role in bone processes.

CDK2 is a serine/threonine protein kinase that controls the G1/S transition in the cell cycle, regulates the exit from S phase, promotes DNA replication, and has been found to promote the G2/M DNA damage response checkpoint [36]. In the G1/S phase, CDK4 and CDK6 complex with cyclin D and initially phosphorylate the retinoblastoma (Rb) protein which is crucial for preventing excessive cell growth. The association of CDK2 with cyclin E then completes the phosphorylation of Rb. CDK2 also complexes with cyclin A to control the transition from S to G2 in the cell cycle. In addition, p21(Cip1) and p27(Kip1), which belong to the Cip/Kip protein family, can form a complex to block CDK2/cyclin E and CDK2/cyclin A kinase activity. Studies have shown that CDK2 is dispensable for cell proliferation [37] and mouse development [38]. In the absence of CDK2, CDK1 can phosphorylate Rb by binding to D-type cyclins and can promote replication as a complex with cyclin E1 and Cyclin A [39]. Subsequent inactivation of the cyclin E-CDK2 complex along with the induction of p21 and activation of Rb influences the fibroblast growth factor (FGF), which is thought to be a negative regulator of chondrocyte growth [40].

CDK4 is a catalytic subunit of the protein kinase complex which plays a role in the regulation of the G1-S transition of the cell cycle. It forms molecular complexes with the members of the D-type cyclin family and is responsible for the phosphorylation of Rb. The activity of CDK4 is negatively inhibited by the CDK inhibitor p16 (INK4a). Additionally, the overexpression/amplification of CDK4 is associated with tumorigenesis of a variety of cancers [41]. Activation of CDK4 phosphorylates the Rb family of proteins, resulting in negative regulation of the passage of cells from G1 to S phase by sequestering transcription factors critical for G1/S transition. The main outcome of CDK4 activation is the inhibition of Rb leading to G1-S cell-cycle transition. CDK4 also directly phosphorylates other proteins which promote cell-cycle progression and inhibit both cell senescence and apoptosis [42]. Studies on the influence of CDK4 in other bone related cells have not been reported. Abella et al. reported that CDK4 is a key regulator of adipocyte differentiation, one of the cell types that an MSC can differentiate into [43].

CDK6 is important for G1 phase progression and G1 to S phase transition of the cell cycle. The activity of this kinase, which initially appears in the middle of G1 phase, is controlled by D-type cyclins and members of the INK4 family of CDK inhibitors (p16, p15, p18, p19). In addition, this kinase has been shown to phosphorylate and regulate the activity of the tumor suppressor protein Rb [44]. Studies have shown that CDK4 and CDK6 in conjunction with cyclin D1 enhances Rb phosphorylation and its related proteins p107 and p130 in the G1 phase of the cell cycle [45]. In vitro studies have shown that CDK6 is one of the key regulators in the differentiation of multiple types of cells [34]. Its downregulation is critical in controlling osteoblasts, osteoclasts, and chondrocyte differentiation [34]. However, the mechanism by which CDK6 controls cell differentiation without influencing the cell cycle is still unknown. Therefore, the possibility of CDK6 being a target in bone regeneration remains to be seen.

Cyclin-dependent kinase inhibitors (CKIs) restrain CDK activity [35]. CKIs are divided into two classes based on their structure and CDK specificity. The INK4 family members—p16, p15, and p19 that primarily target CDK4 and CDK6, and the Cip/Kip family members—p21, p27, and p57 that interfere with the activities of Cyclin A-, D-, and E-dependent kinase complexes.

p15, p18: Mice lacking p15 p19 were reported to have similar phenotype compared to wildtype mice. This suggests that their role in bone development could be compensated for by other cell cycle regulators [71,72,73]. p18 knockout mice were born with similar characteristics to their wildtype counterparts; however, over a period of 3 weeks they became distinctly larger. Mechanisms behind this increase in weight is still unknown and cannot completely be attributed inhibition of p18 alone [74].

p16: p16 (INK4a or CDK2NA) is an important CKI and a tumor suppressor gene that is not only required for the control of unregulated cell growth in most cell types, but also has roles in other cell cycle phases. Its typical role is to check the cell cycle in the early G1 phase and inhibit further transition of the cell cycle from G1 to S phase. p16 binds to CDK4 and inhibits its interaction with cyclin D causing prevention of passage through the G1 phase of the cell cycle [46].The induction of p16 results in a G1 cell cycle arrest by inhibiting phosphorylation of the Rb protein by the cyclin-dependent kinases CDK4 and CDK6 and may also cause inhibition of CDK2 activity [47]. p16 has been reported to play an important role in cell differentiation, cell quiescence, and cell senescence, which makes it a crucial cell regulatory protein for the regulation of terminal differentiation and the aging process [48]. Therefore, further investigation on p16 seems very promising. There have been no studies linking p16 to any bone processes. However, since p16 plays a role in differentiation it is plausible that it could play a role in MSC differentiation as well.

p19: p19 (INK4d) interacts with CDK4 and CDK6 inhibiting them from binding with cyclin D, resulting in the arrest of the cell cycle in the G0/G1 phase. p19 was found to be present at low levels at the onset of G0/G1 and then accumulates at the entry to S phase and remains elevated through S phase into G2 [49]. Induction of this gene was found to contribute to cell cycle arrest, and knockdown of the gene alone found cells to be sensitive to autophagic cell death. p19 is induced to inhibit the proliferation of many kinds of tumor cells like T cell acute lymphoblast leukemia cells. It is also involved in hematopoietic stem cell quiescence and megakaryocyte/granulocytic differentiation which is associated with cell cycle arrest [50]. Like previous INK4 candidates, p19 also has not been reported to directly take part in bone processes. Yet, its involvement in hematopoietic stem cell and inflammatory cell differentiation could contribute to bone healing and regeneration after an injury and is worth exploring.

p21: p21, also known as CIP1 and Waf1, is a CKI that has been involved in cell differentiation, apoptosis and cell proliferation—its inhibition leads to enhanced proliferation of cells. It binds to cyclin-dependent kinase (CDK) 2 and 4 and inhibits its activity. It functions as a regulator of cell cycle progression at the G1 phase. Expression of this gene is controlled by p53 and this protein plays a regulatory role in S phase DNA replication and repair [51]. p21 expression can also be regulated independently of p53 in certain instances like tissues during development and in the adult mouse [52].

Our lab has demonstrated that p21KO mice displayed enhanced bone regeneration capacity after a burr-hole injury in the proximal tibiae measured over 4 weeks [5]. Our results indicate that MSC numbers in bone marrow were not different between the two mouse types, yet, at the site of injury there were significantly more MSCs after 1 week. The osteogenic differentiation capacity of both mice was investigated, and no significant differences were observed. We hypothesize that the increased number of MSCs at the site of injury either play a direct role by enhancing chondrogenesis or an indirect role by expressing trophic factors. Our lab is currently testing this hypothesis by upregulating a downstream effector, E2F1 specifically in chondrocytes. Preliminary results indicate that mice where E2F1 is overexpressed in chondrocytes exhibit enhanced bone healing albeit with reduced bone mineral density [53]. Of all cell cycle regulators, p21 has demonstrated the most promise to be successfully embedded in interventional therapies for bone healing and regeneration. Of significance is also our finding where inhibition of p21 has shown to protect against bone loss in an osteoporotic environment, potentially by overcoming the absence of estrogen; estrogen and p21 have redundant interactions with osteoclasts [54].

p27: p27, known as KIP1, is a CKI that prevents the activation of cyclin E or D, thus controlling cell cycle progression at the G1 phase. The degradation of the protein, triggered by CDK dependent phosphorylation, is required for cell transition from quiescence to the state of proliferation [55]. p27 is an atypical tumor suppressor which regulates G0 to S phase by binding and regulating CDK2, CDK4, and CDK6. In the early G1 phase, p27 translation is at its highest point causing binding and inhibition of cyclin E. Decrease of p27 throughout the G1 phase allows cyclin E and cyclin A to activate gene transcription required for G1-S transition [55, 56]. The p27 pathway has been shown to modulate skeletal growth through bone formation by interacting with the parathyroid hormone-related peptide (Pthrp). When p27 was deleted in Pthrp KI mice, skeletal growth retardation and defective osteoblastic bone formation was rescued [7]. Another study by Yin et al. demonstrated that p27 negatively regulates alveolar bone development, in agreement with the previously described study [57]. p27 plays a role in bone and bone cells and also in osteoprogenitor cells. Bone marrow osteoprogenitor cells from p27KO mice exhibited increased proliferative capacity and formed larger osteoblastic colonies while also differentiating to the mineralization stage [6]. From these studies it is evident that p27 might also be worth considering as a potential target to improve bone related processes like healing and regeneration.

p57: p57 (Kip2) is a strong inhibitor of the G1 cell-cycle CDK complexes. p57 was found to be a negative regulator of cell proliferation [58]. The kinases p21, p27, and p57 have an affinity to bind to, and inhibit, the CDK 2, 4, and 6 [59]. The over expression of p57 leads to cell-cycle arrest in the G1 phase [60]. p57 has also shown that it influences osteoblasts as deletion of p57 can upregulate osteoblasts proliferation and differentiation while also improving bone mineral density [61, 62].

Cyclins function as regulators of CDKs. Cyclins drive the events of the cell cycle by coupling with CDKs to activate them, making it a functional enzyme allowing it to modify target proteins. Different cyclins exhibit distinct expression and degradation patterns which contribute to coordination of each mitotic event.

Cyclin A: Cyclin A can activate two different CDKs (CKD1 and CDK2), making it particularly interesting among the cyclin family. Cyclin A was also found to bind to the Rb gene family, E2F1 and p21 [63, 64]. Cyclin A is needed in the S and the G2 phase of the cell cycle and reaches a maximal level right before mitosis, after which it degrades rapidly. The only study connecting cyclin A with bone processes is through MSCs, where Fei et al., demonstrated that osteoprotegerin (OPG) deficient mice showed the osteogenic growth peptide (OGP) stimulated MSC proliferation and increased the expression of Cyclin A and CDK2 at mRNA and protein levels [65]. OPG triggered the Cyclin A-CDK2 pathway, resulting in the proliferation of MSCs of OPG-deficient mice. It is however unknown if the MSCs that proliferated also differentiated into bone cells.

Cyclin D1: Cyclin D1 forms a complex with, and functions as a regulatory subunit of, CDK4 and CDK6. In the G1 phase of the cell cycle, Cyclin D1 and its CDK partner are responsible for transition into the S phase through phosphorylation of the Rb gene which will then cause the release of transcription factors for the initiation of DNA replication [64]. Cyclin D1 has been shown to interact with the Rb gene family, where the expression of Cyclin D1 is regulated positively by Rb. Cells that lack functional Rb have significantly lower amounts of Cyclin D1 and Cyclin D1-CDK4 complexes, thus exhibiting a negative feedback loop in which Cyclin D1 synthesis and activation leads to Rb phosphorylation, which then decreases Cyclin D1 expression. In mice lacking cyclin D1, a small skeletal phenotype was observed along with a 50% chance of malformation of the jaw caused by misalignment of the incisor [66]. However, it should be noted that the dwarfism phenotype observed could be a result of lower levels of growth hormones or pituitary issues rather than bone development.

Cyclin D2: Cyclin D2, like Cyclin D1 forms a complex with CDK4 and CDK6. CDK4 and CDK6 are associated with the D-type Cyclins during the G1 phase of the cell cycle. Cyclin D2 reaches its maximum activity during the G1 phase and regulates transition into S phase through phosphorylation of the Rb gene [64]. Cyclin D2 inactivates Rb by phosphorylation and induces the release of E2F [67]. In human MSCs, the overexpression of Cyclin D2 promoted proliferation of the cells. Cyclin 2 could be considered a target for increasing MSC numbers, but like with cyclin A, converting these MSCs to bone cells still requires investigation.

The protein encoded by Rb is a negative regulator of cell cycle. It was found to stabilize heterochromatin to maintain the overall chromatin structure. Hypo-phosphorylated forms of this protein bind to the transcription factor E2F1, meaning through under-phosphorylation of Rb, the G1 cell cycle begins to function in an antiproliferative stage [40, 64]. The Rb gene and its relatives p107 and p130 encode proteins which share several properties including one which inhibits cell-cycle progression [68]. Rb plays a role in maintaining quiescence in adult stem cells, when needed Rb is transiently inactivated to allow for self-renewal and differentiation of stem cells [69]. In addition to cell cycle, Rb plays a vital role in cell adhesion. Sosa-Garcia et al., demonstrated that when Rb is inhibited osteoblasts do not form cell–cell contacts but do continue to proliferate [70]. While proliferation of osteoblasts is important in bone development and healing, loss of cell adhesion can result in metastasis rather than development of bone. For these reasons Rb might not be a suitable candidate for targeted bone therapies.

The proteins encoded by p107 and p130 are negative regulators of cell cycle. Hypo-phosphorylated forms of these proteins bind to the transcription factor E2F1 of p107 and p130, which leads to the G1 cell cycle to function in an antiproliferative stage [40, 64]. Cobrinik et al., demonstrated that p107 and p130 play a significant role in limb development by controlling the proliferation of chondrocytes [75]. Proliferative arrest of chondrocytes takes place when they differentiate into hypertrophic chondrocytes accompanied by loss of p107 and p130 [76]. p107 aids in induction of alkaline phosphatase encoding gene Alpl through recruitment of SWI/SNF chromatin remodeling complex [77]. p107 and p130 do present as potential targets to improve bone healing. However, this would require further research into teasing out the role of Rb from p107 and 130 as they form an intricate network.