Craniofacial Bone Tissue Engineering: Current Approaches and Potential Therapy

Craniofacial bone defects can result from various disorders, including congenital malformations, tumor resection, infection, severe trauma, and accidents. Successfully regenerating cranial defects is an integral step to restore craniofacial function. However, challenges managing and controlling new bone tissue formation remain. Current advances in tissue engineering and regenerative medicine use innovative techniques to address these challenges. The use of biomaterials, stromal cells, and growth factors have demonstrated promising outcomes in vitro and in vivo. Natural and synthetic bone grafts combined with Mesenchymal Stromal Cells (MSCs) and growth factors have shown encouraging results in regenerating critical-size cranial defects. One of prevalent growth factors is Bone Morphogenetic Protein-2 (BMP-2). BMP-2 is defined as a gold standard growth factor that enhances new bone formation in vitro and in vivo. Recently, emerging evidence suggested that Megakaryocytes (MKs), induced by Thrombopoietin (TPO), show an increase in osteoblast proliferation in vitro and bone mass in vivo. Furthermore, a co-culture study shows mature MKs enhance MSC survival rate while maintaining their phenotype. Therefore, MKs can provide an insight as a potential therapy offering a safe and effective approach to regenerating critical-size cranial defects.

Moreover, the role of MKs regenerating a critical-size bone defect is an ongoing and active area of research, utilizing in vitro cell culture system [56,112,116,[122][123][124]. However, whether or not MKs can facilitate MSCs proliferation and differentiation to regenerate cranial bone defects is a question yet to be addressed. Particularly, the effects of increasing MK count to enhance viability and differentiation of BMSCs or DPSCs in vivo for cranial bone tissue engineering purposes has not been determined [125].
However, over productions of MKs for craniofacial regeneration purposes can have a downside effect. Studies have shown that increasing MKs, and ultimately platelets can be a potential risk causing bone marrow fibrosis [122,126]. Furthermore, a co-culture study of MKs with MSCs has reported that MKs inhibit MSC differentiation into osteoblast lineage cells by suppressing expression of ALP activity and calcium deposition [125].
While progress has been made in tissue engineering and regenerative medicine field, several challenges remain. For instance, although BMP-2 has demonstrated promising results in regenerating large cranial bone defects, better outcomes are desired. Furthermore, the need for an additional growth factor rises from the excessive effects of BMP-2 and low survival rate of MSCs post-implantation. Herein, we discuss current craniofacial bone tissue engineering approaches. This review aims to identify advantages and challenges of current and proposed solutions in craniofacial bone tissue engineering field. Specifically, we will review methods currently used to restore cranial bone defects, such as natural and synthetic bone graft substitutes, MSCs (BMSCs and DPSCs), and current growth factors commonly used for cranial bone regeneration. Next, we will present a potential therapy; a discussion on rationale of inducing MKs for therapeutic applications to facilitate craniofacial bone regeneration.

Autologous Bone Graft
Bone grafting is a surgical procedure that aims to replace missing bone using tissue harvested from patient's skeleton (autograft) [32,134,135,144], donor (allograft) [32,134,138,145,146] or different species (xenograft) [32,34,[147][148][149]. However, bone autografting has showed positive outcomes regenerating cranial bone defects [140,141]. An autograft is a procedure using bone tissue as a substitute harvested from patient's different anatomical location and transplanted into the defect site [32, [149][150][151]. In other words, bone graft is harvested from one side of patient's skeleton into cranial defect site [147,[150][151][152]. The graft substitute of autologous bone can be harvested from a variety of locations. Common locations are tibia and iliac crest [142,143,147,153,154]. Autograft is a safe surgical procedure moving tissue from one side to another [30,142,143,153]. Using patient's bone tissue minimizes risk of immune system reactions and transferring pathogens from one source to another [32,151,153,155]. In addition, autografts have several advantages: Enabling osteogenesis [25, 145,152,156,157], osteoinduction [25,145,148,152,156,157], and osteoconduction [149,152,156,157]. All of which are essential to promote new bone formation [152,157].
On the other hand, bone autograft may require rearrangement for two surgical procedures [142,153]. As a result, patients face extra pain and possible blood loss due to the required two surgeries [157][158][159][160]. Moreover, the autograft procedure may require an extended hospitalization to a mandatory care service [153,155,161]. Consequently, this may yield a higher cost for patients [162]. Other disadvantages are increasing pain, scar at donor site, and extra damages to surrounding healthy tissues such as nerve, bone, and blood vessels [142,143,147,149,150,153]. Moreover, patients with pre-existing conditions such as diabetes may not be eligible for bone autografting [159,163,164].
Another factor to consider is patient's age and health condition [151]. The age of patient can be disadvantage using autograft reconstruction [151]. For instance, harvesting bone graft for children can cause complications and pain [151]. Moreover, autograft substitute may not be an optimal decision for children unless their cranium is fully established and can better withstand the impact of significant surgery [142,158]. Therefore, children might be less likely suitable candidates for bone autograft procedure [151,158,159,165]. Similarly, aging adults with medical conditions such as neuromuscular scoliosis have shown insufficient bone healing [138,163]. Table 1 summarizes the advantages and disadvantages of bone autograft.

Allogeneic Bone Graft
Although autograft bone is a desired approach to regenerate critical-size cranial bone defect [32, 148,153,174], allograft has been an attractive and alternative method [32,35,148,153,174]. Allograft bone tissue is transplanted to the patient (recipient) from a donor of the same species (human) [23,32,147,153,168]. In the United States, the number of bone allogenic grafts surgery is steadily increasing [33,174]. In the last decades, the number of surgeries involving allogeneic grafting tissue increased more than 15 times [147,166,168,175]. The main advantages of bone allografts are to provide structural support [32, 166,176], decrease surgical time [32, 149,176], and promote cranial healing [149,175,176]. The three most common bone allograft types are cortical, cancellous, and hybrid bone tissue (cortical and cancellous bone tissue) [23, 157,171]. Each one of harvested bone substitute has advantages. For instance, the cancellous bone is a desired bone graft for cranial reconstruction [161,166,167,177]. This is due to bone's elasticity and sufficient pore size that allow for cell infiltration and nutrient and gas exchange [23,172,[178][179][180]. Furthermore, mechanical and structural properties of cancellous bone allow for new bone and blood vessel formation [177,178,181]. On the other hand, cortical bone is a less favorable bone graft due to its low osteoconductivity and resorption rate [172,178,180].
Nevertheless, patients who receive allogeneic transplant from a deceased donor pose some risks [33,168]. Although bone allograft remains safe (the case of viral transmission is low), concerns regarding the safety of allografts remain [168,172]. On a rare occasion, unexpected transmission of pathogen such as Human Immunodeficiency Virus (HIV) from donor to the recipient can occur, despite donor screening to rule out possibility of donor infection [147,168,182]. In addition, potential risk of unwanted immune response, transplant rejection, and allergy may present challenges [35,182,183].
Various methods to prepare allograft bone for transplant have been used. One of the common practices is to debride donated tissue and sterilize it, followed by lyophilizing tissue to destroy any remaining living cells in bone tissue [155,157]. Although this method has shown constructive outcomes, an optimal procedure to clean, sterilize, and remove cellular and biological constituents from bone substitutes is desired [33,178]. For instance, when bone tissue undergoes lengthy cleaning, sterilization, and decellularization processes (such as lyophilization), a meaningful decrease in mechanical strength and structure of bone tissue occurs [168,170]. However, enhancing weight-bearing of allograft bone with a polymer composite is proposed and shows promising results [183]. Table 1 summarizes advantages and disadvantages of allografts.

Xenogeneic Bone Graft
A xenograft is a procedure of transplanted bone tissue harvested from different species to the patient. Bone tissue is prepared by physical or chemical processing and implanted in the patient (recipient) [166,170]. The most common sources of xenogeneic grafts are bovine and natural coral [149,170]. Similar to allogeneic bone grafts, xenogeneic bone graft serves as a structural load-bearing scaffold to facilitate new bone tissue growth and fill the vacant defect [29,184]. Unlike allograft, xenograft reduces the risk of transmitting human diseases caused by transmitted pathogen from the donor to recipient [31, 172,185]. On the other hand, xenogeneic bone grafts present potential risks [173]. For instance, immunological barrier to xenotransplantation and potential of transmitting infectious diseases are a concern for tissue engineering and medical community [31, [185][186][187]. Furthermore, the unique sterilization process, such as exposing harvested bone to a high temperature deteriorates mechanical and structural properties of the bone graft and reduces osteogenic and osteoinductive properties [149,157].
However, despite poor outcomes reported from xenogeneic bone grafts [149], xenotransplantation remains a standard and successful procedure in dental applications [148,188,189]. To overcome challenges presented by xenogeneic bone grafts, bone graft has been combined with growth factors [190,191] and, in other cases, with allogeneic or alloplastic bone substitute to serve as a hybrid scaffold [175,191,192]. This approach has shown promising outcomes by inducing new bone tissue [193][194][195]. Table 1 summarizes advantages and disadvantages of xenograft.

Alloplastic Bone Graft Substitute
An alloplastic bone substitute is a biocompatible material that is produced synthetically by physical or chemical processing. In recent years, alloplastic bone substitutes have gained more attention, mainly in craniofacial bone reconstruction [170,176]. While surgical procedure to repair cranial defects is known as cranioplasty [35,40,177], the term of alloplastic bone substitute is associated with synthetic biomaterials [160,166]. Alloplasty is a procedure that substitutes large missing bone with synthetic biomaterials to bridge the fracture [35, 172,196]. Notable reasons that make alloplastic biomaterials desirable in cranial repair are unlimited availability, elimination of the need for donors, and minimize potential risk of pathogen transmissions [35, 172,190]. Another advantage of alloplastic biomaterials is that patients may not require a second surgery [161,166].
Several organic and inorganic biomaterials meet these requirements, among them are PMMA and CPC. PMMA is used as a biomaterial for craniofacial reconstruction [182,186,202]. PMMA is routinely used in cranioplasty due to its desired mechanical stability in vivo [40, 203,204], and is widely available and affordable [185,186]. Furthermore, PMMA can be molded into shape to match irregular patient defects [38]. Although PMMA has several advantages that make it suitable biomaterial for cranial bone regeneration, other disadvantages are reported. For instance, PMMA is non-biodegradable polymer. However, it has been used for applications that require a permanent implant, such as dental applications [187,205]. Furthermore, PMMA may display insignificant integration with surrounding tissue, including bone tissues [187,205].
On the other hand, CPC is a promising biomaterial that has been studied to repair cranial bone defects [41,42,206]. The desirable prosperities of CPC include ease to shape and contour, as well as ability to enhance osteoconductivity [188,189]. These unique properties make CPC an attractive and alternative biomaterial for craniofacial bone regeneration. However, one of CPC disadvantages is brittleness. Reinforcing CPC with biopolymers to serve as a composite biomaterial is proposed [40,193,194,207]. Nevertheless, combination of alloplastic biomaterials with MSCs has shown encouraging results [61,64,67].
Another aspect affecting MSC differentiation into target cell lines is the interaction between cells and the surface of biomaterials. It has been established that scaffold topogra-phy of biomaterials influences MSC fate, including migration, differentiation, proliferative capacity, and adherence. Recently, two separate studies showed that 60.66 µm and 32.97 nm pore sizes of distinct nano-and microtopography of wet Spongostan are sufficient to facilitate osteogenic differentiation of human stromal cells in vitro [195], as well as to induce new bone formation in a critical-size calvarial rate in vivo [195,208].

Mesenchymal Stromal Cells: Successes and Challenges
Stem cell therapy has been an active research area to overcome challenges face tissue engineering and medical community [196,197,209,210]. Several studies have demonstrated a successful transition of stem cells to the patients. Research studies have enriched the hope that this regenerative approach may become a treatment for a wide range of critical-size craniofacial bone defects. However, researchers are exploring multiple stem cell types to regenerate critical-size craniofacial defects, including MSCs.

Bone Marrow Mesenchymal Stromal Cells (BMSCs)
BMSCs are adult multipotent stem cells derived from bone marrow tissue [47]. BMSCs are a promising cell source due to their self-renewal and multipotency by differentiating into different cell types, such as osteoblasts, chondrocytes, and adipocytes [125,206,209,[223][224][225][226][227][228][229]. BMSCs are attractive MSCs that have high therapeutic potential. BMSCs can proliferate in vitro and can be used in clinical applications without losing their capacity [215,216,230,231]. BMSCs have demonstrated potential to regenerate cranial bone defects. A new bone formation is observed when BMSCs are harvested, culture-expanded, and implanted in calvarial bone defect of rabbit animal model [24,220,232,233]. This procedure demonstrates efficacy in repairing a cranial bone defect. Furthermore, promising results have been shown when autologous BMSCs are used. The transplant of autologous BMSCs is vital to avoid unwanted immune system responses [46,52,215,231,234].
BMSCs act as reservoirs of reparative cells. They have been identified as key players in bone maintenance and repair [215,216,235]. Accordingly, there has been an increasing interest in using BMSCs for cranial bone regeneration. Recently, we have shown that photoencapsulated BMSCs in fast degrading thiol acrylate hydrogels promote new bone formation in rabbit calvarial defects, compared to negative control group, 6 weeks postimplantation [24,47], Figure 3. Other studies have shown similar conclusions using BMSCs to regenerate critical-size bone defects [50, 236,237]. BMSCs have several advantages.
Studies show encouraging outcomes in restoring critical-size cranial fracture utilizing animal models. However, one challenge facing research communities is maintaining BMSCs phenotype in tissue culture dish, particularly during proliferation and passaging in vitro. When BMSCs attach to the surface of tissue culture dish, they tend to activate and upregulate key bone markers [125,[238][239][240]. Furthermore, BMSCs tend to age and lose their proliferation capacity with advanced passage numbers [215,241,242]. Advanced passage numbers of BMSCs show an increase in senescence markers, where BMSCs enter G 1 /S phase of cell cycle arrest [216,243]. Ridzuan et al. have reported rat BMSCs show a decline in cell growth at advanced passage number of four [216]. Moreover, senescence beta-galactosidase stain, an enzyme-based assay that identifies senescent cells in culture, is increased at passage number of five [216]. The authors concluded that advanced passage number of BMSCs meditated cellular senescence by limiting BMSCs growth [216]. Other studies have reached a similar conclusion demonstrating the impact of using prolonged and advanced passage number of MSCs on their potential use for regenerative or research purposes [197,221,[243][244][245][246][247].
One of the limitations of BMSCs for bone tissue regeneration is low survival rate of BMSCs after transplantation [222,235,248,249]. The low survival rate of BMSCs posttransplantation is crucial for researchers and physicians [220,222,235,248]. The harsh native microenvironments such as inflammation and immune system response, mechanical leakage of BMSCs after injection, cell necrosis and apoptosis, and imbalance in radicals and antioxidants can lead to BMSCs loss [235,250]. Moreover, low survival rate of BMSCs can limit their self-renewal capacity due to lack of nutrients, ECM production, and oxygen [235]. Despite challenge, several techniques have been explored to overcome these obstaclesnotably, more effective methods delivering BMSCs.
Using three-dimensional biodegradable hydrogel scaffolds has demonstrated a promising strategy [25,26,144,251]. Hydrogel scaffolds can provide a temporary structure for protection until BMSCs can differentiate and produce their own ECM [25,26,49,144]. Another strategy is a combined administration of BMSCs with growth factors or with other cell types. For instance, adding BMP-2 to BMSCs culture enhances survival rate and induces BMSC differentiation. A higher BMSCs survival rate is observed when combined administration of BMP-2 with immortalized mouse BMSCs are encapsulated in three-dimensional polymeric scaffolds, Figure 4 [47]. Another approach to increase BMSCs viability is demonstrated through co-culture of BMSCs with MKs, Figure 5A [125]. Maintaining BMSC high survival rate and potency after transplantation could increase their efficacy in vivo, therefore increasing new bone formation [220].
Similar to BMSCs, several bone markers are upregulated when DPSCs are differentiated into osteoblasts. Collagen type I, collagen type III, alkaline phosphatase, and osteocalcin are among these markers [48,260]. In contrast, proliferation rate and differentiation capacity can be distinguished between two MSC types [54, 125,[260][261][262][263]. Studies have demonstrated that DPSCs possess a higher metabolic and proliferative capacity than BMSCs [26,55,56,222,264]. A recent co-culture study of DPSCs with MKs shows higher levels of DPSC viability than BMSCs treated under the same condition at day 5, Figure 5B [125]. Similarly, Alge et al. studied differences and similarities between BMSCs and DPSCs harvested from rat animal model [54]. The authors concluded that DPSCs have a higher proliferation rate and higher expression of Alkaline Phosphatase (ALP) activity and calcium deposition than BMSCs [54]. Other research groups have reached similar conclusion [265,266]. Furthermore, DPSCs show no early senescence signs during in vitro expansion and passaging (replicative senescence) [51, 222,267].
In addition, DPSCs have been proposed as suitable MSCs for cranial reconstruction [268,269], perhaps due to their embryonic origin of craniofacial skeleton [54,57,231]. Investigators have shown that DPSCs enhance cranial bone regeneration in vivo when a cranial defect is created in various animal models [3,13,268,[270][271][272]. In a previous study, photoencapsulated-DPSCs in thiol acrylate hydrogels show increased levels of ALP activity compared to photoencapsulated-BMSCs at day 7 [24], Figure 6. In addition, the study shows photoencapsulated-DPSCs in fast degrading thiol acrylate hydrogels demonstrate higher capacity inducing new bone formation in rabbit calvarial defects comparing to positive and negative control groups, 6 weeks post-implantation [24], Figure 3. The results of similar studies [3,13,24,268,[270][271][272] using DPSCs to reconstruct cranial defects have demonstrated that DPSCs can be a reliable source enhancing cranial bone regeneration. PEGDA hydrogels, cultured in OST medium, show increased ALP activity levels at day 7 compared to photoencapsulated-BMSCs in 5 or 15 wt% PEGDA hydrogels at days 1 and 3. (C) photoencapsulated-DPSCs in 5 or 15 wt% PEGDA hydrogels, cultured in BSL medium, show no difference in ALP activity at days 1, 3, and 7. (D) photoencapsulated-DPSCs in 5 or 15 wt% PEGDA hydrogels, cultured in OST medium, show increased ALP activity levels on day 7 compared to days 1 and 3. * and ** indicates that comparison values are significant (* p < 0.05 and ** p < 0.01). Error bars show ± SD [24].

Bone Morphogenetic Protein-Based Therapy
Bone Morphogenetic Proteins (BMPs) are multifunctional growth factors that belong to Transforming Growth Factors (TGF-β) superfamily [299][300][301]. BMPs are growth factors that regulate cellular functions and embryonic development of musculoskeletal tissues, including craniofacial development [282,302]. BMP-2 expresses during facial ectomesenchyme and tooth developments, as well as during early skull development [282,303].
Several types of BMPs are used to heal large bone defects: notably, BMP-2, BMP-6, and BMP-7. BMP-2 is considered a gold standard protein that is frequently used to regenerate critical-size bone defects. Since US Food and Drug Administration (FDA) has approved recombinant-human Bone Morphogenetic Protein-2 (BMP-2) [156,300,304], research studies have shown enhanced bone tissue regeneration in multiple animal models [65, [274][275][276]299,300,305,306]. Culturing MSCs with BMP-2 has showed increased levels in bone markers, indicating that MSCs are differentiating into osteoblasts [251,273,274,283].
Furthermore, an ideal delivery of BMP-2 into defect site is a challenge to overcome in clinical applications. One reason may occur due to short half-life of BMP-2, usually 1 to 4 h [256,281]. In addition, robust release of BMP-2 post-implantation remain a challenge. However, effective methods and new approaches have been proposed and investigated to deliver BMP-2 effectively [281,[307][308][309][310].
Nevertheless, better results are obtained when BMSCs are delivered along with BMP-2 [251,273,274,283]. Another challenge is that BMP-2 has been shown to boost growth of surrounding bone tissues, such as cartilage and tendon [311][312][313][314]. As a result, control release of BMP-2 is desired to eliminate untargeted tissue growth that may interrupt bone regeneration and cause cosmetic deformities.
Despite challenges, BMP-2 has been a desired choice to regenerate large-size cranial defects. Studies have shown encouraging outcomes of BMP-2 in regulating human cranial osteoblasts by inducing MSC differentiation [315]. For instance, we have recently studied delivering immortalized mouse BMSCs using photoencapsulation method, with or without BMP-2, for craniofacial bone engineering applications [47]. Although negative control groups (photoencapsulated-BMSCs without BMP-2 (BMSCs)) and experimental groups (photoencapsulated-BMSCs with BMP-2 (BMBMP2)) are cultured in basal medium, an increased level of ALP activity is observed in experimental group at day 7 [47], Figure 7. Furthermore, expression of c-Fos, associated with cell cycle and growth [316], and confocal microscopy images show elevated viability levels of BMBMP2 compared to control group (BMSCs) [47], Figures 3 and 8, respectively. The study shows ability of BMP-2, not only improving differentiation capacity of immortalized mouse BMSCs, but also enhancing viability.
Furthermore, a recent study shows BMP-2 can be a chemokine recruiting BMSCs in vitro and in vivo. Liu et al. show that BMP-2 stimulates migration of BMSCs by activating migration-related signaling pathways (CDC42/PAK1/LIMK1) in vitro [267]. Similarly, BMP-2 loaded on collagen sponge shows recruitment of BMSCs injected into circulatory system in vivo [267]. Using CDC42, an inhibitory silencing for migration-related signaling pathway, displays a significant decrease in BMSC recruitment [267]. Therefore, study shows ability of BMP-2 to recruit BMSCs and provides further understanding of BMP-2 benefits in vitro and in vivo.  Other related growth factors have been investigated as a potential candidate for cranial bone repair. For instance, Platelet-Derived Growth Factor (PDGF) and Fibroblast Growth Factor (FGF) have been explored in several studies [317][318][319][320]. Studies show increased levels in new tissue formation when Vascular Endothelial Growth Factor (VEGF) is combined with BMPs, due to a short half-life time of VEGF [321,322]. The authors concluded that combined growth factors stimulate osteoprogenitor cell differentiation and enhanced angiogenesis and regeneration of bone fractures [318].
PRP has some limitations in clinical applications. One of these limitations is that PRP, used as a natural gel, requires thrombin and calcium chloride to initiate the gelation in vitro. As a result, thrombin may have unsought effects by increasing the levels of two factors, V and XI, which can cause coagulopathies [291]. In addition, high concentrations of growth factors in PRP raise safety concerns [290]. For instance, adding multiple growth factors into defect sites at one time may increase potential risk of targeting native microenvironment of surrounding bone tissues [290]. Moreover, some studies have reported an inhibitory effect of PRP on osteoblasts [264,327]. Unlike BMP-2, PRP is not an osteoinductive factor [286]. Furthermore, there is a concern that PRP may cause infection during processing in vitro [286,289].
Despite challenges, PRP has proven to be an effective therapy in regenerating bone defects. Histological analysis by Xie et al. show a combined treatment of PRP, bone fragments, and BMSCs show a larger area of newly formed bone tissue compared to each component used alone [293]. Similarly, Oley et al. show higher lamellar bone growth when a large cranial bone defect is created in a rat animal model and scaffold with a hydroxyapatite combined with PRP is implanted [292]. These studies show efficacy of PRP as a potent growth factor regenerating bone defects.
While mechanisms have not yet been elucidated, growing evidence indicates that MKs play role in skeletal system, remodeling, and homeostasis [340]. For example, early research by Yan et al. shows that mice overexpressing TPO have an elevated MK level in bone marrow [341]. Increased MK count is associated with increased levels of bone formation [341]. Similarly, another study using animal model shows that mice with high MK count show an increased bone mass [112].
Moreover, studies have shown that MKs influence osteoblast and osteoclast proliferation and formation [108,[112][113][114][115]. MKs have demonstrated a robust increase in osteoblast proliferation and bone formation [112,113]. A co-culture experiment of MKs with murine calvarial osteoblasts showed improvements in osteoblast proliferation by three-to six-fold, compared to control groups, osteoblasts cultured alone [114]. Other studies have demonstrated that MKs are vital in osteoclast formation [114][115][116][117][118][119]. In addition, osteoclasts are essential in bone remodeling and eliminate necrotic tissue in the early phases of bone repair [120,121]. An experiment investigating the effects of MKs on osteoclast formation shows prevention of osteoclast development in vitro [340]. Therefore, it is believed that MKs enhance bone mass by inhibiting bone resorption via decreasing osteoclastogenesis and increasing osteoblast proliferation, leading to a net increase in overall bone volume [112]. Furthermore, TPO has indirectly enhanced angiogenesis by increasing platelets (thrombocytes) and stimulating endothelial cell proliferation [342].
Although MSCs do not express TPO receptors (Mpl), MKs show a key role in regulating MSCs [125]. Recently, a study showed that prolonged co-culture of MKs with MSCs (BMSCs vs. DPSCs) enhanced MSC proliferation by two-to three-fold, compared to control groups, MSCs cultured alone [125]. However, the results also show MKs inhibit MSC differentiation into osteoblast lineage cells in vitro [125].
Successfully delivering and preserving high MSC count into defect site remains a challenge. Furthermore, immune system response and leakage of MSCs post-implantation led to lower prediction of MSC survival rate. Therefore, enhancing MSC viability postimplantation is desired. The evidence shows using TPO to induce MKs is a desired approach to investigate, to enhance regeneration of large cranial bone defects.
Nevertheless, indirect impact of TPO may have a downside. For instance, directly injecting and delivering a high dose of TPO can increase platelet production [343]. Therefore, there is a potential risk of TPO to induce bone marrow fibrosis by increasing MK and platelet levels [122,126]. Another concern is that MKs have been shown to inhibit MSC differentiation [125]. Previously, we demonstrated prolonged co-culture of MKs with MSCs shows that MKs inhibit MSC differentiation into osteoblast lineages [125]. Furthermore, study reveals BMSCs, Figure 9A,C, and DPSCs, Figure 9B, co-cultured with MKs, have a significantly lower ALP activity expression than control groups, and similarly, a lower calcium deposition compared to control groups, Figure 10 [125]. Therefore, although MKs elevate BMSC and DPSC viabilities, MKs inhibit MSC differentiation into osteoblast linage cells [125]. TPO has demonstrated possibility increasing bone mass in animal models. Furthermore, several studies have shown co-culture of MKs increases osteoblast and MSC viabilities in vitro, although that MKs are relatively low, they account for approximately 0.01 to 0.05% of all nucleated bone marrow cells in humans [344,345]. The question is whether increasing MK count can enhance regeneration of large-size cranial defects in vivo. With evidence-based and promising practice, additional in vitro and in vivo studies are required to understand the effect of MKs on cranial bone regeneration.  Figure 9A,B shows calcium deposition measured on days 1, 7, and 14, while Figure 9C shows calcium deposition measured on days 1, 17, and 21. Error bars reflect standard deviation of the mean. Significant difference between groups is indicated by error bars: * = p < 0.05 and ** = p < 0.01 [125].

Conclusions and Future Insights
Large cranial defects can result from a variety of conditions. Current approach to regenerate craniofacial bone defects is by pursuing tissue engineering approaches using bone graft substitutes combined with stem cells and growth factors. The uprising and rapidly developing field of stem cell technology and progress made in biomaterials science and technology have enabled cranial defect regeneration. Particularly, the use of growth factors, such as BMP-2 and PRP, have multiple advantages that activate MSCs to differentiate into osteoblasts lineage cells, though limitations exist. For instance, the need for a promising growth factor arises from excessive outcomes of BMP-2 and low survival rate of MSCs post-transplantation.
TPO is a megakaryocyte growth and platelet production. Studies have demonstrated that TPO may have a downside effect by inhibiting osteoclastogenesis and delaying MSC differentiation in vitro, as well as possibly inducing bone marrow fibrosis by increasing MK and platelet levels in vivo. However, co-culture studies of MKs show to enhance osteoblasts and MSC viabilities and maintain their phenotype in vitro, as well as increase bone mass in vivo.
Currently, there are multiple thrombopoietic agents (TPO-like agents) that FDA has approved to treat thrombocytopenia. Therefore, a large amount of information is known on the safety profiles of these agents. Although thrombopoietin to induce and increase MK count for craniofacial bone tissue engineering in humans requires FDA approval, it should not be expected to take as long as a newly tested protein for in vivo application. Therefore, inducing MKs using thrombopoietin for cranial bone regeneration may become a reality in the future.