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Craniofacial and Long Bone Development in the Context of Distraction Osteogenesis

Shah, Harsh N. M.P.H.; Jones, Ruth E. M.D.; Borrelli, Mimi R. M.B.B.S., M.Sc.; Robertson, Kiana B.S.; Salhotra, Ankit B.S.; Wan, Derrick C. M.D.; Longaker, Michael T. M.D., M.B.A.

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Plastic and Reconstructive Surgery: January 2021 - Volume 147 - Issue 1 - p 54e-65e
doi: 10.1097/PRS.0000000000007451
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Abstract

The skeleton possesses unprecedented biomedical research potential, and translational research in skeletal biology may significantly benefit patients. The incidence of skeletal dysplasia is one in 5000 live births,1,2 whereas traumatic fracture, osteoporosis, and arthritis are the predominant skeletal disorders during aging.3–5 The annual health care cost of fracture repair resulting from osteoporosis is approximately $17 billion in the United States alone.3,6 With the large burden of skeletal abnormalities permeating all stages of life, uncovering the fundamental biological processes driving skeletal development and regeneration is essential in the development of novel targeted therapies.

The skeleton consists of specialized connective tissues including ossified and nonossified elements, bone marrow stroma, and supportive tissues.7 Numerous cell types make up these tissues, including osteocytes, chondrocytes, and hematologic and stromal cells. The common progenitor cell that gives rise to the bone, cartilage, and stromal elements during development, repair, and regeneration is the skeletal stem cell.8,9 Recent discoveries highlight the significant role of the skeletal stem cell as the enactor of mandibular regeneration during distraction osteogenesis (Table 1)—the process of lengthening bone through endogenous tissue engineering using a guided mechanical environment.10 In addition, expression of morphogens is tightly regulated to provide signaling gradients necessary for skeletal growth. We review the current knowledge of the developmental and regenerative biology of the craniofacial and appendicular skeleton.

Table 1. - Terms Used throughout the Review, with Abbreviations and Definitions
Term Abbreviation Definition
Distraction osteogenesis DO The process of lengthening bone through endogenous tissue engineering using a guided mechanical environment
Skeletal stem cell SSC Reside in the postnatal bone marrow and give rise to cartilage, bone, hematopoiesis-supportive stroma
Neural crest cell NCC Temporary group of cells arising from the embryonic ectoderm cell layer, and giving rise to a diverse cell lineage
Bone morphogenic protein BMP Growth factors consisting of pivotal morphogenetic signals determining tissue architecture throughout the body
Apical ectodermal ridge AER Structure that forms at the distal end of the limb bud acting as a major signaling center to ensure proper development of a limb
Zone of polarizing activity ZPA Area of the developing limb that contains signals that instruct the developing limb bud to form along the anteroposterior axis
Retinoic acid RA A metabolite for vitamin A1 that is required for growth and development
Fibroblast growth factor FGF Cell signaling proteins that are crucial in normal development
Mouse skeletal stem cell mSCC Skeletal stem cells isolated from mouse bones
Mandibular distraction osteogenesis MDO A surgical procedure that lengthens the lower jaw
Focal adhesion kinase FAK Protein tyrosine kinase concentrated in focal adhesions forming among cells attaching to extracellular matrix
Deferoxamine DFO A medication that binds iron and aluminum
Hypoxia inducible factor 1-α HIF-1α Transcription factor that responds to hypoxia
Recombinant human BMP-2 rhBMP-2 BMPs generated using recombinant DNA technology for clinical use
Mesenchymal stem cell MSC Multipotent stromal cells that can differentiate into a variety of cell types
Low-level laser LLL Application of red and near-infrared light over injuries or lesions to improve wound and soft-tissue healing and reduce inflammation
Low-intensity pulsed ultrasound LIPUS Low-intensity and pulsed mechanical waves used to induce regenerative and antiinflammatory effects on bone, cartilage, and tendon
Limb distraction osteogenesis LDO A surgical procedure that lengthens long bones of the appendicular skeleton, including the tibia and femur

DEVELOPMENT OF CRANIAL BONES

Most cranial bones arise from ectodermal neural crest cells, which originate from the dorsal margins of the closing neural tube (Fig. 1). During neurulation, the borders of the neural plate converge at the dorsal midline to form the neural tube. At this point, the neural crest cells from the roof plate undergo epithelial-to-mesenchymal transition during neurulation,11–14 including delamination and migration events (Fig. 1). Delamination begins with the dorsal expression of bone morphogenetic proteins (BMPs), which leads to decreased expression of occludins and cadherins, resulting in reduced cellular adhesion.13,15 Occludins are an integral component of tight junctions, and cadherins are important in the formation of adherens junctions.16,17 Both occludins and cadherins are important in maintaining cell-to-cell adhesion. Concurrently, neural crest cells secrete matrix metalloproteinases, which break down the overlying basal lamina.13,18,19 The permeable basal lamina and decreased cellular attachments enable neural crest cells to migrate throughout the embryo.

Fig. 1.
Fig. 1.:
Embryonic view of bone development. (Above, left) Developing mandible (red box) and lower limb (blue box). (Right) Endothelial-to-mesenchymal transition of cranial neural crest cells. (Below, left) Signaling throughout the limb bud trunk (purple), zone of polarizing activity (yellow), progress zone (orange), and apical endodermal ridge (blue).

Neural crest cells migrate from rostral to caudal because of repulsive guidance between molecular signals, extracellular matrix interactions, and cellular contact inhibition.20,21 Expression of the Eph receptor tyrosine kinase by neural crest cells allows them to bind to the ephrin transmembrane ligand, leading to cytoskeletal rearrangement and cellular repulsion.22 In general, tyrosine kinases catalyze the phosphorylation of tyrosine residues, which cause a functional change in the protein23 (Fig. 2). Both Eph and ephrin ligands are membrane-bound proteins that require direct cell-to-cell interactions for activation. Neural crest cells, in addition, express integrin α5β1, which guides migration by binding to ligands such as collagen, laminin, and fibronectin on the extracellular matrix.24,25 Integrins are transmembrane receptors that activate signal transduction pathways mediating cellular-extracellular matrix interactions and intracellular cytoskeleton rearrangements.26

Fig. 2.
Fig. 2.:
Tyrosine kinase pathway. The single pass, type I receptor tyrosine kinase resides in the plasma membrane. The receptor tyrosine kinase is activated through the binding of a ligand leading to a ligand-induced dimerization with the cytoplasmic tyrosine kinase domain. The dimerization results in autophosphorylation of the tyrosine residues inducing conformational changes that stabilize the active site of the kinase. The phosphotyrosine residues act as recruitment sites for downstream signaling proteins.

After cranial neural crest cells colonize the facial prominences, the cells aggregate, condense, and differentiate in response to signals from the surrounding niche.27 Neural crest cells that colonize the first arch form the maxilla and mandible.21 The transcriptional profile of neural crest cell populations uniquely correlate with their origination on the neural tube anteroposterior axis. For example, Hox gene expression in the pharyngeal arch cell populations increases in the posterior direction.28,29Hox genes are homeobox genes that specify body plan regions along the head-tail axis.30 The Hox proteins ensure the correct structures form in the correct places of the body. Overexpression of Hoxa2 in the first pharyngeal arch limits mandible formation31–33 (Fig. 3, above). Furthermore, Hoxa2 has been shown to suppress the expression of Runx2, which is important in skeletogenesis. Runx2 promotes bone differentiation, and inhibition of Runx2 through Hoxa2 expression limits bone formation34–36 (Fig. 3, center). As craniofacial bone development proceeds through intramembranous ossification, absence of Hoxa2 expression is critical for bone formation in the first pharyngeal arch (Fig. 3, below).35

Fig. 3.
Fig. 3.:
Hoxa2/Runx2 pathway. (Above, left) Mouse embryo with developing pharyngeal arches (PA). Pharyngeal arch 1 (red) gives rise to the muscles of mastication and mandible. Pharyngeal arch 2 (orange) gives rise to the muscles of facial expression and hyoid bone. Pharyngeal arch 3 (yellow) gives rise to the greater horn and lower body of the hyoid bone. Pharyngeal arch 4 (green) gives rise to the thyroid and cricoid cartilage. (Below, left) In the absence of Hoxa2, Runx2 activation will occur, leading to bone formation. In the presence of Hoxa2, Runx2 will be suppressed, limiting bone formation. (Right) The expression of Hoxa2 increases in the caudal direction of the pharyngeal arches, with pharyngeal arch 1 not having expression of Hoxa2, whereas pharyngeal arch 4 possesses a high level of Hoxa2 expression. Analogously, the expression of Runx2 decreases in the caudal direction of the pharyngeal arches, with pharyngeal arch 1 having the greatest expression of Runx2, whereas pharyngeal arch 4 possesses a low level of Runx2 expression.

Intramembranous ossification is a process distinct to bone development of the mandible, clavicle, and most bones of the skull. Intramembranous ossification initiates during fetal development in utero, and the skull and clavicles are not fully ossified at birth.37 These bones fully ossify at different postnatal time points and follow a similar ossification paradigm: (1) formation of ossification center, (2) matrix formation, (3) periosteum weaving, and (4) compact bone formation.37 A concentration of mesenchymal cells differentiate into bone-depositing osteoblasts that cluster to form an ossification center.38 Next, the osteoblasts secrete collagenous matrix proteins, or osteoids, which calcify and confine the osteoblasts. Once the osteoblasts are embedded onto the osteoid, the osteoblasts develop into osteocytes. Synchronously, osteogenic cells from adjacent connective tissue differentiate into osteoblasts on the periphery of the growing bone. Ongoing bone deposition allows collections of osteoids to congregate near capillaries, forming the trabecular matrix of spongy bone. Osteoblasts on the periphery of the spongy bone develop into the periosteum. This newly formed periosteum produces compact bone around the spongy bone, and the spongy bone surrounding nearby blood vessels condenses into bone marrow.37 Intramembranous ossification thus results in formation of the bone without an intermediate cartilaginous anlage.

DEVELOPMENT OF LONG BONES

Long bone development begins with outgrowth of the limb buds from the trunk (Fig. 1) in the presumptive forelimb and hindlimb locations. Cells from the lateral plate mesoderm migrate to create a mass of proliferative bone progenitor cells known as the limb field. Three areas of significance form to pattern the growing limb bud: apical ectodermal ridge, progress zone, and zone of polarizing activity (Fig. 1). The apical ectodermal ridge is a structurally distinct ridge of epithelium located at the distalmost extent of the limb bud. The apical ectodermal ridge bisects the dorsal and ventral aspects of the growing limb bud and is necessary for limb outgrowth.39 Second, the progress zone is a mass of cells found underneath the apical ectodermal ridge.40,41 The progress zone is necessary for limb type specification, with cells in this zone harboring intrinsic properties to determine limb type. The third structure is the zone of polarizing activity, which is restricted to the posterior aspect of the bud and provides signals directing limb bud growth along the anteroposterior axis.42

Morphogenetic signaling gradients are central to direct limb length and patterning during development. For example, proximal-to-distal specification relies on the antagonistic relationship between retinoic acid and fibroblast growth factor (FGF)-8.43,44 Cell fates are influenced by a proximal source of retinoic acid originating from the embryonic trunk and a distal source of FGF-8 originating from the apical ectodermal ridge.45 Anteroposterior specification occurs through Sonic hedgehog, BMP, and Gremlin signaling, which originates in the zone of polarizing activity domain,42,46–48 whereas dorsal-to-ventral specification relies on a gradient of WNT and BMP.49 Long bone development further continues through endochondral ossification.

Endochondral ossification is responsible for bone formation of all skeletal elements other than the craniofacial bones and clavicle. This begins when progenitor cells differentiate into chondrocytes and synthesize extracellular matrix abundant in type II collagen.37,50–54 This cartilaginous model prefigures the shape of ossified bone and enlarges through chondrocyte proliferation. The chondrocytes are divided into three zones during this process. First is the zone of proliferation, located in the center and which contains rapidly dividing chondrocytes. These cells stop proliferating in the zone of maturation. The outermost, hypertrophic zone is composed of chondrocytes that secrete a distinct matrix containing type X collagen.37,50,54–59 Concomitantly, the hypertrophic chondrocytes direct the cells in the perichondrium to differentiate into osteoblasts.60–62 Moreover, angiogenesis of the hypertrophic zone and perichondrium allow ossification of the cartilage matrix by the invading osteoblasts.11,63–67

Our understanding of the cellular basis in limb development has greatly advanced with the recent discovery of the skeletal stem cell. These cells were first identified in the femoral growth plates of mice, and possess the ability to self-renew and differentiate into bone, cartilage, and stromal subtypes.8 Further evidence supporting the intrinsic ability of the mouse skeletal stem cell and its downstream progenitor cells to generate these tissue types included production of ossicle, cartilage, and marrow after transplantation of purified cells into a kidney capsule niche8 (Fig. 4, above). The corresponding human skeletal stem cell was subsequently isolated from the femoral growth plate, exhibiting similar properties of self-renewal and differentiation into each skeletal tissue type9 (Fig. 4, center).

Fig. 4.
Fig. 4.:
Skeletal stem cell hierarchy. (Left) The skeletal stem cell hierarchy in mice beginning with a self-renewing mouse skeletal stem cell (mSSC) differentiating to lineage-restricted bone, cartilage, and stromal cells through a bone, cartilage, stromal progenitor (BCSP) cell (Chan CK, Seo EY, Chen JY, et al. Identification and specification of the mouse skeletal stem cell. Cell 2015;160:285–298. 10.1016/j.cell.2014.12.002). (Right) The skeletal stem cell hierarchy in humans beginning with a self-renewing human skeletal stem cell (hSSC) differentiating to lineage-restricted bone, cartilage, and stromal cells through a bone, cartilage, stromal progenitor (BCSP) cell (Chan CKF, Gulati GS, Sinha R, et al. Identification of the human skeletal stem cell. Cell 2018;175:43–56.e21).

The identification of the skeletal stem cell has illuminated important skeletal biology. For example, downstream progenitor subsets that are derived from the mouse skeletal stem cell have been shown to execute long bone fracture repair.68 Following femoral fracture, these cells exhibited increased cell frequency, viability, and enhanced osteogenic function. Intriguingly, the injury-responsive mouse skeletal stem cell transcriptional profile showed up-regulation of the same genes and signaling morphogens important in long bone embryogenesis, such as BMP and Hedgehog.68 These data highlight the molecular overlap between long bone embryogenesis and regeneration.

With regard to the role of the skeletal stem cell in regenerative contexts, a recent study explored the behavior of mouse skeletal stem cells during mandibular distraction osteogenesis. This work revealed that the bone regenerate was clonally derived from mouse skeletal stem cells.10 The pathway by which mouse skeletal stem cells respond to the mechanical force of mandibular distraction osteogenesis involves up-regulation of focal adhesion kinase signaling10 (Fig. 5). The underlying genetic programs responding to focal adhesion kinase signal transduction in mouse skeletal stem cells included activation of transcriptional elements characteristic of primitive neural crest cells.10,69 This genetic reversion of mandibular mouse skeletal stem cells back to primitive neural crest cell characteristics underscores the importance of understanding bone embryogenesis as it applies to bone regeneration biology. Furthermore, the fact that mandibular distraction osteogenesis can proceed in patients with type II collagenopathy harken back to intramembranous ossification, which occurs without a cartilage intermediate.70 In fact, mouse skeletal stem cells are capable of forming new mandibular bone through intramembranous ossification during mandibular distraction osteogenesis, thereby recapitulating developmental processes during regeneration.10

Fig. 5.
Fig. 5.:
Focal adhesion kinase (FAK) signaling pathway. Cytoplasmic tyrosine kinase focal adhesion kinase becomes activated after interacting with transmembrane integrin proteins, allowing focal adhesion kinase to form a complex with Src family kinase. The complex initiates downstream signaling pathways through the phosphorylation of other proteins such as ERK/MAPK.

ADJUVANT THERAPIES FOR MANDIBULAR DISTRACTION OSTEOGENESIS

Mandibular distraction osteogenesis is an advantageous strategy to treat mandibular hypoplasia, which is a feature of multiple clinical problems related to mandibular deficiencies. Although generally performed in children, mandibular distraction osteogenesis may be applied to adult populations as well.71,72 Mandibular distraction osteogenesis is a dynamic process rather than a single intervention. Common corticotomies used include the oblique body/angle cut, vertical ramus cut, and inverted-L ramus cut, which vary in use based on age and mandibular anatomy.71,73 Postoperatively, the distraction protocol begins with a variable period of 0 to 5 days. After latency, distraction occurs with a total rate of 1 to 2 mm of distraction per day divided over a frequency of one to four times per day. The final phase of mandibular distraction osteogenesis involves consolidation of the regenerate over a period of 6 to 12 weeks.71,74 The overall complication rate ranges from 20 to 40 percent, and a technical learning curve may exist, with increased complication rates noted earlier in a surgeon’s experience.75 Major postoperative compilations of mandibular distraction osteogenesis include malunion/nonunion, premature consolidation, and relapse.75

The ability of adjuvant therapies to enhance the mandibular distraction osteogenesis operations has been studied using various animal models. Deferoxamine accelerates bone consolidation in rats undergoing mandibular distraction osteogenesis76 by chelating iron, which results in the stimulation of the hypoxia inducible factor 1-α (HIF-1α) pathway. HIF-1α is a subunit of the heterodimeric transcription factor HIF-1, which is considered the master transcriptional regulator for cellular and developmental response toward hypoxia.77–79 With regard to regeneration, the up-regulation of HIF-1α led to improved wound healing of damaged tissue in mice, whereas down-regulation of HIF-1α resulted in diminished wound closure.80 Specifically, in terms of bone regeneration, mouse skeletal stem cell chromatin architecture sequencing revealed that the HIF-1α transcriptional network plays a substantial role during mandibular distraction osteogenesis.10 In addition, a recent case report showed improved pterygomaxillary area and density in a patient receiving deferoxamine during mandibular distraction osteogenesis after irradiation, highlighting the potential clinical relevance of deferoxamine as an adjuvant therapy during mandibular distraction osteogenesis.81

Another prominent molecule that supports mandibular distraction osteogenesis bone regeneration is BMP, with high levels of expression confirmed during distraction and subsequent decline during consolidation.82 BMP interacts with cell surface receptors known as BMP receptors. The interaction leads to signal transduction resulting in the mobilization of members from the SMAD family of proteins, which are essential for fracture repair and bone growth.83 Recombinant human BMP-2 (rhBMP-2) has been approved for patient administration to improve fracture repair in the tibia and for specific spinal indications. Contraindications for use include any type of anterior cervical spine fusion and soft-tissue swelling near the esophagus and trachea, given the propensity of rhBMP to cause swelling in this region. Other reports have also highlighted problems with ectopic bone growth and variability in dosage delivered with current carrier systems.84 Aside from BMP, vascular endothelial growth factor and FGF-2 expression have also been found to increase during consolidation.85,86 Exogenous growth factor administration along similar timelines holds potential to promote improved bone formation during mandibular distraction osteogenesis.87

Alternatively, the mesenchymal stem cell has been studied as a cellular therapeutic to enhance bone consolidation in the setting of mandibular distraction osteogenesis. Sheep hemimandibles treated with mesenchymal stem cells on the first day of consolidation had a greater total and compact bone ratio in the regenerate zone.88 Another study examined endogenous recruitment of mesenchymal stem cells to the site of bone formation using a rat model of mandibular distraction osteogenesis; the stromal cell–derived factor-1/chemokine receptor-4 pathway activation was found to promote migration of mesenchymal stem cells to the distraction site.89 However, these studies were not able to determine contribution efficiency of the recruited mesenchymal stem cells to the distraction regenerate.

Noninvasive therapies that aid in bone formation during mandibular distraction osteogenesis include low-level laser therapy and low-intensity pulsed ultrasound. Low-level laser therapy consists of daily laser treatments using an 800-nm wavelength laser during distraction and/or consolidation, and has shown improved bone regeneration in rabbits.90 How low-level laser promotes bone formation from a mechanistic standpoint, however, remains poorly understood. Compared to low-level laser therapy, there is more insight into the cellular mechanism of low-intensity pulsed ultrasound in promotion of bone formation. The technology uses low-intensity and pulsed mechanical waves to induce regenerative and antiinflammatory effect on bone, cartilage, and tendon.91 The mechanism by which low-intensity pulsed ultrasound induces regenerative effects is unknown; however, one theory is the nonthermal phenomenon, where the mechanical waves cause changes in cellular physiology.92–94 As such, focal adhesion kinase, which was shown to be up-regulated in mouse skeletal stem cells during mouse mandibular distraction osteogenesis, may have an important role as mechanical signals are transduced into cellular signals.10

ADJUVANT THERAPIES FOR LIMB DISTRACTION OSTEOGENESIS

Just as mandibular distraction osteogenesis may be used to address a wide range of underlying abnormality, limb distraction osteogenesis is applied to various bone and soft-tissue deficits of the appendicular skeleton. These include limb length discrepancies, oncologic resection, traumatic deformity correction, and treatment of ankle osteoarthritis.95–98 Clinical procedures for limb distraction osteogenesis are similar to mandibular distraction osteogenesis, and preoperative imaging and planning are the first steps. Careful evaluation of bone vascular health and surrounding soft tissue is important for limb distraction osteogenesis.99 Postoperative protocols also mirror mandibular distraction osteogenesis distraction strategies, with 5 to 7 days of latency and subsequent distraction carried out at 0.75 to 1.0 mm/day, paired with ongoing physical therapy into the consolidation phase.100,101 The complication rate of appendicular distraction appears to be higher than that of mandibular distraction. The most frequent complications are frame-related, followed by nonunion.99 The data are heterogenous, with some retrospective studies noting the total number of complications exceeding the number of patients enrolled in the study.102,103

Similar to mandibular distraction osteogenesis, bone marrow mesenchymal stem cells have been studied to determine their therapeutic role in reducing the treatment time of limb distraction osteogenesis. One rabbit study used autogenous bone marrow mesenchymal stem cells from the tibia, with transplantation of 1 million cells into the distraction gap after four to six ex vivo passages.104 Another study in dogs used allogenic bone marrow mesenchymal stem cells from the tibia and transplanted 1 million cells into the distraction gap after three passages.105 Although mesenchymal stem cell administration led to faster bone formation, heterogenous cell culture and administration methodology precludes clinical application at this time.

Low-intensity pulsed ultrasound has also been investigated in the setting of tibial distraction osteogenesis. In the study, 20 minutes of therapy at a frequency of 1.5 MHz and impulse length of 200 μsec daily throughout the distraction period demonstrated an increased radiologic callus density by 33 percent.106 Another study applied the same parameters of low-intensity pulsed ultrasound therapy during both distraction and consolidation, which led to faster healing.107 Although the use of low-intensity pulsed ultrasound as a potential adjuvant therapy in limb distraction osteogenesis is promising, quantification of bone formation is inconsistent, impairing direct comparisons.

Overall, data surrounding the use of adjuvant therapies to augment limb distraction osteogenesis are not robust, and many strategies are extrapolated from their use in mandibular distraction osteogenesis. For example, low-level laser therapy has not been studied in the context of limb distraction osteogenesis. However, given its promising results in mandibular distraction osteogenesis, low-level laser therapy holds potential to enhance bone formation during limb distraction osteogenesis. Overall, understanding the biological basis of distraction osteogenesis will continue to illuminate potential translational therapies to improve bone regeneration and thus clinical outcomes. Furthermore, interpreting these strategies from the perspective of resident skeletal stem cells in long bone, from which they were first described, will be key for appreciating how these approaches may be clinically translated.

CONCLUSIONS

Although progress has been achieved in skeletal biology research, the fundamental understanding of regulatory mechanisms governing bone development and regeneration remains elusive. Studies have uncovered the morphogens and transcription factors necessary for skeletal growth; however, the role of these factors in bone regeneration has yet to be determined. Discovery of the skeletal stem cell has improved our understanding of the cellular underpinnings of bone regeneration, highlighting the recapitulation of developmental processes in various postnatal processes. Harnessing these cells as therapeutic targets may prove to be a powerful tool in addressing the clinical challenges of both mandibular distraction osteogenesis and limb distraction osteogenesis, and other skeletal disorders. Further research should seek to better understand the molecular biology of skeletal stem cells, along with their precise behavior during tissue production, maintenance, and repair. Developmental mapping of skeletal tissue, including characterization of cellular, molecular, and genetic patterns giving rise to craniofacial and long bones, is crucial for understanding bone regenerative processes and the role of adjuvant therapies during treatment. Current data examining adjuvant therapies to enhance bone formation during distraction osteogenesis are preliminary; continued work to determine the biological basis of distraction osteogenesis will inform the development of innovative surgical techniques and adjunctive treatments. Furthermore, the biological understanding of mandibular distraction osteogenesis is more advanced than limb distraction osteogenesis with regard to development, regeneration, and adjuvant therapy outcomes, which presents a key opportunity to advance the scientific knowledge surrounding limb distraction osteogenesis. Advances in our grasp of skeletal regenerative and developmental biology hold potential for translation of clinical interventions to provide patients with improved solutions for skeletal defects and injury.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants R01DE026730 (to M.T.L.), R01DE027323 (to M.T.L.), R01DE027346 (to D.C.W.), and 5T32GM119995-02 (to H.N.S.); the Hagey Lab for Pediatric Regenerative Medicine (to M.T.L.); and a generous gift from Carmelita Ko and Keith Tsu (to D.C.W.). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in this article.

REFERENCES

1. Orioli IM, Castilla EE, Barbosa-Neto JG. The birth prevalence rates for the skeletal dysplasias. J Med Genet. 1986;23:328–332.
2. Krakow D. Skeletal dysplasias. Clin Perinatol. 2015;42:301–319, viii.
3. Cauley JA. Public health impact of osteoporosis. J Gerontol A Biol Sci Med Sci. 2013;68:1243–1251.
4. Kanis JA, Oden A, Johansson H, Borgström F, Ström O, McCloskey E. FRAX and its applications to clinical practice. Bone. 2009;44:734–743.
5. Unnanuntana A, Gladnick BP, Donnelly E, Lane JM. The assessment of fracture risk. J Bone Joint Surg Am. 2010;92:743–753.
6. Ray NF, Chan JK, Thamer M, Melton LJ III. Medical expenditures for the treatment of osteoporotic fractures in the United States in 1995: Report from the National Osteoporosis Foundation. J Bone Miner Res. 1997;12:24–35.
7. Karsenty G. The complexities of skeletal biology. Nature. 2003;423:316–318.
8. Chan CK, Seo EY, Chen JY, et al. Identification and specification of the mouse skeletal stem cell. Cell. 2015;160:285–298.
9. Chan CKF, Gulati GS, Sinha R, et al. Identification of the human skeletal stem cell. Cell. 2018;175:43–56.e21.
10. Ransom RC, Carter AC, Salhotra A, et al. Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration. Nature. 2018;563:514–521.
11. Runyan CM, Gabrick KS. Biology of bone formation, fracture healing, and distraction osteogenesis. J Craniofac Surg. 2017;28:1380–1389.
12. Huang X, Saint-Jeannet JP. Induction of the neural crest and the opportunities of life on the edge. Dev Biol. 2004;275:1–11.
13. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014;15:178–196.
14. Theveneau E, Mayor R. Neural crest delamination and migration: From epithelium-to-mesenchyme transition to collective cell migration. Dev Biol. 2012;366:34–54.
15. Bolós V, Peinado H, Pérez-Moreno MA, Fraga MF, Esteller M, Cano A. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: A comparison with Snail and E47 repressors. J Cell Sci. 2003;116:499–511.
16. Saitou M, Furuse M, Sasaki H, et al. Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell. 2000;11:4131–4142.
17. Hulpiau P, van Roy F. Molecular evolution of the cadherin superfamily. Int J Biochem Cell Biol. 2009;41:349–369.
18. Nistico P, Bissell MJ, Radisky DC. Epithelial-mesenchymal transition: General principles and pathological relevance with special emphasis on the role of matrix metalloproteinases. Cold Spring Harb Perspect Biol. 2012;4:a011908.
19. Sauka-Spengler T, Bronner-Fraser M. A gene regulatory network orchestrates neural crest formation. Nat Rev Mol Cell Biol. 2008;9:557–568.
20. Köntges G, Lumsden A. Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development. 1996;122:3229–3242.
21. Kulesa PM, Fraser SE. In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches. Development. 2000;127:1161–1172.
22. Smith A, Robinson V, Patel K, Wilkinson DG. The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells. Curr Biol. 1997;7:561–570.
23. Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 2008.5th edNew York: Freeman.
24. Alfandari D, Cousin H, Gaultier A, Hoffstrom BG, DeSimone DW. Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin. Dev Biol. 2003;260:449–464.
25. McLennan R, Kulesa PM. In vivo analysis reveals a critical role for neuropilin-1 in cranial neural crest cell migration in chick. Dev Biol. 2007;301:227–239.
26. Clark EA, Brugge JS. Integrins and signal transduction pathways: The road taken. Science. 1995;268:233–239.
27. Minoux M, Holwerda S, Vitobello A, et al. Gene bivalency at polycomb domains regulates cranial neural crest positional identity. Science. 2017;355eaal2913.
28. Couly G, Grapin-Botton A, Coltey P, Ruhin B, Le Douarin NM. Determination of the identity of the derivatives of the cephalic neural crest: Incompatibility between Hox gene expression and lower jaw development. Development. 1998;125:3445–3459.
29. Gendron-Maguire M, Mallo M, Zhang M, Gridley T. Hoxa-2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell. 1993;75:1317–1331.
30. Holland PW, Booth HA, Bruford EA. Classification and nomenclature of all human homeobox genes. BMC Biol. 2007;5:47.
31. Rijli FM, Mark M, Lakkaraju S, Dierich A, Dollé P, Chambon P. A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene. Cell. 1993;75:1333–1349.
32. Gavalas A, Studer M, Lumsden A, Rijli FM, Krumlauf R, Chambon P. Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch. Development. 1998;125:1123–1136.
33. Kanzler B, Kuschert SJ, Liu YH, Mallo M. Hoxa-2 restricts the chondrogenic domain and inhibits bone formation during development of the branchial area. Development. 1998;125:2587–2597.
34. Dobreva G, Chahrour M, Dautzenberg M, et al. SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation. Cell. 2006;125:971–986.
35. Grammatopoulos GA, Bell E, Toole L, Lumsden A, Tucker AS. Homeotic transformation of branchial arch identity after Hoxa2 overexpression. Development. 2000;127:5355–5365.
36. Pasqualetti M, Ori M, Nardi I, Rijli FM. Ectopic Hoxa2 induction after neural crest migration results in homeosis of jaw elements in Xenopus. Development. 2000;127:5367–5378.
37. Betts JG, Desaix P, Johnson E, et al. Bone formation and development. In: Anatomy and Physiology. 2017.Houston: OpenStax.
38. Stricker S, Mundlos S. FGF and ROR2 receptor tyrosine kinase signaling in human skeletal development. Curr Top Dev Biol. 2011;97:179–206.
39. Pizette S, Abate-Shen C, Niswander L. BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb. Development. 2001;128:4463–4474.
40. Tickle C, Wolpert L. The progress zone: Alive or dead? Nat Cell Biol. 2002;4:E216–E217.
41. Tickle C. How the embryo makes a limb: Determination, polarity and identity. J Anat. 2015;227:418–430.
42. Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993;75:1401–1416.
43. Mariani FV, Ahn CP, Martin GR. Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning. Nature. 2008;453:401–405.
44. Thaller C, Eichele G. Identification and spatial distribution of retinoids in the developing chick limb bud. Nature. 1987;327:625–628.
45. Cornell RA, Kimelman D. Activin-mediated mesoderm induction requires FGF. Development. 1994;120:453–462.
46. Büscher D, Bosse B, Heymer J, Rüther U. Evidence for genetic control of Sonic hedgehog by Gli3 in mouse limb development. Mech Dev. 1997;62:175–182.
47. Roberts DJ, Johnson RL, Burke AC, Nelson CE, Morgan BA, Tabin C. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development. 1995;121:3163–3174.
48. Sagai T, Masuya H, Tamura M, et al. Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh). Mamm Genome. 2004;15:23–34.
49. Parr BA, McMahon AP. Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature. 1995;374:350–353.
50. Berendsen AD, Olsen BR. Bone development. Bone. 2015;80:14–18.
51. Nusspaumer G, Jaiswal S, Barbero A, et al. Ontogenic identification and analysis of mesenchymal stromal cell populations during mouse limb and long bone development. Stem Cell Reports. 2017;9:1124–1138.
52. Gentili C, Cancedda R. Cartilage and bone extracellular matrix. Curr Pharm Des. 2009;15:1334–1348.
53. Heinegård D. Fell-Muir lecture: Proteoglycans and more. From molecules to biology. Int J Exp Pathol. 2009;90:575–586.
54. Aszódi A, Bateman JF, Gustafsson E, Boot-Handford R, Fässler R. Mammalian skeletogenesis and extracellular matrix: What can we learn from knockout mice? Cell Struct Funct. 2000;25:73–84.
55. Egawa S, Miura S, Yokoyama H, Endo T, Tamura K. Growth and differentiation of a long bone in limb development, repair and regeneration. Dev Growth Differ. 2014;56:410–424.
56. Mariani FV, Martin GR. Deciphering skeletal patterning: Clues from the limb. Nature. 2003;423:319–325.
57. Grabowski P. Physiology of bone. Endocr Dev. 2015;28:33–55.
58. Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell. 2002;2:389–406.
59. Kronenberg HM. The role of the perichondrium in fetal bone development. Ann N Y Acad Sci. 2007;1116:59–64.
60. Caplan AI. Bone development and repair. Bioessays. 1987;6:171–175.
61. Pazzaglia UE, Congiu T, Sibilia V, Pagani F, Benetti A, Zarattini G. Relationship between the chondrocyte maturation cycle and the endochondral ossification in the diaphyseal and epiphyseal ossification centers. J Morphol. 2016;277:1187–1198.
62. Shapiro IM, Adams CS, Freeman T, Srinivas V. Fate of the hypertrophic chondrocyte: Microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate. Birth Defects Res C Embryo Today. 2005;75:330–339.
63. White A, Wallis G. Endochondral ossification: A delicate balance between growth and mineralisation. Curr Biol. 2001;11:R589–R591.
64. Vu TH, Shipley JM, Bergers G, et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell. 1998;93:411–422.
65. Marie PJ. Transcription factors controlling osteoblastogenesis. Arch Biochem Biophys. 2008;473:98–105.
66. Karelina TV, Goldberg GI, Eisen AZ. Matrix metalloproteinases in blood vessel development in human fetal skin and in cutaneous tumors. J Invest Dermatol. 1995;105:411–417.
67. Mackie EJ, Ahmed YA, Tatarczuch L, Chen KS, Mirams M. Endochondral ossification: How cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol. 2008;40:46–62.
68. Marecic O, Tevlin R, McArdle A, et al. Identification and characterization of an injury-induced skeletal progenitor. Proc Natl Acad Sci USA. 2015;112:9920–9925.
69. Bell S, Terentjev EM. Focal adhesion kinase: The reversible molecular mechanosensor. Biophys J. 2017;112:2439–2450.
70. Garza RM, Alyono JC, Dorfman DW, Wan DC. Mandibular distraction in a patient with type II collagenopathy. J Craniofac Surg. 2017;28:2073–2075.
71. Ow AT, Cheung LK. Meta-analysis of mandibular distraction osteogenesis: Clinical applications and functional outcomes. Plast Reconstr Surg. 2008;121:54e–69e.
72. McCarthy JG, Grayson B, Williams JK, Turk A. Distraction of the Mandible. 2017.New York: Springer Nature.
73. McCarthy JG, Schreiber J, Karp N, Thorne CH, Grayson BH. Lengthening the human mandible by gradual distraction. Plast Reconstr Surg. 1992;89:1–8; discussion 9–10.
74. Guerrero CA, Bell WH, Contasti-Bocco GI, Rodriguez AM, Contasti RV. Intraoral Mandibular Distraction. 2017.New York: Springer Nature.
75. Master DL, Hanson PR, Gosain AK. Complications of mandibular distraction osteogenesis. J Craniofac Surg. 2010;21:1565–1570.
76. Donneys A, Deshpande SS, Tchanque-Fossuo CN, et al. Deferoxamine expedites consolidation during mandibular distraction osteogenesis. Bone. 2013;55:384–390.
77. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA. 1995;92:5510–5514.
78. Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev. 1998;12:149–162.
79. Smith TG, Robbins PA, Ratcliffe PJ. The human side of hypoxia-inducible factor. Br J Haematol. 2008;141:325–334.
80. Zhang Y, Strehin I, Bedelbaeva K, et al. Drug-induced regeneration in adult mice. Sci Transl Med. 2015;7:290ra92.
81. Momeni A, Rapp S, Donneys A, Buchman SR, Wan DC. Clinical use of deferoxamine in distraction osteogenesis of irradiated bone. J Craniofac Surg. 2016;27:880–882.
82. Rauch F, Lauzier D, Croteau S, Travers R, Glorieux FH, Hamdy R. Temporal and spatial expression of bone morphogenetic protein-2, -4, and -7 during distraction osteogenesis in rabbits. Bone. 2000;27:453–459.
83. Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors. 2004;22:233–241.
84. Agrawal V, Sinha M. A review on carrier systems for bone morphogenetic protein-2. J Biomed Mater Res B Appl Biomater. 2017;105:904–925.
85. Hu J, Zou S, Li J, Chen Y, Wang D, Gao Z. Temporospatial expression of vascular endothelial growth factor and basic fibroblast growth factor during mandibular distraction osteogenesis. J Craniomaxillofac Surg. 2003;31:238–243.
86. Warren SM, Mehrara BJ, Steinbrech DS, et al. Rat mandibular distraction osteogenesis: Part III. Gradual distraction versus acute lengthening. Plast Reconstr Surg. 2001;107:441–453.
87. Makhdom AM, Hamdy RC. The role of growth factors on acceleration of bone regeneration during distraction osteogenesis. Tissue Eng Part B Rev. 2013;19:442–453.
88. Aykan A, Ozturk S, Sahin I, et al. Biomechanical analysis of the effect of mesenchymal stem cells on mandibular distraction osteogenesis. J Craniofac Surg. 2013;24:e169–e175.
89. Cao J, Wang L, Du ZJ, et al. Recruitment of exogenous mesenchymal stem cells in mandibular distraction osteogenesis by the stromal cell-derived factor-1/chemokine receptor-4 pathway in rats. Br J Oral Maxillofac Surg. 2013;51:937–941.
90. Kan B, Tasar F, Korkusuz P, et al. Histomorphometrical and radiological comparison of low-level laser therapy effects on distraction osteogenesis: Experimental study. Lasers Med Sci. 2014;29:213–220.
91. El-Bialy T, Tanaka E, Aizenbud D, eds. Therapeutic Ultrasound in Dentistry: Applications for Dentofacial Repair, Regeneration, and Tissue Engineering. 2018.New York: Springer.
92. Hagiwara T, Bell WH. Effect of electrical stimulation on mandibular distraction osteogenesis. J Craniomaxillofac Surg. 2000;28:12–19.
93. Andrade Gomes do Nascimento LE, Sant’anna EF, Carlos de Oliveira Ruellas A, Issamu Nojima L, Goncalves Filho AC, Antonio Pereira Freitas S. Laser versus ultrasound on bone density recuperation after distraction osteogenesis: A cone-beam computer tomographic analysis. J Oral Maxillofac Surg. 2013;71:921–928.
94. Khanna A, Nelmes RT, Gougoulias N, Maffulli N, Gray J. The effects of LIPUS on soft-tissue healing: A review of literature. Br Med Bull. 2009;89:169–182.
95. Chim H, Sontich JK, Kaufman BR. Free tissue transfer with distraction osteogenesis is effective for limb salvage of the infected traumatized lower extremity. Plast Reconstr Surg. 2011;127:2364–2372.
96. de Baat P, de Baat C, Bessems JH. Distraction osteogenesis in orthopaedics (in Dutch). Ned Tijdschr Tandheelkd. 2008;115:306–313.
97. Papakostidis C, Bhandari M, Giannoudis PV. Distraction osteogenesis in the treatment of long bone defects of the lower limbs: Effectiveness, complications and clinical results. A systematic review and meta-analysis. Bone Joint J. 2013;95-B:1673–1680.
98. Sabharwal S, Nelson SC, Sontich JK. What’s new in limb lengthening and deformity correction. J Bone Joint Surg Am. 2015;97:1375–1384.
99. Watson JT. Distraction osteogenesis. J Am Acad Orthop Surg. 2006;14(Spec XXX No.):S168–S174.
100. Herzenberg JE, Standard SC. Flynn JM, Sankar WN, Wiesel SW, eds. Tibial lengthening with circular external fixation. In: Operative Techniques in Pediatric Orthopaedic Surgery. 20162nd ed. Philadelphia: Wolters Kluwer; 558–566.
101. Herzenberg JE, Standard SC. Flynn JM, Sankar WN, Wiesel SW, eds. Femoral lengthening with external fixation. In: Operative Techniques in Pediatric Orthopedic Surgery. 20162nd edPhiladelphia: Wolters Kluwer; 538–549.
102. Liantis P, Mavrogenis AF, Stavropoulos NA, et al. Risk factors for and complications of distraction osteogenesis. Eur J Orthop Surg Traumatol. 2014;24:693–698.
103. Vargas Barreto B, Caton J, Merabet Z, Panisset JC, Pracros JP. Complications of Ilizarov leg lengthening: A comparative study between patients with leg length discrepancy and short stature. Int Orthop. 2007;31:587–591.
104. Harada Y, Nakasa T, Mahmoud EE, et al. Combination therapy with intra-articular injection of mesenchymal stem cells and articulated joint distraction for repair of a chronic osteochondral defect in the rabbit. J Orthop Res. 2015;33:1466–1473.
105. Zeng JJ, Guo P, Zhou N, Xie QT, Liao FC. Treatment of large bone defects with a novel biological transport disc in non-vascular transport distraction osteogenesis. Int J Oral Maxillofac Surg. 2016;45:670–677.
106. Salem KH, Schmelz A. Low-intensity pulsed ultrasound shortens the treatment time in tibial distraction osteogenesis. Int Orthop. 2014;38:1477–1482.
107. Song MH, Kim TJ, Kang SH, Song HR. Low-intensity pulsed ultrasound enhances callus consolidation in distraction osteogenesis of the tibia by the technique of lengthening over the nail procedure. BMC Musculoskelet Disord. 2019;20:108.
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