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
||The process of lengthening bone through endogenous tissue engineering using a guided mechanical environment
|Skeletal stem cell
||Reside in the postnatal bone marrow and give rise to cartilage, bone, hematopoiesis-supportive stroma
|Neural crest cell
||Temporary group of cells arising from the embryonic ectoderm cell layer, and giving rise to a diverse cell lineage
|Bone morphogenic protein
||Growth factors consisting of pivotal morphogenetic signals determining tissue architecture throughout the body
|Apical ectodermal ridge
||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
||Area of the developing limb that contains signals that instruct the developing limb bud to form along the anteroposterior axis
||A metabolite for vitamin A1 that is required for growth and development
|Fibroblast growth factor
||Cell signaling proteins that are crucial in normal development
|Mouse skeletal stem cell
||Skeletal stem cells isolated from mouse bones
|Mandibular distraction osteogenesis
||A surgical procedure that lengthens the lower jaw
|Focal adhesion kinase
||Protein tyrosine kinase concentrated in focal adhesions forming among cells attaching to extracellular matrix
||A medication that binds iron and aluminum
|Hypoxia inducible factor 1-α
||Transcription factor that responds to hypoxia
|Recombinant human BMP-2
||BMPs generated using recombinant DNA technology for clinical use
|Mesenchymal stem cell
||Multipotent stromal cells that can differentiate into a variety of cell types
||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
||Low-intensity and pulsed mechanical waves used to induce regenerative and antiinflammatory effects on bone, cartilage, and tendon
|Limb distraction osteogenesis
||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.
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
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
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).
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
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.
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.
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.
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