INTRODUCTION

Scientific advances in the 21st century have helped us to understand molecular factors that regulate condylar growth.1 Genetic disorders in the craniofacial region lead to a number of craniofacial anomalies. For example, micrognathia and hemifacial microsomia are such conditions where impaired condylar growth requires comprehensive surgical intervention. Currently, the available approaches include distraction osteogenesis, orthognathic surgery, bone grafting using autogenous bone, allogenic bone grafts, and others. Osteoinductive matrices are limited by their disadvantages such as bone graft resorption, their association with lack of volume, donor site morbidity, potential for antigenicity, and disease transmission. Most would agree that the current patient care regimen is inefficient, expensive, and painful, and therefore needs to be improved.2–5

The rapid development of recombinant DNA technology has led to the development of growth factor-based approaches. Using the specific gene encoding the proteins, we are now able to synthesize large quantities of the therapeutic proteins for treatment purposes.67 Local administration of insulin-like growth factor (IGF-I) in the mandibular condyle of rats has been shown to induce actual bone formation.89

Although these findings are promising, the possible applications of these growth factors are limited by their short biological half-life which requires repeated administrations and expensive dosages.10 Recent advances in molecular biology have led to fast progress in the development of gene therapy and the most promising novel approaches to maximally stimulate bone formation in animals as well as in humans.11 Since Baum and O'Connell12 first described the potential impact of gene therapy on dentistry, gene therapy has forged its own position as a novel strategy to induce bone formation. Earlier, we reviewed advances of gene therapy for the repair of craniofacial bonedefects.3 By means of this technique, genes can be therapeutically used to produce osteoinductive growth factors in the local environment to heal experimental craniofacial defects.13–15 This has direct and profound implications for the treatment of craniofacial deformities. Most recently, we were able to construct a delivery vehicle where potential therapeutic genes could be delivered to the condyle.16

Vascular endothelial growth factor (VEGF) has been shown to play an important role in mandibular condylar growth.117–19 VEGF is a potent regulator of neovascularization expressed during endochondral ossification of the condyle.117 Chondrocytes of the mandibular condyle express VEGF which stimulates neovascularization and marks the onset of endochondral ossification.

Some successful gain of function studies with recombinant VEGF proteins or gene therapy has yielded significant increases in vascularization and bone regeneration in a defects model.2021 Local administration of recombinant human vascular endothelial growth factor (rhVEGF) was found to enhance the amount of tooth movement.2223 Moreover, rhVEGF administration leads to enhanced blood vessel formation and ossification in bone defects.20 In vivo gene therapy with adenovirus mediated VEGF proved to modify bone defect healing.2124 Thus, VEGF has possible clinical applications for inducing bone formation. More recently, Rabie et al25 successfully established recombinant adeno-associated virus (rAAV) mediated VEGF delivery system and identified transgene distribution in the condylar cartilage and significant increase in the expression of chondrogenic and osteogenic markers. Therefore, the aim of this study is to further investigate the morphological changes in the mandibular condyles treated by VEGF gene therapy.

MATERIALS AND METHODS

Morphological Measurement

Gross morphological analysis was carried out as described in detail previously.26 Digital photos of the lateral view of the left mandibles were taken using a true color video camera (JVC TK-C1380, Tokyo, Japan) to allow for the angular and linear measurements.26 The condylar head in particular was separated from the mandible and inserted into a homemade columniform abutment for photo taking.27 The photos were taken through the digital picture capture system with a ruler to standardize the amplification. To increase the accuracy of the small morphological measurement changes, the images were magnified two times the original size with the known scale and were traced with selected landmarks and distance (Figure 1, Table 1). Measurement was blind and evaluated by two independent tracings, which were carried out at an interval of at least 2 weeks. Ten species were randomly selected for the evaluation of method error, which was calculated with Dahlberg's formula Me = Σd²/2n, where d represents the difference between two registrations and n is the number of duplicate registrations. The method error determined was 0.13 mm for linear measurement and 1.4° for angular measurement, which were both statistically insignificant (P > .05).

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Table 1.  Definition of Linear and Angular Measurements²⁶

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RESULTS

Changes in Body Weight During the Experimental Period

A gradual increase in body weight was recognized during the experimental period in both experimental and control groups (Figure 2). However, no significant difference in body weight was detected among the one experimental group and two control groups (P > .05).

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Morphological Analysis

The length (C-D) and width (Q-R) of the condylar head increased significantly from day 30 to day 60. Moreover, the length of the condylar process (B-F) together with the dependent mandibular length (A-F) significantly increased from day 30 to day 60. The distance from the posterior part of the condylar head (D) to the mandibular plane (GH plane) increased on day 60 followed by the increased distance between the midpoint of the condylar surface (F) to the GH plane. However, the change of position of the point F did not affect the angle of BF/GH. The length of mandibular base (A-B) and the distance between the reference point on the most anterior surface of the condyle (C) and the GH plane remained unchanged in both groups throughout the whole period (Table 2).

Table 2.  Values of Linear and Angular Measurements of Mandibular Morphology in Experimental and Control Groups at Different Time Points^a

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DISCUSSION

In the present study, we injected the mandibular condyle with rAAV mediated VEGF and demonstrated significant condylar growth. Such growth lasted for the duration of the experiment, which was 60 days after injection. This work should be considered as “proof of principle” that a key regulatory growth factor can stimulate mandibular growth at will.

Growth of mandibular condylar cartilage is partly genetically determined, but is also influenced by exogenic factors including mechanical factors and numerous systemic and local mediators.28 Furthermore, the effects of mechanical and systemic factors is, in part, mediated by growth factors, which play a central role in the local regulation of cartilage growth and metabolism.129 This gives gene therapy a critical role in the future for management of anomalies related to growth. Gene therapy offers new possibilities to either correct a defective gene in a given tissue or organs or to deliver the lacking growth factors in a given tissue to stimulate a function.

Osteogenesis (the formation of new bone) and angiogenesis (the invasion of new blood vessels) are closely correlated.30 VEGF, the best-characterized angiogenic factor, has been shown to play an important role in long bone and mandibular condylar growth.131 When VEGF was inactivated in mice through the systemic administration of a soluble receptor chimeric protein (sFlt-1), blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone.31 Some reports demonstrated a successful effect of recombinant VEGF and in vivo gene therapy on bone formation.2021 The advance of virus research has brought rAAV into the limelight of viral gene therapy. The major advantages of rAAV include the potential for long-term transgene expression, less pathogenicity, low immunogenicity, and the ability to infect a broad range of host tissues including both dividing and nondividing cells.3 Most recently, we first reported that transgene can be transferred to the mandibular condyle in vivo by rAAV delivery.16

The reporter gene of eGFP, an exogenous intracellular molecule, was used for in situ identification of delivered gene32 and to eliminate the role of delivery vehicle. The local delivery of rAAV-VEGF in the present study resulted in elongated mandibular condylar growth in the posterior part of the condyle. This was due to the fact that VEGF was locally delivered into the mandibular condyle through the posterior attachment. The rAAV-VEGF vector (22–25 nm in size) was small enough to diffuse from the injected joint space to the cell surface layers, and then deeper to infect chondrocytes and hypertrophic chondrocytes and to make them secrete the growth factor, VEGF, to regulate the microenvironment in the condyle.25 Moreover, in the previous study, we extracted RNA from the remote organs of liver, kidney, heart, and spleen of rAAV-VEGF injected animals for reverse transcription polymerase chain reaction (RT-PCR) analysis. The exogenous VEGF was not detected, thereby reducing the prospects of systemic adverse effects.25

Treated by VEGF gene therapy, the growth of the condyle ultimately resulted in the increase in mandibular length (A-F); no increase was identified in the length of mandibular base (A-B); and the distances of point F as well as point D to mandibular plane demonstrated an increase, but the position of point C was kept stable. Moreover, the changed position of point F did not affect the angle of condylar process to the mandibular plane (BF/GH). Thus, it pointed out that mandibular condylar growth occurred in a backward and upward direction.

After continuous bite-jumping treatment, the most prominent cellular response was documented in posterior surface of the condyle, but not in the anterior surface. The condyle expressed its adaptability in terms of directional changes in mandibular growth,1 the distance from point F and D to GH was reduced, together with the reduction of the angle of the condylar process to the mandibular plane, which displaced the condylar process backwards but downwards.26 Hence, the directional remodeling of mandibular condyle by gene therapy and bite-jumping treatment was different in the vertical direction, but similar in the sagittal direction. Gene therapy is still in its infancy stage for experimental growth of the mandibular condyle. We hope that in the near future we can target cells in the superior layer of the condyle that could result in a more upward growth of the condyle and thus help in regulating direction of growth.

One interesting finding in this study was the delayed effect on morphological change. Five time points, 7, 14, 21, 30, and 60 days, were selected because each of these time points demonstrate the peak expression of one of the growth factors expressed during induced growth of the mandible.118 For example, VEGF expression was evident on day 14, while chondrogenesis peaked on day 21, and osteogenesis on day 30 during mandibular advancement. Day 60 was added to determine whether the gene expression was still evident after the injection for a longer time. On day 7, 14, and 21, all the measured morphological parameters of the rats' mandibles showed no significant difference among the rAAV-VEGF delivery, rAAV-eGFP, and PBS injected group. From day 30, parameters of condylar width and length demonstrated a significant increase. Up to day 60, more parameters showed significant change. Since it was known that the rAAV vector exists as a single stranded DNA virus, while the viral genome is transported to the nucleus within minutes after infection,33 in vivo transduction takes days to weeks as a consequence of lagging second-strand synthesis.34 On the other hand, VEGF expressed on the basis of extractable protein from mandibular condyle was significantly increased from day 14, and the biochemical analysis showed that osteogenic markers of alkaline phosphatase activity and osteocalcin content significantly increased from day 30.25 Taken together, it was consistent with the earlier reports that VEGF expression was closely correlated to mandibular condylar growth, which preceded the peak of bone formation.11927 In the near future, it is necessary to know whether these genetically treated rats need reinjection or inhibition to avoid relapse or overgrowth. Therefore, the long-term effect on mandibular condylar growth by VEGF gene therapy needs to be investigated further.

CONCLUSIONS

• The present study provides further evidence that local rAAV mediated VEGF gene transfer enhances the size of mandibular condyle leading to mandibular condylar growth. It provides the basis to regulate mandibular condylar growth for future clinical practice.