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Nanomechanical Properties of Facial Sutures and Sutural Mineralization FrontTissue Engineering Laboratory (MC 841), Departments of Orthodontics and Bioengineering, 801 S. Paulina Street, University of Illinois at Chicago, Chicago, IL 60612-7211, USA; Correspondence: * corresponding author, jmao2{at}uic.edu
The mechanical properties of craniofacial sutures have rarely been investigated. Three facial sutures—the pre-maxillomaxillary (PMS), the nasofrontal (NFS), and the zygomaticotemporal (ZTS)—and their corresponding sutural mineralization fronts in 8 young New Zealand White rabbits were subjected to nano-indentation with atomic force microscopy as a test of the hypothesis that they have different mechanical properties. The average elastic modulus of the PMS was 1.46 ± 0.24 MPa (mean ± SD), significantly higher than both the ZTS (1.20 ± 0.20) and NFS (1.16 ± 0.18). The average elastic moduli of sutural mineralization fronts 30 µm away were significantly higher than their corresponding sutures and had the same distribution pattern: the PMS (2.07 ± 0.24 MPa) significantly higher than both the ZTS (1.56 ± 0.29) and NFS (1.71 ± 0.22). Analysis of these data suggests that facial sutures and their immediately adjacent sutural mineralization fronts have different capacities for mechanical deformation. The elastic properties of sutures and sutural mineralization fronts are potentially useful for improving our understanding of their roles in development.
Key Words: sutures • bone • mineralization • mechanical • atomic force microscopy
Whereas the appendicular skeleton lengthens by endochondral ossification of epiphyseal plates, craniofacial bones are enlongated by bone apposition in sutures. Sutures and epiphyseal plates, therefore, both facilitate longitudinal skeletal growth and have different adult tissue phenotypes, sutures being usually fibrous connective tissue and epiphyseal plate being hyaline cartilage (Pritchard et al., 1956; Martin et al., 1998; Opperman, 2000). The first step toward understanding a biological tissue from an engineering standpoint is to characterize its material properties. Whereas the epiphyseal plates have been characterized with regard to their material characteristics (e.g., Cohen et al., 1992, 1994; Radhakrishnan et al., 2004), there are only scarce data on the biomechanical properties of facial sutures, largely attributable to technical limitations. Recent advances in submicron imaging by atomic force microscopy have allowed for characterization of not only topographic properties of the articular surface, but also regional nanomechanical properties (Jurvelin et al., 1996; Hu et al., 2002; Patel and Mao, 2003; Allen and Mao, 2004). In the present study, nano-indentation was used to characterize compressive mechanical properties of 3 craniofacial sutures, the zygomaticotemporal, nasofrontal, and pre-maxillomaxillary sutures (cf. Fig. 1A  ), that have distinct morphological characteristics. First, they connect different facial and cranial bones, and therefore can be considered to have different local morphogenetic environments. Second, whereas the rabbit nasofrontal and pre-maxillomaxillary sutures interdigitate to different degrees (cf. Figs. 1C and 1D, respectively), the zygomaticotemporal suture is a straight-edge (end-to-end) suture with little interdigitation (cf. Fig. 1B). Third, whereas the pre-maxillomaxillary suture is directly adjacent to incisor bite forces (cf. "M.I." for the maxillary incisor in Fig. 1A), both the nasofrontal and zygomaticotemporal sutures are distant from either incisor or molar loading.
There are prior reports of tensile properties of isolated cranial sutures. Under tensile test, the elastic moduli of the interparietal sutures of infant human, porcine, and rat specimens have been found to vary by several orders of magnitude from 0.64 to 1371 MPa (Margulies and Thibault, 2000; McLaughlin et al., 2000; Tanaka et al., 2000). In addition to the wide range of the reported elastic moduli, these data obtained from tensile tests cannot be used to indicate sutures’ mechanical properties under compression, a mechanical condition that many sutures are known to experience (Hylander and Johnson, 1997; Rafferty and Herring, 1999; Mao et al., 1999; Herring et al., 2001; Kopher et al., 2003). In parallel with the above scarce information on the elastic properties of isolated cranial sutures in response to macroscopic tensile tests, biomechanical properties of facial sutures have not been characterized under tension, compression, or shear stresses. Mechanical stresses play important roles in suture development at different levels of organization (Mao, 2002). Although tensile stresses hypothetically resulting from brain enlargement have long been attributed to allowing the separation of cranial sutures and subsequent sutural growth, analysis of recent data showing anabolic sutural responses upon compressive stresses generated by either mastication or exogenous forces suggests the diverse nature of mechanical stimuli capable of activating sutural growth (Rafferty and Herring, 1999; Herring et al., 2001; Mao, 2002; Kopher and Mao, 2003; Mao et al., 2003). The present work tested a hypothesis that significant differences exist in the elastic properties of 3 rabbit facial sutures—the zygomaticotemporal, nasofrontal, and pre-maxillomaxillary—upon compressive testing by nano-indentation with atomic force microscopy.
Measurements of Sutural Interdigitation Three facial sutures—the zygomaticotemporal (ZTS), nasofrontal (NFS), and pre-maxillomaxillary (PMS)—were harvested from both the left and right sides in each of 8 six-week-old, male New Zealand White rabbits within 1 hr of death. Rabbits were used, because these are growing animals in which sutural development is of interest. The degree of sutural interdigitation of the ZTS, the NFS, and the PMS was quantified unilaterally in 4 rabbits, following a commonly used method (Herring, 1972; Jaslow, 1990). Upon skin incision and deflection of subcutaneous connective tissue, these sutures were readily exposed and removed en bloc from the skull by means of an electrical surgical saw (Hall Surgical, Largo, FL, USA) and stained with methylene blue (Am Regent Labs, Melville, NY, USA). Digital photographs of the whole suture specimens were subjected to computerized image analysis. The actual course of the entire suture on its subcutaneous surface was measured to the decimal point nearest 0.1 mm. We calculated the degree of sutural interdigitation by dividing the linear sutural distance over the actual sutural course.
Sample Preparation for Atomic Force Microscopy
Nano-indentation, Topographic and Force-volume Imaging
where E is the Young’s modulus, F is the applied nanomechanical load, v is the Poisson’s ratio, R is the radius of curvature of the AFM tip, and
Data Analysis
Representative topographic images of the ectocranial surfaces of the 3 facial sutures and their corresponding sutural mineralization fronts are presented in Fig. 2 , each with 5 x 5 µm horizontal scan size. The ZTS demonstrated a characteristically smooth gradient topographic distribution (Fig. 2A), whereas the NFS (Fig. 2C) and PMS (Fig. 2E) both had robust topographic variation in surface contour. Although the ZTS had a smooth surface topography, its variation in magnitude in the Z plane was similar to that of both the NFS and PMS (Figs. 2A, 2C, 2E). The ZTS sutural mineralization front (Fig. 2B) demonstrated notably robust topography, similar in magnitude to the topography of sutural mineralization fronts of the NFS (Fig. 2D) and PMS (Fig. 2F).
The elastic moduli of all 3 sutures and their corresponding sutural mineralization fronts are presented in Fig. 3 . The average elastic moduli of the ZTS, NFS, and PMS were 1.20 ± 0.20 (mean MPa ± SD), 1.16 ± 0.18, and 1.46 ± 0.24, respectively. The average elastic modulus of the PMS was significantly higher than the average elastic moduli of both the ZTS and NFS (p < 0.01) (blank histograms in Fig. 3), suggesting that the pre-maxillomaxillary suture is capable of resisting higher mechanical stresses such as those experienced in incision.
The elastic moduli of sutural mineralization fronts 30 µm away from the suture-bone boundary (cf. dots across each suture in Figs. 1B  , 1C, 1D) followed the same distribution pattern as the elastic moduli of their corresponding sutures: The PMS-SMF (2.07 ± 0.24 MPa) (mean MPa ± SD) was significantly higher than both the ZTS-SMF (1.56 ± 0.29 MPa) and the NFS-SMF (1.71 ± 0.22 MPa) (Fig. 3). The average elastic moduli of the sutural mineralization fronts were significantly higher than their corresponding sutures (solid histograms in Fig. 3) (p < 0.01). Of particular interest was a lack of statistically significant differences in the average elastic moduli between not only the ZTS and NFS but also between their corresponding sutural mineralization fronts (Fig. 3).
Structural variation in the degree of sutural interdigitation, calculated as the ratio of the actual sutural length by linear sutural distance, is presented in the Table
The present findings demonstrate that the elastic moduli of 3 rabbit facial sutures—namely, the zygomaticotemporal, nasofrontal, and pre-maxillomaxillary—are, on average, 1.20, 1.16, and 1.46 MPa, respectively, upon nano-indentation by atomic force microscopy. These values are similar to the elastic moduli of collagen fibers in the linear elastic region of up to approximately 3 Newtons (Fratzl et al., 1998), and are similar to the elastic moduli in the rat interparietal suture under macroscopic tensile tests (McLaughlin et al., 2000; Tanaka et al., 2000), but are lower than ligaments and tendons of the upper and lower extremities (Woo and Levine, 1998), although care must be exercised when the absolute elastic moduli among studies are compared. The relative distribution of the elastic moduli identified here is perhaps of more interest. First, the significantly higher elastic modulus of the interdigitating PMS in comparison with that of the straight-edge ZTS is of particular interest, because, whereas the rabbit PMS is directly adjacent to the maxillary incisor, the ZTS is adjacent to the masseter attachment and the jaw joint (cf. Fig. 1 ). In addition to incisal bite forces, the mechanical properties of the PMS may also be affected by presumed mechanical stresses generated from incisor eruption and nasal septum cartilage. From its straight-edge morphology and its location in the skull, the rabbit ZTS can be predicted to experience primarily tensile or shear loads, which forms a hypothesis testable by bone strain experiments. It then becomes interesting to consider whether compressed sutures are associated with higher elastic moduli than tensed sutures, like different elastic properties of opposing tensile and compressive cortical surfaces in long bones (Takano et al., 1999). Second, the lack of significant differences in the elastic moduli between not only the ZTS and NFS, but also their sutural mineralization fronts, despite their different degrees of sutural interdigitation, suggests that sutural interdigitation alone may not account for their different sutural elastic properties. The elastic moduli of the sutural mineralization front 30 µm away from the suture-bone boundary as defined in the present study are of tremendous interest. First, the elastic moduli of the sutural mineralization front 30 µm away from the suture-bone boundary in the average range of 1.56 to 2.07 MPa are an order of magnitude lower than those of mineralized bone (e.g., Ashman et al., 1984). This is likely attributable to the present nano-indentation of the presumed osteogenic front or osteoid formed by osteogenic cells, instead of fully mineralized bone. Second, the distribution of the elastic moduli of the SMF follows the same pattern as the elastic moduli of their corresponding sutures: The SMF of the PMS is significantly higher than those of the ZTS and NFS, suggesting that factors regulating sutural elastic properties also regulate their mineralization. Third, significantly higher elastic properties of SMF 30 µm away from the suture-bone boundary compared with its corresponding suture indicate that sutural mineralization increases considerably within this small distance. Hence, factors that regulate sutural mineralization must operate between the present focal point of sutural imaging and the focal point of SMF imaging, which are 30 µm apart. At present, it is unclear how a cascade of known growth factors and their receptors regulates sutural mineralization (Opperman, 2000; Mao, 2002). Equally lacking is substantial experimental evidence that forces transmitted through the suture regulate sutural mineralization (Mao, 2002), although there is indirect in vitro evidence that mineralization increases upon application of biomechanical strain (Camacho et al., 1995; Wozniak et al., 2000). At the sutural mineralization front, collagen fibrils are thickened and arranged in a stacked platelet pattern (Weismann et al., 1998). Despite presently unknown mechanisms, it appears that sutural mineralization fronts are modulated in much the same way as sutural elastic properties. The smooth topographic surface of the ZTS, in contrast to the robust topography of the NFS and PMS, can be related to its macroscopic characteristics. First, the rabbit ZTS likely experiences tensile loads during mastication, a biomechanical trait that tends to stretch collagen fibrils (Meikle et al., 1984; Fratzl et al., 1998). It would be of further interest to determine whether tensile loads are associated with smooth sutural surface topography in other sutures. Second, the straight-edge rabbit ZTS likely permits the two opposing bones, the zygomatic and the temporal, to slide against each other and therefore experience shear stresses. It is presently unknown, however, why a lower elastic modulus is associated with a suture such as the ZTS, that likely permits both tensile and shear loads to occur. Nevertheless, the present findings on the ZTS indicate the need for study of other straight-edge skull sutures. The present data should be interpreted with the following caveats. For instance, the Poisson ratios for the suture and sutural bone were assumed from previous work on sutural collagen and bone. This is compensated for by the present goal of investigating relative patterns of mechanical properties, instead of absolute values. A probable limitation of nano-indentation is its potential bias of small sampling areas. We partially compensated for this in the present study by verifying similar elastic moduli between homologous sutures on the sutures of the left and right sides (data not shown), and also by our previous calibration effort (Hu et al., 2001; Patel and Mao, 2003; Allen and Mao, 2004). Removal of craniofacial bone from the in vivo environment inevitably has an impact on the physiological relevance of the explant, despite the fact that AFM is known to require minimum sample preparation. Within the constraints of the present approaches, analysis of the present data suggests that facial sutures and their immediately adjacent sutural mineralization front have different capacities to withstand mechanical stresses. The elastic properties of sutures and sutural mineralization front are potentially useful for understanding their roles in development.
We are grateful to Drs. Walter Greaves and Robert Scapino for their constructive criticism on earlier versions of the manuscript. We thank two anonymous reviewers whose insightful comments helped to improve our manuscript. This research was supported by a Biomedical Engineering Research Grant from the Whitaker Foundation RG-01-0075, and by USPHS Research Grants DE13964 and DE15391 from the National Institute of Dental and Craniofacial Research, as well as EB02332 from the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health. Received for publication July 30, 2003. Revision received April 8, 2004. Accepted for publication April 19, 2004.
Journal of Dental Research, Vol. 83, No. 6,
470-475 (2004) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
is the amount of nano-indentation. 
0.05 was considered to indicate statistical significance. 
