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Regional Alterations in Fiber Type Distribution, Capillary Density, and Blood Flow after Lower Jaw Sagittal Advancement in Pig Masticatory Muscles
1 Department of Orthodontics, Technical University, Dresden, Germany; Correspondence: *corresponding author, rbau{at}mti-n.mti.uni-jena.de
Muscular remodeling is known to be a prerequisite for permanent correction of mandibular-maxillary malocclusion. The objective of this study was to clarify if an increase in type I fiber number is accompanied by an increased capillary density and improved muscular blood flow. Juvenile pigs received build-ups on the molars, which induced a protrusion of 7.6 + 1.5 mm. After 4 weeks of treatment, chronic lower jaw protrusion induced a marked muscle blood flow increase in the anterior and medial regions of the superficial part of the masseter and in the medial pterygoid muscle (P < 0.05). Furthermore, an increase in capillary density and in the amount of type I fibers was found in all regions of masticatory muscles with an increased muscle blood flow (P < 0.05). Finally, the capillary-to-fiber ratio increased (P < 0.05). Muscle blood flow and capillary density showed a strong linear correlation (r = 0.89, P < 0.01). These changes suggest a complex muscle adaptation for long-term, fatigue-resistant activity during the early corrective period of mandibular-maxillary malocclusion treatment.
Key Words: muscle fiber types • capillarization • masticatory muscle blood flow • juvenile pigs
A primary challenge of Angle Class II1 treatment is still the permanent correction of occlusal and skeletal discrepancies (between maxilla and mandible). The therapeutic success of functional appliances is crucially related to sufficient remodeling of temporomandibular joint structures (Watted et al., 2001). Muscular adaptation to the target position appears to be of similar relevance for permanent correction of mandibular-maxillary malocclusion (Thuer et al., 1992). Muscular adaptation includes adequate correction in length and temporomandibular joint stabilization during rest as well as temporomandibular joint guidance during quite different mandibular movements. It is suggested that muscular strain mainly induces underlying adaptive processes in the (neuro) muscular masticatory system (Ingervall and Bitsanis, 1986). Nevertheless, controversial opinions still exist on the specific roles of different muscular alterations appearing during treatment with functional orthodontic appliances (Eckardt et al., 2001). This also may result from deficient disclosure underlying causal mechanisms of morpho-functional modification. Recently, experimental studies have shown that at least during the initial phase of sagittal mandibular advancement, e.g., within a period of 4 wks, a marked conversion of muscle fibers occurred in masticatory muscles with a significant increase in the proportion of slow-twitch type I fibers (Gedrange et al., 2001a). This suggests an increased long-term activity of the respective regions of the masticatory muscles. Furthermore, these intramuscular alterations induced by sagittal mandibular advancement seem to be accompanied by increased oxidative stress, indicated by an altered intramuscular glutathione metabolism (Gedrange et al., 2001b). However, because type I muscle fibers are not only responsible for maintenance functions through increased fatigue resistance but also sensitive to oxygen shortages, a persisting maintenance of the remodeled masticatory muscle function needs an appropriate adaptation in blood supply. To clarify how an expected increase in oxygen and substrate demand of converted masticatory muscles is warranted, we determined the effects of sagittal mandibular advancement on regional blood flow and capillary density, together with assumed muscle fiber conversion, as shown previously (Gedrange et al., 2001a). It was hypothesized that an increase in type I fiber number may be accompanied by an increased capillary density and improved muscular blood flow. Juvenile pigs were used because there are several craniofacial similarities with humans, e.g., both are omnivores and exhibit a comparable dentition, vertical occlusion, and stroke orientation, as well as relatively shortened faces (Ciochon et al., 1997). In addition, development dynamics and mature patterns of muscle fiber distribution and muscle function are comparable (Tuxen and Rostrup, 1993; Anapol and Herring, 2000). Therefore, the juvenile pig represents a valuable nonprimate model for studying morpho-functional aspects of corrective influences on malocclusion.
All surgical and experimental procedures were approved by the Ethics Committee of the Thuringian State Government on Animal Research. Twelve pigs of mixed German domestic breed (aged 10 wks) were used in the study.
Procedures to Induce Long-term Lower Jaw Sagittal Advancement
Muscle Blood Flow Measurement Then, the regional blood flow was measured with colored microspheres. The application of this method in piglets has been validated and described in detail (Bauer et al., 1996; Walter et al., 1997). Briefly, yellow microspheres (3 x 106 in 1 mL saline and 0.01% Tween 80) were injected within 10-15 sec into the left atrium (Dye Trak, Triton Technology, San Diego, CA, USA). During injection, an arterial reference sample was withdrawn from the abdominal aorta at a rate of 3.53 mL • min–1 in 120 sec, starting 15 sec before microsphere injection (syringe pump SP210iw, World Precision Instruments Inc., Sarasota, FL, USA). After microsphere injection, an arterial blood sample was taken for measurement of oxygen saturation, blood hemoglobin, and blood gases (OSM2, ABL 50, Radiometer, Copenhagen, Denmark). The animals were then killed by intravenous injection of saturated potassium chloride solution, and masticatory muscles were taken and carefully isolated. Specimens of the right masticatory muscles were taken for histological evaluation. The left and remaining right masticatory muscles were used for muscle blood flow measurement. Three regions of the masseter muscle were differentiated, i.e., the anterior (M1) and medial (M2) regions of the superficial part and the posterior region of the profound part (M3), and were dissected from animals of both groups. Additionally, the anterior (TP1) and posterior (TP2) parts of the temporal muscle together with samples of the medial pterygoid muscle (PM) and the geniohyoid muscle (GH) were investigated. Muscle and arterial reference blood samples were digested in 4 mol potassium chloride containing 4% Tween 80 and filtered through an 8-µm-pore polyester-membrane filter. We recovered the dye from the microspheres by adding 150 µL N,N-dimethylformamide and measured the absorption spectra of the dye solution with a UV/VIS spectrophotometer (Model 7500, Beckman Instruments, Fullerton, CA, USA). We used specific absorbencies of the individual dyes to quantify the colored microspheres (MISS software package; Triton Technology, San Diego, CA, USA) of each tissue sample. We calculated regional muscle blood flows (mL • min–1 • 100 g–1) using the equation: (flow ratetissue = number of CMStissue • (flow ratereference/number of CMSreference)
Immunohistochemistry Specimens were sectioned (thickness 8 µm) at 20°C by means of a microtome (model 2055, Leica, Bensheim, Germany). Serial sections were collected on poly-L-lysine-coated glass slides, dried, and stored at room temperature, and then processed in parallel. Cross-sections were pre-incubated in TRIS-buffered saline and then exposed to 3.0% hydrogen peroxide in 60% methanol-40% buffered saline for 20 min at room temperature. To block nonspecific binding of antibodies, we incubated the cross-sections in 0.1 M TRIS-buffered saline containing 1% bovine serum albumin and 10% normal serum for 4 hrs at room temperature. Primary antibodies were applied at 37°C for 45 min. After being washed in TRIS-buffered saline for 10 min with biotinylated secondary antibodies, the serial sections were detected by incubation for 30 min with a commercially available antimouse goat antibody (Dako, Glostrup, Denmark; dilution, 1:400). Sections were then incubated for 60 min with avidin-biotinylated enzyme complex solution (Vectastain kit, Vector, Burlingame, CA, USA). The color was developed with diaminobenzidine (Aldrich Chemical Company, Inc., Milwaukee, WI, USA), and the sections were counterstained with hematoxylin. As negative controls, the primary antibodies were replaced by TRIS-buffered saline or non-relevant monoclonal IgG.
Morphometric Characteristics of Muscle Tissue Cross-sections The fiber ratio was measured from the surfaces of type I fibers (stained) and type II fibers (unstained). Capillary density was defined as the number of capillaries per millimeter squared. The number of capillaries was counted for all fibers in the field of vision. For fibers in the periphery of the field cut by the frame of the vision field, an area corresponding to one-half of the area of the marginal fiber cross-section was added. In addition, fiber density (fiber number per millimeter squared) was determined from the anterior region of the superficial part of the masseter muscle (M1), and the capillary per muscle fiber ratio was calculated in both groups.
Statistical Analyses
Sagittal advancement of the lower jaw amounted to 6 ± 0.9 mm on the first day. After 28 days with build-ups on the molars and premolars, a sagittal advancement of the lower jaw (protrusion) by 7.6 ± 1.5 mm had occurred, whereas the untreated animals retained a neutrocclusion. However, tooth preparation did not alter the amount of food intake (Table  ). Furthermore, there were similar physiologic parameters in both groups during blood flow measurement of the masticatory muscles (Table).
Regional muscle blood flow was quite similar in masticatory muscles of untreated juvenile pigs (Fig. 1 ), whereas chronic lower jaw protrusion induced a marked blood flow increase in the anterior and medial regions of the superficial part of the masseter and in the medial pterygoid muscle (P < 0.05). The remaining regions of the masticatory muscles studied showed no significant blood flow alterations after chronic protrusion. Furthermore, regional distributions of capillary density as well as type I fibers in masticatory muscles were quite similar in untreated juvenile pigs (Fig. 1). After chronic lower jaw protrusion, a marked increase in capillary density was found in all regions of masticatory muscles with an increased blood flow as well as in the posterior part of the temporal muscle (Fig 1, P < 0.05). The amount of type I fibers of masticatory muscles was increased in the same regions, which was reflected in an increased capillary density (Fig 1, P < 0.05). However, total muscle fiber density was similar in all muscles of treated and untreated animals. Consequently, the capillary-to-fiber ratio, estimated in M1, increased markedly (group 1, 0.6 + 0.13; group 2, 1.3 + 0.25, P < 0.01).
Masticatory muscle blood flow under resting conditions and capillary density exhibited a strong linear correlation (Fig. 2 , r = 0.89, P < 0.01).
Data presented here demonstrate for the first time that sagittal mandibular advancement is accompanied by a tuned response in muscle fiber conversion, increased capillary density, and elevated resting blood flow of certain parts of the masticatory muscles. The amount and regional distribution of muscle fiber conversion support previous findings, which were obtained under consistent conditions but with different methods of muscle fiber typing (Gedrange et al., 2001a). In the present study, we used the immunohistochemical determination of slow-twitch muscle fibers by means of myosin heavy-chain type I antibodies, which are highly specific in skeletal muscles of the pig (Lefaucheur et al., 1995, 2002). The fiber composition, e.g., slow to fast fiber relation of masticatory muscles, presented in this study is in good concurrence with those found in masseters of pigs of comparable age (Suzuki and Cassens, 1980; Anapol and Herring, 2000). Combined characterization of simultaneously increased numbers of type I fibers, increased capillary density, and enhanced blood flow in distinct regions of the masticatory muscles resulting from a certain period of artificial mandibular protrusion suggest a coordinated structural and functional change in these muscle regions. However, the causal mechanisms responsible for morphological and functional changes in masticatory muscles during the induction of lower jaw sagittal advancement have yet to be studied. Findings reported from skeletal muscles have shown that augmented conversion to slow-twitch, "fatigue-resistant" type I fibers is influenced in vivo by impulse activity as determined by the nerve (Salmons and Sreter, 1976), by the level of physical activity (Salmons and Henriksson, 1981), and by passive stretch (Goldspink et al., 1992). During fast-to-slow transformation, genes encoding slow isoforms of myosin heavy- and light-chains as well as genes encoding enzymes involved in the oxidative metabolism are up-regulated (Pette and Vrbova, 1992). Recently, it has been shown that an intracellular signaling pathway dependent on calcineurin is crucially involved in the control of fiber-type specific gene expression in skeletal muscles (Chin et al., 1998; Olson and Williams, 2000). Interestingly, the temporal behavior of chronic muscle activity appears to be responsible for the effectiveness of fast-to-slow transformation of contractile proteins. It has been shown that initiation of a fast-to-slow transformation process requires a minimum duration of the activation period used during each stimulation cycle, and the total number of stimuli applied is not the decisive parameter for the fiber type switch, which is caused by a nuclear accumulation of specific transcription factors (Kubis et al., 2002). However, calcineurin alone is not sufficient to mediate the complete transformation, because mitochondrial and cytosolic enzymes of energy metabolism are not regulated by this pathway (Meissner et al., 2001).
Fast-to-slow fiber remodeling of the masticatory muscles also encompasses an increase in capillary density in identical muscle regions (Fig. 1 We determined the regional distribution of capillary density in masticatory muscles and found a similar distribution for slow-twitch muscle fibers. Furthermore, the time course of capillarization and conversion in the oxidative metabolism during remodeling is obviously quite different. As shown in skeletal muscles, angiogenesis occurred just a few days after the beginning of low-frequency muscle stimulation and reached a plateau after 2 wks. During this period, vascular endothelial growth factor activity was markedly enhanced. However, citrate synthase activity, a marker of aerobic-oxidative metabolic potential, was first registered after 1 wk and increased gradually throughout the seven-week observation period, indicating that increases in capillarization precede increases in the aerobic-oxidative potential of the energy metabolism (Skorjanc et al., 1998).
Capillary density and capillary-to-fiber ratio in masticatory muscles have not been determined before. Nevertheless, similar values in the size of the capillary bed were reported in the canine gracilis muscle, with a similar percentage of type I fibers under normal conditions and a comparable conversion index after chronic stimulation (Hudlicka et al., 1987). The strong linear correlation between capillary density and regional blood flow of masticatory muscles (Fig. 2 In conclusion, we have shown that chronic lower jaw sagittal advancement in the juvenile pig induced an orchestrated remodeling of distinct parts of masticatory muscles with transformation of fast, fatigable muscles toward slower, fatigue-resistant ones, together with an increase in size of the capillary bed and an associated increase in regional blood flow. These changes suggest a complex muscle adaptation for long-term, fatigue-resistant activity during the early corrective period of mandibular-maxillary malocclusion.
The financial support of the Deutsche Forschungsgemeinschaft (German Research Council, GE 1154/2-1/2-2) and Bundesministerium für Bildung und Forschung (R.B. FKZ01 ZZ0105) is gratefully acknowledged. The authors thank U. Jäger, R.-M. Zimmer, and L. Wunder for skillful technical assistance. Received for publication October 23, 2002. Revision received April 24, 2003. Accepted for publication April 29, 2003.
Journal of Dental Research, Vol. 82, No. 7,
570-574 (2003)
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