Adaptation of the Masseter and Temporalis Muscles Following Alteration in Length, with Or Without Surgical Detachment

THE ANATOMICAL RECORD 200:127-137 (1981)

Adaptation of the Masseter and Temporalis Muscles Following Alteration in Length, With or Without Surgical Detachment

LEO C. MAXWELL, DAVID S. CARLSON, JAMES A. McNAMARA. JR., AND JOHN A. FAULKNER Departments of Physiology (L.C.M.,J.A.F.) and Anntomy (D.S.C.,JA.M.1, and The Center for Human Growth and Development (L.C.M.,D.S.C., JA.M.1, University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT Histochemical properties, muscle fiber cross-sectional area, muscle fiber length, and the oxidative capacity of masticatory muscles of female rhesus monkeys were assessed following alteration in functional length by an intraoral appliance or by detachment of the muscle. Experimental groups received the appliance only (A); the appliance and subsequent detachment of the masseter (AD); the appliance and detached masseter, but with surgical reattachment of the masseter to the pterygomasseteric sling (ADR); no appliance, but detachment and reattachment of masseter (DR); or an appliance which was removed after 24 weeks to study posttreatment responses (F’T). Animals were sacrificed and the muscles were studied at intervals from 4 to 48 weeks after initiation of the experimental period. The results of these studies led to the following conclusions: (1) Stretching the masseter and temporalis muscles within physiological limits did not significantly alter the proportion of fiber types, although oxidative capacity of the fibers was reduced. (2) Fibers with “intermediate” myofibrillar AT- Pase activity were no more prevalent in experimental than control muscles. (3) The cross-sectional area of Type I fibers of masseter muscles decreased following some experimental procedures, indicating that recruitment of these fibers is the most sensitive to altered jaw function. (4) Minimal alteration of muscle capillarity was induced by any of the experimental procedures. (5)The lengths of masseter muscle fibers in Group PT and of temporalis muscle fibers in groups AD and ADR were greater than in control animals.

Skeletal muscle fibers may undergo both Nearly all previous studies of muscle ad- morphological and physiological adaptation aptation following alteration of function have during growth and in response to altered func- been undertaken in mammalian limb muscles. tion due to changes in length, position, and There is relatively little known concerning the behavior. Morphologically, alteration in func- effects of alteration of function on the muscles tional demand could lead to changes in muscle of mastication. This is especially unfortunate fiber geometry, cross-sectional area, the since clinicians routinely alter the length, po- amount and distribution of connective tissue sition, and habitual function of the perioral within the muscle and its terminal ends, and muscles and the muscles of mastication during the length of the individual muscle fibers due the course of orthodontic treatment and surg- to either an addition or deletion of sarcomeres. ical intervention in both growing and adult Physiologically, fibers of limb skeletal muscles individuals. The capability of the muscles of have been shown to undergo adaptive change with respect to oxidative capacity, and there Dr. Maxwell’s currentaddress is University of Texas Health Science is evidence to suggest that fibers may undergo Center at San Antonio, Department of Physiology, 7703 Floyd Curl conversion between Type I and Type 11 myos- Drive, San Antonio, Texas 78284. ins during growth or following alteration of function (Maxwell et al., 1979, 1980a). Received December 10, 1979; accepted November 7, 1980

0003-276X/81/2002-0127$03.500 1981 ALAN R. LISS, INC. 128 LEO C. MAXWELL ET AL mastication and perioral muscles to adapt ison with experimental results. Experimental morphologically and physiologically to alter- groups were defined in order to evaluate (1) ations in function concomitant with muscle the effect of increased muscle functional lengthening andlor detachment following re- length, (2) the effect of surgical detachment, positioning of skeletal elements is not known with or without reattachment, and (3) the ef- at this time, nor is it clear what effect the fect of decreased functional length on the phys- adaptation of these muscles has upon post- iological and morphological characteristics of treatment skeletal relapse which often occurs the masseter and temporalis muscles. following therapeutic intervention for the cor- rection of dental skeletal disharmony (Chris- Group A-appliance only. Six group A monkeys received the bite-opening appliance tiansen, 1975; McNamara et al., 1978). and were subjected to no other experimental The purpose of this study is to determine the manipulations except routine radiological ex- nature and degree of adaptation which takes amination. place within the superficial masseter and temporalis muscles in response to alterations in Group AD-applianceldetachment. In or- the functional length or surgical detachment der to evaluate the effects of muscle detach- of these muscles. Specifically, we determined ment on the masseter muscle following alterations relative to control muscles in the stretching, the masseter muscles of eight mon- histochemical properties, fiber cross-sectional keys were bilaterally surgically detached from area, fiber length, and oxidative capacity of their insertions along the ramus of the man- the masseter muscle and the temporalis mus- dible 1day after cementation of a bite-opening cle in response to altered functional length, appliance on the maxillary arch. They were with or without myotomy of the masseter mus- not surgically reattached to the pterygomas- cle. seteric sling, but were allowed to form spon- taneous reattachments. EXPERIMENTAL DESIGN Group ADR-upplianceldetachmentireat- Appliance design tachment. An appliance was cemented to the An intraoral appliance was developed to pro- maxillary arch of six animals 1 day prior to duce an alteration in length of the muscles of surgery and each masseter muscle was de- mastication without the necessity of surgical tached, then immediately reattached to the intervention (McNamara, 1975). An occlusal pterygomasseteric sling. splint which displaced the mandible infero- Group DR-detachedlreattached. The mas- posteriorly was cast in Ticonium and cemented seter muscles of six animals were bilaterally to the maxillary arch. The effect of the splint detached and reattached surgically, but no was to open the bite approximately 2 cm in- bite-opening appliance was used. Thus, group cisally, thereby bilaterally stretching the mas- DR animals permitted separation of the effects seter, medial pterygoid, and temporalis mus- of opened bite from the effects of surgery in cles. The appliance caused a 15% lengthening the determination of the adaptations in the of masseter (Carlson, 1977) and temporalis masseter muscle to altered function. (unpublished data). The animals adapted read- ily to the bite-opening appliance and experi- Group PT-posttreatment. Bite-opening enced no significant problems in feeding be- appliances were cemented onto the maxillary havior. arch of five monkeys in group PT, and following a 24-week period of treatment the appliances were removed. This design initiated an Experimental groups experimental period to evaluate the effects of Thirty-one adult female rhesus monkeys decreased muscle functional length on the (Macaca mulatta) were assigned to five exper- morphology and physiology of a muscle pre- imental groups. Each of the monkeys exhibited viously subjected to chronic stretch. a full permanent dentition, with the exception of incomplete eruption of the third molars in METHODS some cases, indicating that each was at least Surgical procedures 4.5 years of age (Hurme and Van Wagenen, 1961). Previously published data from the Masseter myotomy. Animals were pre- muscles of seven adult female rhesus monkeys anesthetized with an intramuscular injection (Maxwell et al., 1979) were used for compar- of phencyclidine hydrochloride (1.5 mg/kg). MASTICATORY MUSCLE ADAPTATION TO ALTERED LENGTH 129

Pentobarbital sodium (5 mg/kg) was admin- mately 300 to 400 mg were prepared for bio- istered intravenously in the operating room to chemical analysis of oxidative capacity. obtain a surgical level of anesthesia. An incision approximately 3.5 cm to 4.0 cm Histochemistry. Cross sections 14 pm thick in length was made through the skin along the were cut from a frozen block in the cryostat at inferoposterior border of the mandible. The - 20°C. Sections were incubated for myofi- subcutaneous fat and platysma muscle were brillar ATPase (Chayen et al., 1973) with or sharply dissected to expose the masseter mus- without preincubation for 15 minutes at pH cle at the gonial angle. Following blunt dis- 10.2 - 10.4, succinic acid dehydrogenase (SDH) section of the soft tissues surrounding the mas- (Nachlas et al., 1957), and for capillary mem- seteric fascia, the masseter muscle was incised brane phosphatase (Maxwell et al., 1977) ac- from the posterior border of the ramus of the tivities (Figs. 1- 3). mandible and along the inferior mandibular border to the region of the antegonial notch. Myofibrillar ATPase. Without alkaline pre- A periosteal elevator was then used to strip incubation, muscle fibers of the masseter mus- the entire masseter muscle from the inferior cle could be differentiated on the basis of the border of the ramus proximally to the level of activity of myofibrillar ATPase, but low-activ- the sigmoid notch. The region was packed with ity fibers were nonuniform in reaction intensi- gauze to establish hemostasis and the proce- ty (Fig. 2) and some fibers had only slightly dure was repeated on the other side. less intense ATPase than high-activity fibers. In group AD animals, the masseter muscle This is in contrast to the excellent differentia- was realigned as closely as possible along the tion observed in limb muscles and in the tem- ramus, but no attempt was made to reattach poralis muscle (Maxwellet al., 1980b).Alkaline it. In group ADR and DR animals, the mas- preincubation inhibited the low-activity fibers seter muscle was reapproximated in its pre- such that they became more uniform in reac- operative position and 3-0 chromic suture was tion intensity and were more easily distin- used to reattach it. The platysma layer was guished from the high-activity fibers. The then closed using 3-0 chromic suture and the myofibrillar ATPase activity demonstrated by skin was closed using 5-0 silk suture. each type of muscle fiber could be completely There were no significant or unusual seque- inhibited by fixation of sections in calcium for- lae in any of the monkeys who underwent mol or by inclusion of parahydroxy-mercuri- surgical intervention. Each resumed her nor- benzoate (PHMB)in the incubation medium. mal dietary behavior within 7 to 10 days fol- A section of each muscle sample incubated lowing surgery. for myofibrillar ATPase was projected at x 1,000 and the outlines of individual fibers in Biopsy. Animals were sacrificed at selected each of three or four 25 cm X 40 cm sample times after operative procedures. No animal areas were traced. At least 100 fibers were was sacrificed at less than 4 weeks postoper- traced from each muscle, and the cross- atively. Regression analysis revealed no sig- sectional area of each determined by nificant alterations in measured parameters planimetry. Fibers completely within the sam- following 4 weeks; thus adaptations were com- ple area and fibers which crossed the top and plete within 4 weeks. At the time of sacrifice, right boundaries and top corners of the sample monkeys were preanesthetized with ketamine area were included in the sample. Fibers cross- (10 mglkg IM) followed by an intravenous in- ing the other boundaries and corners of the jection of pentobarbital sodium to induce surg- sample area were excluded. ical anesthesia. The right superficial masseter In cat gastrocnemius muscle, muscle fibers and temporalis muscles were exposed and sam- with low myofibrillar ATPase contract slowly ples for histochemical and biochemical anal- whereas those with high myofibrillar ATPase yses were excised from standardized sampling contract quickly (Burke et al., 1971). This cor- sites (Maxwell et al., 1979, 1980a,b) near the relation of physiological and histochemical anterior and posterior border of the superficial characteristics has not yet been performed for lamina of these muscles. Samples were also fibers of the masticatory muscles of rhesus obtained from the midline portion of the an- monkeys. Furthermore, histochemical reac- terior belly of the digastric muscle. Histochem- tions for myofibrillar ATPase, particularly fol- ical samples (approximately 4 x 4 x 4 mm) lowing acid preincubation, differ for mastica- were quick frozen in isopentane cooled with tory compared to limb muscles of monkeys dry ice. Muscle samples weighing approxi- (Maxwell et al., 1980b). Due to these limita- 130 LEO C. MAXWELL ET AL. MASTICATORY MUSCLE ADAPTATION TO ALTERED LENGTH 131 tions, we classified masticatory muscle fibers ferential respirometry, essentially as de- only as Type I or Type I1 (Fig. 2) based on scribed by Potter (1964). Protein content of the myofibrillar ATPase activity (Brooke and Kai- homogenate was determined by the method of ser, 19701, rather than as slow or fast in ac- Lowry et al. (1951) and succinate oxidase ac- cordance with Burke et al. (1971), as we had tivity was expressed as nl of oxygen consumed done previously. mg homogenate protein - minute - ’. SDH. A serial section incubated for SDH ac- Fiber length. At sacrifice, after removal of tivity was then projected, the same sample histochemical and biochemical samples, the fibers were identified, and Type I1 fibers were animals were perfused with a fixative solution further classified as Type IIa or Type IIb. Fi- of neutral-buffered formalin. The heads were bers classified IIa had distinct SDH activity removed and placed in 10% formalin for stor- and subsarcolemmal aggregates of diforma- age. The masseter and temporalis muscles zan, especially near capillaries. Fibers classi- were then removed intact from the skull. The fied IIb had very little SDH activity in sub- deep portion of the masseter muscle was sep- sarcolemmal regions of the fibers. Both arated from the superficial masseter and the localization and intensity of activity were used muscles were stored in 10% formalin for at in determining fiber classification. Type I, least 1 week. Fixed muscles were then im- Type IIa, and Type IIb fibers were identified mersed in 30% nitric acid to digest connective (Fig. 1). tissue and release muscle fiber bundles (Wil- Capillarity. A third serial section incu- liams and Goldspink, 1971; Maxwell et al. bated for capillary membrane phosphatase ac- 1974). The acid was then replaced with 50% tivity (Fig. 3) was projected and the location glycerol and small bundles of muscle fibers of capillaries relative to the same sample fi- were carefully teased free. The length of 20 bers were drawn on tracings. The number of such bundles was determined by measurement capillaries within the sample area, as well as under a dissecting microscope fitted with an the number of capillaries adjacent to each fiber ocular micrometer. Fibers from these bundles in the sample, were counted. were then mounted in 50% glycerol and ex- amined at x 1,000 under oil immersion. The Fiber area. Fiber area was determined by length of 10 sarcomere units was determined planimetry of fiber outlines drawn from pro- for at least 20 different fibers. Dividing mean jection of sections incubated for myofibrillar fiber length by mean sarcomere length yields ATPase. Mean fiber area, the percentage of an estimate of the numbers of sarcomeres com- each type of fiber in the sample (percent comprising individual muscle fibers. position), fibers/mm2, capillaries/mm2, and capillary to fiber ratio (capillaries/mm2 di- Statistical evaluation. Animals were sac- vided by fibers/mm2)were calculated. rificed at selected intervals greater than 4 weeks after initiation of treatment. When ana- Oxidative capacity. Muscle samples for bio- lyzed by linear regression, no significant re- chemical procedures were blotted to remove lationship with the duration of treatment was excess moisture, weighed, and homogenized in observed. Therefore, data from all muscles 19 volumes of ice cold 0.01 M phosphate buffer within an experimental group were pooled and (pH 7.4) using a Polytron tissue homogenizer. statistical measures were calculated. Statis- Succinate oxidase activity for duplicate ali- tical analyses included analysis of variance to quots of homogenate was determined by dif- detect significant differences within the

Fig. 1. A photomicrograph of a cross section of an anterior masseter muscle incubated for succinate dehydrogenase activity shows variability in reaction amongst fibers. Selected examples representing three types of muscle fiber are identified for comparison with Figures 2 and 3. Magnification is x 200.

Fig. 2. A photomicrograph of a serial cross section of an anterior masseter muscle incubated for myofibrillar ATPase activity without prior alkaline incubation illustrates Type I (low-activity) and Type I1 (high-activity) fibers. No fibers with “intermediate”ATPase activity are present in this sample. The same representative fibers are identified as in Figure 1, and illustrate the histochemical profiles of three fiber types. Magnification is x 200.

Fig. 3. Capillarity of a cross section of a serial cross section of an anterior messeter muscle is demonstrated by capillary membrane phosphatase activity. Magnification is x 200. 132 LEO C. MAXWELL ET AL. grouped data for each variable, and the New- poralis or diagastric muscles in any experi- man-Keuls multiple comparison test to iden- mental group. tify differences between specific groups. In a few cases it was desirable to pool data into two Fiber area major groups to test specific hypotheses. In The mean cross-sectional areas of Type I fi- these cases, differences were tested by t-test. bers in group ADR animals was significantly Significance was accepted at theP < 0.05 level. reduced relative to controls in the anterior masseter muscle (Table 1).This effect did not RESULTS occur in posterior masseter, anterior or posterior temporalis, or digastric muscles of these Fiber type animals. A variable proportion of muscle fibers in the anterior regions of some masseter and tem- Capillarity poralis muscles exhibited myofibrillar ATPase Determination of the number of adjacent activity greater than expected for Type I fi- capillaries for individual muscle fibers re- bers, but less than the activity of Type I1 fibers. vealed no significant alteration from control When fibers with this “intermediate” level of value for the muscle of the experimental myofibrillar ATPase activity were classified, groups (Table 3). Capillary density values no consistent pattern for the frequency of oc- (Table 3) were occasionally greater than the currence was observed, and many muscles of control value, reflecting changes in mean fiber control and of experimental animals had no area of the muscle samples. Capillarity seemed fibers with the intermediate level of activity. to be only minimally affected by the experi- It was often difficult to distinguish whether a mental procedures. fiber was truly “intermediate” or was a Type I fiber with nonhomogeneous reaction inten- Fiber length sity. Therefore, fibers with intermediate my- The number of sarcomeres comprising mus- ofibrillar ATPase activity were pooled with the cle fibers of deep and superficial masseter mus- Type I fibers. cles was not significantly altered from control No difference from control data in the pro- values by experimental procedures, whether portion of Type I fibers was observed for mus- the functional length of the masseter was in- cles in any experimental group. Although creased by the appliance, the masseter was Type I1 fibers in muscles of experimental an- detached, or detached and reattached (Table imals had less intense SDH activity than mus- 4). Due to trends in opposing directions, fibers cle fibers of control animals, many Type I1 fi- of masseter muscles were longer in groups PT bers retained the pattern of localization of than ADR animals. Significantly more sar- SDH activity classifying them as Type IIa. comeres per temporalis muscle fiber were ob- Thus, in masticatory muscles of experimental served in those animals which had surgical relative to control animals, there was reduced manipulation of masseter muscle in addition oxidative capacity but no significant alteration to a bite-opening appliance (groups AD, ADR) in the proportion of muscle fibers of the three than in those animals which did not (control, classifications (Table 1). A, DR, PT). Neither surgical manipulation of the masseter alone (group DR) nor the appli- Oxidative capacity ance alone (group A) were sufficient to induce Succinate oxidase activity of muscle homog- significant adaptation from control, but a com- enates (Table 2) supports the observations on bination of the two stimuli was required for the histochemical demonstration of SDH ac- the effect. tivity. Masseter muscles of groups A, AD, ADR, and DR had significantly less succinate DISCUSSION oxidase activity than the muscles of controls Histochemical demonstration of myofibril- or Group PT animals. In anterior portions of lar ATPase activity of masticatory muscle fi- temporalis muscles from animals in which an bers does not exactly fit the pattern normally appliance was present at sacrifice (group A, seen for three types of muscle fibers in limb AD, ADR) succinate oxidase activity was less muscles (Maxwell et al., 1980a). Specifically, than for the muscles of animals which did not when incubated without prior treatment, some (control and groups DR, PT). No differences of the fibers in masticatory muscles demon- from control values were observed for the suc- strate an activity relatively greater than usu- cinate oxidase activity of the posterior tem- ally seen for Type I fibers, but less than for MASTICATORY MUSCLE ADAPTATION TO ALTERED LENGTH 133

TABLE I. Percentage Composition and Mean Fiber Area For Masseter, Temporalis, and digastric muscles (data are mean t SEMI

Percentage composition Mean fiber area (kmZ) I IIa IIb I IIa IIb Anterior masseter Control 89.3 t 4.8 11.0 t 3.9 3.1 i 2.6 2,330 t 120 1,080 f 200 1,300 t 210 A 84.6 ? 7.4 15.4 i 7.4 0 2,220 -c 450 1,480 f 350 - AD 83.5 ? 5.8 14.9 c 5.8 1.6 i 1.4 1,900 ? 180 1,170 f 270 1,860 t 609 ADR 65.0 f 13.5 22.0 ? 14.6 13.0 f 9.2 1,600 i 260* 1,100 t 350 1,330 f 330 DR 80.3 f 5.8 16.0 t 5.4 4.0 % 2.4 1,920 2 230 750 5 120 980 i 310 PT 77.0 f 9.1 19.8 i 6.2 3.2 f 3.2 1,670 t 140 880 f 260 1,200 f 50 Posterior masseter Control 40.0 t 6.8 38.4 f 6.9 21.6 f 9.0 2,240 t 160 1,900 t 190 2,140 f 260 A 53.8 f 7.0 23.0 f 6.4 22.9 f 8.7 2,260 ? 410 1,210 t 160 1,410 t 230 AD 29.4 t 4.8 44.0 f 10.3 27.0 f 6.4 2,100 f 340 1,830 t 230 1,850 t 280 ADR 30.0 c 4.9 27.7 i 8.8 37.2 t 11.6 1,750 f 330 1,930 t 310 2,310 t 420 DR 37.4 ? 10.5 42.5 ? 10.0 20.0 _t 7.5 2,210 t 62 1,980 f 280 1,960 f 330 PT 45.3 f 11.2 18.4 f 3.6 36.3 f 9.3 1,400 ? 100 1,270 t 130 1,840 f 320 Anterior temporalis Control 50.0 t 8.0 27.0 f 5.7 23.0 f 8.8 2,420 t 230 1,620 f 180 1,680 t 170 A 53.8 t 7.0 23.0 t 6.4 22.9 IT 8.7 2,260 t 410 1,220 t 160 1,450 f 250 AD 40.4 t 4.9 18.1 f 3.8 41.5 f 4.8 2,300 t 210 1,580 f 150 1,700 t 180 ADR 41.7 t 7.2 14.4 f 5.7 38.7 f 1.7 2,270 f 480 1,740 t 220 2,080 * 200 DR 49.2 t 4.0 24.9 ? 5.8 25.8 f 4.9 2,700 f 420 1,530 t- 180 2,040 t 300 PT 32.7 t 9.7 20.2 t 4.5 47.1 f 8.2 1,930 t 250 1,820 t 430 1,860 f 400 Posterior temporalis Control 9.4 ? 2.4 27.8 7.4 62.8 f 6.3 1,800 t 170 1,600 ? 220 1,960 ? 190 A 11.9 f 3.0 5.0 f 2.5 83.1 t 5.4 1,610 f 310 1,180 f 120 1,610 f 130 AD 17.8 f 2.0 19.2 f 5.9 63.8 2 5.4 2,060 f 260 1,390 t 140 1,760 f 180 ADR 9.4 t 3.0 20.0 f 4.4 69.6 t 5.8 1,800 f 360 1,430 f 210 2,040 f 290 DR 10.2 t 4.5 16.6 f 5.3 73.2 f 8.0 1,930 f 308 1,500 2 160 1,960 & 140 PT 16.9 t 6.5 18.1 * 9.2 65.0 t 15.5 1,650 ? 160 1,500 t 150 1,780 f 190 Digastric Control 44.0 ? 6.8 28.7 f 8.4 27.0 t 8.7 2,370 i 210 3,190 t 160 4,370 t 730 A 52.21 c 0.8 12.6 t 6.2 35.2 f 3.3 2,390 t 290 2,400 t 630 3,820 t 470 AD 41.3 t 3.1 29.0 t 4.4 30.0 t 6.0 2,040 i 210 2,840 t 330 3,260 f 370 ADR 41.2 t 9.6 23.4 f 7.4 35.3 4 5.3 1,870 f 310 2,700 t 390 2,920 t 460 DR 43.4 i 7.1 20.4 f 3.5 35.4 f 8.2 1,860 f 330 2,580 t 410 3,270 t 480 PT 42.4 ? 1.8 42.9 t 5.5 14.7 f 3.7 2,180 ? 270 2,830 t 280 4,000 t 320

* Significantly different from control (P < 0.05).

TABLE 2. Succinate oxidase activity of muscle homogenates (nl Odmg homogenate protein-llmin-ll (data are mean c SEM) Masseter Temporalis

Anterior Posterior Anterior Posterior Digastric

Control 441 f 24 416 t 33 330 f 24 175 t 26 218 ? 126 A 170 f 28* - 144 f 31* 76 * 12 169 f 32 AD 205 f 28* - 180 t 28* 106 i 21 234 f 38 ADR 158 IT 39* - 177 t 42* 134 f 38 227 i 77 DR 225 t 51’ - 212 t 36 85 ? 13 136 c 45 FT 387 t 37 - 296 c 82 171 f 44 344 f 27

* Significantly different from control or FC value iP < 0.05).

Type 11 fibers. It has been suggested that fibers Kugelberg, 1976; Maxwell et al., 1980b). Ac- which exhibit an intermediate intensity of cording to this hypothesis greater proportions myofibrillar ATPase activity may be transi- of “intermediate” fibers might be expected fol- tional between Type I and Type I1 fiber types lowing alterations of muscle function which which are responding to alterations in func- result in significant differences in the propor- tional demand (Ringqvist, 1973, 1974, 1977; tion of Type I and Type I1 fibers. However, in 134 LEO C. MAXWELL ET AL

TABLE 3. Capillary of mseter, temporalis, and digastric muscles (data are wan f SEMI

Number of adjacent capillaries Capillaries Capillary to muscle I IIa IIb per mmz fiber ratio

Anterior masseter Control 5.29 f 0.43 3.49 _t 0.70 3.09 i 0.91 863 f 68 2.30 f 0.21 A 5.16 i 0.46 3.63 f 0.69 - 793 s 94 1.79 f 0.610 AD 5.79 f 0.74 3.71 f 0.97 - 972 f 97 2.06 f 0.25 ADR 3.90 f 0.50 2.99 f 0.43 3.34 ? 0.48 825 f 73 1.38 f 0.22 DR 6.01 f 0.76 3.57 f 0.37 3.02 -c 0.50 931 i 135 2.10 f 0.33 IT 4.03 f 0.46 2.37 -c 0.24 - 778 f 153 1.37 f 0.17 Posterior masseter Control 4.59 f 0.47 3.60 i 0.49 3.73 i 0.72 566 ? 82 1.45 i 0.17 A 4.35 ? 0.38 3.48 f 0.26 3.40 f 0.22 753 f 89 1.49 -c 0.08 AD 4.76 f 0.56 4.14 f 0.82 4.13 f 0.87 778 f 38 1.75 f 0.28 ADR 4.02 f 1.22 3.99 f 1.23 3.55 f 0.72 932 -c 99* 2.15 ? 0.39 DR 5.45 f 0.77 4.94 ? 0.43 4.07 f 0.81 991 i 91* 2.36 f 0.21 PT 4.20 f 0.50 3.24 f 0.19 3.06 -+ 0.29 720 i 137 1.26 f 0.22 Anterior temporalis Control 5.38 -c 0.48 3.35 f 0.49 - 645 ? 90 1.86 f 0.27 A 4.52 f 0.44 3.15 f 0.47 3.30 f 0.40 746 f 96 1.60 i 0.17 AD 5.44 ? 0.56 3.23 i 0.34 3.52 i 0.25 823 f 68 1.71 f 0.12 ADR 4.49 ? 0.63 3.66 f 0.65 3.59 f 0.10 608 f 126 1.34 ? 0.14 DR 5.93 ? 1.07 4.03 f 0.33 4.12 f 0.39 677 f 93 1.64 ? 0.21 FT - - - - Posterior temporalis Control 3.97 f 0.36 3.31 ? 0.27 2.84 0.26 567 ? 37 1.35 f 0.14 A - - - - - AD 5.12 f 0.75 3.70 f 0.26 3.42 f 0.34 962 f 20 1.67 ? 0.16 ADR - - - - DR 4.00 f 0.32 4.00 f 0.33 3.51 f 0.25 - IT - - - - Digastric Control 4.88 k 0.65 6.04 i 0.08 - 646 f 103 2.11 i 0.10 A - - - - - AD 4.83 i 0.92 5.45 i 1.30 4.60 f 0.68 1017 i 109 2.86 2 0.50 ADR - - - - - DR 4.37 f 0.86 4.75 f 1.29 4.51 f 0.61 873 f 18 2.30 f 0.49 FT - - - - -

* Significantly different from control (P < 0.05).

the current research no differences in the pro- and limb muscles from rhesus monkeys (Max- portion of Type I and Type I1 fibers were found well et al., 1980b). between any of the experimental groups and Since fibers with “intermediate” myofibril- controls. Thus, if fibers with “intermediate” lar ATPase activity were not observed in every myofibrillar ATPase activity are transitional muscle, pooling of fibers which have lower fibers in which the type of myosin is altering, myofibrillar ATPase activity than Type I1 fi- it could be that at any point in time a small bers into one group as Type I fibers simplifies but equal number of fibers are converting from results. This approach is justified because (1) Type I to Type I1 as from Type I1 to Type I. the intermediate level of activity more closely Alternatively, it is possible that the muscles resembles the activity of Type I than Type I1 of mastication contain a different fiber type fibers; (2) preincubation at pH 10.2-10.4 par- which is actually intermediate in myofibrillar tially inhibits the activity of these fibers, thus ATPase activity, or which requires different displaying alkaline lability characteristic of conditions for optimal expression of myofibril- Type I fibers (Brooke and Kaiser, 1970); (3) lar ATPase. This possibility receives added im- there was no difference in capillarity, oxida- petus from a recent analysis demonstrating tive capacity, or fiber area between the fibers significant differences in pH lability of myofi- with intermediate ATPase activity and Type brillar ATPase between fibers of masticatory I fibers; and (4) the sum of the proportions of MASTICATORY MUSCLE ADAPTATION TO ALTERED LENGTH 135

TABLE 4. Number of sarcomeres per muscle fiber (data are mean f SEM)

Superficial Deep masseter masseter Temporalis

Control 5,908 ? 327 4,241 f 646 7,149 f 471 A 5,758 f 434 4,410 f 427 7,224 f 472 AD 6,440 f 333 4,433 -c 451 7,943 f 396 ADR 5,226 f 506' 4,388 f 451 8,468 ? 421 DR 5,875 s 372 4,972 f 519 6,700 f 510 FT 8,200 t 1906 4,380 f 941 7,287 5 656 C, DR, PT 7,038 f 310 AD,ADR 8,162 f 288**

* Group ADR significantly different from group PT. ** Pooled data for groups AD, ADR significantly different from pled data for groups C, DR, PT (P < 0.05). fibers with intermediate activity and Type I actly the opposite of the situation in the control fibers was consistent both within and between female animals. groups. Nevertheless, failure to observe an altera- Several recent histochemical studies of the tion in the proportion of the different types of muscles of mastication demonstrate changes muscle fibers with the experimental tech- in fiber cross-sectional area and in percentage niques in this report is not totally unexpected. composition of fiber types in response to alter- Limb muscles respond to increased functional ations in functional demand. An analysis of length due to growth without alteration of the histochemical characteristics of the mas- myofibrillar ATPase (Maxwell et al., 1973). seter and temporalis muscles in normal juve- Furthermore, no alteration of myofibrillar AT- nile and adult rhesus monkeys, for example, Pase was observed following an endurance found significant differences in percentage training program (Maxwell et al., 1973). Thus, composition and cross-sectional area of specific there are examples in the literature of altered fiber types between juveniles and adults, and functional length or altered activity level between adult males and females (Maxwell et which do not cause changes in myofibrillar al., 1979). It was suggested that these varia- ATPase. Chronic electrical stimulation exper- tions in histochemical properties of the mus- iments suggest that activity induced by bursts cles of mastication actually derive from alter- of impulses at a frequency of lO/sec once each ation in jaw function associated with the second will induce the transformation of developing occlusion and secondary sex char- myosin from Type I1 to Type I (Salmons and acteristics of the adult dentofacial complex. In Sreter, 1976). Thus, the experimental proce- a study of denture wearers, Ringqvist (1974) dures in the current study may not have sig- noted a reduced percentage and reduced cross- nificantly altered the pattern of impulses to sectional area of Type I1 fibers in the tempor- the muscle from motor neurons. It is perhaps alis muscle of individuals who described their the frequency of impulses within a burst of dentures as deficient, relative both to individ- activity, rather than the frequency of bursts uals with a natural dentition and to individ- of activity, which is important in the regula- uals with well-fitted dentures. An experimen- tion of the type of myosin in muscle fibers. If tal study of the masseter and temporalis so, changes in overall activity level without muscles of female monkeys who were eden- change in the frequency of impulses within tulous for a period of 4.5 years (Maxwell et al., bursts would result in no change in myofi- 1980a) demonstrated a pronounced change in brillar ATPase. histochemical composition and fiber area in Oxidative capacity of muscle fibers (Max- the edentulous monkeys relative to controls. well et al., 1973) does respond to changes in The edentulous animals had significantly the overall level of activity. Reduced intensity fewer Type I fibers and more Type IIb fibers of SDH activity in muscle fibers and the re- in the posterior masseter and anterior and pos- duced succinate oxidase activity in muscle ho- terior temporalis. Moreover, the Type I fibers mogenates in groups A, AD, ADR, and DR all were smaller in mean cross-sectional area suggest a reduction relative to control in the than Type IIb fibers at all sample sites--ex- overall activity of these muscles following 136 LEO C. MAXWELL ET AL. placement of an intraoral appliance or mas- animals. Thus, it appears that when both masticatory muscle surgery. As demonstrated by seter and temporalis muscles can contribute the data from group PT, the effect due to the to the support and function of the mandible, appliance is reversible, and return toward con- any adaptation to the increased functional trol values occurs following removal of the ap- length must have occurred through geometr- pliance. Lack of significant change in the ox- ical rearrangement of temporalis muscle fi- idative capacity of digastric muscle bers. However, when the masseter muscle in- demonstrates that the effect of the experimen- sertion was surgically detached and the tal procedures is selective, occurring in specific masseter muscle was less able to contribute to muscles, and that demonstrated differences mandibular movement and support, sarcom- from control represent effects of experimental eres were added to the fibers of temporalis procedures rather than general effects on mus- muscle. This effect occurred only in response cle resulting from age, time in captivity, or to the combined stimulus of the appliance and handling of animals. detachment of the masseter, whether or not The placement of a bite-opening appliance masseter was surgically reattached (groups in the mouth causes masticatory muscle fibers AD, ADR). Neither the appliance alone (group to function at greater than optimal length and A), nor surgical detachment and reattachment at decreased mechanical advantage due to the of masseter muscle alone (group DR), were ef- altered position of the mandible (Carlson, fective. The average 15%increase in the num- 1977). However, no change in mean fiber area ber of sarcomeres of temporalis muscle fibers was observed in response to the stretch in- in groups AD and ADR corresponds well to the duced by the appliance alone (group A). Surg- increase in functional length of the temporalis ical detachment of the insertion of masseter muscle. muscle relieves the stretch of the muscle. A decrease relative to control of Type I fiber cross-sectional area was observed in those an- CONCLUSIONS imals in which the masseter was surgically Alterations in the functional length of the detached and reattached (group ADR). masseter and temporalis muscles, with and Muscle fibers could adapt to alterations in without masseter myotomy, resulted in only functional length by addition or deletion of subtle morphological and physiological adap- sarcomeres to muscle fibers, by rearrangement tations. Nevertheless, certain general conclu- of muscle fiber orientation, or both. In limb sions can be drawn from the data which con- muscles immobilized by plaster casts, Tabary tribute to our understanding of muscle et al. (1972) and Goldspink et al. (1974) have adaptation following surgical interventions or demonstrated addition of sarcomeres to muscle alterations in length. fibers when immobilization length exceeded 1. Stretching the masseter and temporalis the normal rest length and loss of sarcomeres muscles within physiological limits did not sig- when muscles were immobilized at less than nificantly alter the proportion of fiber types; rest length. These changes in sarcomere num- however, it did appear to cause a reduction in ber in response to functional length changes the oxidative capacity of Type I and Type IIa occur within 4 weeks even in denervated mus- fibers. Removal of the bite-opening appliance, cles (Goldspink et al., 1974). Inclusion of the thus alleviating the stretch of the masseter appliance in the mouths of group A animals and temporalis muscles, resulted in a “recov- produced approximately a 15% increase in ery” of the potential for oxidative phosphoryl- functional length of masseter (Carlson, 1977) ation to within normal limits. and temporalis muscles. However, no altera- 2. Fibers with intermediate intensity of my- tion in the number of sarcomeres per muscle ofibrillar ATPase activity were no more prev- fiber were observed in group A animals. Ani- alent in muscles of experimental animals than mals in group A were smaller (4.82 2 0.32 kg) in controls. than control animals (6.13 * 0.42 kg). Perhaps 3. Surgical detachment of the masseter mus- this masked an alteration of fiber length in cle with surgical reattachment resulted in a this group. This suggestion receives support significant decrease in the mean cross-sec- from the observation of greater than control tional area of Type I fibers in the anterior length of masseter muscles in group PT, which masseter muscle. These data indicate that the had also been subjected to stretch by the ap- cross-sectional area of Type I fibers, which are pliance. No alteration of the number or sar- probably most active during postural activity comeres per muscle fiber was observed in tem- and sustained mastication, is most sensitive poralis muscles of control or group A, DR, FT to alterations in jaw function. MASTICATORY MUSCLE ADAPTATION TO ALTERED LENGTH 137

4. There was minimal alteration of muscle Hume, V.O., and E.G. Van Wagenen (1961) Basic data on capillarity, regardless of the experimental pro- the permanent teeth in the rhesus monkey. hc.Am. Philos. Soc., 105: 105-140. tocol followed. Thus, it appears that neither Kugelberg, E. (1976) Adaptive transformation of rat soleus alteration in functional length nor surgical in- motor units during growth. J. Neurol. Sci., 27: 269-289. tervention of the sort described in this analysis Lo-, O.H., N.J. Rosebrough, A.L. Faro, and R.J. Crandall have any significant direct effect on the cap- (1951) Protein measurement with the Folin phenol re- agent. J. Biol. Chem., 193: 265-275. Arch. Neurol., 23; illarity of masseter and temporalis muscles. 369-379. 5. The length of individual fibers within the Maxwell, L.C., J.A. Faulkner, and D.A. Lieberman (1973) temporalis muscle increased following muscle Histochemical manifestations of age and endurance train- lengthening in conjunction with masseter my- ing in skeletal muscle fibers. Am. J. Physiol., 344; 356361. otomy (i.e., groups AD and ADR), probably due Maxwell, L.C., J.A. Faulkner, and G.J. Hyatt (1974) Esti- to an addition of sarcomeres at the terminal mation of the number of fibers in guinea pig skeletal ends of the fibers. There was no increase in muscles. J. Appl. Physiol., 37: 259-264. fiber length within either the masseter muscle Maxwell, L.C., J.K. Barclay, D.E. Mohrman, and J.A. Faulkner (1977) Physiological characteristics of skeletal or the temporalis muscle following muscle muscles of dogs and cats. Am. J. Physiol., 233: C14-Cl8. stretching alone (group A); nor was there any Maxwell, L.C., D.S. Carlson, J.A. McNamara, Jr., and J.A. increase in fiber length in either muscle as- Faulkner (1979)Histochemical characteristics of the mas- sociated with masseter myotomy alone (group seter and temporalis muscles of the rhesus monkey (Ma- caca Mulatta). Anat. Rec., 193: 389401. DR). These data suggest that compensatory Maxwell, L.C., J.A. McNamara, Jr., P.S. Carlson, and J.A. activity of the temporalis muscle (1)following Faulkner (1980a) Histochemical characteristics of fibers masseter myotomy and (2) from a length which of masseter and temporalis muscles of edentulous rhesus is greater than its normal resting length, lead monkeys (Macaca Mulatta) Arch. Oral Biol., Arch Oral Biol25 87-93. to longitudinal growth of the temporalis mus- Maxwell, L.C., D.S. Carlson, and C.E. Brangwyn (1980b) cle fibers. Increase in the length of the tem- Lack of acid reversal of myofibrillar ATPase in masti- poralis fibers through addition of sarcomeres catory muscles fibers of rhesus monkeys. Histochem J. reestablishes the optimal overlap between the 12; 209-219. actin and myosin filaments within each sar- McNamara, J.A., Jr. (1975) An experimental study of increased vertical dimension in the growing face. Am. J. comere, increasing the tension developed by Orthod., 71: 382395. temporalis muscle fibers at the longer length McNamara, J.A., Jr., D.S. Carlson, G.M. Yellich, and R.P. and in the absence, however transient, of mas- Hendricksen (1978) Musculoskeletal adaptation follow- seter function. ing orthognathic surgery. In: Muscle Adaptation in the Craniofacial Region. D.S. Carlson and J.A. McNamara, ACKNOWLEDGMENTS Jr., eds. Center for Human Growth and Development, The University of Michigan, Ann Arbor, Michigan. pp. This work was supported by United States 91-132. Public Health Service grants DE 04227 and by Nachlas, M., M.K. Tsou, E. deSouza, C. 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