Material and methods

The custom-engineered loading device used a voice coil connected to a power supply and function generator to produce cyclic forces ( Fig 1 ). A load cell on the device measured the magnitude of force output. The device was placed superior to the NFS and parallel to the midsagittal axis. Two vertically extended plates were fitted with holes and screws that engaged loading attachments on the skull. Calibration with known loads allowed conversion of output voltage into kilograms.

The loading device, showing how it engaged the loading attachments on the skull. It weighed 420 g and measured 14 × 4 × 6.3 cm (diagram not drawn to scale). F-Att , Frontal bone attachment; N-Att , nasal bone attachment.

Loading attachments were installed on the frontal and nasal bones. To provide sufficient contrast with physiologic strain during mastication (–1600 microstrain [μɛ] ⁸ ), the imposed cyclic strain was tensile, but the system is capable of compressive loading as well. Frequency was set at 2 to 3 Hz to mimic the rate of a pig’s chewing. To avoid injuring the sutural tissue, the desired NFS strain magnitude was 800 to 1000 μɛ, about the mean for sutures during mastication.

Four pig heads of varying ages and provenances (D-1 to D-4; Table I ) were used to establish the load output necessary to achieve the targeted strain at the NFS. Initially, stainless steel screws were used as the loading attachment. However, these screws lacked adequate retention and were changed to stainless steel loading plates with a central loading post ( Fig 2 ). The heads were instrumented with single-element strain gauges (EP-08-125BT-120; Micro-Measurements, Wendell, NC) over the loaded and contralateral NFS. Bilateral NFS were exposed, the periosteum was removed, and the sites prepared using the BAK-200 Basic Application Kit (Micro-Measurements) before the strain gauges were affixed with cyanoacrylate glue. The lead wires from the strain gauges were connected to the strain conditioner/amplifiers (model 2110; Micro-Measurements), which were linked to a computer for data acquisition. Strain information was collected at intervals throughout the loading session and digitized using the MP150 system and Acqknowledge 3.9 software (BIOPAC Systems, Goleta, Calif). The load output from the loading device was also sent through a strain gauge conditioner/amplifier as a separate channel. Strain and load output values were simultaneously collected in Acqknowledge in voltage, transferred into Excel (Microsoft, Redmond, Wash), and converted to microstrains and kilograms, respectively. Magnitudes of the strain and load outputs were obtained by subtracting the baseline values from the peak values.

Design of the 2-screw plate loading attachment. This custom attachment, made of stainless steel and weighing 0.94 g, was used in the ex-vivo heads (H-1 to H-6) and in-vivo pigs 1 and 2 (P-1, P-2). The stainless steel screws were 1.5 mm in diameter and 7.9 mm long.

Six heads (H-1 to H-6) from freshly slaughtered pigs (Kapowsin Meats, Graham, Wash), sex unknown and assumed to be about 6 months old, were used. Loading attachments (2-screw plates) were fastened on both sides of the right NFS. Single-element strain gauges were placed bilaterally on the NFS and coronal sutures, and on the internasal and interfrontal sutures as described previously. Three-element 45° stacked rosette gauges (WA-13-060WR-120; Micro-Measurements) were placed on the right nasal and frontal bones ( Fig 3 ), with the middle element perpendicular to the midsagittal plane. Cyclic loading was carried out for 30 minutes, with mean load output and strains for the beginning, middle, and final minutes of the session determined from averages of 20 waveforms each.

Pig head H-3 showing placement of the 2-screw plate loading attachments and single-element ( white rectangles ) and 3-element 45° stacked rosette ( white squares ) gauges.

Five mixed-breed pigs ( Sus scrofa ; 4 male, 1 female) approximately 3 months old (Progressive Swine Farms, Woodinville, Wash) were used (P-1 to P-5, Table II ). All experimental procedures followed the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the University of Washington Institutional Animal Care and Use Committee. The animals were acclimated for approximately 1 week. Pigs P-1 and P-2 received the 2-screw plate attachments but because these became loose, the design was changed for pigs P-3 to P-5. The new design ( Fig 4 ) had a custom titanium plate with 4 flanges, which could be contoured to the natural skull curvature. Each flange was stabilized by 2 stainless steel cortex self-tapping 2-mm screws (DePuy Synthes Vet, West Chester, Pa). These produced comparable load outputs to the 2-screw design in trials using dry skulls.

Design of the 8-screw plate titanium loading attachment. This custom attachment, made of titanium and weighing 1.63 g, was used for in-vivo pigs (P-3 to P-5). The screws were self-tapping stainless steel, 2.0 mm in diameter and 6.0 to 8.0 mm long (DePuy Synthes Vet, West Chester, Pa).

To affix the attachments, anesthesia was used with a cocktail consisting of midazolam, xylazine, and butorphanol delivered intramuscularly and maintained with isoflurane inhalation. Under aseptic conditions, arcuate incisions were made unilaterally on the right frontal and nasal bones. The NFS was not exposed. Flaps were raised with the periosteum left intact. Pilot holes were made for the screws, and the loading attachments were placed. The incisions were closed with interrupted sutures so that the loading posts projected from the skin. After 1 week of healing, cyclic loading sessions began. Pig P-2 had loose attachments and was considered a sham control. Daily for 5 consecutive days, the pigs were anesthetized as described previously, placed in a prone position, and the loading device was engaged to the loading attachments. On days 1 and 3, mineralization labels were administered (described below). Loading was conducted for approximately 30 minutes per day. In the sham control, the loading device was engaged, but no loads were applied. On the final (5th) day, before loading, under anesthesia, the periosteal flaps were elevated to expose the nasofrontal, internasal, interfrontal, and coronal sutures. Strain gauges were affixed as in the ex-vivo study, except for the unloaded sham control. Because bone strain levels recorded in the ex-vivo experiments had been low, no gauges were placed on the nasal and frontal bones. Load output and strain data were collected during cyclic loading as for the ex-vivo study. Data were analyzed by 1 investigator (S.H.S.).

Fluorochrome labels of mineral apposition were dissolved in sterile saline solution, adjusted to a neutral pH, filtered at 0.22 μm, and administered intravenously on days 1 (calcein, 12.5 mg/kg; Sigma-Aldrich, St Louis, Mo) and 3 (alizarin complexone, 12.5 mg/kg; Sigma-Aldrich) of loading.

After the loading session on day 5, the animals were injected intravenously with heparin and then killed with pentobarbital (Beuthanasia-D Special; Merck, Madison, NJ). This was followed by perfusion through the ascending aorta with approximately 4 L of saline solution and then approximately 4 L of 20% glyoxal fixative (derived from Prefer Concentrate; ANATECH, Battle Creek, Mich, diluted with reagent alcohol and deionized water). Instrumented sutures were removed and separated into 2 blocks, one for assessment of sutural width and mineral apposition, and the other for future immunohistochemistry. In addition, a randomly chosen NFS was removed from each of 4 additional pig specimens of comparable breed mix (C-1 to C-4, histologic controls; Table II ). These animals had been used in an unrelated study involving tooth mobility and periodontal disease and had received bone labels on a slightly different schedule.

The undecalcified samples were dehydrated in an ethanol series and infiltrated with Micro-Bed resin (Electron Microscopy Sciences, Hatfield, Pa) before curing for approximately 21 days in a thermal oven set at 35°C to 36°C. Sections perpendicular to the sutures at 50-μm intervals were obtained using a saw microtome (SP1600; Leica Biosystems, Buffalo Grove, Ill) and mounted on slides. One to 3 sections were selected per suture based on clarity and completeness, and examined under epifluorescent illumination (Eclipse E400; Nikon, Tokyo, Japan). Using a digital camera (SPOT RT3 2Mp Slider; Diagnostic Instruments, Sterling Heights, Mich), the same microscopic field was separately captured for calcein and alizarin. Images were then merged to show images with double fluorescent labels via the MetaVue software (Molecular Devices, Sunnyvale, Calif). Histomorphometric analysis was performed with MetaMorph software (Molecular Devices). Suture width was calculated by dividing the area between both sutural margins over the average length of the 2 sutural margins. Mineral apposition rate was calculated by measuring the area between the calcein and alizarin bone fronts and dividing it by the average suture length and the number of days between labels to give mineral apposition rates in micrometers per day. Mineral apposition rate values from each side of the suture were averaged to give the mean mineral apposition rates ( Fig 5 ). Intraexaminer reliability was determined by remeasuring 5 randomly selected samples. Selection of slides and measurements were all made by 1 investigator blinded to the identity of the samples.

Entire interfrontal suture of pig P-3 under epifluorescent illumination. The endocranial (more interdigitated) side is toward the bottom of the Figure. A, Double-labeled interfrontal suture. The first label was calcein ( green ), and the second label was alizarin complexone ( red ); the labels were given on days 1 and 3 of loading. B, Suture gap highlighted in yellow . Width was calculated as the gap area divided by the average true length of the sutural bony margins. C, Newly mineralized bone on each side of the suture highlighted in magenta and blue . Scale bar = 993 μm.

Mann Whitney U-tests and unpaired t tests were used for statistical analyses, and statistical significance was determined at P <0.05 (GraphPad Prism, La Jolla, Calif).

Results

Ex vivo, the 2-screw plate loading attachments remained intact and stable, but they were unsuccessful in vivo (pigs P-1 and P-2). The 8-screw plate loading attachments in pigs P-3 to P-5 were stable, except for the nasal attachment in pig P-4 that was somewhat moveable, although it still permitted loading and strain recording.

In the ex-vivo study on the transmission of strain in pig heads H-1 to H-6, the loading device produced a mean output of 1.55 ± 0.1 kg. Strains matching the loading frequency of 2.5 Hz were registered at working gauge locations. Load output and strains tended to reduce in magnitude during the session. The highest level of strain was seen in the loaded right NFS, with a mean tensile strain of +898 ± 233 μɛ ( Fig 6 ; Table III ). The contralateral NFS also showed substantial, but lower tensile strain (+660 ± 205 μɛ). Coronal sutures experienced very low compressive strain, averaging –47 ± 30 μɛ on the loaded side and –17 ± 16 μɛ on the contralateral side. The interfrontal suture was usually in tension, with 1 exception (H-6, –85 μɛ). The internasal suture was less consistent, showing tension or compression in an equal number of pig heads, ranging from –210 to +230 μɛ.

Strain patterns produced by cyclic loading of the right NFS on pig heads H-1 to H-6. Tensile strains are indicated by divergent arrowheads and compressive strains by convergent arrowheads . The values are given in Table III . Arrows of type A indicate strain data from the single-element strain gauges. Arrows of type B indicate the rosette gauge principal strains. Arrows of type C indicate strain data from individual working elements of rosette gauges when principal strains were indeterminate. Note the difference in scale for the arrow types.

Table III

Strain transmission in ex-vivo heads: mean strains and load output during 30 minutes of cyclic loading of the right NFS

Number	Right nasal bone rosette gauge				Right frontal bone rosette gauge				Load output (kg)	Suture single-element gauges (μɛ)
	Principal strain (μɛ)			Max angle (°)	Principal strain (μɛ)			Max angle (°)		Right NFS loaded	Left NFS	Right CS loaded-side	Left CS	IFS	INS
	Max	Min	Shear		Max	Min	Shear
H-1	Indeterminate				Indeterminate				1.55	1272	693	−54	−15	15	81
H-2	15	−37	52	64	38	−84	121	94	1.52	684	490	0	−3	86	26
H-3	Indeterminate				Indeterminate				1.60	737	664	−28	−15	53	190
H-4	Indeterminate				Indeterminate				1.64	720	508	−47	−44	20	−100
H-5	46	−34	80	128	Indeterminate				1.64	1063	1045	–80	0	213	−192
H-6	27	−46	73	57	26	−55	80	111	1.37	914	558	−75	−27	−85	−17
Mean (SD)	29 (15)	−39 (6)	68 (14)	83 (39)	32 (8)	−69 (21)	101 (29)	103 (12)	1.55 (0.10)	898 (233)	660 (205)	−47 (30)	−17 (16)	∗	∗

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