In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

Summary

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e. geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

Abstract

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

Introduction

Several experimental methods continue to be used to investigate the biomechanics of diarthrodial and fibrous joints. Methods specific to the tooth organ biomechanics include the use of strain gauges^1-3, photoelasticity methods^4,5, Moiré interferometry^6,7, electronic speckle pattern interferometry⁸, and digital image correlation (DIC)^9-14. In this study, the innovative approach includes noninvasive imaging using X-rays to expose the internal structures of a fibrous joint (mineralized tissues and their interfaces consisting of softer zones, and interfacing tissues such as ligaments) at loads equivalent to in vivo conditions. An in situ loading device coupled to a micro-X-ray microscope will be used. The load-time and load-displacement curves will be collected as the molar of interest within a freshly harvested rat hemi-mandible is loaded. The main goal of the approach presented in this study is to emphasize the effect of three-dimensional morphology of tooth-bone by comparing conditions at: 1) no load and when loaded, and when 2) concentrically and eccentrically loaded. Eliminating the need for cut specimens, and to perform experiments on whole intact organs under wet conditions will allow for maximum preservation of the 3D stress state. This opens a new area of investigation in understanding dynamic processes of the complex under various loading scenarios.

In this study, the methods for testing PDL biomechanics within an intact fibrous joint of a Sprague Dawley rat, a joint considered as an optimum bioengineering model system will be detailed. Experiments will include simulation of mastication loads under hydrated conditions in order to highlight three important features of the joint as they relate to organ level biomechanics. The three points will include: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. The three fundamental readouts of the proposed technique can be applied to investigate the adaptive nature of joints in vertebrates either due to changes in functional demands, and/or disease. Changes in the aforementioned readouts, specifically the correlation between reactionary loads with displacement, and resulting reactionary load-time and load-displacement curves at different loading rates can be applied to highlight overall changes in joint biomechanics. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint.

Protocol

Animal housing and euthanasia: All animals used in this demonstration were housed under pathogen-free conditions in accordance to the guidelines of the Institutional Animal Care and Use Committee (IACUC) and the National Institute of Health (NIH).

Provide animals with standard hard-pellet rat chow and water ad lib. Euthanize animals via a two-step method of carbon dioxide asphyxiation, bilateral thoracotomy in accordance with the standard protocol of UCSF as approved by IACUC. Perform biomechanical testing within 24 hours of animal sacrifice to avoid tissue degradation.

1. Preparation and Dissection of a Rat Mandible or Maxilla

1. Remove rat mandibles by gently severing membranous tissue and muscle tissue attachments while preserving the entire mandible, including the coronoid process and the condylar process (Figure 1)¹⁵.
2. Separate hemimandibles by carefully cutting the fibrous tissue of mandibular symphysis with a scalpel blade.
   Note: The coronary and condylar processes, and ramus of the mandible (Figure 1) should be removed if they physically obstruct biomechanical testing of the 2^nd molar.
3. Cut the incisors without exposing the pulp chamber as not to hinder loading of the molar.

2. Specimen Preparation for in situ Compressive Loading (Figure 2)

1. Immobilize the specimen on a steel stub by using a material that is significantly stiffer than the experimental specimen prior to loading it in an in situ loading device (Figure 2A).
   Note: Polymethylmethacrylate (PMMA) was used to immobilize the specimen in this study and excess, if any, was removed using a dental explorer.
2. Align the occlusal surface of the molar(s) of interest parallel with the AFM metal specimen disc using a straight edge in both planes (i.e. mesial-distal and buccal-lingual).
3. Create a trough with a blunt instrument surrounding the molars.
   Note: This space should serve as a “moat” to contain excess liquid and maintain tissue hydration during in situ loading.
4. Prepare the tooth surface to build up for concentric (Figure 2B) or eccentric (Figure 2C) loading using a dental composite. Etch the surface of the tooth of interest with 35% phosphoric acid gel on occlusal surface for 15 sec.
5. Rinse the etchant thoroughly with deionized water and dry the surface using an air/water syringe or a compressed-air canister. With an explorer, spread a drop of the bonding agent into open cusps in a thin layer. Cure the composite with a dental curing light.
   Note: All steps involving composites should be performed without direct light from a lamp. Such conditions would undesirably accelerate the polymerization process, and could prevent proper placement of the composite. Room lighting is acceptable.
6. Remove excess bonding agent from adjacent teeth with a fine scalpel or razor blade.
7. Place flowable dental composite on the surface following the preparation of the surface and spread it into grooves of the molar(s) of interest using a dental explorer.
8. Expose the composite to dental curing light for 30 sec.
9. Mold an occlusal buildup of about 3-4 mm using a dental resin composite, from the occlusal plane of the molar(s) of interest and light cure for 30 sec.
10. Reduce the top of the composite buildup to a flat surface parallel to enable a consistent loading scheme across all specimens by using a straight edge and a high speed hand piece.
    Note: During biomechanical testing, other specimens should be stored in tris-phosphate buffered solution (TBS) with 50 mg/ml penicillin, and streptomycin¹⁵.

3. Loading Device Drift and Stiffness, Material Property Differentiating Capability, in situ Loading of the Fibrous Joint

1. Secure the specimen with the composite buildup on the anvil of the loading stage, and test for uniform loading as shown in Figure 2B.
2. Place an articulating paper on the surface of the composite followed by loading the specimen to a finite load to check for concentric or eccentric loading (Figures 2B and 2C).
3. Place TBS-soaked Kimwipe around the specimen to ensure specimen hydration. Make a trough around the specimen and fill it with TBS to keep the organ hydrated during imaging.
4. Input peak load and displacement rate into the Deben software to compress the molar to a desired peak load at a displacement rate following immobilization of the hemimandible.
   Note: Typical readouts should include a reactionary load as the material is compressed over time (load transducer sensitivity = 0.1 N). From load-time and displacement-time, a load-displacement curve for the compressed material should be obtained^16-18. Using the data collected from the loading cycles, various properties of the joint can also be determined. The stiffness of the joint should be calculated by taking the slope of the linear portion (approximately the last 30% of the data) of the loading phase of the load vs. displacement curve¹⁹.

4. Staining of Soft Tissue, the PDL, with Phosphotungstic Acid (PTA)

Note: To enhance X-ray attenuation contrast, the PDL should be stained with 5% PTA solution²⁰.

1. Backfill PTA staining solution into a clean 1.8 ml glass carpule and place loaded carpule into syringe.
2. Inject solution slowly (5 min/carpule) into the PDL-space of adjacent teeth to prevent structural damage to periodontal tissues surrounding molar of interest.
   Note: The above steps should be repeated until about 5 full carpules (9 ml) of solution are injected and allowed to flow into the surrounding tissues. The prepped specimens can also be soaked overnight in the remaining PTA solution (8 hr).

5. Recommended μ-XCT Scanning Settings

Perform m-XCT with the following scanning settings:

Objective Magnification	4X, 10X
	1,800 images
X-ray tube voltage	75 kVp (50 kVp for PTA stained samples)
	8 W
Exposure Time	~8-25 sec*
	~4 μm (4X objective),~2 μm (10X objective) **

* exposure time can vary based on the geometry and optical density of the specimen and X-ray tube voltage.
** actual pixel resolution will slightly differ based on the configuration of the source, specimen, and detector.

Results

Estimation of loading device “backlash”, “pushback”, stiffness, and system drift under a constant load

Backlash: Between loading and unloading portions of the cycle, there exists a pause of 3 sec during which gears reverse within the motor before true unloading commences, i.e. as the specimen pulls away from the top jaw (Figure 3). This period is referred to as a backlash in the system, which represents a ...

Discussion

The first step in establishing this protocol involved evaluating the stiffness of the loading frame by using a rigid body. Based on the results, the stiffness was significantly higher enabling the use of the loading device for further testing of specimens with significantly lower stiffness values. The second step highlighted the ability of the instrument to distinguish different stiffness values by using two phases of the loading-unloading curve generated by using a rigid body, PDMS materials of different crosslink densi...

Materials

Name	Company	Catalog Number	Comments
Bard Parker Blade	BD	MEDC-001054
AFM metal disk	Ted Pella	16218
Polymethyl methacrylate	GC America	N/A
Uni-Etch	Bisco	E5502EBM
Optibond Solo Plus	Kerr Corp	N/A
Filtek Flow	3M	N/A
Hurculite Ultra	Kerr	34346
Tris buffer	Mediatech Inc.	N/A
Articulating paper	Parkell Inc.
Phosphotungstic Acid	Sigma Aldrich	HT152