Enhanced study of facial soft tissues using a novel large scale histology technique

Abstract The safety and effectiveness of facial cosmetic surgery procedures are dependent on detailed 3D understanding of the complex surgical anatomy of the face. Traditional, small sample size anatomical dissection studies have limitations in providing definitive clarification of the fascial layers of the face, and especially in their relationship with the facial nerve, and their reaction to surgical manipulation. The objective study of large tissue areas is required to effectively demonstrate the broader architecture. Conventional histology techniques were modified to handle extraordinarily large tissue samples to fulfill this requirement. Full‐thickness soft tissue samples (skin to bone) of maximum length 18 cm, width 4 cm, and tissue thickness 1 cm, were harvested from 20 hemifaces of 15 fresh human cadavers (mean age at death = 81 years). After fixation, the samples were processed with an automated processor using paraffin wax for 156 h, sectioned at 30 μm, collected on gelatin‐chromium‐coated glass slides, stained with a Masson's Trichrome technique and photographed. Using this technique, excellent visualization was obtained of the fascial connective tissue and its relationship with the facial mimetic muscles, muscles of mastication and salivary glands in 73 large histological slides. The resulting slides improved the study of the platysma and superficial musculo‐aponeurotic system (SMAS), the spaces and ligaments, the malar fat pad, and the facial nerve in relations to the deep fascia. Additionally, surgically induced changes in the soft‐tissue organization were successfully visualized. This technique enables improved insight into the broad structural architecture and histomorphology of large‐scale facial tissues.


Abstract
The safety and effectiveness of facial cosmetic surgery procedures are dependent on detailed 3D understanding of the complex surgical anatomy of the face. Traditional, small sample size anatomical dissection studies have limitations in providing definitive clarification of the fascial layers of the face, and especially in their relationship with the facial nerve, and their reaction to surgical manipulation. The objective study of large tissue areas is required to effectively demonstrate the broader architecture. Conventional histology techniques were modified to handle extraordinarily large tissue samples to fulfill this requirement. Full-thickness soft tissue samples (skin to bone) of maximum length 18 cm, width 4 cm, and tissue thickness 1 cm, were harvested from 20 hemifaces of 15 fresh human cadavers (mean age at death = 81 years). After fixation, the samples were processed with an automated processor using paraffin wax for 156 h, sectioned at 30 μm, collected on gelatin-chromium-coated glass slides, stained with a Masson's Trichrome technique and photographed. Using this technique, excellent visualization was obtained of the fascial connective tissue and its relationship with the facial mimetic muscles, muscles of mastication and salivary glands in 73 large histological slides. The resulting slides improved the study of the platysma and superficial musculo-aponeurotic system (SMAS), the spaces and ligaments, the malar fat pad, and the facial nerve in relations to the deep fascia. Additionally, surgically induced changes in the softtissue organization were successfully visualized. This technique enables improved insight into the broad structural architecture and histomorphology of large-scale facial tissues. The lack of a complete anatomical overview with details of the facial soft tissue architecture may contribute to the shortcoming of current surgical techniques (Hamra, 2002). Moreover, adoption of these techniques by inexperienced surgeons is hindered by the absence of clear and unambiguous descriptions of the anatomy pertaining to the layers and commonly used dissection planes. Knowledge of anatomical structural features, such as tissue layers, gliding planes, ligaments, spaces, fusion zones, and so forth in the face still remains largely based on traditional anatomical dissection and surgical experience, occasionally leading to conflicting descriptions (Mendelson & Wong, 2016Pessa, 2016;Zins & Hashem, 2016).
Objective technical investigations, such as histology, computed tomography (CT) and magnetic resonance imaging (MRI), are required to complement dissection findings, but have their limitations, which reduce their value in the study of facial soft tissues. Both CT and MRI lack the spatial resolution for the required microscopic visualization of the facial nerve branches in relation to the different fascial sheets.
Histologic investigations, while ideal to demonstrate the structural architecture of soft tissues, are restricted by sample size. Standard histology techniques focus on small biopsy sizes 1-2 cm (L/W) Â 0.5 cm (D) to allow optimal fixation and processing during tissue preparation.
Traditional methods for embedding and sectioning of larger specimens (e.g., in nitrocellulose) are hazardous, time-consuming, and increasingly expensive (Gray, 1954;Mann, 1902). More recently described methods to study larger samples of soft tissues still fall short of the necessary size needed to study the complex soft tissue architecture of the face (Bryant et al., 2019).
To overcome this limitation, numerous adjustments were made to our routine histological methods to enable production of large-scale samples using conventional and affordable materials, allowing more labs to use this technique for their research. The goal of this publication is to provide the detailed methodology and histological technique to encourage clinical anatomists to investigate and report on large facial samples, of up to L 18 Â W 4 Â D 1 cm.

| MATERIALS AND METHODS
Ethical approval for the project was granted by the University Human Research Ethics Committee of the Queensland University of Technology (Project number LR 2021-4306-4761). The authors state that every effort was made to follow all local and international ethical guidelines and laws that pertain to the use of human cadaveric donors in anatomical research. From September 2021 to February 2022, 20 hemifaces of 15 human cadavers were investigated (n = 15; male = 9; female = 6; mean age at death = 81 years). Two fresh-frozen bodies were used for methodology optimization and nine fresh non-frozen bodies, one embalmed body and three fresh-frozen bodies were used to produce histological slides using the described histological method. Five cadavers underwent a surgical intervention to the unilateral face prior to harvesting to allow comparison with the unaltered side.

| Harvesting and preserving
The hemiface is dissected, sutured to firm cardboard, and then submerged in formalin (10% Neutral Buffered Formalin) at a 10:1 volume ratio of chemical fixative to facial tissue. Following 5 days of fixation, manual sub-sectioning of each hemiface into equally long but thinner samples is performed using a brain knife. The resulting samples, L 5-18 Â W 1-4 Â D 0.7-1 cm, are individually sutured flat to cardboard at four corners and resubmerged in formalin for another 48 h (Video S1).

| Processing
Fixed tissue samples are washed in slow running distilled water for 2 h at room temperature to remove excess fixative remaining from storage in the formalin solution. Afterwards, tissue samples are placed into 30% ethanol, followed by 50% ethanol for 2 h each at room temperature.
The tissue samples are loaded into processing trays and processed through a Leica ASP300S Pathcentre. A 6-day automated cycle is set with processing times detailed in Table 1. Samples are processed through ascending concentrations of ethanol, cleared in xylene, and infiltrated with molten paraffin wax for 24 h in each solution, while under a vacuum of À70 kPa and impregnation pressure of 35 kPa. All steps of the process occur at 35 C, except for the wax stations, which are at 65 C. Subsequently, the wax infiltrated samples are stored in zip-lock bags at 4 C.
The mounting stages feature two parts: a mounting plate of 2-4 mm thick aluminum sheet metal with 5 mm perforations to allow wax penetration and air bubble release, and a mounting base of 7.5 Â 7.5 Â 2 cm square aluminum metal that can be screwed onto the mounting plates prior to embedding. Each sample is embedded to obtain a longitudinal sectioning orientation parallel and close to the surface of the wax block face. Molten paraffin wax is poured slowly and incrementally into the mold allowing the lower portion of the mold to partially thicken while the sample is held in place with forceps. The mounting stage is positioned on top and, together, they are carefully transferred onto a cooling plate. Once the wax has set and is firmly attached to the mounting stage, the silicone mold is removed, and the sample block is stored at 4 C prior to sectioning (Video S2).

| Sectioning and staining
Embedded blocks are placed into the stage lock clamp of a Microm HM 430 sliding microtome. The blade holder is positioned at a 5 angle, and a disposable blade is inserted (Epredia, Bio-Strategies, Aus).
Sections are cut at 30 μm, gathered with the use of forceps and a paintbrush, and floated onto a warm gelatinized water bath at 42 C containing thymol preservative (Video S3).
Sections are collected onto large size gelatin-chromium-coated glass slides (12.5 Â 17.5 cm). Coating of these plain glass slides is done prior, by dipping in a gelatin-chromium adhesive solution (1.25% gelatin and 0.125% chromium potassium sulfate in distilled water) and allowing to dry at room temperature for a minimum of 24 h. Specimen slides are dried overnight at room temperature and then baked in a 37 C oven for 30 min prior to staining. The sections are stained with a Masson's Trichrome stain, summarized in Table 2.
Coverslips are mounted with a non-aqueous hard setting mounting medium (DPX Neutral Mounting Medium). The slides are placed into a 37 C oven to harden prior to imaging.

| Imaging
Excess mounting medium on the slides is removed with a straight edge blade and slides are cleaned with ethanol. The slides are imaged F I G U R E 1 Equipment used for this technique. From left to right: Custom made silicon molds and mounting stages composed of a plate and a base, Pathcentre trays with tissues, sliding microtome. Aluminum mounting plates were made in two sizes to accommodate the varying tissue lengths, a 10 Â 20 cm plate for larger specimens and a 6.5 Â 15 cm plate for smaller specimens. The processor, Leica ASP300S Pathcentre, is not shown in this image T A B L E 2 Modified Masson's Trichrome staining technique for large histological samples a

Main steps
Step-by-step actions 3 | RESULTS

| Histological results
Extended processing times resulted in shrinkage of the tissues ( Figure 2). This was noticed after the first processing run and, for the remaining tissues, the length and width were measured before and after tissue processing to permit quantitation of tissue shrinkage. In the 22 tissues measured, the length decreased with a mean of 6.4% (range 3.6%-14%) and the width decreased with a mean of 11.6% (range 7.1%-16.7%).

| Anatomical results
Using demonstrating this structure nor its connecting role between these two muscles (Macchi et al., 2009).
The early work of Barton et al. and Gosain et al. demonstrated that it is possible to conduct larger-scale histology on facial soft tissues (Barton, 1992;Gosain et al., 1993). However, the absence of a reproducible histological technique has hindered the use of these techniques by less histology-experienced researchers. Our histological methodology presented here, provides the clinical anatomist sufficient information to conduct structural and histomorphological investigation of facial architecture on large tissue samples from embalmed, freshfrozen or fresh cadavers. Upcoming publications by the authors will report on the detailed analysis of such large histological samples produced using this technique on the areas of the deep fascia, the superficial musculo-aponeurotic system (SMAS), the spaces and ligaments, the malar fat pad, and the platysma. The detailed anatomy of the jowl area using this technique was recently published (Minelli et al., 2022).
Conventional computed tomography (CT) and magnetic resonance (MR) imaging, while able to study larger areas, lack the spatial resolution to perform microscopic differentiation. A new micro-CT technique with improved spatial resolution has recently been intro-

| Harvesting and preserving
Minimum sample thickness is limited by (1) manual sub-sectioning which is prone to failure when cutting thinner than 7 mm and (2) required thickness for later microtomy sectioning along entire length of specimen allowing some alignment errors and tissue curling issues. Warping of samples is avoided by suturing the whole hemiface to rigid carboard prior to submersion in formalin, with sub-sectioning into thinner samples 5 days later, after fixation has occurred.

| Staining
Masson's Trichrome stain was selected for its clear differentiation of collagen and muscle, necessary for demonstration of soft tissue architecture (Masson, 1929). Initial staining trials using an unmodified Masson's staining protocol produced understained muscle fibers and overstained collagen fibers. Subsequently, Biebrich-scarlet red and aniline blue staining times were modified to optimize stain intensity of muscle and collagen, and acetic acid differentiation time was increased to improve the blue hue of the aniline blue. Some collagen fibers stained purple due to an overlapping false positive staining effect of Biebrich-scarlet red. Phosphotungstic acid and phosphomolybdic acid differentiation times therefore were increased to ensure collagen fibers were adequately stripped of Biebrich-scarlet red prior to aniline blue staining. The final protocol optimized for 30 μm sections of facial tissue is included in  Figure 3A) or embalmed ( Figure 3B). While these demonstrated equally good results, embalmed cadavers cannot be used to study the surgical manipulated architecture of soft tissue. The reason for this limitation is not because embalming prevents the surgical manipulation (soft embalming regimens allow surgical manipulation), but rather because previous embalming prevents subsequent fixation of surgically manipulated tissues in a fixed position.

| Limitations
This study and technique have certain limitations. The technique is limited to samples that can fit the processing tray (L 17.5 Â W 13.0 cm). This limitation cannot be overcome by manual processing, which would be too slow due to the absence of the vacuum and pressure steps. Our research has not been able to explain why the staining

INFORMED CONSENT
For this type of study informed consent is not required. F I G U R E 4 Differences in Masson's Trichrome staining in human cheek at varying section thickness. Four images of the same human cheek sample were initially compared to determine the optimum thickness for sectioning and staining of the soft tissue layer and muscle. Section thicknesses (top to bottom) were (i) 12, (ii) 20, (iii) 25, and (iv) 30 μm. The 30 μm section thickness was selected as the experimental section thickness due to the visualization of more intact retinacula cutis septa fibers compared to 12, 20, and 25 μm