3D printing from micro-CT images of the trochlea of the superior oblique muscle and its future applications

This study investigated the determination of detailed microstructure modeling of the trochlea of the superior oblique muscle (SOM) using micro-computed tomography (micro-CT) and modeling of a potential prototype for a trochlea implant using three-dimensional (3D) printing. We dissected 15 intact orbits of 15 embalmed cadavers. The trochleae of the SOM were detached from the periosteum. The specimens were stained by immersion in a 15% Lugol’s solution. Images were reconstructed using conventional scanner software. Measurement points were determined for the middle cross section. Points P1 and P2 were selected where the SOM adjoined the curvature of the inner trochlea. They defined the inner contact points of the SOM in the inner part of the trochlea curvature. On the back of the trochlea, points P3 and P4 were selected at the uppermost and lowest points in the inner parts of the straight trochlea, respectively. Origin O was defined on the arcuate line of P1P2^\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\widehat{P1P2}$$\end{document} to generate the smallest-diameter circle consisting of P1, O, and P2. We then measured the angle from OP1¯toOP2¯\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{OP1 } to \overline{ OP2 }$$\end{document}, and from OP3¯toOP4¯.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{OP3 } to \overline{OP4 }.$$\end{document} We also measured the distances OP1¯,OP2¯,OP3¯,andOP4¯\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{OP1 }, \overline{OP2 }, \overline{OP3 }, and \overline{OP4 }$$\end{document} for the design of a potential trochlea implant prototype using 3D-printing and micro-CT-based modeling. The distances OP1¯,OP2¯,OP3¯,andOP4¯\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{OP1 }, \overline{OP2 }, \overline{OP3 }, and \overline{OP4 }$$\end{document} were 2.2 ± 0.7, 1.4 ± 0.5, 2.7 ± 0.9, and 2.5 ± 0.4 mm (mean ± SD), respectively. The angles from OP1¯toOP2¯\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{OP1 } to \overline{OP2 }$$\end{document}, from OP2¯toOP4,-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{OP2 } to \stackrel{-}{OP4,}$$\end{document} and from OP3¯toOP4¯\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{OP3 } to \overline{OP4 }$$\end{document} were 100.7 ± 14.4, 66.3 ± 18.0, and 98.9 ± 24.9 degrees, respectively. The present investigation demonstrates that the high-resolution CT is a powerful imaging technique for defining the true 3D geometry of a specimen and can potentially be used to create a 3D-printed trochlea implant.


Introduction
The trochlea of the superior oblique muscle (SOM) is attached to the anteromedial orbital roof. The SOM tendon passes through the trochlea, which redirects it to become the functional origin of the SOM for ocular movement [1]. Because of the characteristic location of the trochlea at the anterior margin of the orbital roof, trochlea injury can occur during blepharoplasty [2,3], orbital surgery [4], and following traumatic eyelid injuries [5,6]. Injury to the trochlea occurs infrequently, but it can result in devastating complications such as diplopia, abnormal head posture, and ocular motility disturbances [7,8]. Understanding the detailed anatomy of the trochlea is therefore important to plan the comprehensive therapeutic management of injury to this structure.
Previous studies have mostly focused on the intracranial course of the trochlear nerve (in fourth nerve palsy) and SOM damage in the orbit, with inadequate consideration of damage to the trochlea itself. Its morphology has been described previously as a U-shaped piece of cartilage attached to the orbital plate of the frontal bone. However, our study group previously reported the anatomic location of the trochlea with reference to soft landmarks and found that the trochlea has a complex three-dimensional (3D) structure and that its morphology varies between individuals [9]. The 3D structure and morphology of the trochlea observed using high-resolution imaging may therefore be helpful in defining individual variations in trochlear anatomy.
Micro-computed tomography (micro-CT) imaging is an emerging tool within the biomedical field, which was developed to scan small samples at a high resolution. Micro-CT allows the noninvasive visualization of structure morphology (e.g., shape and size) with a high degree of precision and accuracy [10]. Micro-CT is also able to provide quantified data sets of the structure to allow precise anatomic models for 3D printing. 3D printing based on micro-CT-imaged specimens can provide a better understanding of these structures, new insights into craniofacial surgical applications including possible prosthetics, and can be used in anatomy education [11].
This study used cadaveric dissection with the purpose of determining the detailed microstructure modeling of the trochlea using micro-CT. We attempted to design a trochlea implant prototype using 3D printing. Such information may provide a comprehensive overview to the learner and also assist clinicians to develop new concepts such as trochlea replacement for the treatment of strabismus caused by trochlea injury.

Materials and methods
We dissected 15 intact orbits of 15 embalmed adult Asian cadavers (7 male and 8 female orbits, 8 right and 7 left orbits), aged 41-86 years at death (mean, 71.7 years). None of the cadaveric specimens had eyelid or orbital abnormalities. This study was performed in accordance with the principles outlined in the Declaration of Helsinki. Appropriate consent and approval were obtained before using the specimens.

Trochlea specimen sampling
The medial corner between the external eyelid surface and orbit was dissected meticulously. Fibrous septae connecting the SOM tendon with the globe and those connecting the SOM belly with the adjacent periorbita were cut. The trochlea and the SOM were detached from the periosteum, and the connective tissue inside the trochlea was then carefully removed. Special care was taken to ensure that the cartilaginous trochlear saddle was not disturbed (Fig. 1).

Image acquisition using micro-CT
Before performing micro-CT scanning, the specimens were stained by immersion in 15% Lugol's solution (10 g of potassium iodide and 5 g of iodine in 100 ml of water) for 24 h. The trochlea was then washed with alcohol to remove free iodine, blotted dry, and scanned (SkyScan 1176, Bruker, Kontich, Belgium). The micro-CT scanning was conducted under the conditions: 0.01188 mm of layer thickness, T Fig. 1 Cadaveric dissection demonstrating the location and shape of the trochlea (T). The trochlea is attached to the anteromedial orbital roof. The tendon of the superior oblique muscle (SOM) passes through the inside of the trochlea 0.01188 × 0.01188 mm 2 of resolution, 70 kV of voltage, and 141 mA of X-ray tube current.
For image segmentation, the Hounsfield unit (HU) values for the localized area were determined using the local thresholding method. The segmentation of trochlea was carried out using region glow and split mask. Images were reconstructed using the scanner software (NRecon 1.6.6.0, SkyScan) and converted to Digital Imaging and Communications in Medicine (DICOM) format for analysis (Fig. 2, Supplementary Video 1). Images were imported in commercial software (Mimics version 21, Materialise, Leuven, Belgium) and reconstructed following the steps of thresholding, region growing, and semiautomatic image segmentation with manual editing where required, and the final volume meshes were exported as stereolithography (STL) files.

Measured parameters
We defined the part of trochlea that is close to the eye and in contact with superior oblique muscle as the anterior and the part that is attached to the orbital plate of the frontal bone as posterior. In order to design a generalized trochlea model, extracted STL files were loaded into Materialise 3-matic software (version 15, Materialise) to be measured. Measurement points were determined using the middle cross section of the trochlea model as shown in Fig. 3A. Firstly, points P1 and P2 were selected where the SOM adjoined the curvature of the inner trochlea of anterior part. Point O located on the trochlea curvature was then determined for generating the smallest-diameter circle consisting of P1, O, and P2. Point O was the origin in the proposed trochlea model. These points of the trochlea and the circle were the muscle path, and were designated as P1 and P2, respectively (Fig. 3A). OP1, OP2, and the angle from OP1toOP2(∠P1OP2 ) were measured based on point O. Among the parts corresponding to the posterior part of trochlea, the uppermost part was set as P3, and the lowest part was set as P4;OP3, OP4, and the angles from OP2toOP4(∠P2OP4) and from OP3toOP4(∠P3OP4) were then measured.   Table 1 lists the averaged values obtained among 15 trochlea models. The distances OP1, OP2, OP3, andOP4 were 2.2 ± 0.7, 1.4 ± 0.5, 2.7 ± 0.9, and 2.5 ± 0.4 mm (mean ± SD), respectively, while angles ∠P1OP2 , ∠P2OP4 , and ∠P3OP4 were 100.7 ± 14.4, 66.3 ± 18.0, and 98.9 ± 24.9°, respectively. Based on the averages of the measured data, a generalized trochlea middle crosssection model was plotted on the coordinates using the analytical geometry method as shown in Fig. 3B (green line). OP2 was positioned on the y-axis, and the curvature connecting P1, O, and P2 was implemented by calculating the equation of the circle.
The trochlea is hollow and cylindrical in shape, but an artificial trochlea implant will probably have to be longer and accommodate an internal groove for the SOM. As shown in Fig. 3B, points S1 and S2 were set by drawing a line perpendicular to the tangents at points P1 and P2, respectively. A passage connecting points P1-P4, S1, and S2 was made (blue line in the figure). Based on the standardized cross-sectional design, the trochlea implant was 1.5 mm thick.
The plate to fix the trochlea implant into the orbit is shown in Fig. 4A. The implant was designed with a thickness of 1 mm and a micro screw diameter of 1.5 mm. For the robust equipping on the curvature of the orbit, the plate was bent into a right angle rather than a straight line. The generalized trochlea models with the plate are shown in Fig. 4B-D. The averaged value for 15 trochleae in Table 1

Discussion
The primary role of the trochlea is to change the vector of the movements produced by the SOM and to act as a pulley. When the eye moves upward and inward, the SOM normally relaxes and the tendon passes smoothly through the trochlea. Any restriction in this passage will impair the elevation in adduction. Mechanical restriction of the trochlea has been treated using various surgical procedures, including superior oblique tenectomy [12], superior oblique tenotomy [13], and a superior oblique tendon silicone expander [14]. Manipulating the trochlea has generally been avoided due to the fear of postoperative complications. However, Mombaerts et al. [15] shifted the surgical target from the SOM tendon to the trochlea. They introduced trochlear luxation surgery through the upper eyelid crease incision. Kokubo et al. [16] performed trochlea reconstruction surgery using a step-cut and resuturing technique. In this regard, we considered expanding the surgical concept from reconstruction to replacement of the trochlea. The aim of the present study was to design a generalized trochlea model using a conventional micro-CT system for future application in trochlea replacement therapy. The most notable characteristic of the model is a posterolaterally directed flange that guides the reflected tendon of the SOM (Fig. 5). We quantitatively assessed the inner and outer radii of the trochlea after dividing it into its anterior and posterior regions, and estimated the encountered angles of O. We found that the trochlea has a unilateral asymmetric shape: anterior part of trochlea always had a smaller anterior radius than the posterior part of trochlea. We hypothesize that there was no significant difference between the sizes of the posterior radii of the medial and lateral part of trochlea, indicating a bilateral symmetric shape.
The trochlea is located along the superomedial margin of the orbit and is firmly attached to the bone. Our study group previously demonstrated that the detailed location of the trochlea is in the superomedial orbit. The results of that study indicated that the superolateral tip of the trochlea was located 15.8 mm superior and 1.6 mm lateral to the apex of the lacrimal caruncle. It was also located 11.4 mm inferior to the top of the supraorbital notch/foramen (Fig. 6). These data of the exact location of the trochlea could be used together with the information of the structure of the trochlea for future trochlea replacement surgery.
The trochlea can only be palpated along the superomedial margin of the orbit by highly skilled experts, because Fig. 5 Illustration of the trochlea replacement in a cadaver (trochlea model was produced by replicating it to make it stand out). The 3D-printed trochlea was fixed to the superomedial orbit using the plate. The SOM passed through the inside of the trochlea and was attached to the eyeball (a slit-like gap was made at the lateral portion of the 3D printed trochlea so that the SOM could be inserted) Fig. 6 Trochlea location in the superomedial orbit (reprinted with permission). 1 MCL, medial canthal ligament; SOF, superior orbital fissure; SON, superior orbital notch it is small cartilaginous tissue. For this reason, its anatomy is not familiar to even strabismus surgeons. Virtual and printed models are currently used for didactic and research purposes in other areas of medicine. These data and 3D-printed models can therefore potentially a valuable resource for teaching trochlea anatomy to medical students and trainees, and for illustrating its 3D complexity [17].
Tissue replacement has gradually become a common operation, especially in orthopedic surgery. The increasing number of reports about 3D printing technology such as patient-specific implants play important roles in supporting operations, especially in complex cases [18][19][20]. Although it is still at the primary stage, our novel modeling of a 3D-printed trochlea introduces a new concept of trochlear surgery to clinicians and will expand our ability to understand structures.
Even if a trochlea implant is constructed, in order for the SOMs to move without damage after long periods, the material must be biocompatible and sufficient hard to reduce friction. In this regard, 3D bioprinting is a promising approach for repairing cartilage tissue after damage due to injury or disease, or to construct load-bearing connective tissue such as cartilage [21]. Therefore, in order for this technology to be realized in the future, development of cartilage tissue engineering needs to be followed based on the availability of bioinks.
We have demonstrated that a standard 3D X-ray micro-CT imaging technique can be used to measure human cartilage with high precision and few accuracy errors. This is consistent with the study of Kim et al. [22], in which histological grading systems for evaluating structures, cells, safranin-O staining, and tidemark integrity were compared with 3D measurements of cartilage volume and thickness using micro-CT to provide complementary information. One potential obstacle to using micro-CT is the high water content of cartilage. Particularly in smaller samples, the evaporation rate can be very high, and the subsequent dehydration can cause motion artifacts during acquisition especially when imaging at a high resolution [23]. In air, cartilage can be imaged with good contrast but is highly sensitive to dehydration. In order to resolve the dehydration problem, Choo et al. [24] used a customized humidification chamber for mouse cartilage that could limit the shrinkage rate to 3% over a 16-min imaging period.
The limitations of the present study include the use of embalmed cadavers rather than it being performed in vivo. Formalin fixation typically reduced the width of soft-tissue specimens by around 4% [25]. However, the trochlea comprises dense fibrous connective tissue, and so would be minimally affected by formalin fixation; we therefore believe that the size and morphology of the trochlea would be similar in vivo.

Conclusion
In this study, detailed 3D microstructure modeling of the trochlea was carried out using high-resolution CT scanning which is a powerful imaging technique. Fifteen trochleae detached from periosteum of fifteen embalmed cadavers were scanned by micro-CT, then segmentation and image reconstruction were performed to obtain a final volume mesh in STL file. Five specified points for quantifying the scanned trochlea were suggested in the middle cross section of trochlea to determine the generalized modeling. The trochlea implant prototype was designed and 3D printed for validation. The quantitative data presented herein can potentially be used to create a 3D-printed trochlea implant for patients with iatrogenic or posttraumatic trochlear damage. Further studies are needed to determine if these techniques will be safe and efficacious in clinical applications.