Abstract
Autogenous bone graft represents the gold standard for mandibular reconstruction. The authors used a beagle mandibular defect model and reconstructed with iliac crest and ulna graft. Healing masseter entheses were harvested 24 weeks after surgery and analyzed by histology and Raman microspectroscopy. The intensity ratio of 960/2940 was to document mineral-to-collagen ratio as degree of mineralization. Pearson correlation was used to evaluate the association between the intensity ratios of 960/2940 and the tendon-to-bone insertion site. In the normal control group ( n = 4) and the experimental control group with detached masseter muscle ( n = 4), the degree of mineralization at the insertion site increased linearly from tendon to bone. In the iliac graft ( n = 4) and ulna graft groups ( n = 4), healing entheses were far less mature than controls and a linear trend was not observed. There was no significant correlation between degree of mineralization and insertion site in the ulna group ( r spearman = 0.519, P > 0.001). These results indicate that transplanted bone plays a critical role in healing of entheses and healing enthesis to reconstructed mandible is inferior to normal. Raman spectroscopy provides quantitative information about different healing entheses and gives valuable insight into mechanical properties of entheses in functional mandibular reconstruction.
An enthesis is the region where a tendon, ligament or joint capsule attaches to the bone; it is an attachment or insertion site. Tendon entheses can be classed as fibrous or fibrocartilaginous according to the tissue present at the skeletal attachment site. The former can be bony or periosteal, depending on whether the tendon is attached directly to the bone or indirectly via the periosteum. The structure of tendon entheses relates to the need to dissipate stress away from the interface and into the tendon and/or the bone itself. Although the tendon and the bone have similar tensile strength, the elastic modulus of bone is approximately 10 times larger than that of tendon. Hence, a primary function of entheses must be to balance such widely different elastic moduli. According to Suresh’s study, gradients at interface regions smooth stress distribution, eliminate singularities in stress, reduce stress concentration, improve the strength of the bonding and decrease the risk of fracture (i.e. failure). Thomopoulos et al., studied the supraspinatus tendon to bone insertion site of rat and demonstrated that the tendon to bony insertion site varied dramatically along its length in terms of its viscoelastic properties, collagen structure, and extracellular matrix composition. These results indicated that molecular and mechanical gradient was the basic biomechanical property of enthesis for stress dissipation.
Entheses are of particular concern to orthopaedic surgeons because of the common need to re-attach tendon or ligament to bone, with a high risk of early failure and high incidence of recurrence of injury. Tendon-to-bone healing occurs through bone growth and subsequent remodelling of tissues at the interface under mechanical strain. The literature presents different findings about the healing tendon-to-bone interface following surgical reattachment. Some authors think the reestablishment of tendon-to-bone interface could be referred to as a normal physiological enthesis, while others described the reestablishment of a collagen continuum between the tendon and bone but not the reconstruction of the original enthesis. Current research on enthesis mainly concentrates on histological and biomechanical studies. Histological studies including decalcified and undecalcified sections are used to describe the tissue and cell morphology of the healing insertion site and to provide intuitive information for morphological analysis. The decalcified method probably leads to the loss of some valuable information and the undecalcified method is time-consuming and expensive. Biomechanical study is focused on the load to failure of tendon-to-bone interface and provides quantitative results for comparative study, but it is a destructive study and sometimes the failures do not occur at the tendon-to-bone interface but in the muscle-tendon unit. To elucidate the healing bone-to-tendon interface a non-invasive and time-saving method is preferred.
Raman microspectroscopy is a well-established analytical tool based on the interaction of electromagnetic radiation with the molecules in the samples. It has been widely used in the biomedical field in recent decades owing to its versatility for various samples in which hydrated tissue specimens are also included since water contributes slightly to the spectrum.
One of the great advantages of this technique is its ability to analyze biomedical samples nondestructively including cultured cells, the lens of the human eye, bone and coronary arteries. Therefore, it is feasible to undertake a histological analysis of the sample after Raman spectroscopy measurement. In addition, its rapid acquisition times would imply the possibility of real time tissue diagnosis using biochemical tissue signatures. Raman microspectroscopy can provide simultaneous biochemical information about the organic and inorganic constituents of samples with micro-level spatial resolution. Wopenka et al. applied Raman spectroscopy to monitor the distribution of mineral and the degree of mineralization across the tendon-bone insertion site in the shoulders of rats and showed that the mineral-to-collagen ratio at the insertion increased linearly from tendon to bone.
It was also concluded that quantification of the mineral component was more convincing when peak intensities rather than peak areas were interpreted. These findings of mineral gradation help to understand the material and biomechanical properties of tendon-to-bone interface.
The entheses of masticatory muscles are the basis of normal mastication, ensuring the contractile forces generated by the muscle are transmitted to the skeleton. There are entheses of different structure in the same attachment zone, and attachments with periosteal insertion are the major part of masticatory muscle. Detached entheses of masticatory muscles usually occur during surgical treatment for mandible disease such as trauma, inflammation, benign or malignant tumours, prominent mandibular angle and masseter muscle hypertrophy, but research about reattachment of masticatory muscle is mainly limited to mandibular angle surgery. Ramus and angle of the mandible is the part likely to be involved following mandible surgery such as mandible resection and reconstruction.
Autogenous bone graft is a generally accepted treatment strategy for mandibular reconstruction. There have been no reports on the reattachment of masticatory muscle to the transplanted bone and whether its mechanical properties could satisfy the requirements of oral function after mandibular reconstruction. In the present study, the authors used a beagle dog mandibular defect model and reconstructed with iliac crest and ulna graft. The purpose of the study was to investigate the reattachment of masticatory muscle to cancellous bone and cortical bone graft using Raman spectroscopy and histological methods, as well as the association between biochemical alterations and histological appearance of healing entheses.
Material and methods
Sixteen adults, conditioned beagle dogs weighing 13–18 kg were used for the study. Animal selection and management, surgical protocol, and preparation were approved by the Animal Care and Use Committee Sun Yat-sen University. The dogs were randomly divided into four groups of four dogs each. In the normal control group (NC group), no surgery was performed on either side of each dog’s mandible. In the experimental control group (EC group), the masseter muscle on alternate sides was detached, preserving the periosteum. In experimental group one (iliac group), a segmental defect was created in one side of each dog’s mandible and the defect was restored with iliac bone. In experimental group two (ulna group), the defect was restored with ulna graft.
The animals were anesthetized using an intramuscular injection of ketamine hydrochloride (25 mg/kg), and anaesthesia was maintained by intraperitoneal injection of sodium pentobarbital (Sigma, 3 mg/kg). After endotracheal intubation, the animals were placed in a lateral position with the operated side upward. Antibiotics were administered preoperatively to all dogs intravenously.
In iliac group, with an aseptic technique and local anaesthesia using 2% lidocaine, 5 ml, a wide and curved flap was lifted from one side of the submandible and the attachment of the masseter muscle was exposed. The short masseter aponeurosis was incised and the muscle was detached with a periosteal elevator and left free. The hemimandible was dissected free from surrounding fascia both medially and laterally. A segmental defect in the hemimandible, 5 cm long was designed. Osteotomy was performed using an oscillating saw. The medial cut was perpendicular to the inferior border of the mandible body and medial to the first molar. The distal cut was made perpendicular to the posterior margin of the mandible ramus and between the angle and condyle. The iliac bone in the ipsilateral side was harvested and transferred to the defect. The graft was fixed to the mandibular defect with rigid fixation. The miniplate was placed to secure the graft at both the proximal and the distal margins of the defect. The incision was closed in layers. In the ulna group, the same size mandible segmental defect as in iliac group was created and the ulna in the contralateral side was used as a bone graft. In the EC group, the attachment of the masseter muscle was detached in the same manner and the surgical wound was closed in layers.
Postoperatively, all dogs received antibiotics intramuscularly to prevent infection for 5 days. In addition, the animals were given dexamethasone (0.75 mg orally) thrice daily and painkillers for 3 days after surgery. All dogs were fed a gruel diet for the first week postoperatively, and then introduced to moistened dry food, and finally dry kibble.
After 24 weeks of healing, animals were anaesthetized and under aseptic conditions the bone in the reconstructed area and attached masseter muscles were removed en bloc. Harvested samples were rinsed with 0.9% sodium chloride and stored in a deep freezer (−80 °C) until spectroscopic analysis. Then, the dogs were killed.
Raman spectroscopy
Each tendon-to-bone sample was sliced to 1 mm thickness using a low-speed saw (Isomet, Buehler, USA). The tissue samples underwent no chemical treatment except for the use of phosphate-buffered saline solution to keep the sample hydrated. The unstained, unfixed section was placed on a glass slide for Raman analysis.
A Renishaw inVia Raman microscope (Renishaw, UK) was used for this study with a 514 nm laser beam. The output power is 20 mW. The laser was focused on each point of interest through a 50× long-working-distance objective (Olympus, Japan). All spectral acquisitions were performed in the 400–4000 cm −1 range and measured according to the method described by Wopenka et al. Briefly, a tissue sample was placed on a glass microscope slide that sat on the platform of a computer-controlled x – y – z stage, which allowed easy positioning of the sample spot in the focal plane. Under microscopy the tendon-to-bone interface was identified and about 10 analysis spots along a traverse across the interface were selected for analysis ( Fig. 1 ). The minimal distance from one spot to another was 10 μm. Spectral acquisition time per analysis spot was 60 s and the Wire 2 software provided by Renishaw was used to remove the sample background at each acquisition. All measurements were made at room temperature (24 ± 1 °C).
Histological examination
After Raman spectroscopic measurement, each sample was fixed in 10% neutral buffered formalin for 2 days and decalcified for 1 week. After being embedded in paraffin, 4 μm thick sections were prepared and stained with haematoxylin and eosin (H–E). A light microscope (Zeiss, Hamburg, Germany) linked to image analysis software (Axiovision, Zeiss, Hamburg, Germany) was used for histological analysis.
Data analysis
All spectral processing (i.e. baseline correction, peak intensity calculations) was performed with Software Origin 8.0 (OriginLab, USA). According to the study of Wopenka et al., the intensities of the 960 cm −1 symmetric P O stretch and the 2940 cm −1 C H stretch were used to interpret the presence and relative concentration within the excitation volume of the mineral and collagens components, respectively. The intensity of the 960 cm −1 for apatite normalized to 2940 cm −1 was used as an indicator of the abundance of mineral. The intensity ratio of the two peaks was used to document changes in the organic and inorganic components from point to point across the tendon-to-bone insertion zone. To illustrate the gradual mineralization across the enthesis sample, a coordinate system was created. The y -axis showed the intensity ratios of 960/2940 in sample divided by 960/2940 in bone. The degree of mineralization of pure bone was defined as 1 and the degree of mineralization of pure tendon was defined as 0. The x -axis showed the distance across tendon-to-bone insertion site and the pure tendon site was defined as 0.
The data were evaluated using the Statistical Package Social Sciences (SPSS, Version 16.0 for Windows; SPSS, Chicago, IL, USA). Pearson correlation was used to evaluate the association between the intensity ratios of 960/2940 and the tendon-to-bone insertion site. P values of 0.01 were considered to indicate statistical significance.
Results
All the dogs tolerated the anaesthesia and the surgical procedures well and experienced no major complications during the experimental period.
Raman spectroscopy
The authors first characterized pure bone and pure tendon (i.e. tissue away from the insertion site) in Fig. 2 .
For the normal/control enthesis there was a gradual change in degree of mineralization across the approximately 150 μm wide tendon-to-bone transition of the normal masseter enthesis ( r spearman = 0.896, P < 0.000). The ratio I 960/2940 at the insertion site increased linearly ( R 2 = 0.79 for four samples) over the distance from tendon to bone ( Fig. 3 a).
Healing enthesis in the EC group was similar to normal enthesis, there was a gradual change in degree of mineralization across the approximately 180 μm wide tendon-to-bone transition of the healing masseter enthesis ( r spearman = 0.915, P < 0.000). The ratio I 960/2940 at the insertion site increased linearly ( R 2 = 0.837 for four samples) over the distance from tendon to bone ( Fig. 3 b).
Regarding healing enthesis in the iliac group, there was a trend of gradual change in degree of mineralization across the approximately 200 μm wide tendon-to-bone transition of the healing masseter enthesis ( r spearman = 0.638, P < 0.000). The linear trend of ratio I 960/2940 ( R 2 = 0.47 for four samples) at the insertion site over the distance from tendon to bone was not as obvious as for the control and EC groups ( Fig. 3 c).
Regarding healing enthesis in the ulna group, it was difficult to orient the tendon-to-bone transition site. The distribution of mineralization across the approximately 200 μm wide tendon-to-bone transition of the healing masseter enthesis was irregular ( r spearman = 0.519, P > 0.001, R 2 = 0.27 for four samples) ( Fig. 3 d).
Histological findings
The two types of normal enthesis of masseter muscle found in this study were periosteal ( Fig. 4 a) and bony ( Fig. 4 b). The former was mainly found at the ramus and angle of the mandible. The latter was usually found at the lateral surface of the angle of mandible. At the former, the fibres from the tendon were inserted directly into the outer fibrous layer of the periosteum and interwoven with periosteal collagen fibres that were arranged in parallel to the bone surface. The flattened periosteal cells were usually seen at the surface adjacent to the bone. There was a thin layer of woven bone between the osteogenic layer of the periosteum and bone surface. The bone surface was smooth, dense and flat. At the latter, the periosteum was absent and the dense fibrous connective tissue anchored the tendon to the bone with an angle of about 45-90°.
