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Healing masseter entheses of mandibular reconstruction with autograft—Raman spectroscopic and histological study
Int. J. Oral Maxillofac. Surg. 2013; 42: 915–922 http://dx.doi.org/10.1016/j.ijom.2012.12.010, available online at http://www.sciencedirect.com
Research Paper Bone Biology
Healing masseter entheses of mandibular reconstruction with autograft—Raman spectroscopic and histological study
L. Wang1, Y.-x. Su1, G.-s. Zheng1, G.-q. Liao1, W.-h. Zhang2 1
Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China; 2 Instrumental Analysis and Research Center, Sun Yat-sen University, Guangzhou, China
L. Wang, Y.-x. Su, G.-s. Zheng, G.-q. Liao, W.-h. Zhang: Healing masseter entheses of mandibular reconstruction with autograft—Raman spectroscopic and histological study. Int. J. Oral Maxillofac. Surg. 2013; 42: 915–922. # 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. 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-tocollagen 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 (rspearman = 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 0901-5027/070915 + 08 $36.00/0
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.1 The structure of
Key words: enthesis; Raman spectroscopy; mandible defect; mandibular reconstruction; autogenous bone graft; masseter. Accepted for publication 12 December 2012 Available online 29 January 2013
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
# 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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modulus of bone is approximately 10 times larger than that of tendon.2 Hence, a primary function of entheses must be to balance such widely different elastic moduli. According to Suresh’s study,3 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.,4 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.5 Tendon-to-bone healing occurs through bone growth and subsequent remodelling of tissues at the interface under mechanical strain.6 The literature presents different findings about the healing tendon-to-bone interface following surgical reattachment. Some authors7 think the reestablishment of tendon-tobone interface could be referred to as a normal physiological enthesis, while others8,9 described the reestablishment of a collagen continuum between the tendon and bone but not the reconstruction of the original enthesis.10 Current research on enthesis mainly concentrates on histological and biomechanical studies.11 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-tobone interface but in the muscle-tendon unit.12 To elucidate the healing bone-totendon interface a non-invasive and timesaving method is preferred. Raman microspectroscopy is a wellestablished analytical tool based on the interaction of electromagnetic radiation with the molecules in the samples.13 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.14 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.15 Wopenka et al.16 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.2 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.17 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.18 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
Healing masseter entheses of mandibular reconstruction with autograft 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 8C) 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.16 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 mm. Spectral acquisition time per analysis spot was 60 s and the Wire 2 software provided by Renishaw was used to remove the
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Fig. 1. A magnified view of the tendon-to-bone insertion site was used to track the position of the Raman microprobe traverse (red circles) in individual tissue samples. B, bone and T, tendon.
sample background at each acquisition. All measurements were made at room temperature (24 1 8C). 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 mm 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.
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
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.,16 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
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 mm wide tendon-to-bone transition of the normal masseter enthesis (rspearman = 0.896, P < 0.000). The ratio I960/ 2940 at the insertion site increased linearly (R2 = 0.79 for four samples) over the distance from tendon to bone (Fig. 3a). Healing enthesis in the EC group was similar to normal enthesis, there was a gradual change in degree of mineralization across the approximately 180 mm
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Fig. 2. Baseline-corrected Raman spectra of pure bone and pure tendon obtained with 514 nm excitation.
wide tendon-to-bone transition of the healing masseter enthesis (rspearman = 0.915, P < 0.000). The ratio I960/2940 at the insertion site increased linearly (R2 = 0.837 for four samples) over the distance from tendon to bone (Fig. 3b). Regarding healing enthesis in the iliac group, there was a trend of gradual change in degree of mineralization across the approximately 200 mm wide tendon-tobone transition of the healing masseter enthesis (rspearman = 0.638, P < 0.000). The linear trend of ratio I960/2940 (R2 = 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. 3c). Regarding healing enthesis in the ulna group, it was difficult to orient the tendonto-bone transition site. The distribution of mineralization across the approximately
Fig. 3. Gradual change in degree of mineralization across the tendon-to-bone transition as evaluated by peak height ratio 960/2940 versus distance. Pure tendon site was defined as 0 of x-axis. Peak height ratio 960/2940 in pure bone was defined as 1 and pure tendon was defined as 0. (a) Normal enthesis (n = 4); (b) healing enthesis in EC group (n = 4); (c) healing enthesis in iliac group (n = 4); and (d) healing enthesis in ulna group (n = 4).
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Fig. 4. H–E stained section of normal masseter enthesis and healing enthesis in EC group. (a) periosteal insertion in normal enthesis; 20; (b) bony insertion in normal enthesis, 20; (c) periosteal and bony reattachment in EC group, 10; and (d) bony reattachment in EC group, 20. T, tendon; B, bone; P, periosteum; and WB, woven bone.
200 mm wide tendon-to-bone transition of the healing masseter enthesis was irregular (rspearman = 0.519, P > 0.001, R2 = 0.27 for four samples) (Fig. 3d). Histological findings
The two types of normal enthesis of masseter muscle found in this study were periosteal (Fig. 4a) and bony (Fig. 4b). 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 45-908. Regarding healing enthesis in EC group, two types of healing entheses were
observed. For periosteal reattachment (Fig. 4c), the structure between the tendons and the outer fibrous layer of the periosteum resembled that of normal periosteal attachment. Compared to the normal, the periosteal fibres towards the end of the tendon were less parallel to the bone surface, whereas the bone surface was similar to normal. For bony reattachment (Fig. 4d), the reattachment fibres from the ends of tendon could be traced into the underlying bone and the fibres resembled the normal but were not organized as normal enthesis. The layer of newly formed woven bone was thicker than that of normal enthesis. Regarding healing enthesis in iliac group, there were two types of histological findings associated with different osteogenic activity of grafted bone. For grafted bone predominated by bone absorption with slight osteogenesis, there were thin and disorganized perforating collagen fibres between the surface of grafted bone and tendon (Fig. 5a). For grafted bone with active osteogenesis, new woven bone was formed on the bone surface. Perforating collagen fibres appeared, extending from the tendon and penetrating directly into new woven bone, even bone absorption was still obvious (Fig. 5b). The
demarcation between tendon and periosteum was not as clear as that of normal enthesis and periosteal reattachment. Healing enthesis in the ulna group revealed two types of histological findings associated with different osteogenic activities of grafted bone. Firstly, bone absorption on cortical bone was more severe than that in iliac group. Few perforating collagen fibres were observed between the bone surfaces and surrounding tendon (Fig. 5c). Secondly, depression on the bone surface caused by bone absorption and newly formed woven bone appeared simultaneously. Obvious perforating collagen fibres penetrated directly into the new woven bone. The demarcation between tendon and periosteum was not as clear as that of normal enthesis and periosteal reattachment. It seemed that the bone absorption would spread to the newly formed reattachment (Fig. 5d). Discussion
A mandibular defect may result from trauma, inflammatory disease and benign or malignant tumours. It can create a significant cosmetic deformity and lead to functional deficit including loss of mastication, deglutition, and speech. Although
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Fig. 5. H–E stained section of healing enthesis in iliac group and ulna group 20 (a) grafted bone predominated by bone absorption in iliac group; (b) grafted bone with active osteogenesis in iliac group; (c) few perforating fibres formed in ulna group; and (d) obvious perforating collagen fibres formed in ulna group. T, tendon; B, bone; and WB, woven bone.
immediate mandibular reconstruction aims to restore facial symmetry, arch alignment, and stable occlusion, masticatory function often remains compromised.18 Masticatory impairment of patients with mandibular reconstruction is often related to an altered biomechanical relationship that results from muscular imbalance.19 Adequate masitcatory muscle attachment is one of the prerequisite for muscular imbalance. In the present study, the authors investigated the reattachment of masticatory muscle to transplanted bone in a beagle dog mandibular defect and reconstruction model. To the best of the authors’ knowledge, this is the first biochemical histological analysis of healing enthesis of transplanted bone using Raman spectroscopy. According to the study of Wopenka et al.,16 because the Raman peaks arising from the organic materials in both tendon and bone are primarily due to contributions from the same organic molecule (i.e. type I collagen), wavelengths of around 2940 cm 1 and 1003 cm 1 were used as the references of normalization. In addition, 960 cm 1 was used as the indicator of mineralization since it is influenced to a minor extent by environmental factors compared to the other phosphate vibrational modes. In the present authors’ experiment, the intensity of 1003 cm 1 was not as high and stable as
2940 cm 1 in the bone-graft groups. To avoid errors when the spectral of bone-graft groups were processed including baseline correction and deconvolution, only the ratio 960–2940 cm 1 was used in the authors’ experiment. Although the histological morphology of masseter enthesis is different from that of the rotator cuff which exhibits four different zones (tendon, fibrocartilage, mineralized fibrocartilage, and bone),16 it fulfils a biomechanical function similar to the rotator cuff, namely adapting the different elasticity moduli of bone and tendon tissues. The present study has shown that the mineral-to-collagen ratio at the normal masseter enthesis increases linearly over a distance of approximately 150 mm from tendon to bone and the authors’ findings confirmed that mineral gradation serves to minimize stress concentrations that otherwise would arise between compliant tendon and stiff mandible. As for healing the enthesis of detached masseter muscle, histological analysis demonstrated that it resembles normal but has a differing appearance including disorganized reattachment fibres and a thicker layer of newly formed woven bone. The Raman spectroscopic study showed that over the distance from tendon to bone the relative change in mineralization is linear with a linear
correlation coefficient R2 of 0.837. This finding suggested that the mechanical properties of a healing enthesis could adapt to functional demands and a functional enthesis is not only dependent on the replication of the original enthesis morphology, but the biochemical gradient in the insertion site. This is consistent with the results of a long-term morphologic study of patellar tendon reattachment in a sheep model.10 It is worth noting that the normal enthesis of masseter muscle in this study appeared as periosteal and bony insertion whereas the rotator cuff is a fibrocartilaginous enthesis. Unlike the masseter enthesis, the rotator cuff is prone to injury and generally heals poorly. The possible explanation is that fibrocartilaginous enthesis is more susceptible to injury and harder to cure than periosteal or bony insertion, but this requires further study. Autogenous bone graft represents the gold standard for management of mandible defects. It possesses biologic advantages over heterologous and synthetic bone substitutes because of its excellent combination of osteogenic, osteoinductive, and osteoconductive properties.20 Different types of autogenous bone grafts have variable properties associated with structural anatomy. Iliac crest bone is a typical grafted cancellous bone and ulna
Healing masseter entheses of mandibular reconstruction with autograft bone is commonly used as grafted cortical bone. The trabecular structure of iliac bone results in a large surface area compared with ulna graft. This allows for a high number of cellular components including mesenchymal stem cells, immature and mature osteoblasts to be incorporated, which explains its excellent osteogenic and osteoinductive capabilities. Furthermore, the trabecular structure allows for easy revascularization and rapid incorporation at the host site.21 In contrast, cortical bone has fewer osteoblasts and osteocytes, fewer growth factors, and less surface area per unit weight, and its structure constitutes a barrier to vascular ingrowth and remodelling.22 Therefore, the early stage of osteointegration with revascularization of iliac graft is followed by graft remodelling with active bone formation and resorption of necrotic bone after 4 weeks, whereas revascularization of cortical bone graft takes approximately 2 months.20 Based on the above reasons healing masseter enthesis in mandibular reconstruction with autograft is more complicated than reattachment to intact mandible because of the healing process of transplanted bone. The present study showed the woven bone layer on the bone surface plays a critical role in anchoring collagen fibres at the transplanted bone. The bigger area woven bone occupied, the larger reattached interface was formed. The thicker the woven bone layer, the deeper reattached collagen fibres perforated. The quality and quantity of woven bone was determined by the osteogenic activity and structure stability of transplanted bone. Cancellous bone has greater cellular diversity and activity than cortical bone, whereas cortical grafts have enhanced mechanical properties. This factor could underlie the histological finding in the present study that healing enthesis to iliac graft was superior to ulna graft. Additionally, it was confirmed with Raman spectroscopy that the mineral content increased gradually in the iliac group but not in the ulna group. It was also noted in this study that both healing masseter entheses in the ulna and iliac groups were inferior to the muscle detachment group. One week after tendon retraction, a reattachment to the surrounding connective tissues was observed.23 Because of remodelling of transplanted bone, a stable platform for tendon-bone healing could not be promptly set up. Resorption of the bony surface that the tendon is apposed to may prevent the incorporation of collagen fibres into the mineralized tissue. Additionally, according to Gimbel et al.,24 delayed reattachment of
masseter to transplanted bone could lead to increased repair tension and compromise the healing of enthesis. Besides delayed reattachment, the physical environment influences the development, remodelling and healing of enthesis.25 Using a clinically relevant model of flexor tendon injury and repair, Thomopoulos et al. found that muscle loading was beneficial to healing and there was likely a balance that must be reached during tendon-to-bone healing between too much loading (leading to damage at the repair) and too little loading (leading to insufficient cell stimulation).26 Chen et al.27 studied the effect of mechanical force on parathyroid hormone-related protein (PTHrP) expression in a number of fibrous insertion sites and identified that mechanical force seems to be an important regulator of PTHrP expression which may influence the recruitment and/or activities of underlying bone cell populations. Therefore, compromised masseter loading caused by delayed enthesis healing to transplanted bone seems to have a side-effect on healing of enthesis. Consistent with the histological findings, Raman spectroscopy reflected the localized biochemical composition of the healing masseter enthesis. The histological structure of normal masseter enthesis involves three discrete zones (tendon, new-formed woven bone and bone) or four zones (tendon, periosteum, new-formed woven bone and bone). There is increasing mineralization in this sequence and in the structure of healing enthesis of detachment muscle. As for healing enthesis to transplanted bone, both histological structure and biochemical composition showed a much more irregular distribution in mineral content from tendon to bone due to remodelling of transplanted bone involving bone resorption and bone formation. Although both methods could display the healing of enthesis, Raman microspectroscopy demonstrated several advantages over the histological method for tissue characterization.16 Firstly, this technique requires almost no special sample preparation in contrast to histological analysis which requires significant manipulation of the sample. Secondly, it has excellent spatial resolution for documenting quantitative changes in the degree of mineralization across the tendon-to-bone insertion zone and to optimize the quantification algorithm that effectively can be applied in future comparative study. Thirdly, there is a close relationship between the mechanical properties and the biochemical composition of musculoskeletal tissue. In the present Raman spectroscopic study, the reattachment of the experimental control group showed a
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similar linear trend as the normal group but the two experimental groups (iliac and ulna groups) did not show a linear trend. The present findings suggest that although anatomical replication is not acquired the mineral-to-collagen ratio at the insertion site increasing linearly from tendon to bone indicates functional reconstruction. Therefore, Raman microspectroscopy is able to act as the bridge between biomechanical and morphological analysis. Conclusion
This study showed healing of masseter enthesis via collagen fibres into a new layer of woven bone that forms on top of transplanted bone. The maturity of reattachment is dependent on the quality and quantity of new-formed woven bone. Both healing masseter entheses in cancellous graft and in cortical graft are inferior to that of muscle detachment and the types of transplanted bone for mandibular reconstruction play a critical role in the healing of entheses. Raman spectroscopy provides quantitative information about different healing entheses and gives valuable insight into the mechanical properties of enthesis in functional mandibular reconstruction. Funding
This study was supported by grants from National Natural Science Foundation of China (No. 30901682), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20090171120105), Natural Science Foundation of Guangdong Province (No. 9451008901001993), the Fundamental Research Funds for the Central Universities (No. 10ykpy18), Medical Science Research Grant of Guangdong Province (No. 2009221) and the priming scientific research foundation for the junior teachers of medicine in Sun Yat-sen University (No. 3171914). Competing interests
None declared. Ethical approval
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11. Thomopoulos S, Marquez JP, Weinberger B, Birman V, Genin GM. Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations. J Biomech 2006;39:1842–51. 12. Hibino N, Hamada Y, Sairyo K, Yukata K, Sano T, Yasui N. Callus formation during healing of the repaired tendon-bone junction. J Bone Joint Surg Br 2007;89B:1539–44. 13. Shen J, Fan L, Yang J, Shen AG, Hu JM. A longitudinal Raman microspectroscopic study of osteoporosis induced by spinal cord injury. Osteoporos Int 2010;21:81–7. 14. Petry R, Schmitt M, Popp J. Raman spectroscopy – a prospective tool in the life sciences. ChemPhysChem 2003;4:14–30. 15. Xu C, Yao X, Walker MP, Wang Y. Chemical/molecular structure of the dentin-enamel junction is dependent on the intratooth location. Calcif Tissue Int 2009;84:221–8. 16. Wopenka B, Kent A, Pasteris JD, Yoon Y, Thomopoulos S. The tendon-to-bone transition of the rotator cuff: a preliminary Raman spectroscopic study documenting the gradual mineralization across the insertion in rat tissue samples. Appl Spectrosc 2008;62:1285–94. 17. Zhu S, Cui J, Gao Y, Zhang B, Hu J. Changes of masseter muscles after mandibular angle ostectomy in rhesus monkeys. Ann Plast Surg 2009;63:670–5. 18. Goh BT, Lee S, Tideman H, Stoelinga PJ. Mandibular reconstruction in adults: a review. Int J Oral Maxillofac Surg 2008;37:597–605. 19. Curtis DA, Plesh O, Hannam AG, Sharma A, Curtis TA. Modeling of jaw biomechanics in the reconstructed mandibulectomy patient. J Prosthet Dent 1999;81:167–73. 20. Pape HC, Evans A, Kobbe P. Autologous bone graft: properties and techniques. J Orthop Trauma 2010;24:S36–40. 21. Khan MA. Update on spondyloarthropathies. Ann Intern Med 2002;136:896–907.
22. Burchardt H. Biology of bone transplantation. Orthop Clin North Am 1987;18:187– 96. 23. Laurin J, Gondin J, Dousset E, Decherchi P. Effect of tenotomy on metabosensitive afferent fibers from tibialis anterior muscle. Exp Brain Res 2008;186:87–92. 24. Gimbel JA, Van Kleunen JP, Williams GR, Thomopoulos S, Soslowsky LJ. Long durations of immobilization in the rat result in enhanced mechanical properties of the healing supraspinatus tendon insertion site. Biomech Eng Trans ASME J 2007;129:400–4. 25. Thomopoulos S, Kim HM, Rothermich SY, Biederstadt C, Das R, Galatz LA. Decreased muscle loading delays maturation of the tendon enthesis during postnatal development. J Orthop Res 2007;25: 1154–63. 26. Thomopoulos S, Zampiakis E, Das R, Silva MJ, Gelberman RH. The effect of muscle loading on flexor tendon-to-bone healing in a canine model. J Orthop Res 2008;26:1611–7. 27. Chen XS, Macica C, Nasiri A, Judex S, Broadus AE. Mechanical regulation of PTHrP expression in entheses. Bone 2007;41:752–9.
Address: Gui-qing Liao Department of Oral and Maxillofacial Surgery Guanghua School of Stomatology Sun Yat-sen University 56 Lingyuanxi Road Guangzhou 510055 China Tel: +86 20 83862579; Fax: +86 20 83822807; Mobile: +86 13500020072 E-mail: [email protected]
Research Paper Bone Biology
Healing masseter entheses of mandibular reconstruction with autograft—Raman spectroscopic and histological study
L. Wang1, Y.-x. Su1, G.-s. Zheng1, G.-q. Liao1, W.-h. Zhang2 1
Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China; 2 Instrumental Analysis and Research Center, Sun Yat-sen University, Guangzhou, China
L. Wang, Y.-x. Su, G.-s. Zheng, G.-q. Liao, W.-h. Zhang: Healing masseter entheses of mandibular reconstruction with autograft—Raman spectroscopic and histological study. Int. J. Oral Maxillofac. Surg. 2013; 42: 915–922. # 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. 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-tocollagen 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 (rspearman = 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 0901-5027/070915 + 08 $36.00/0
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.1 The structure of
Key words: enthesis; Raman spectroscopy; mandible defect; mandibular reconstruction; autogenous bone graft; masseter. Accepted for publication 12 December 2012 Available online 29 January 2013
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
# 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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modulus of bone is approximately 10 times larger than that of tendon.2 Hence, a primary function of entheses must be to balance such widely different elastic moduli. According to Suresh’s study,3 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.,4 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.5 Tendon-to-bone healing occurs through bone growth and subsequent remodelling of tissues at the interface under mechanical strain.6 The literature presents different findings about the healing tendon-to-bone interface following surgical reattachment. Some authors7 think the reestablishment of tendon-tobone interface could be referred to as a normal physiological enthesis, while others8,9 described the reestablishment of a collagen continuum between the tendon and bone but not the reconstruction of the original enthesis.10 Current research on enthesis mainly concentrates on histological and biomechanical studies.11 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-tobone interface but in the muscle-tendon unit.12 To elucidate the healing bone-totendon interface a non-invasive and timesaving method is preferred. Raman microspectroscopy is a wellestablished analytical tool based on the interaction of electromagnetic radiation with the molecules in the samples.13 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.14 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.15 Wopenka et al.16 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.2 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.17 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.18 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
Healing masseter entheses of mandibular reconstruction with autograft 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 8C) 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.16 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 mm. Spectral acquisition time per analysis spot was 60 s and the Wire 2 software provided by Renishaw was used to remove the
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Fig. 1. A magnified view of the tendon-to-bone insertion site was used to track the position of the Raman microprobe traverse (red circles) in individual tissue samples. B, bone and T, tendon.
sample background at each acquisition. All measurements were made at room temperature (24 1 8C). 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 mm 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.
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
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.,16 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
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 mm wide tendon-to-bone transition of the normal masseter enthesis (rspearman = 0.896, P < 0.000). The ratio I960/ 2940 at the insertion site increased linearly (R2 = 0.79 for four samples) over the distance from tendon to bone (Fig. 3a). Healing enthesis in the EC group was similar to normal enthesis, there was a gradual change in degree of mineralization across the approximately 180 mm
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Fig. 2. Baseline-corrected Raman spectra of pure bone and pure tendon obtained with 514 nm excitation.
wide tendon-to-bone transition of the healing masseter enthesis (rspearman = 0.915, P < 0.000). The ratio I960/2940 at the insertion site increased linearly (R2 = 0.837 for four samples) over the distance from tendon to bone (Fig. 3b). Regarding healing enthesis in the iliac group, there was a trend of gradual change in degree of mineralization across the approximately 200 mm wide tendon-tobone transition of the healing masseter enthesis (rspearman = 0.638, P < 0.000). The linear trend of ratio I960/2940 (R2 = 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. 3c). Regarding healing enthesis in the ulna group, it was difficult to orient the tendonto-bone transition site. The distribution of mineralization across the approximately
Fig. 3. Gradual change in degree of mineralization across the tendon-to-bone transition as evaluated by peak height ratio 960/2940 versus distance. Pure tendon site was defined as 0 of x-axis. Peak height ratio 960/2940 in pure bone was defined as 1 and pure tendon was defined as 0. (a) Normal enthesis (n = 4); (b) healing enthesis in EC group (n = 4); (c) healing enthesis in iliac group (n = 4); and (d) healing enthesis in ulna group (n = 4).
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Fig. 4. H–E stained section of normal masseter enthesis and healing enthesis in EC group. (a) periosteal insertion in normal enthesis; 20; (b) bony insertion in normal enthesis, 20; (c) periosteal and bony reattachment in EC group, 10; and (d) bony reattachment in EC group, 20. T, tendon; B, bone; P, periosteum; and WB, woven bone.
200 mm wide tendon-to-bone transition of the healing masseter enthesis was irregular (rspearman = 0.519, P > 0.001, R2 = 0.27 for four samples) (Fig. 3d). Histological findings
The two types of normal enthesis of masseter muscle found in this study were periosteal (Fig. 4a) and bony (Fig. 4b). 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 45-908. Regarding healing enthesis in EC group, two types of healing entheses were
observed. For periosteal reattachment (Fig. 4c), the structure between the tendons and the outer fibrous layer of the periosteum resembled that of normal periosteal attachment. Compared to the normal, the periosteal fibres towards the end of the tendon were less parallel to the bone surface, whereas the bone surface was similar to normal. For bony reattachment (Fig. 4d), the reattachment fibres from the ends of tendon could be traced into the underlying bone and the fibres resembled the normal but were not organized as normal enthesis. The layer of newly formed woven bone was thicker than that of normal enthesis. Regarding healing enthesis in iliac group, there were two types of histological findings associated with different osteogenic activity of grafted bone. For grafted bone predominated by bone absorption with slight osteogenesis, there were thin and disorganized perforating collagen fibres between the surface of grafted bone and tendon (Fig. 5a). For grafted bone with active osteogenesis, new woven bone was formed on the bone surface. Perforating collagen fibres appeared, extending from the tendon and penetrating directly into new woven bone, even bone absorption was still obvious (Fig. 5b). The
demarcation between tendon and periosteum was not as clear as that of normal enthesis and periosteal reattachment. Healing enthesis in the ulna group revealed two types of histological findings associated with different osteogenic activities of grafted bone. Firstly, bone absorption on cortical bone was more severe than that in iliac group. Few perforating collagen fibres were observed between the bone surfaces and surrounding tendon (Fig. 5c). Secondly, depression on the bone surface caused by bone absorption and newly formed woven bone appeared simultaneously. Obvious perforating collagen fibres penetrated directly into the new woven bone. The demarcation between tendon and periosteum was not as clear as that of normal enthesis and periosteal reattachment. It seemed that the bone absorption would spread to the newly formed reattachment (Fig. 5d). Discussion
A mandibular defect may result from trauma, inflammatory disease and benign or malignant tumours. It can create a significant cosmetic deformity and lead to functional deficit including loss of mastication, deglutition, and speech. Although
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Fig. 5. H–E stained section of healing enthesis in iliac group and ulna group 20 (a) grafted bone predominated by bone absorption in iliac group; (b) grafted bone with active osteogenesis in iliac group; (c) few perforating fibres formed in ulna group; and (d) obvious perforating collagen fibres formed in ulna group. T, tendon; B, bone; and WB, woven bone.
immediate mandibular reconstruction aims to restore facial symmetry, arch alignment, and stable occlusion, masticatory function often remains compromised.18 Masticatory impairment of patients with mandibular reconstruction is often related to an altered biomechanical relationship that results from muscular imbalance.19 Adequate masitcatory muscle attachment is one of the prerequisite for muscular imbalance. In the present study, the authors investigated the reattachment of masticatory muscle to transplanted bone in a beagle dog mandibular defect and reconstruction model. To the best of the authors’ knowledge, this is the first biochemical histological analysis of healing enthesis of transplanted bone using Raman spectroscopy. According to the study of Wopenka et al.,16 because the Raman peaks arising from the organic materials in both tendon and bone are primarily due to contributions from the same organic molecule (i.e. type I collagen), wavelengths of around 2940 cm 1 and 1003 cm 1 were used as the references of normalization. In addition, 960 cm 1 was used as the indicator of mineralization since it is influenced to a minor extent by environmental factors compared to the other phosphate vibrational modes. In the present authors’ experiment, the intensity of 1003 cm 1 was not as high and stable as
2940 cm 1 in the bone-graft groups. To avoid errors when the spectral of bone-graft groups were processed including baseline correction and deconvolution, only the ratio 960–2940 cm 1 was used in the authors’ experiment. Although the histological morphology of masseter enthesis is different from that of the rotator cuff which exhibits four different zones (tendon, fibrocartilage, mineralized fibrocartilage, and bone),16 it fulfils a biomechanical function similar to the rotator cuff, namely adapting the different elasticity moduli of bone and tendon tissues. The present study has shown that the mineral-to-collagen ratio at the normal masseter enthesis increases linearly over a distance of approximately 150 mm from tendon to bone and the authors’ findings confirmed that mineral gradation serves to minimize stress concentrations that otherwise would arise between compliant tendon and stiff mandible. As for healing the enthesis of detached masseter muscle, histological analysis demonstrated that it resembles normal but has a differing appearance including disorganized reattachment fibres and a thicker layer of newly formed woven bone. The Raman spectroscopic study showed that over the distance from tendon to bone the relative change in mineralization is linear with a linear
correlation coefficient R2 of 0.837. This finding suggested that the mechanical properties of a healing enthesis could adapt to functional demands and a functional enthesis is not only dependent on the replication of the original enthesis morphology, but the biochemical gradient in the insertion site. This is consistent with the results of a long-term morphologic study of patellar tendon reattachment in a sheep model.10 It is worth noting that the normal enthesis of masseter muscle in this study appeared as periosteal and bony insertion whereas the rotator cuff is a fibrocartilaginous enthesis. Unlike the masseter enthesis, the rotator cuff is prone to injury and generally heals poorly. The possible explanation is that fibrocartilaginous enthesis is more susceptible to injury and harder to cure than periosteal or bony insertion, but this requires further study. Autogenous bone graft represents the gold standard for management of mandible defects. It possesses biologic advantages over heterologous and synthetic bone substitutes because of its excellent combination of osteogenic, osteoinductive, and osteoconductive properties.20 Different types of autogenous bone grafts have variable properties associated with structural anatomy. Iliac crest bone is a typical grafted cancellous bone and ulna
Healing masseter entheses of mandibular reconstruction with autograft bone is commonly used as grafted cortical bone. The trabecular structure of iliac bone results in a large surface area compared with ulna graft. This allows for a high number of cellular components including mesenchymal stem cells, immature and mature osteoblasts to be incorporated, which explains its excellent osteogenic and osteoinductive capabilities. Furthermore, the trabecular structure allows for easy revascularization and rapid incorporation at the host site.21 In contrast, cortical bone has fewer osteoblasts and osteocytes, fewer growth factors, and less surface area per unit weight, and its structure constitutes a barrier to vascular ingrowth and remodelling.22 Therefore, the early stage of osteointegration with revascularization of iliac graft is followed by graft remodelling with active bone formation and resorption of necrotic bone after 4 weeks, whereas revascularization of cortical bone graft takes approximately 2 months.20 Based on the above reasons healing masseter enthesis in mandibular reconstruction with autograft is more complicated than reattachment to intact mandible because of the healing process of transplanted bone. The present study showed the woven bone layer on the bone surface plays a critical role in anchoring collagen fibres at the transplanted bone. The bigger area woven bone occupied, the larger reattached interface was formed. The thicker the woven bone layer, the deeper reattached collagen fibres perforated. The quality and quantity of woven bone was determined by the osteogenic activity and structure stability of transplanted bone. Cancellous bone has greater cellular diversity and activity than cortical bone, whereas cortical grafts have enhanced mechanical properties. This factor could underlie the histological finding in the present study that healing enthesis to iliac graft was superior to ulna graft. Additionally, it was confirmed with Raman spectroscopy that the mineral content increased gradually in the iliac group but not in the ulna group. It was also noted in this study that both healing masseter entheses in the ulna and iliac groups were inferior to the muscle detachment group. One week after tendon retraction, a reattachment to the surrounding connective tissues was observed.23 Because of remodelling of transplanted bone, a stable platform for tendon-bone healing could not be promptly set up. Resorption of the bony surface that the tendon is apposed to may prevent the incorporation of collagen fibres into the mineralized tissue. Additionally, according to Gimbel et al.,24 delayed reattachment of
masseter to transplanted bone could lead to increased repair tension and compromise the healing of enthesis. Besides delayed reattachment, the physical environment influences the development, remodelling and healing of enthesis.25 Using a clinically relevant model of flexor tendon injury and repair, Thomopoulos et al. found that muscle loading was beneficial to healing and there was likely a balance that must be reached during tendon-to-bone healing between too much loading (leading to damage at the repair) and too little loading (leading to insufficient cell stimulation).26 Chen et al.27 studied the effect of mechanical force on parathyroid hormone-related protein (PTHrP) expression in a number of fibrous insertion sites and identified that mechanical force seems to be an important regulator of PTHrP expression which may influence the recruitment and/or activities of underlying bone cell populations. Therefore, compromised masseter loading caused by delayed enthesis healing to transplanted bone seems to have a side-effect on healing of enthesis. Consistent with the histological findings, Raman spectroscopy reflected the localized biochemical composition of the healing masseter enthesis. The histological structure of normal masseter enthesis involves three discrete zones (tendon, new-formed woven bone and bone) or four zones (tendon, periosteum, new-formed woven bone and bone). There is increasing mineralization in this sequence and in the structure of healing enthesis of detachment muscle. As for healing enthesis to transplanted bone, both histological structure and biochemical composition showed a much more irregular distribution in mineral content from tendon to bone due to remodelling of transplanted bone involving bone resorption and bone formation. Although both methods could display the healing of enthesis, Raman microspectroscopy demonstrated several advantages over the histological method for tissue characterization.16 Firstly, this technique requires almost no special sample preparation in contrast to histological analysis which requires significant manipulation of the sample. Secondly, it has excellent spatial resolution for documenting quantitative changes in the degree of mineralization across the tendon-to-bone insertion zone and to optimize the quantification algorithm that effectively can be applied in future comparative study. Thirdly, there is a close relationship between the mechanical properties and the biochemical composition of musculoskeletal tissue. In the present Raman spectroscopic study, the reattachment of the experimental control group showed a
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similar linear trend as the normal group but the two experimental groups (iliac and ulna groups) did not show a linear trend. The present findings suggest that although anatomical replication is not acquired the mineral-to-collagen ratio at the insertion site increasing linearly from tendon to bone indicates functional reconstruction. Therefore, Raman microspectroscopy is able to act as the bridge between biomechanical and morphological analysis. Conclusion
This study showed healing of masseter enthesis via collagen fibres into a new layer of woven bone that forms on top of transplanted bone. The maturity of reattachment is dependent on the quality and quantity of new-formed woven bone. Both healing masseter entheses in cancellous graft and in cortical graft are inferior to that of muscle detachment and the types of transplanted bone for mandibular reconstruction play a critical role in the healing of entheses. Raman spectroscopy provides quantitative information about different healing entheses and gives valuable insight into the mechanical properties of enthesis in functional mandibular reconstruction. Funding
This study was supported by grants from National Natural Science Foundation of China (No. 30901682), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20090171120105), Natural Science Foundation of Guangdong Province (No. 9451008901001993), the Fundamental Research Funds for the Central Universities (No. 10ykpy18), Medical Science Research Grant of Guangdong Province (No. 2009221) and the priming scientific research foundation for the junior teachers of medicine in Sun Yat-sen University (No. 3171914). Competing interests
None declared. Ethical approval
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