- Email: [email protected]
Detection of sonic hedgehog in patients undergoing orthognathic surgery
International Journal of Surgery Open 5 (2016) 1–4
Contents lists available at ScienceDirect
International Journal of Surgery Open j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j s o
Research Paper
Detection of sonic hedgehog in patients undergoing orthognathic surgery Yuki Kunisada, Tsuyoshi Shimo *, Masanori Masui, Norie Yoshioka, Soichiro Ibaragi, Kenichi Matsumoto, Tatsuo Okui, Naito Kurio, Shohei Domae, Koji Kishimoto, Akiyoshi Nishiyama, Akira Sasaki Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan 700-8525
A R T I C L E
I N F O
Article history: Received 6 April 2016 Accepted 10 September 2016 Available online 14 September 2016 Keywords: Sonic hedgehog ELISA Osteotomy
A B S T R A C T
Purpose: Sonic Hedgehog (SHH) is a regulatory protein involved in bone fracture healing. Orthognathic surgery involves surgical osteotomy of the mandible or maxilla to restore the proper anatomic and functional position in patients with dentofacial deformity. The purpose of this study was to analyze SHH local blood serum concentrations after osteotomy to gain further understanding of the molecular regulation of the initial stage of osteotomy healing. Methods: Serum samples (local drainage and peripheral venous) of 34 patients (24 females and 10 males, mean age was 23.4 (16–42) years) who underwent orthognathic surgery were isolated from patients at different time points during the perioperative period. The levels of SHH, soluble receptor activator of nuclear factor-κB ligand (sRANKL) and osteoprotegerin (OPG) were measured using ELISA. Results: SHH was detected in the local drainage immediately after osteotomy (309.5 ± 58.2 pg/ml), and decreased for 2 days after the operation (197.5 ± 43.6 pg/ml). The sRANKL local serum concentrations were at the maximum level immediately after the operation (141.4 ± 22.6 pg/ml) and decreased for 2 days (110.1 ± 23.4 pg/ml). On the other hand, the OPG concentration in the local serum was at a minimum after osteotomy (59.4 ± 4.6 pg/ml) and reached its maximum (181.5 ± 17.8 pg/ml, P < 0.01) at 2 days after osteotomy. SHH and OPG local serum levels on day 2 were associated with the amount of bleeding during the operation. The local drainage serum level of SHH of maxillary/mandibular osteotomy had a tendency to be higher than that of mandible-only osteotomy at 2 days after operation. Conclusions: Elevated levels of SHH in local serum after osteotomy, especially during the initial stage of healing, indicates its importance in osteotomy healing. © 2016 The Authors. Published by Elsevier Ltd on behalf of Surgical Associates Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Mandibular osteotomy healing is a unique process that leads to bone regeneration, and several cytokines and growth factors are known to be involved in the critical initial stage of fracture healing. However, local and systemic concentrations of certain factors during the initial stage of mandible osteotomy are not well understood. Fracture healing involves a delicate balance of bone formation and resorption [1]. Sonic Hedgehog (SHH) is a regulatory protein involved as a morphogen of embryonic development [2], fracture healing [3] and socket healing after tooth extraction [4]. SHH indirectly induces osteoclast formation by upregulating parathyroid hormone-related peptide (PTHrP) [5] and receptor activator for
nuclear factor-κB ligand (RANKL) in osteoblasts and bone stromal cells [6]. RANKL stimulates differentiation and maturation of osteoclasts, leading to bone resorption. Osteoprotegerin (OPG), a decoy receptor of RANKL, is made by osteoblasts and blocks osteoclast formation and bone resorption. Soluble RANKL (sRANKL) and OPG in plasma and drainage fluid is involved in the fracture-healing process after surgical treatment of proximal humeral fracture [7]. However, it is not yet established whether the local serum concentration of SHH is increased after mandibular osteotomy. In this study, we evaluated the local levels of SHH expression after mandible osteotomy along with RANK and OPG expression for better understanding of the role of SHH in the process of mandible osteotomy healing. 2. Materials and methods
* Corresponding author. Department of Oral and Maxillofacial Surgery, OkayamaUniversityGraduateSchoolofMedicine,DentistryandPharmaceuticalSciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. Tel.: +81 86 235 6702; fax: +81 86 235 6704. E-mail address: [email protected] (T. Shimo).
2.1. Patients and specimens Between 2012 and 2013, 34 patients (24 females and 10 males, mean age 23.4 (16–42) years) were admitted to our institution for
http://dx.doi.org/10.1016/j.ijso.2016.09.002 2405-8572/© 2016 The Authors. Published by Elsevier Ltd on behalf of Surgical Associates Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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3. Results 3.1. SHH, RANKL and OPG serum concentrations in patients with mandible osteotomy The SHH local serum concentration was 309.5 ± 58.2 pg/ml immediately after osteotomy, and decreased time independently after osteotomy (Fig. 1A, day 1, 258.8 ± 55.9 pg/ml; day 2, 197.5 ± 43.6 pg/ ml). sRANKL local serum concentrations remained unchanged during the considered time points (Fig. 1B, immediately, after osteotomy, 141.4 ± 22.6 pg/ml; day 1, 142.0 ± 30.8 pg/ml; day 2, 110.1 ± 23.4 pg/ml). On the contrary, OPG local serum levels significantly increased timedependently after osteotomy (Fig. 1C, day 1, 85.7 ± 10.5 pg/ml, P < 0.01; day 2, 185.1 ± 17.8 pg/ml, P < 0.01).
3.2. Relationships of SHH, sRANKL and OPG local serum concentrations with the amount of bleeding during surgery SHH local serum concentrations of patients with osteotomy were significantly associated with the amount of bleeding during the surgery on day 2 (Fig. 2A, r = 0.4415). At these time points, sRANKL local serum concentrations were not correlated with the amount of the bleeding (Fig. 2B). OPG local serum concentrations of patients were weakly related to the amount of bleeding on day 2, with statistical significance (Fig. 2C, r = 0.3062).
Fig. 1. Detection of SHH, sRANKL and OPG local drainage serum concentrations in mandible osteotomy. SHH (A) sRANKL (B) and OPG (C) measurements in local drainage serum for each considered time point. Values shown are means and S.E.s. Statistically significant differences (*P < 0.01) between the indicated groups are marked by asterisks.
orthognathic surgery (22 maxillary/mandibular, 12 mandibleonly). Human serum from local drainage from the mandible osteotomy and peripheral venous blood was isolated from patients at different time points during the perioperative period (local drainage: day 0, 1, 2 and peripheral venous: day −1, 1, 3, 7). The protocols were reviewed and approved by The Human Research Ethics Committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1327). 2.2. Serum measurements Serum samples were stored after collection at −80 °C until analysis. SHH concentrations in the serum were determined using a sonic hedgehog N terminus (Human Shh-N) enzyme-linked immunosorbent assay (ELISA) kit from RayBiotech (Norcross, GA, USA). Total sRANKL (free and bound sRANKL) concentrations in the serum were determined using a sandwich enzyme-linked immunosorbent assay (ELISA) kit from BioVendor (Brno, Czech Republic). OPG concentrations in the serum were determined using an enzyme-linked immunosorbent assay (ELISA) kit from RayBiotech (Norcross, GA, USA). All measurements were performed in duplicate. 2.3. Statistical analysis Data were analyzed using Tukey’s test or Spearman’s rank– order correlation coefficient. Results were expressed as means ± S.E.s P values < 0.01 were considered to indicate statistical significance.
Fig. 2. Relationship between SHH, sRANKL and OPG local drainage serum concentrations and amount of bleeding during the surgery. The amount of bleeding and the concentrations of SHH (A), sRANKL (B), and OPG (C) in local drainage serum for each considered time point. Spearman’s rank–order correlation was used to identify the strength of the relationship between the amount of bleeding and the factor measurements.
Y. Kunisada et al. / International Journal of Surgery Open 5 (2016) 1–4
3.3. Comparison of SHH, sRANKL and OPG local serum concentrations in the bilateral mandible indwelling drain between mandibular osteotomy and maxillary/mandibular osteotomy Local SHH levels from the bilateral mandible indwelling drain decreased time-dependently over 2 postoperative days in both mandible-only osteotomy and maxillary/mandibular osteotomy; the SHH levels were 0.58 and 0.75 times lower than immediately after osteotomy on days 1 and 2 individually (Fig. 3A). The local mandible drainage SHH levels of mandible-only osteotomy had a tendency to be lower than that of maxillary/mandibular osteotomy in the 2 days after operation (immediately post-operation, 312.7 ± 75.8 and 386.8 ± 109.1 pg/ml; day 1, 269.0 ± 81.3 and 332.3 ± 104.4 pg/ml; day 2, 179.6 ± 61.3 and 288.7 ± 87.1 pg/ml), sRANKL levels remained statistically unchanged during the 2 postoperative days (Fig. 3B, immediately post-operation, 170.7 ± 41.8 pg/ml and 128.4 ± 27.3 pg/ml; day 1, 161.8 ± 52.7 and 133.2 ± 39.5 pg/ml; day 2, 143.2 ± 40.2 and 95.3 ± 28.8 pg/ml). Immediately after and day 1 after operation, OPG levels remained statistically unchanged; however, on day 2 the local drainage OPG levels were 3.2 times (mandibular osteotomy) and 3.1 times (maxillary/mandibular osteotomy) higher than the levels measured at immediately after operation (Fig. 3C, immediately postoperation, 50.7 ± 7.7 vs. 64.2 ± 5.6 pg/ml; day 1, 74.3 ± 11.8 and 91.9 ± 15.0 pg/ml; day 2, 161.5 ± 23.1 and 198.0 ± 24.4 pg/ml.).
Fig. 3. Comparison between mandibular osteotomy and maxillary/mandibular osteotomy in SHH, sRANKL and OPG local serum concentrations collected in the indwelling drain of the bilateral mandible. SHH (A), sRANKL (B) and OPG (C) concentrations in local drainage serum after mandible-only osteotomy (solid bar) or maxillary/mandibular osteotomy (open bar).
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4. Discussion In this study, we report the local serum concentrations of SHH after mandibular osteotomy. Previous studies showed that numerous local growth factors were involved in fracture healing [8,9]. The results of those studies indicated that local growth factors are released into the peripheral circulation after fracture and are associated with systemic reactions that might partly be attributed to bioactive molecules [10]. SHH appears to be released immediately into local serum after osteotomy (309.5 ± 58.2 pg/ml). However, at no time point do the levels of SHH in peripheral blood statistically exceed pre-surgery levels (pre-operation, 277.5 ± 52.3 pg/ml; day 1, 232.7 ± 52.3 pg/ml; day 3, 281.8 ± 58.2 pg/ml; day 5, 238.9 ± 67.3 pg/ml). These results indicate that no significant alteration was observed in the systemic distribution of SHH for 1, 3 and 7 days. Increased expressions of other growth factors such as TGF-β, PDGF, VEGF and M-CSF in peripheral blood early after fracture have been reported in other studies [11]. The increases may partly be attributed to the absorption of factors from the fracture site into the circulation in systemic responses [8]. The SHH concentration in local osteotomy sites immediately after surgery were higher than that in peripheral blood, indicating the local release of SHH during the immediate response in the bone marrow microenvironment. The maximum local serum SHH level in the early healing processes suggests that the hedgehog pathway plays an important role in bone repair via enhancing differentiation of periosteal progenitors and that activation of the hedgehog pathway at the onset of mandible healing could be beneficial for repair and regeneration [12]. The expression patterns of sRANKL and OPG are particularly enhanced in the bone-remodeling processes [13]. However, there are few reports about the concentrations of RANKL and OPG in the osteotomy healing processes. OPG was significantly increased in the local serum for 1 and 2 days after osteotomy, indicating a high level of activation of cells of the osteoblastic lineage [14]. However, we found no significant systemic distribution of OPG in the peripheral blood (before surgery: 29.8 ± 3.1 pg/ml; day 1, 28.1 ± 2.6 pg/ml; day 3, 25.3 ± 2.4 pg/ml; day 7, 20.8 ± 2.4 pg/ml). On the contrary, sRANKL showed a constant level in local serum after the surgery, indicating that increasing the number of osteoblasts and progenitor cells is more important to the early healing processes than bone resorption. On the contrary, sRANKL showed a tendency to increase in the peripheral blood (before surgery: 174.2 ± 29.4 pg/ml; day 1, 224.4 ± 52.6 pg/ml; day 3, 207.2 ± 33.84 pg/ml; day 7, 282.8 ± 40.1 pg/ml). The results suggested that sRANKL plays a more important role in systemic bone metabolism than in the local osteotomy region in the early stage of bone healing. Another question addressed in the study was whether SHH levels differ according to the amount of bleeding during the operation and the type of osteotomy. Data were collected for mandible-only osteotomy and maxillary/mandibular osteotomy. The local drainage was set at the mandible region after the mandible osteotomy, which is performed after the maxillary osteotomy in the latter case. Therefore, the amount of bleeding depends on whether or not maxillary osteotomy is performed. SHH local serum concentration on day 2 was statistically correlated with the amount of bleeding (r = 0.4415) and the levels were higher in the group in which maxilla osteotomy preceded the mandible osteotomy (Fig. 3A). On the other hand, for SHH levels in the peripheral blood, no significant difference was recognized between the mandible-only osteotomy and maxillary/ mandibular osteotomy (data not shown). These results suggest that the growth factors which stimulate SHH production were most likely produced from the maxilla after the osteotomy, and that these would be strong candidates to reflect the upregulation of SHH in the local serum concentration near the mandible. However, the levels of growth factors such as PDGF, VEGF, IGF-I, and TGF-β were
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observed after day 3 postoperation of bone fracture, not soon enough to stimulate SHH [11]. The increase in circulating levels of sRANKL is significantly correlated with the amount of bleeding (r = 0.6923), and this suggests the possible role of sRANKL in systemic bone metabolism given the invasiveness of an osteotomy. OPG shows no significant correlation with the amount of bleeding in the peripheral blood but is significantly related to local serum on day 2 (r = 0.3062). The OPG local serum levels show a similar tendency to that of SHH, indicating that OPG expression might be regulated by SHH in the osteoblasts at the osteotomy region [15]. In conclusion, our study can be considered the first comprehensive evaluation of SHH, sRANKL, and OPG levels during orthognathic surgery mandible healing, which may provide clues to understanding the molecular regulation involved; also, these biomarkers may become tools to predict bone healing. Further investigation is needed to clarify the role of SHH in relation to clinical outcomes of orthognathic surgery. Ethical approval The protocols were reviewed and approved by The Human Research Ethics Committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1327). Funding This work was partly supported by a Grant-in-Aid for scientific Research (B) (#26293428 to T.S.) and Grant-in-Aid for Challenging Exploratory Research (#24659892 to T.S.) from the Ministry of Education, Cultures, Sports, Science, and Technology of Japan. Author contribution Study design: TS Data collections: NY, SI, KM, SD, KK, AN, AS Data analysis: YK, MM, TO, NK Writing: TS Conflict of interest None of the authors have any conflicts of interest.
Guarantor Tsuyoshi Shimo Research registration UIN N/A References [1] Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat Rev Rheumatol 2015;11:45–54. [2] Briscoe J, Thérond PP. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 2013;14:416–29. [3] Horikiri Y, Shimo T, Kurio N, Okui T, Matsumoto K, Iwamoto M, et al. Sonic hedgehog regulates osteoblast function by focal adhesion kinase signaling in the process of fracture healing. PLoS ONE 2013;8:e76785. [4] Pang P, Shimo T, Takada H, Matsumoto K, Yoshioka N, Ibaragi S, et al. Expression pattern of sonic hedgehog signaling and calcitonin gene-related peptide in the socket healing process after tooth extraction. Biochem Biophys Res Commun 2015;467:21–6. [5] Mak KK, Bi Y, Wan C, Chuang P-T, Clemens T, Young M, et al. Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression. Dev Cell 2008;14:674–88. [6] Honami T, Shimo T, Okui T, Kurio N, Hassan NMM, Iwamoto M, et al. Sonic hedgehog signaling promotes growth of oral squamous cell carcinoma cells associated with bone destruction. Oral Oncol 2012;48:49–55. [7] Colombini A, Lombardi G, Galliera E, Dogliotti G, Randelli P, Meerssemann A, et al. Plasma and drainage fluid levels of soluble receptor activator of nuclear factor-kB (sRANK), soluble receptor activator of nuclear factor-kB ligand (sRANKL) and osteoprotegerin (OPG) during proximal humerus fracture healing. Int Orthop 2011;35:777–82. [8] Sarahrudi K, Thomas A, Mousavi M, Kaiser G, Köttstorfer J, Kecht M, et al. Elevated transforming growth factor-beta 1 (TGF-β1) levels in human fracture healing. Injury 2011;42:833–7. [9] Giannoudis PV, Pountos I, Morley J, Perry S, Tarkin HI, Pape H-C. Growth factor release following femoral nailing. Bone 2008;42:751–7. [10] Chapurlat RD, Confavreux CB. Novel biological markers of bone: from bone metabolism to bone physiology. Rheumatology (Oxford) 2016;in press. [11] Pountos I, Georgouli T, Henshaw K, Bird H, Giannoudis PV. Release of growth factors and the effect of age, sex, and severity of injury after long bone fracture. A preliminary report. Acta Orthop 2013;84:65–70. [12] Wang Q, Huang C, Zeng F, Xue M, Zhang X. Activation of the Hh pathway in periosteum-derived mesenchymal stem cells induces bone formation in vivo: implication for postnatal bone repair. Am J Pathol 2010;177:3100–11. [13] Eriksen EF. Cellular mechanisms of bone remodeling. Rev Endocr Metab Disord 2010;11:219–27. [14] Martin TJ, Sims NA. RANKL/OPG; critical role in bone physiology. Rev Endocr Metab Disord 2015;16:131–9. [15] Matsumoto K, Shimo T, Kurio N, Okui T, Obata K, Masui M, et al. Expression and role of sonic hedgehog in the process of fracture healing with aging. In Vivo 2016;30:99–105.
Contents lists available at ScienceDirect
International Journal of Surgery Open j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j s o
Research Paper
Detection of sonic hedgehog in patients undergoing orthognathic surgery Yuki Kunisada, Tsuyoshi Shimo *, Masanori Masui, Norie Yoshioka, Soichiro Ibaragi, Kenichi Matsumoto, Tatsuo Okui, Naito Kurio, Shohei Domae, Koji Kishimoto, Akiyoshi Nishiyama, Akira Sasaki Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan 700-8525
A R T I C L E
I N F O
Article history: Received 6 April 2016 Accepted 10 September 2016 Available online 14 September 2016 Keywords: Sonic hedgehog ELISA Osteotomy
A B S T R A C T
Purpose: Sonic Hedgehog (SHH) is a regulatory protein involved in bone fracture healing. Orthognathic surgery involves surgical osteotomy of the mandible or maxilla to restore the proper anatomic and functional position in patients with dentofacial deformity. The purpose of this study was to analyze SHH local blood serum concentrations after osteotomy to gain further understanding of the molecular regulation of the initial stage of osteotomy healing. Methods: Serum samples (local drainage and peripheral venous) of 34 patients (24 females and 10 males, mean age was 23.4 (16–42) years) who underwent orthognathic surgery were isolated from patients at different time points during the perioperative period. The levels of SHH, soluble receptor activator of nuclear factor-κB ligand (sRANKL) and osteoprotegerin (OPG) were measured using ELISA. Results: SHH was detected in the local drainage immediately after osteotomy (309.5 ± 58.2 pg/ml), and decreased for 2 days after the operation (197.5 ± 43.6 pg/ml). The sRANKL local serum concentrations were at the maximum level immediately after the operation (141.4 ± 22.6 pg/ml) and decreased for 2 days (110.1 ± 23.4 pg/ml). On the other hand, the OPG concentration in the local serum was at a minimum after osteotomy (59.4 ± 4.6 pg/ml) and reached its maximum (181.5 ± 17.8 pg/ml, P < 0.01) at 2 days after osteotomy. SHH and OPG local serum levels on day 2 were associated with the amount of bleeding during the operation. The local drainage serum level of SHH of maxillary/mandibular osteotomy had a tendency to be higher than that of mandible-only osteotomy at 2 days after operation. Conclusions: Elevated levels of SHH in local serum after osteotomy, especially during the initial stage of healing, indicates its importance in osteotomy healing. © 2016 The Authors. Published by Elsevier Ltd on behalf of Surgical Associates Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Mandibular osteotomy healing is a unique process that leads to bone regeneration, and several cytokines and growth factors are known to be involved in the critical initial stage of fracture healing. However, local and systemic concentrations of certain factors during the initial stage of mandible osteotomy are not well understood. Fracture healing involves a delicate balance of bone formation and resorption [1]. Sonic Hedgehog (SHH) is a regulatory protein involved as a morphogen of embryonic development [2], fracture healing [3] and socket healing after tooth extraction [4]. SHH indirectly induces osteoclast formation by upregulating parathyroid hormone-related peptide (PTHrP) [5] and receptor activator for
nuclear factor-κB ligand (RANKL) in osteoblasts and bone stromal cells [6]. RANKL stimulates differentiation and maturation of osteoclasts, leading to bone resorption. Osteoprotegerin (OPG), a decoy receptor of RANKL, is made by osteoblasts and blocks osteoclast formation and bone resorption. Soluble RANKL (sRANKL) and OPG in plasma and drainage fluid is involved in the fracture-healing process after surgical treatment of proximal humeral fracture [7]. However, it is not yet established whether the local serum concentration of SHH is increased after mandibular osteotomy. In this study, we evaluated the local levels of SHH expression after mandible osteotomy along with RANK and OPG expression for better understanding of the role of SHH in the process of mandible osteotomy healing. 2. Materials and methods
* Corresponding author. Department of Oral and Maxillofacial Surgery, OkayamaUniversityGraduateSchoolofMedicine,DentistryandPharmaceuticalSciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. Tel.: +81 86 235 6702; fax: +81 86 235 6704. E-mail address: [email protected] (T. Shimo).
2.1. Patients and specimens Between 2012 and 2013, 34 patients (24 females and 10 males, mean age 23.4 (16–42) years) were admitted to our institution for
http://dx.doi.org/10.1016/j.ijso.2016.09.002 2405-8572/© 2016 The Authors. Published by Elsevier Ltd on behalf of Surgical Associates Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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3. Results 3.1. SHH, RANKL and OPG serum concentrations in patients with mandible osteotomy The SHH local serum concentration was 309.5 ± 58.2 pg/ml immediately after osteotomy, and decreased time independently after osteotomy (Fig. 1A, day 1, 258.8 ± 55.9 pg/ml; day 2, 197.5 ± 43.6 pg/ ml). sRANKL local serum concentrations remained unchanged during the considered time points (Fig. 1B, immediately, after osteotomy, 141.4 ± 22.6 pg/ml; day 1, 142.0 ± 30.8 pg/ml; day 2, 110.1 ± 23.4 pg/ml). On the contrary, OPG local serum levels significantly increased timedependently after osteotomy (Fig. 1C, day 1, 85.7 ± 10.5 pg/ml, P < 0.01; day 2, 185.1 ± 17.8 pg/ml, P < 0.01).
3.2. Relationships of SHH, sRANKL and OPG local serum concentrations with the amount of bleeding during surgery SHH local serum concentrations of patients with osteotomy were significantly associated with the amount of bleeding during the surgery on day 2 (Fig. 2A, r = 0.4415). At these time points, sRANKL local serum concentrations were not correlated with the amount of the bleeding (Fig. 2B). OPG local serum concentrations of patients were weakly related to the amount of bleeding on day 2, with statistical significance (Fig. 2C, r = 0.3062).
Fig. 1. Detection of SHH, sRANKL and OPG local drainage serum concentrations in mandible osteotomy. SHH (A) sRANKL (B) and OPG (C) measurements in local drainage serum for each considered time point. Values shown are means and S.E.s. Statistically significant differences (*P < 0.01) between the indicated groups are marked by asterisks.
orthognathic surgery (22 maxillary/mandibular, 12 mandibleonly). Human serum from local drainage from the mandible osteotomy and peripheral venous blood was isolated from patients at different time points during the perioperative period (local drainage: day 0, 1, 2 and peripheral venous: day −1, 1, 3, 7). The protocols were reviewed and approved by The Human Research Ethics Committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1327). 2.2. Serum measurements Serum samples were stored after collection at −80 °C until analysis. SHH concentrations in the serum were determined using a sonic hedgehog N terminus (Human Shh-N) enzyme-linked immunosorbent assay (ELISA) kit from RayBiotech (Norcross, GA, USA). Total sRANKL (free and bound sRANKL) concentrations in the serum were determined using a sandwich enzyme-linked immunosorbent assay (ELISA) kit from BioVendor (Brno, Czech Republic). OPG concentrations in the serum were determined using an enzyme-linked immunosorbent assay (ELISA) kit from RayBiotech (Norcross, GA, USA). All measurements were performed in duplicate. 2.3. Statistical analysis Data were analyzed using Tukey’s test or Spearman’s rank– order correlation coefficient. Results were expressed as means ± S.E.s P values < 0.01 were considered to indicate statistical significance.
Fig. 2. Relationship between SHH, sRANKL and OPG local drainage serum concentrations and amount of bleeding during the surgery. The amount of bleeding and the concentrations of SHH (A), sRANKL (B), and OPG (C) in local drainage serum for each considered time point. Spearman’s rank–order correlation was used to identify the strength of the relationship between the amount of bleeding and the factor measurements.
Y. Kunisada et al. / International Journal of Surgery Open 5 (2016) 1–4
3.3. Comparison of SHH, sRANKL and OPG local serum concentrations in the bilateral mandible indwelling drain between mandibular osteotomy and maxillary/mandibular osteotomy Local SHH levels from the bilateral mandible indwelling drain decreased time-dependently over 2 postoperative days in both mandible-only osteotomy and maxillary/mandibular osteotomy; the SHH levels were 0.58 and 0.75 times lower than immediately after osteotomy on days 1 and 2 individually (Fig. 3A). The local mandible drainage SHH levels of mandible-only osteotomy had a tendency to be lower than that of maxillary/mandibular osteotomy in the 2 days after operation (immediately post-operation, 312.7 ± 75.8 and 386.8 ± 109.1 pg/ml; day 1, 269.0 ± 81.3 and 332.3 ± 104.4 pg/ml; day 2, 179.6 ± 61.3 and 288.7 ± 87.1 pg/ml), sRANKL levels remained statistically unchanged during the 2 postoperative days (Fig. 3B, immediately post-operation, 170.7 ± 41.8 pg/ml and 128.4 ± 27.3 pg/ml; day 1, 161.8 ± 52.7 and 133.2 ± 39.5 pg/ml; day 2, 143.2 ± 40.2 and 95.3 ± 28.8 pg/ml). Immediately after and day 1 after operation, OPG levels remained statistically unchanged; however, on day 2 the local drainage OPG levels were 3.2 times (mandibular osteotomy) and 3.1 times (maxillary/mandibular osteotomy) higher than the levels measured at immediately after operation (Fig. 3C, immediately postoperation, 50.7 ± 7.7 vs. 64.2 ± 5.6 pg/ml; day 1, 74.3 ± 11.8 and 91.9 ± 15.0 pg/ml; day 2, 161.5 ± 23.1 and 198.0 ± 24.4 pg/ml.).
Fig. 3. Comparison between mandibular osteotomy and maxillary/mandibular osteotomy in SHH, sRANKL and OPG local serum concentrations collected in the indwelling drain of the bilateral mandible. SHH (A), sRANKL (B) and OPG (C) concentrations in local drainage serum after mandible-only osteotomy (solid bar) or maxillary/mandibular osteotomy (open bar).
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4. Discussion In this study, we report the local serum concentrations of SHH after mandibular osteotomy. Previous studies showed that numerous local growth factors were involved in fracture healing [8,9]. The results of those studies indicated that local growth factors are released into the peripheral circulation after fracture and are associated with systemic reactions that might partly be attributed to bioactive molecules [10]. SHH appears to be released immediately into local serum after osteotomy (309.5 ± 58.2 pg/ml). However, at no time point do the levels of SHH in peripheral blood statistically exceed pre-surgery levels (pre-operation, 277.5 ± 52.3 pg/ml; day 1, 232.7 ± 52.3 pg/ml; day 3, 281.8 ± 58.2 pg/ml; day 5, 238.9 ± 67.3 pg/ml). These results indicate that no significant alteration was observed in the systemic distribution of SHH for 1, 3 and 7 days. Increased expressions of other growth factors such as TGF-β, PDGF, VEGF and M-CSF in peripheral blood early after fracture have been reported in other studies [11]. The increases may partly be attributed to the absorption of factors from the fracture site into the circulation in systemic responses [8]. The SHH concentration in local osteotomy sites immediately after surgery were higher than that in peripheral blood, indicating the local release of SHH during the immediate response in the bone marrow microenvironment. The maximum local serum SHH level in the early healing processes suggests that the hedgehog pathway plays an important role in bone repair via enhancing differentiation of periosteal progenitors and that activation of the hedgehog pathway at the onset of mandible healing could be beneficial for repair and regeneration [12]. The expression patterns of sRANKL and OPG are particularly enhanced in the bone-remodeling processes [13]. However, there are few reports about the concentrations of RANKL and OPG in the osteotomy healing processes. OPG was significantly increased in the local serum for 1 and 2 days after osteotomy, indicating a high level of activation of cells of the osteoblastic lineage [14]. However, we found no significant systemic distribution of OPG in the peripheral blood (before surgery: 29.8 ± 3.1 pg/ml; day 1, 28.1 ± 2.6 pg/ml; day 3, 25.3 ± 2.4 pg/ml; day 7, 20.8 ± 2.4 pg/ml). On the contrary, sRANKL showed a constant level in local serum after the surgery, indicating that increasing the number of osteoblasts and progenitor cells is more important to the early healing processes than bone resorption. On the contrary, sRANKL showed a tendency to increase in the peripheral blood (before surgery: 174.2 ± 29.4 pg/ml; day 1, 224.4 ± 52.6 pg/ml; day 3, 207.2 ± 33.84 pg/ml; day 7, 282.8 ± 40.1 pg/ml). The results suggested that sRANKL plays a more important role in systemic bone metabolism than in the local osteotomy region in the early stage of bone healing. Another question addressed in the study was whether SHH levels differ according to the amount of bleeding during the operation and the type of osteotomy. Data were collected for mandible-only osteotomy and maxillary/mandibular osteotomy. The local drainage was set at the mandible region after the mandible osteotomy, which is performed after the maxillary osteotomy in the latter case. Therefore, the amount of bleeding depends on whether or not maxillary osteotomy is performed. SHH local serum concentration on day 2 was statistically correlated with the amount of bleeding (r = 0.4415) and the levels were higher in the group in which maxilla osteotomy preceded the mandible osteotomy (Fig. 3A). On the other hand, for SHH levels in the peripheral blood, no significant difference was recognized between the mandible-only osteotomy and maxillary/ mandibular osteotomy (data not shown). These results suggest that the growth factors which stimulate SHH production were most likely produced from the maxilla after the osteotomy, and that these would be strong candidates to reflect the upregulation of SHH in the local serum concentration near the mandible. However, the levels of growth factors such as PDGF, VEGF, IGF-I, and TGF-β were
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observed after day 3 postoperation of bone fracture, not soon enough to stimulate SHH [11]. The increase in circulating levels of sRANKL is significantly correlated with the amount of bleeding (r = 0.6923), and this suggests the possible role of sRANKL in systemic bone metabolism given the invasiveness of an osteotomy. OPG shows no significant correlation with the amount of bleeding in the peripheral blood but is significantly related to local serum on day 2 (r = 0.3062). The OPG local serum levels show a similar tendency to that of SHH, indicating that OPG expression might be regulated by SHH in the osteoblasts at the osteotomy region [15]. In conclusion, our study can be considered the first comprehensive evaluation of SHH, sRANKL, and OPG levels during orthognathic surgery mandible healing, which may provide clues to understanding the molecular regulation involved; also, these biomarkers may become tools to predict bone healing. Further investigation is needed to clarify the role of SHH in relation to clinical outcomes of orthognathic surgery. Ethical approval The protocols were reviewed and approved by The Human Research Ethics Committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1327). Funding This work was partly supported by a Grant-in-Aid for scientific Research (B) (#26293428 to T.S.) and Grant-in-Aid for Challenging Exploratory Research (#24659892 to T.S.) from the Ministry of Education, Cultures, Sports, Science, and Technology of Japan. Author contribution Study design: TS Data collections: NY, SI, KM, SD, KK, AN, AS Data analysis: YK, MM, TO, NK Writing: TS Conflict of interest None of the authors have any conflicts of interest.
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