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The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits
Journal of Cranio-Maxillo-Facial Surgery xxx (xxxx) xxx
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The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits _ Azgın a, *, Hamdi Arbag b, Mehmet Akif Eryılmaz b, Zeliha Esin Çelik c Isa a
Health Sciences University, Konya Training and Research Hospital, Department of Otorhinolaryngology Head and Neck Surgery, Konya, Turkey Necmettin Erbakan University, Meram Medical Faculty, Department of Otorhinolaryngology Head and Neck Surgery, Konya, Turkey c Selçuk University, Medical Faculty, Department of Medical Pathology, Konya, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 10 July 2019 Accepted 25 January 2020 Available online xxx
Objective: This study aimed to determine whether administration of topical and intraperitoneal zinc for maxillofacial fractures has any impact on the bone healing process. Material and method: Thirty-two New Zealand rabbits were randomly assigned to four groups of eight each. The first group was the control group; fracture lines were fixed using titanium microplates and no medication was administered. The second group received fixations using zinc-coated titanium microplates. A single dose of 3 mg/kg zinc was administered intraperitoneally to the third group following fixations with titanium microplates. A single dose of 3 mg/kg zinc was administered intraperitoneally to the fourth group following fixations with zinc-coated titanium microplates. Zinc coating on to the titanium microplates was achieved using the physical vapor deposition technique. A fracture line was created in the nasal bones of all subjects and fixed with five-hole flat microplates and three 5-mm micro screws. All work groups were sacrificed at the end of the sixth week. Results: Histological examination showed that the number of osteoblasts were significantly higher in zinc-coated group (Group 2) than zinc uncoated, control group (Group 1), (415.6 ± 46.7 vs 366.3 ± 11.8) (p < 0.001). It was observed that intraperitoneal zinc treatment alone (Group 3) did not significantly increase in the osteoblast count compared to zinc un-coated group (Group 1), (390.6 ± 83.2 vs 366.3 ± 11.8), (p ¼ 0.341). The immunoreactivity scores for IGF-1 were significantly higher in the zinccoated group compared to control group (Group 2 vs 1), (9.3 ± 2.8 vs 3.7 ± 1.9) (p < 0.05). It was observed that intraperitoneal zinc treatment did not cause a significant difference in the aspect of IGF-1 for zinc-coated groups (Group 2 vs 4) (9.3 ± 2.8 vs 9.6 ± 2.2) (p ¼ 0.791). The difference in the immunoreactivity score among whole groups for TGF-b was not statistically significant (Group 1 vs 2, 3.2 ± 1.7 vs 4.4 ± 2.3, p ¼ 0.256; Group 1 vs 3, 3.2 ± 1.7 vs 3.8 ± 2.8, p ¼ 0.524; Group 1 vs 4, 3.2 ± 1.7 vs 2.8 ± 1.3, p ¼ 0.717; Group 2 vs 3, 4.4 ± 2.3, vs 3.8 ± 2.8, p ¼ 0.610; Group 2 vs 4, 4.4 ± 2.3, vs 2.8 ± 1.3, p ¼ 0.124; Group 3 vs 4, 3.8 ± 2.8, vs 2.8 ± 1.3, p ¼ 0.311). Conclusion: The local use of titanium microplates coated with zinc by PVD technique was found effective for fracture healing. Zinc coating of titanium microplates used in fracture treatment can accelerate fracture healing. It may be concluded that clinical studies should be performed now in order to explore if comparable results can be achieved in humans. © 2020 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Maxillofacial trauma Fracture healing Zinc IGF-1 TGF- b
1. Introduction
* Corresponding author. Department of Otorhinolaryngology, Head and Neck Surgery, Health Sciences University, Konya Training and Research Hospital, Meram Yeniyol Caddesi No:97, PK: 42090, Meram, Konya, Turkey. Tel.: þ90 533 552 5216, fax: þ90 332 323 67 23. _ Azgın). E-mail address: [email protected] (I.
Due to increased traffic and work accidents, bone fractures have become a significant health problem in society. Maxillofacial trauma can cause serious clinical and cosmetic problems (Malara et al., 2006). Bone fractures can lead to significant damage to a person's social and economic situation, in addition to the economic losses a country suffers through labor loss.
https://doi.org/10.1016/j.jcms.2020.01.013 1010-5182/© 2020 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
_ Azgın et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (xxxx) xxx I.
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Various metal plates and screws have been used in the treatment of bone fractures for many years. Today, titanium plates are mostly used due to their superiority over the other metal alloys and as they are tissue friendly. In addition to the need for methods of increasing the effect of titanium, the need for systemic and local treatment methods which will accelerate the bone healing has also increased. It is known that zinc, a trace element, has many functions at the cellular level with regard to the development of animals and humans (Koekkoek and vanZanten, 2016; Tapiero and Tew, 2003; Abrisham et al., 2010). Zinc increases the number of osteoblasts by stimulating protein synthesis in the osteoblastic cells. In addition, it serves an important function in the preservation of the bone structure, inhibiting bone resorption, and stimulating bone formation (Notomi et al., 2015; Wey et al., 2014). Zinc also has antioxidant effects (Dede et al., 2003). Coating with thermal evaporation physical vapor deposition (PVD) is the deposition of metal atoms on another targeted material by evaporation and using a vacuum. The metals coated with PVD technique showed significant mechanical properties and resistance to abrasion, erosion, and corrosion. Physical vapor deposition is at the core of a wide variety of applications (Prabu et al., 2013; Harsha, 2006). While there are studies with zinc-coated titanium implants in the dental field (Kellesarian et al., 2017; Yu et al., 2017; Ferraris and Spriano, 2016; Liang et al., 2015; He et al., 2018) and orthopedic (Zhao et al., 2017, 2019; Li et al., 2014; Tao et al., 2016; Wang et al., 2011) applications, we could not find any study with local (zinccoated titanium plates by PVD technique) and intraperitoneal zinc treatment for maxillofacial fractures. We aimed to determine the effects of local and intraperitoneal zinc treatments on the maxillofacial fracture healing process. This study aims to investigate the effects of intraperitoneal and local zinc treatment on the healing of maxillofacial fractures. 2. Material and methods The protocol of this experimental study was approved by Necmettin Erbakan University Experimental Medicine Research and Application Center (Permission no:2011/57). The animals were followed by housing two animals in each cage where 12 h of dark and 12 h of light environment was provided at 20e22 C under the control of a veterinary physician. The feed and water needs of animals in all groups were regularly supplied in equal amounts. 2.1. Working Groups Thirty-two male New Zealand white rabbits (mean weights ranged 2300 ± 200 g and mean 9 months old) were randomly assigned into four groups with eight rabbits in each group. In the control group, the fracture lines were fixed with titanium microplates (Trimed®Ti6AI4V) and no medication was applied. In the second group, fixation was made with zinc (Ted Pella® Zinc Pieces) coated titanium microplates. In the third group, intraperitoneal 3 mg/kg zinc sulphate (ZnSO4) (Fluka®) was given following the fixation with titanium microplates. The fourth group was given intraperitoneal 3 mg/kg zinc sulphate (ZnSO4) following the fixation with zinc-coated titanium microplates (Table 1). All rabbits were sacrificed at the end of the sixth week and surgical specimens were taken for histopathological evaluation.
Table 1 Demonstration of animal study groups. Group 1 (Control) Group 2 Group 3
Group 4
Group with only titanium microplate fixation Group with fixation with zinc plated titanium microplates A single dose of 3 mg/kg intraperitoneal zinc group followed by titanium microplate fixation A single dose of 3 mg/kg intraperitoneal zinc group followed by fixation with zinc plated titanium microplate
was 2.0 mm, the width was 3.0 mm and thickness was 0.5 mm. The micro screws used were 5.0 mm long and 1.6 mm in diameter. The zinc coating procedure on the titanium microplates was conducted with the technique of PVD. In this technique, the titanium microplates were placed into the device of a MB-200B Modular Glove Box Workstation (MBraun®) and Thermal evaporator (Leybold® PVD systems). Then, the zinc parts (Ted Pella® Zinc Pieces) were placed into the tungsten pot. In the thermal evaporator device, the zinc metal was evaporated by applying current to the tungsten pot under 106 millibar pressure and the titanium microplates were coated with zinc (Fig. 1). 2.3. Animal model and surgical operation The combination of 25 mg/kg Ketamin (Ketalar®, Pfizer, Turkey) and 2 mg/kg Xylazine (Rompun®, Bayer, Turkey) was used as anesthetic combination intramuscularly. After shaving the nasal areas of rabbits, they were dyed with povidone iodine (Batticon®, ADEKA, Turkey). A 2-cm longitudinal incision was applied under the skin frontonasally. The periosteum was lateralized with the help of an elevator and the nasal bone was exposed. Approximately 1 cm2 bone window was removed using 0.1 mm cutting microtur (Fig. 2A). The removed bone window was reinserted and fixed with a five-hole flat microplate and three 5-mm micro screws (Fig. 2B). Then, the operation was terminated by suturing the skin with 5.0 Prolene® and the periosteum with 5.0 vicryl®. The third and fourth groups received 3 mg/kg ZnSO4 intraperitoneally after the operation. The rabbits that received surgery were followed up for 6 weeks postoperatively by housing two animals in each cage. 2.4. Statistical analysis The SPSS® 21 program was used for the analysis of data. For the analysis of quantitative data, their conformity with the normal distribution was reviewed by KolmogoroveSmirnov test, ShapiroeWilk test and coefficients of variation taken into consideration; the parametric methods were used in the analysis of variables which had a normal distribution, and non-parametric
2.2. Preparation of zinc-coated titanium plates Perforated straight titanium microplates (Trimed®Ti6AI4V) were used. The sizes of plates were as follows: the hole diameter
Fig. 1. Zinc-uncoated (top) and zinc-coated (bottom) titanium microplates.
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
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Fig. 2. A, the nasal bone is exposed and the window formed in the bone. B, fixation of bone fracture with zinc coated titanium microplate.
methods were used in the analysis of variables which did not have a normal distribution. One-Way Anova test was used in the comparison of independent multiple groups with each other and LSD tests were used for Post Hoc analyses. After taking the main factors of quantitative data under control, Partial Correlation test was used in order to review the correlation of variables. The quantitative data was referred to as the average ± std (standard deviation) values in the tables. The categorical data was referred to as n(number) and percentages (%). The data was reviewed with 95% confidence level and p value was accepted as lower than 0.05 and significant. 3. Results 3.1. Histopathological and histomorphometric findings Callus formation, woven bone tissue, percentage of bone to whole bone area, adipose tissue in the interstitial area, edema and osteoblast numbers were evaluated for healing of fracture line. While calculating the osteoblast numbers, the incipient osteoblasts
around the bone in an area were calculated under 100 magnification and multiplied with the total area number, and the results were taken into evaluation. There were callus formation and osteoblastic activity in Group-1 (control group). The woven bone tissue was observed at a very small amount and its ratio to whole bone was determined as 5e10%. The adipose tissue in the interstitial area was remarkable and there were young collagen tissue formation, edema and congested vascular structures (Fig. 3A). The callus formation and woven bone tissue was distinct in Group-2 (the group to which fixation was made with zinc coated titanium microplates) and its ratio to whole bone tissue and its ratio to whole bone tissue was observed as 60e70%. The adipose tissue in the interstitial area was less but the edema was remarkable (Fig. 3B). The callus tissue was in calcific form occasionally in Group-3 (the group to which intraperitoneal zinc was given following titanium microplate fixation) and the ratio of woven bone structure to whole bone structure was observed as 50e60%. The interstitial
Fig. 3. A, Osteoblastic activity (arrow) and callus formation (star) are seen (Group 1, 100). B, osteoblastic activity (arrow) and woven bone structure (star) appearance (Group 2, 100). C, woven bone structure (star) (Group 3, 100) is observed together with callus texture in calcified structure in places. D, Occasionally, there is lamellar bone formation (star) and osteoblastic activity (arrow) is the most prominent appearance (Group 4, 400), sections were stained with haematoxylin and eosin.
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
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adipose tissue was still available at a small amount but edema was also observed (Fig. 3C). The callus tissue was in calcific form occasionally in Group-4 (the group to which intraperitoneal zinc was given following zinc-coated titanium microplate fixation) and this was the group at which the woven bone structure was the densest and its ratio to whole bone structure was observed as 80e85%. The adipose tissue in the interstitial area was at a very small amount and the proliferous fibroblasts were observed distinctly (Fig. 3D). The average osteoblast numbers were 366.3 ± 11.8; 415.6 ± 46.7; 390.6 ± 83.2; 508.8 ± 148, respectively in Group-1, Group-2, Group3 and Group-4 (Table 2). When the groups were compared in terms of osteoblast numbers, there was a statistically significant difference between Group-1 and Group-2 (p < 0.01) and Group-1 and Group-4 (p < 0.01). No statistically significant difference was determined between Group-1 and Group-3 (p ¼ 0.341). There was a statistically significant difference between Group-2 and Group-1 and Group-2 and Group-4 (p < 0.05). A statistically significant difference was not obtained between Group-2 and Group-3 (p ¼ 0.124).
immunoreactivity score (Fig. 5). The differences between the groups were not statistically significant in terms of TGF-b (Group 1 vs Group 2, p ¼ 0.256; Group 1 vs Group 3, p ¼ 0.524; Group 1 vs Group 4, p ¼ 0.717; Group 2 vs Group 3, p ¼ 0.610; Group 2 vs Group 4,p ¼ 0.124; Group 3 vs Group 4, p ¼ 0.311) (Table 4). Also, the correlation between IRS values for IGF-1 antibody and TGF-b antibody was evaluated with Partial Correlation test and a weak correlation was determined among IRS values (r ¼ 0.175). This correlation was not statistically significant (p ¼ 0.374). 4. Discussion
When the groups which had been dyed with IGF-1 antibody were reviewed, Group-1 was the group in which IRS average was the lowest (3.7 ± 1.9) (Fig. 4A). There was a statistically significant difference between Group-1 and other groups (p < 0.05). IRS average of Group-2 (9.3 ± 2.8) was higher than Group-1 and Group3 but lower than Group-4. IRS average of Group-3 (7.1 ± 2.9) was higher than Group-1 but lower than Group-2 and Group-4. Group-4 was the group in which IRS average was the highest (9.6 ± 2.2) (Fig. 4B). There was a statistically significant difference between Group-1 and Group-2 (p < 0.001), Group-1 and Group-4(p < 0.001), and Group-3 and Group-4 (p ¼ 0.049). Any statistically significant difference was not determined between Group-2 and Group-4 (p ¼ 0.791) (Table 3). When the groups dyed with TGF-b antibody were reviewed, it was seen that in general, all groups were dyed with low
Physical vapor deposition (PVD) is the deposition of the coating process on a particular substance targeted using a vacuum by condensation from a stream of neutral or ionized metal atoms. The transition of the metallic component from a solid phase to the vapor phase is affected by heating the evaporation source. The metals coated with PVD technique showed significant resistance to abrasion, erosion, corrosion and mechanical properties. By using PVD technologies, a large number of inorganic metals, alloys, compounds, mixtures and some organic materials can be coated. The flexibility of the coating process, especially the PVD method, which is well supported by the superior and controllable properties of modern coatings, has enabled the application of coated tools worldwide. Physical vapor deposition is at the core of a wide range of applications in the medical, aerospace, automotive, sporting goods industry, optics, electronics and related defense fields. The use of rigid and abrasion resistant PVD coatings has been increased widely in global production, reducing production costs and increasing productivity (Prabu et al., 2013; Harsha, 2006). Titanium (Ti) is a bioinert metal commonly used in orthopedic surgery for decades. Unlike other metals such as cobalt-chromiummolybdenum (CoCrMo) or surgical steel (CrNiMo), it has no allergenic or immunogenic potential in vivo, has excellent corrosion resistance and promotes osseointegration when applied to and fixed to the osseous tissue. However, metal implants are susceptible to wear, leading to loosening, erosion, and poor loading of the implant, which may result in a reduction in the implant's ability to
Table 2 Osteoblast counts and statistical data of the groups.
Table 3 Immuno reactivity score (IRS) data of groups for IGF-1.
3.2. Immunohistochemical findings
Mean ± SD p values Group 1 Group 2 Group 3
Group 1
Group 2
Group 3
Group 4
366.3 ± 11,8
415.6 ± 46.7
390.6 ± 83.2
508.8 ± 148
-
p < 0.01 -
p ¼ 0.341 p ¼ 0.124 -
p < 0.01 p < 0.01 p < 0.01
Mean ± SD P values Group 1 Group 2 Group 3
Group 1
Group 2
Group 3
Group 4
3.7 ± 1.9
9.3 ± 2.8
7.1 ± 2.9
9.6 ± 2.2
-
p < 0.001 -
p ¼ 0.024 p ¼ 0.093 -
p < 0.001 p ¼ 0.791 p ¼ 0.049
Fig. 4. A, there is a lesser osteoblastic differentiation and a cross-section of Group-1 with a lower IRS for IGF-1 (Anti-IGF-1, 400). B, there is an osteoblastic differentiation and a cross-section of Group 4 with high IRS for IGF-1 (Anti-IGF-1, 400).
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
_ Azgın et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (xxxx) xxx I.
Fig. 5. A cross-section of Group-4 is observed with little staining with anti-TGF-ß (400).
Table 4 Immuno reactivity score (IRS) data of groups for TGF-b.
Mean ± SD P values Group 1 Group 2 Group 3
Group 1
Group 2
Group 3
Group 4
3.2 ± 1.7
4.4 ± 2.3
3.8 ± 2.8
2.8 ± 1.3
-
0.256 -
0.524 0.610 -
0.717 0.124 0.311
support due to reduced bone mass. In contrast to its excellent osteoconductive properties, the reduction in titanium hardness shows low wear resistance and limits its application to articulated parts of artificial joints. It is generally accepted that the biocompatibility of titanium can be increased by coating with bioactive materials and the presence of micro- and nano-scale structures. Various strategies have been explored to overcome these problems. Coating the surface with bioactive components seems to be a promising way to improve the osseointegration of metal implants. In recent years, titanium surfaces have been modified by incorporating different ions to improve the mechanical fixation of implants €ger et al., 2017). to bone (Ja Zinc is one of the indispensable metabolic elements for immune system, glucose control, normal functioning of cognitive processes, wound healing and oxidative stress responses. It has also been shown that zinc is necessary for the development of the skeletal system. Considering the mechanism of action at the cellular level, zinc is involved in the proliferation and activity of osteoblastic cells and inhibits osteoclastic activity. Zinc supplementation has been reported to be beneficial in fracture healing. In 60 patients with traumatic fractures, oral zinc supplementation significantly increased callus formation compared to the control group (Sadighi et al., 2008). In a study of zinc-deficient young rats, it was observed that bone growth was reduced and that less force was needed to produce fractures (Rossi et al., 2001). Zinc can stimulate protein synthesis that activates aminoacyl-tRNA synthetase in osteoblastic cells (Igarashi and Yamaguchi, 2001). Micro-CT and histological analyses revealed greater bone formation in calvarial defects for rats treated with zinc hydroxyapatite scaffold compared to commercially available collagen membranes (Bernstein et al., 2010). While there are studies with zinc-coated titanium implants in the field of dental (Kellesarian et al., 2017; Yu et al., 2017; Ferraris and Spriano, 2016; Liang et al., 2015; He et al., 2018) and orthopedic (Zhao et al., 2017, 2019; Li et al., 2014; Tao et al., 2016; Wang
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et al., 2011), sciences, we could not find any study with zinc-coated titanium implants for maxillofacial fractures by PVD technique. Defect healing is the process of reconstruction of the bone tissue at the site of injury (Walsh et al., 1997). Bone is constantly being resorbed by osteoclasts and then replaced by osteoblasts in a process called bone remodelling (Caetano-Lopes et al., 2007). Osteoblasts are derived from multipotent mesenchymal cells that also give rise to chondrocytes, myocytes, adipocytes, tendon cells and various types of fibroblasts (Tanaka et al., 2005). In addition to bone formation, osteoblasts regulate osteoclast differentiation and resorption activity by the secretion of cytokines or by direct cell contact (Neve et al., 2011). In our study, the number of osteoblasts reflecting osteoblastic activity showed a statistically significant difference between zinc-coated and uncoated titanium-plated groups (Group 2 vs 1) in favor of the zinc-coated group (Group 2). But such statistical significance was not detected between only titanium plate group (Group 1) and single dose intraperitoneal zinc plus titanium plate group (Group 3). In terms of osteoblastic activity, in the cases treated with titanium plates, addition of single dose intraperitoneal zinc therapy may not produce the desired results. On the other hand, in cases of zinc coated titanium microplates (Group 2), addition of single dose intraperitoneal zinc (Group 4) may provide the desired results. The fracture healing process is also regulated by numerous systemic and local growth factors. Local growth factors have been shown to contribute significantly by affecting the type and rate of fracture and defect repair (Blumenfeld et al., 2002). During fracture repair, a number of growth factors, cytokines and their receptors are present at high levels in and around the fracture site. Many of these proteins are normally expressed in skeletal tissue, and others are released from related inflammatory cells at the site of injury (Igarashi and Yamaguchi, 2001). In addition to the release of local growth factors in the fracture site, a systemic reaction has been suggested to trigger local effects. An insufficient supply of systemic growth factor is associated with loss of bone matter and decrease in differentiation of osteoblasts (Zimmermann et al., 2005). IGF-1, mesenchymal cells, periost cells, osteoblasts and chondrocytes play a role in cell proliferation or differentiation and stimulates the synthesis of bone matrix in vivo (Okazaki et al., 2003). IGF-1 secreted from osteoblasts in the bone tissue has been demonstrated to be a potent chemotactic factor that might play a major role in the recruitment of osteoblasts during bone formation (Nakasaki et al., 2008). IGF-1 acts in an autocrine/paracrine fashion to increase bone formation by stimulating the differentiation and activity of skeletal cells, thus playing an important role in the accumulation and maintenance of bone mass (Zhang et al., 2011). Local and systemic recombinant human IGF-1 treatment increases bone formation. Also, increased osteoblastic activity was found after systemic administration of IGF-1 (Reible et al., 2018; Spencer et al., 1991; Blumenfeld et al., 2002; Srouji et al., 2005). In a study of experimentally femoral fractured rats, IGF-1 levels were significantly increased in the group supplemented with zinc before corrective surgery (Igarashi and Yamaguchi, 2002). In our study, when IGF-1 and IRS values were compared in the study groups and control groups, when systemic zinc was given, more statistically significant levels of IGF-1 expression were detected in Group 3 (titanium plate plus systemic zinc-treated group) than Group 1 (only titanium plate group). This means that when the patients systemically received zinc in addition to the titanium plate, the treatment success may be higher than in only titanium plate-treated patients. Also there was a statistically significant difference between Group 2 (zinc coated titanium plate group) and the control group (only titanium plate group). There is a nuance that statistical significance between Group 1 vs Group 2 was p < 0,01 and was p ¼ 0.024 between Group 1 vs Group 3. In
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
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addition, when compared between Group 2 and Group 4, there was not a statistical significance. So, addition of systemic zinc onto the locally zinc coated titanium plate group when compared to the zinc coated titanium plate group has no statistical significance. Here, from our study, it may be hypothesized that local zinc coating onto titanium plates may be superior to single dose intraperitoneal zinc therapy for fractured cases. TGF-b causes the proliferation and differentiation of osteoblasts. It is locally produced by osteoblasts and acts locally as a paracrine agent (Poniatowski et al., 2015; Arnott et al., 2008; Srouji et al., 2005). TGF-b has a special importance in fracture healing due to its involvement in bone formation, resorption and endochondral ossification processes (Blumenfeld et al., 2002). TGF-b is one of the most potent mitogens in the stimulation of osteoprogenitor cells and promotes bone formation by collagen formation (Farhadieh et al., 1999; Nielsen et al., 1994). Following local TGF-b injection, increased in both bone strength of the tibial bone fracture site and in the cross-sectional area of the callus has been reported (Blumenfeld et al., 2002). TGF-b is a protein-carrying molecule that plays a role in the early stages of bone healing. A region containing any new bone defects may show high activity for TGF-b. TGF-b concentrations begin to reach a plateau between 4 and 8 weeks and decrease. This decrease in TGF-b serum concentration may be due to increased mechanical stability of the fracture (Sarahrudi et al., 2011). In our study, we found that there was no correlation between coating of titanium plates with zinc and single dose intraperitoneal zinc application and TGF-b levels in fractured tissues at the end of the experiment. This finding supports that TGF-b increases during early fracture but decreases in the late recovery phase. This study has some limitations. The first matter is the mechanical loading test. Mechanical loading tests are important for the strength of bones. So, further studies are needed to clarify whether the treatment of zinc with the technique mentioned in this study has the effect on mobile bone fracture, such as mandible with mechanical loading test. Second, the systemic zinc therapy has been given only one dose intraperitoneally. Perhaps with daily application of intraperitoneal zinc during the six weeks of experiment period, the results may be stronger. More studies are required for daily intraperitoneal zinc administration. In conclusion, it was revealed that the local use of zinc-coated titanium microplates was efficient for bone fracture healing. By contrast, with the administration of only a single dose of intraperitoneal zinc, the desired effect on fracture healing was not obtained. Local zinc coating may accelerate fracture healing in cases that are planned to use titanium microplate for fracture treatment. Based on the results of the present study, clinical studies should be performed now in order to explore if comparable results can be achieved in humans. Funding This work was supported by the Necmettin Erbakan University, Medical Faculty [Grant Number: 121518016]. Declaration of Competing Interest None. Acknowledgements The authors owe a debt of gratitude to Necmettin Erbakan University and Necmettin Erbakan University Experimental Medicine Research and Application Center due to their support and intensive effort to our project.
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The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits _ Azgın a, *, Hamdi Arbag b, Mehmet Akif Eryılmaz b, Zeliha Esin Çelik c Isa a
Health Sciences University, Konya Training and Research Hospital, Department of Otorhinolaryngology Head and Neck Surgery, Konya, Turkey Necmettin Erbakan University, Meram Medical Faculty, Department of Otorhinolaryngology Head and Neck Surgery, Konya, Turkey c Selçuk University, Medical Faculty, Department of Medical Pathology, Konya, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 10 July 2019 Accepted 25 January 2020 Available online xxx
Objective: This study aimed to determine whether administration of topical and intraperitoneal zinc for maxillofacial fractures has any impact on the bone healing process. Material and method: Thirty-two New Zealand rabbits were randomly assigned to four groups of eight each. The first group was the control group; fracture lines were fixed using titanium microplates and no medication was administered. The second group received fixations using zinc-coated titanium microplates. A single dose of 3 mg/kg zinc was administered intraperitoneally to the third group following fixations with titanium microplates. A single dose of 3 mg/kg zinc was administered intraperitoneally to the fourth group following fixations with zinc-coated titanium microplates. Zinc coating on to the titanium microplates was achieved using the physical vapor deposition technique. A fracture line was created in the nasal bones of all subjects and fixed with five-hole flat microplates and three 5-mm micro screws. All work groups were sacrificed at the end of the sixth week. Results: Histological examination showed that the number of osteoblasts were significantly higher in zinc-coated group (Group 2) than zinc uncoated, control group (Group 1), (415.6 ± 46.7 vs 366.3 ± 11.8) (p < 0.001). It was observed that intraperitoneal zinc treatment alone (Group 3) did not significantly increase in the osteoblast count compared to zinc un-coated group (Group 1), (390.6 ± 83.2 vs 366.3 ± 11.8), (p ¼ 0.341). The immunoreactivity scores for IGF-1 were significantly higher in the zinccoated group compared to control group (Group 2 vs 1), (9.3 ± 2.8 vs 3.7 ± 1.9) (p < 0.05). It was observed that intraperitoneal zinc treatment did not cause a significant difference in the aspect of IGF-1 for zinc-coated groups (Group 2 vs 4) (9.3 ± 2.8 vs 9.6 ± 2.2) (p ¼ 0.791). The difference in the immunoreactivity score among whole groups for TGF-b was not statistically significant (Group 1 vs 2, 3.2 ± 1.7 vs 4.4 ± 2.3, p ¼ 0.256; Group 1 vs 3, 3.2 ± 1.7 vs 3.8 ± 2.8, p ¼ 0.524; Group 1 vs 4, 3.2 ± 1.7 vs 2.8 ± 1.3, p ¼ 0.717; Group 2 vs 3, 4.4 ± 2.3, vs 3.8 ± 2.8, p ¼ 0.610; Group 2 vs 4, 4.4 ± 2.3, vs 2.8 ± 1.3, p ¼ 0.124; Group 3 vs 4, 3.8 ± 2.8, vs 2.8 ± 1.3, p ¼ 0.311). Conclusion: The local use of titanium microplates coated with zinc by PVD technique was found effective for fracture healing. Zinc coating of titanium microplates used in fracture treatment can accelerate fracture healing. It may be concluded that clinical studies should be performed now in order to explore if comparable results can be achieved in humans. © 2020 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Maxillofacial trauma Fracture healing Zinc IGF-1 TGF- b
1. Introduction
* Corresponding author. Department of Otorhinolaryngology, Head and Neck Surgery, Health Sciences University, Konya Training and Research Hospital, Meram Yeniyol Caddesi No:97, PK: 42090, Meram, Konya, Turkey. Tel.: þ90 533 552 5216, fax: þ90 332 323 67 23. _ Azgın). E-mail address: [email protected] (I.
Due to increased traffic and work accidents, bone fractures have become a significant health problem in society. Maxillofacial trauma can cause serious clinical and cosmetic problems (Malara et al., 2006). Bone fractures can lead to significant damage to a person's social and economic situation, in addition to the economic losses a country suffers through labor loss.
https://doi.org/10.1016/j.jcms.2020.01.013 1010-5182/© 2020 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
_ Azgın et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (xxxx) xxx I.
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Various metal plates and screws have been used in the treatment of bone fractures for many years. Today, titanium plates are mostly used due to their superiority over the other metal alloys and as they are tissue friendly. In addition to the need for methods of increasing the effect of titanium, the need for systemic and local treatment methods which will accelerate the bone healing has also increased. It is known that zinc, a trace element, has many functions at the cellular level with regard to the development of animals and humans (Koekkoek and vanZanten, 2016; Tapiero and Tew, 2003; Abrisham et al., 2010). Zinc increases the number of osteoblasts by stimulating protein synthesis in the osteoblastic cells. In addition, it serves an important function in the preservation of the bone structure, inhibiting bone resorption, and stimulating bone formation (Notomi et al., 2015; Wey et al., 2014). Zinc also has antioxidant effects (Dede et al., 2003). Coating with thermal evaporation physical vapor deposition (PVD) is the deposition of metal atoms on another targeted material by evaporation and using a vacuum. The metals coated with PVD technique showed significant mechanical properties and resistance to abrasion, erosion, and corrosion. Physical vapor deposition is at the core of a wide variety of applications (Prabu et al., 2013; Harsha, 2006). While there are studies with zinc-coated titanium implants in the dental field (Kellesarian et al., 2017; Yu et al., 2017; Ferraris and Spriano, 2016; Liang et al., 2015; He et al., 2018) and orthopedic (Zhao et al., 2017, 2019; Li et al., 2014; Tao et al., 2016; Wang et al., 2011) applications, we could not find any study with local (zinccoated titanium plates by PVD technique) and intraperitoneal zinc treatment for maxillofacial fractures. We aimed to determine the effects of local and intraperitoneal zinc treatments on the maxillofacial fracture healing process. This study aims to investigate the effects of intraperitoneal and local zinc treatment on the healing of maxillofacial fractures. 2. Material and methods The protocol of this experimental study was approved by Necmettin Erbakan University Experimental Medicine Research and Application Center (Permission no:2011/57). The animals were followed by housing two animals in each cage where 12 h of dark and 12 h of light environment was provided at 20e22 C under the control of a veterinary physician. The feed and water needs of animals in all groups were regularly supplied in equal amounts. 2.1. Working Groups Thirty-two male New Zealand white rabbits (mean weights ranged 2300 ± 200 g and mean 9 months old) were randomly assigned into four groups with eight rabbits in each group. In the control group, the fracture lines were fixed with titanium microplates (Trimed®Ti6AI4V) and no medication was applied. In the second group, fixation was made with zinc (Ted Pella® Zinc Pieces) coated titanium microplates. In the third group, intraperitoneal 3 mg/kg zinc sulphate (ZnSO4) (Fluka®) was given following the fixation with titanium microplates. The fourth group was given intraperitoneal 3 mg/kg zinc sulphate (ZnSO4) following the fixation with zinc-coated titanium microplates (Table 1). All rabbits were sacrificed at the end of the sixth week and surgical specimens were taken for histopathological evaluation.
Table 1 Demonstration of animal study groups. Group 1 (Control) Group 2 Group 3
Group 4
Group with only titanium microplate fixation Group with fixation with zinc plated titanium microplates A single dose of 3 mg/kg intraperitoneal zinc group followed by titanium microplate fixation A single dose of 3 mg/kg intraperitoneal zinc group followed by fixation with zinc plated titanium microplate
was 2.0 mm, the width was 3.0 mm and thickness was 0.5 mm. The micro screws used were 5.0 mm long and 1.6 mm in diameter. The zinc coating procedure on the titanium microplates was conducted with the technique of PVD. In this technique, the titanium microplates were placed into the device of a MB-200B Modular Glove Box Workstation (MBraun®) and Thermal evaporator (Leybold® PVD systems). Then, the zinc parts (Ted Pella® Zinc Pieces) were placed into the tungsten pot. In the thermal evaporator device, the zinc metal was evaporated by applying current to the tungsten pot under 106 millibar pressure and the titanium microplates were coated with zinc (Fig. 1). 2.3. Animal model and surgical operation The combination of 25 mg/kg Ketamin (Ketalar®, Pfizer, Turkey) and 2 mg/kg Xylazine (Rompun®, Bayer, Turkey) was used as anesthetic combination intramuscularly. After shaving the nasal areas of rabbits, they were dyed with povidone iodine (Batticon®, ADEKA, Turkey). A 2-cm longitudinal incision was applied under the skin frontonasally. The periosteum was lateralized with the help of an elevator and the nasal bone was exposed. Approximately 1 cm2 bone window was removed using 0.1 mm cutting microtur (Fig. 2A). The removed bone window was reinserted and fixed with a five-hole flat microplate and three 5-mm micro screws (Fig. 2B). Then, the operation was terminated by suturing the skin with 5.0 Prolene® and the periosteum with 5.0 vicryl®. The third and fourth groups received 3 mg/kg ZnSO4 intraperitoneally after the operation. The rabbits that received surgery were followed up for 6 weeks postoperatively by housing two animals in each cage. 2.4. Statistical analysis The SPSS® 21 program was used for the analysis of data. For the analysis of quantitative data, their conformity with the normal distribution was reviewed by KolmogoroveSmirnov test, ShapiroeWilk test and coefficients of variation taken into consideration; the parametric methods were used in the analysis of variables which had a normal distribution, and non-parametric
2.2. Preparation of zinc-coated titanium plates Perforated straight titanium microplates (Trimed®Ti6AI4V) were used. The sizes of plates were as follows: the hole diameter
Fig. 1. Zinc-uncoated (top) and zinc-coated (bottom) titanium microplates.
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
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Fig. 2. A, the nasal bone is exposed and the window formed in the bone. B, fixation of bone fracture with zinc coated titanium microplate.
methods were used in the analysis of variables which did not have a normal distribution. One-Way Anova test was used in the comparison of independent multiple groups with each other and LSD tests were used for Post Hoc analyses. After taking the main factors of quantitative data under control, Partial Correlation test was used in order to review the correlation of variables. The quantitative data was referred to as the average ± std (standard deviation) values in the tables. The categorical data was referred to as n(number) and percentages (%). The data was reviewed with 95% confidence level and p value was accepted as lower than 0.05 and significant. 3. Results 3.1. Histopathological and histomorphometric findings Callus formation, woven bone tissue, percentage of bone to whole bone area, adipose tissue in the interstitial area, edema and osteoblast numbers were evaluated for healing of fracture line. While calculating the osteoblast numbers, the incipient osteoblasts
around the bone in an area were calculated under 100 magnification and multiplied with the total area number, and the results were taken into evaluation. There were callus formation and osteoblastic activity in Group-1 (control group). The woven bone tissue was observed at a very small amount and its ratio to whole bone was determined as 5e10%. The adipose tissue in the interstitial area was remarkable and there were young collagen tissue formation, edema and congested vascular structures (Fig. 3A). The callus formation and woven bone tissue was distinct in Group-2 (the group to which fixation was made with zinc coated titanium microplates) and its ratio to whole bone tissue and its ratio to whole bone tissue was observed as 60e70%. The adipose tissue in the interstitial area was less but the edema was remarkable (Fig. 3B). The callus tissue was in calcific form occasionally in Group-3 (the group to which intraperitoneal zinc was given following titanium microplate fixation) and the ratio of woven bone structure to whole bone structure was observed as 50e60%. The interstitial
Fig. 3. A, Osteoblastic activity (arrow) and callus formation (star) are seen (Group 1, 100). B, osteoblastic activity (arrow) and woven bone structure (star) appearance (Group 2, 100). C, woven bone structure (star) (Group 3, 100) is observed together with callus texture in calcified structure in places. D, Occasionally, there is lamellar bone formation (star) and osteoblastic activity (arrow) is the most prominent appearance (Group 4, 400), sections were stained with haematoxylin and eosin.
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
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adipose tissue was still available at a small amount but edema was also observed (Fig. 3C). The callus tissue was in calcific form occasionally in Group-4 (the group to which intraperitoneal zinc was given following zinc-coated titanium microplate fixation) and this was the group at which the woven bone structure was the densest and its ratio to whole bone structure was observed as 80e85%. The adipose tissue in the interstitial area was at a very small amount and the proliferous fibroblasts were observed distinctly (Fig. 3D). The average osteoblast numbers were 366.3 ± 11.8; 415.6 ± 46.7; 390.6 ± 83.2; 508.8 ± 148, respectively in Group-1, Group-2, Group3 and Group-4 (Table 2). When the groups were compared in terms of osteoblast numbers, there was a statistically significant difference between Group-1 and Group-2 (p < 0.01) and Group-1 and Group-4 (p < 0.01). No statistically significant difference was determined between Group-1 and Group-3 (p ¼ 0.341). There was a statistically significant difference between Group-2 and Group-1 and Group-2 and Group-4 (p < 0.05). A statistically significant difference was not obtained between Group-2 and Group-3 (p ¼ 0.124).
immunoreactivity score (Fig. 5). The differences between the groups were not statistically significant in terms of TGF-b (Group 1 vs Group 2, p ¼ 0.256; Group 1 vs Group 3, p ¼ 0.524; Group 1 vs Group 4, p ¼ 0.717; Group 2 vs Group 3, p ¼ 0.610; Group 2 vs Group 4,p ¼ 0.124; Group 3 vs Group 4, p ¼ 0.311) (Table 4). Also, the correlation between IRS values for IGF-1 antibody and TGF-b antibody was evaluated with Partial Correlation test and a weak correlation was determined among IRS values (r ¼ 0.175). This correlation was not statistically significant (p ¼ 0.374). 4. Discussion
When the groups which had been dyed with IGF-1 antibody were reviewed, Group-1 was the group in which IRS average was the lowest (3.7 ± 1.9) (Fig. 4A). There was a statistically significant difference between Group-1 and other groups (p < 0.05). IRS average of Group-2 (9.3 ± 2.8) was higher than Group-1 and Group3 but lower than Group-4. IRS average of Group-3 (7.1 ± 2.9) was higher than Group-1 but lower than Group-2 and Group-4. Group-4 was the group in which IRS average was the highest (9.6 ± 2.2) (Fig. 4B). There was a statistically significant difference between Group-1 and Group-2 (p < 0.001), Group-1 and Group-4(p < 0.001), and Group-3 and Group-4 (p ¼ 0.049). Any statistically significant difference was not determined between Group-2 and Group-4 (p ¼ 0.791) (Table 3). When the groups dyed with TGF-b antibody were reviewed, it was seen that in general, all groups were dyed with low
Physical vapor deposition (PVD) is the deposition of the coating process on a particular substance targeted using a vacuum by condensation from a stream of neutral or ionized metal atoms. The transition of the metallic component from a solid phase to the vapor phase is affected by heating the evaporation source. The metals coated with PVD technique showed significant resistance to abrasion, erosion, corrosion and mechanical properties. By using PVD technologies, a large number of inorganic metals, alloys, compounds, mixtures and some organic materials can be coated. The flexibility of the coating process, especially the PVD method, which is well supported by the superior and controllable properties of modern coatings, has enabled the application of coated tools worldwide. Physical vapor deposition is at the core of a wide range of applications in the medical, aerospace, automotive, sporting goods industry, optics, electronics and related defense fields. The use of rigid and abrasion resistant PVD coatings has been increased widely in global production, reducing production costs and increasing productivity (Prabu et al., 2013; Harsha, 2006). Titanium (Ti) is a bioinert metal commonly used in orthopedic surgery for decades. Unlike other metals such as cobalt-chromiummolybdenum (CoCrMo) or surgical steel (CrNiMo), it has no allergenic or immunogenic potential in vivo, has excellent corrosion resistance and promotes osseointegration when applied to and fixed to the osseous tissue. However, metal implants are susceptible to wear, leading to loosening, erosion, and poor loading of the implant, which may result in a reduction in the implant's ability to
Table 2 Osteoblast counts and statistical data of the groups.
Table 3 Immuno reactivity score (IRS) data of groups for IGF-1.
3.2. Immunohistochemical findings
Mean ± SD p values Group 1 Group 2 Group 3
Group 1
Group 2
Group 3
Group 4
366.3 ± 11,8
415.6 ± 46.7
390.6 ± 83.2
508.8 ± 148
-
p < 0.01 -
p ¼ 0.341 p ¼ 0.124 -
p < 0.01 p < 0.01 p < 0.01
Mean ± SD P values Group 1 Group 2 Group 3
Group 1
Group 2
Group 3
Group 4
3.7 ± 1.9
9.3 ± 2.8
7.1 ± 2.9
9.6 ± 2.2
-
p < 0.001 -
p ¼ 0.024 p ¼ 0.093 -
p < 0.001 p ¼ 0.791 p ¼ 0.049
Fig. 4. A, there is a lesser osteoblastic differentiation and a cross-section of Group-1 with a lower IRS for IGF-1 (Anti-IGF-1, 400). B, there is an osteoblastic differentiation and a cross-section of Group 4 with high IRS for IGF-1 (Anti-IGF-1, 400).
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
_ Azgın et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (xxxx) xxx I.
Fig. 5. A cross-section of Group-4 is observed with little staining with anti-TGF-ß (400).
Table 4 Immuno reactivity score (IRS) data of groups for TGF-b.
Mean ± SD P values Group 1 Group 2 Group 3
Group 1
Group 2
Group 3
Group 4
3.2 ± 1.7
4.4 ± 2.3
3.8 ± 2.8
2.8 ± 1.3
-
0.256 -
0.524 0.610 -
0.717 0.124 0.311
support due to reduced bone mass. In contrast to its excellent osteoconductive properties, the reduction in titanium hardness shows low wear resistance and limits its application to articulated parts of artificial joints. It is generally accepted that the biocompatibility of titanium can be increased by coating with bioactive materials and the presence of micro- and nano-scale structures. Various strategies have been explored to overcome these problems. Coating the surface with bioactive components seems to be a promising way to improve the osseointegration of metal implants. In recent years, titanium surfaces have been modified by incorporating different ions to improve the mechanical fixation of implants €ger et al., 2017). to bone (Ja Zinc is one of the indispensable metabolic elements for immune system, glucose control, normal functioning of cognitive processes, wound healing and oxidative stress responses. It has also been shown that zinc is necessary for the development of the skeletal system. Considering the mechanism of action at the cellular level, zinc is involved in the proliferation and activity of osteoblastic cells and inhibits osteoclastic activity. Zinc supplementation has been reported to be beneficial in fracture healing. In 60 patients with traumatic fractures, oral zinc supplementation significantly increased callus formation compared to the control group (Sadighi et al., 2008). In a study of zinc-deficient young rats, it was observed that bone growth was reduced and that less force was needed to produce fractures (Rossi et al., 2001). Zinc can stimulate protein synthesis that activates aminoacyl-tRNA synthetase in osteoblastic cells (Igarashi and Yamaguchi, 2001). Micro-CT and histological analyses revealed greater bone formation in calvarial defects for rats treated with zinc hydroxyapatite scaffold compared to commercially available collagen membranes (Bernstein et al., 2010). While there are studies with zinc-coated titanium implants in the field of dental (Kellesarian et al., 2017; Yu et al., 2017; Ferraris and Spriano, 2016; Liang et al., 2015; He et al., 2018) and orthopedic (Zhao et al., 2017, 2019; Li et al., 2014; Tao et al., 2016; Wang
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et al., 2011), sciences, we could not find any study with zinc-coated titanium implants for maxillofacial fractures by PVD technique. Defect healing is the process of reconstruction of the bone tissue at the site of injury (Walsh et al., 1997). Bone is constantly being resorbed by osteoclasts and then replaced by osteoblasts in a process called bone remodelling (Caetano-Lopes et al., 2007). Osteoblasts are derived from multipotent mesenchymal cells that also give rise to chondrocytes, myocytes, adipocytes, tendon cells and various types of fibroblasts (Tanaka et al., 2005). In addition to bone formation, osteoblasts regulate osteoclast differentiation and resorption activity by the secretion of cytokines or by direct cell contact (Neve et al., 2011). In our study, the number of osteoblasts reflecting osteoblastic activity showed a statistically significant difference between zinc-coated and uncoated titanium-plated groups (Group 2 vs 1) in favor of the zinc-coated group (Group 2). But such statistical significance was not detected between only titanium plate group (Group 1) and single dose intraperitoneal zinc plus titanium plate group (Group 3). In terms of osteoblastic activity, in the cases treated with titanium plates, addition of single dose intraperitoneal zinc therapy may not produce the desired results. On the other hand, in cases of zinc coated titanium microplates (Group 2), addition of single dose intraperitoneal zinc (Group 4) may provide the desired results. The fracture healing process is also regulated by numerous systemic and local growth factors. Local growth factors have been shown to contribute significantly by affecting the type and rate of fracture and defect repair (Blumenfeld et al., 2002). During fracture repair, a number of growth factors, cytokines and their receptors are present at high levels in and around the fracture site. Many of these proteins are normally expressed in skeletal tissue, and others are released from related inflammatory cells at the site of injury (Igarashi and Yamaguchi, 2001). In addition to the release of local growth factors in the fracture site, a systemic reaction has been suggested to trigger local effects. An insufficient supply of systemic growth factor is associated with loss of bone matter and decrease in differentiation of osteoblasts (Zimmermann et al., 2005). IGF-1, mesenchymal cells, periost cells, osteoblasts and chondrocytes play a role in cell proliferation or differentiation and stimulates the synthesis of bone matrix in vivo (Okazaki et al., 2003). IGF-1 secreted from osteoblasts in the bone tissue has been demonstrated to be a potent chemotactic factor that might play a major role in the recruitment of osteoblasts during bone formation (Nakasaki et al., 2008). IGF-1 acts in an autocrine/paracrine fashion to increase bone formation by stimulating the differentiation and activity of skeletal cells, thus playing an important role in the accumulation and maintenance of bone mass (Zhang et al., 2011). Local and systemic recombinant human IGF-1 treatment increases bone formation. Also, increased osteoblastic activity was found after systemic administration of IGF-1 (Reible et al., 2018; Spencer et al., 1991; Blumenfeld et al., 2002; Srouji et al., 2005). In a study of experimentally femoral fractured rats, IGF-1 levels were significantly increased in the group supplemented with zinc before corrective surgery (Igarashi and Yamaguchi, 2002). In our study, when IGF-1 and IRS values were compared in the study groups and control groups, when systemic zinc was given, more statistically significant levels of IGF-1 expression were detected in Group 3 (titanium plate plus systemic zinc-treated group) than Group 1 (only titanium plate group). This means that when the patients systemically received zinc in addition to the titanium plate, the treatment success may be higher than in only titanium plate-treated patients. Also there was a statistically significant difference between Group 2 (zinc coated titanium plate group) and the control group (only titanium plate group). There is a nuance that statistical significance between Group 1 vs Group 2 was p < 0,01 and was p ¼ 0.024 between Group 1 vs Group 3. In
Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013
_ Azgın et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (xxxx) xxx I.
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addition, when compared between Group 2 and Group 4, there was not a statistical significance. So, addition of systemic zinc onto the locally zinc coated titanium plate group when compared to the zinc coated titanium plate group has no statistical significance. Here, from our study, it may be hypothesized that local zinc coating onto titanium plates may be superior to single dose intraperitoneal zinc therapy for fractured cases. TGF-b causes the proliferation and differentiation of osteoblasts. It is locally produced by osteoblasts and acts locally as a paracrine agent (Poniatowski et al., 2015; Arnott et al., 2008; Srouji et al., 2005). TGF-b has a special importance in fracture healing due to its involvement in bone formation, resorption and endochondral ossification processes (Blumenfeld et al., 2002). TGF-b is one of the most potent mitogens in the stimulation of osteoprogenitor cells and promotes bone formation by collagen formation (Farhadieh et al., 1999; Nielsen et al., 1994). Following local TGF-b injection, increased in both bone strength of the tibial bone fracture site and in the cross-sectional area of the callus has been reported (Blumenfeld et al., 2002). TGF-b is a protein-carrying molecule that plays a role in the early stages of bone healing. A region containing any new bone defects may show high activity for TGF-b. TGF-b concentrations begin to reach a plateau between 4 and 8 weeks and decrease. This decrease in TGF-b serum concentration may be due to increased mechanical stability of the fracture (Sarahrudi et al., 2011). In our study, we found that there was no correlation between coating of titanium plates with zinc and single dose intraperitoneal zinc application and TGF-b levels in fractured tissues at the end of the experiment. This finding supports that TGF-b increases during early fracture but decreases in the late recovery phase. This study has some limitations. The first matter is the mechanical loading test. Mechanical loading tests are important for the strength of bones. So, further studies are needed to clarify whether the treatment of zinc with the technique mentioned in this study has the effect on mobile bone fracture, such as mandible with mechanical loading test. Second, the systemic zinc therapy has been given only one dose intraperitoneally. Perhaps with daily application of intraperitoneal zinc during the six weeks of experiment period, the results may be stronger. More studies are required for daily intraperitoneal zinc administration. In conclusion, it was revealed that the local use of zinc-coated titanium microplates was efficient for bone fracture healing. By contrast, with the administration of only a single dose of intraperitoneal zinc, the desired effect on fracture healing was not obtained. Local zinc coating may accelerate fracture healing in cases that are planned to use titanium microplate for fracture treatment. Based on the results of the present study, clinical studies should be performed now in order to explore if comparable results can be achieved in humans. Funding This work was supported by the Necmettin Erbakan University, Medical Faculty [Grant Number: 121518016]. Declaration of Competing Interest None. Acknowledgements The authors owe a debt of gratitude to Necmettin Erbakan University and Necmettin Erbakan University Experimental Medicine Research and Application Center due to their support and intensive effort to our project.
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Please cite this article as: Azgın I_ et al., The effects of local and intraperitoneal zinc treatments on maxillofacial fracture healing in rabbits, Journal of Cranio-Maxillo-Facial Surgery, https://doi.org/10.1016/j.jcms.2020.01.013