INTRODUCTION
Bone healing, also called bone turnover or restoration of the bone microarchitecture, after various acquired defects or congenital deformities of the cranium, such as traumatic injuries, congenital deformities, decompressive craniectomies, or bone flap loss due to infections still represents a significant challenge in neurosurgery. Although numerous studies have been conducted to develop biomaterials displaying osteoconductive and/or osteoinductive properties to date, utilisation of autogenous bone for repair of the cranial defects seems to be the most appropriate option, possibly due to its biosafety and cost advantage [29]. However, risks of additional morbidity to the patient related to obtaining the graft and resorption of the autogenous bone grafts limit their utilisation in the repair of the cranial defects. As a result, there is still no consensus on which agent to be used in cranioplasty is better in reconstruction of the cranial defects in humans. To improve the bone regeneration and quality of the bone, therefore, use of various agents such as topical or systemic application of growth factors, cytokines and melatonin (MEL) has attracted the attention of scientists [3–5, 7–9, 14, 16, 21, 22, 25, 26, 30, 33–37].
In normal remodelling cycle of the bone, which is balanced between bone-forming osteoblasts and bone resorbing osteoclasts, the resorption phase is followed by bone formation phase, including osteoid formation and then mineralisation [1, 2, 10]. In recent years, it has been reported that MEL can have a pivotal role in this process when age-related osteoporosis is manifested, possibly due to an imbalance in bone turnover related to decreased level of the circulating MEL hormone; thus, MEL could act as an autacoid or a local growth factor for the bone cells [4, 6, 7, 30, 36]. Importantly, it has also been reported that the proliferation of osteoblasts and the expression of type I collagen and biochemical markers of bone turnover, including bone sialoprotein (BSP) are also promoted by MEL [7, 9, 17, 25, 26, 35]. Despite promising results, however, very few studies have been conducted to investigate the effects of exogenous administration of MEL as an agent that promotes bone regeneration to date and there is still no consensus on which agent to be used in cranioplasty is better in reconstruction of the cranial defects in humans.
In this experimental study, our main purpose was to investigate the effects of exogenous MEL administration on bone healing and connective tissue alterations surrounding the bone defect histopathologically after production of a burr hole in the calvarium of the rats exposed to functional pinealectomy (Px).
MATERIALS AND METHODS
In this study, 30 adult 200 g female Wistar-Albino rats were used. The experimental protocol was approved by the Ethical Committee of Aydın Adnan Menderes University (HADYEK 64583101/2014/063). Animals were housed in rat cages in standard conditions (24 ± 2°C and 50 ± 5% humidity), exposed to 12:12-h light/dark cycle, fed with standardised rodent chow and tap water ad libitum.
The rats were divided into three groups, with 10 animals per group. The first group of animals formed the control group (CO), which was exposed to 12:12-h light/dark cycle. The second and third groups were exposed to 24-h continuous light for 12 weeks in order to produce light-induced functional Px, as described in a previous study [23]. Next, all rats underwent ketamine/xylasine (50 and 5 mg/kg, respectively) anaesthesia, and a burr hole (diameter: 3.0 mm) was produced using an electric drill (CLASS Mini Grinder sets, PRC) on the right side of the calvarium. Then the wound was closed (Fig. 1). The animals in the second and third groups were moved back to the continuous light-burning room; meanwhile exogenous MEL treatment (Px+MEL) was applied to only the third group.
Melatonin (N-acetyl-5-methoxytryptamine, cat. no: M-5250, Sigma Chemical Co., St. Louis, MO, USA) was first dissolved in pure ethanol and kept in –20°C. The second dilution of this stock was freshly prepared in saline to have 5% ethanol concentration [13]. In the third group, 20 mg/kg/day of MEL was given intraperitoneally as a single injection for 12 weeks.
At the end of 12 weeks, all the rats were decapitated under the ketamine/xylasine (50 and 5 mg/kg, respectively) anaesthesia. The calvarial area which underwent the burr hole operation was removed for further histological study.
Histological examination of calvaria
Following sacrification, calvaria were collected into 10% formalin solution for at least 24 h before placement in decalcification solution containing 10% formaldehyde and 20% formic acid for 2 months, softening of the tissue was controlled by punching irregularly. Tissues were embedded to block in blue-beaded paraffin solution after processing in alcohol and xylol series. 5 µm sections were obtained by Leica RM 2145 microtome, transferred to water bath at 45oC and microwaved to ensure adhesion and to remove any paraffin remaining. Haematoxylin and eosin (H&E), Mallory Azan staining and anti-BSP (bs-4729R, Bioss, 1:100) were applied to the related calvarial area and the surgical specimens were evaluated at 10–20× magnification using an Olympus C5050 camera attached to Olympus BX51 light microscope.
Histomorphometry
For histomorphometric analysis, counting and measurements were made for hole diameter, fibrotic scar areas, ossification areas and number of vessels in the sections taken from calvaria samples from each subject in the experimental groups [15, 18, 42]. Measurement for new ossification areas and fibrotic scar areas was calculated in square micrometres (µm2) using Image Pro Express 4.5 (Media Cybernetics, Inc., Rockville, MD, USA) programme.
Statistical analysis
Data analysis was carried out in IBM SPSS (Statistical Package for Social Sciences) Statistics for Windows, Version 25.0 (IBM Corp. Released 2017, Armonk, NY) package programme. Group comparisons were performed using one-way analysis of variance method. Normality control was performed by Shapiro-Wilk test that obtained from estimated error. Since there was no problem of adaptation to normal distribution, the study continued with one-way analysis of variance. Variance homogeneities were examined by Levene test; Tukey HSD test was used as a post hoc method, since there was no homogeneity problem. All hypothesis tests were performed at the 0.05 significance level, thus p < 0.05 was considered significant.
RESULTS
Macroscopic examination
When the skin on the calvarium was dissected, drilled holes were detected on the bone and these closed holes were observed macroscopically (Fig. 1). It was found that the scar tissue in the Px+MEL samples were closer to normal when compared with the other two groups (Fig. 2).
Microscopic examination
In the microscopic sections, the orientation of the hole in the bone was adjusted and serial sections were taken. These sections were stained in H&E and Mallory Azan in order to show the general structure and were applied in anti-BSP immunohistochemistry. Histologically, the holes were found in the sections. Importantly, it was found that these hole structures were filled with irregular dense connective tissue called fibrotic in both control group and Px group (Fig. 3).
When the hole diameters were evaluated statistically, it was seen that the Px group had the largest hole diameter. No statistical significance was found in the comparison of the groups with each other (Fig. 4A). When evaluated in terms of fibrotic scar area, it was calculated that the most scar area was in the Px group, although no binary statistical significance was found between the CO and Px+MEL groups. Importantly, a statistical significance was found when comparing these two groups with the Px group (p < 0.001) (Fig. 4B). When evaluated in terms of ossification areas, it was observed that the most ossification was in the Px+MEL group, while the highest ossification was found in the Px group. However, there was no statistical significance between the groups (p = 0.306) (Fig. 4C). In terms of vascularisation, it was observed that the most vascular structure was found in the Px+MEL group among the scar areas and ossification areas, while the vascularisation was the least in the Px group. When the CO and Px groups were compared, a statistically significant reduction was observed (p < 0.001). There was also a statistical significance difference between Px and Px+MEL groups (p < 0.001) (Fig. 4D).
When this connective tissue structure was examined at larger magnification, it was found that it contains fibroblast-like cells and type I collagen bundles. In Px group, however, new bone splices were found that they were localised to different sites located within the connective tissue, possibly formed by intramembranous ossification. More importantly, in Px+MEL group, it was noted that this connective tissue area was small and ossification developed in most of the structure. With this appearance, it was the experimental group, which was closest to the normal histological structure of the other groups. Mallory Azan staining showed that the type I collagen bundles were stained blue while the newly formed bone areas were red (Fig. 5).
DISCUSSION
This experimental study evidenced that MEL replacement is related to the improvement of bone healing, in contrast to functional Px group, possibly due to increased osteoblastic activity and modulation of connective tissue alterations surrounding the bone defect driven by exogenous MEL administration in adult rats.
Previous studies reported that matrix cellular proteins, such as BSP, also called SPARC or BM40, osteopontin, tenascin C, and thrombospondin-1 and -2, were the critical regulators of bone healing process [1, 10]. BSP is composed of mineralised tissues such as bone, dentin, cementum and calcified cartilage. It binds to hydroxyapatite crystals and an integral part of the mineral matrix in bone tissue. Now, it is known to be an important component of the extracellular matrix of bone and forms all non-collagen proteins found in bone and cementum. In our study, the cell characterisation of the osteoblast lineage was via immunostaining for BSP. The CO and Px+MEL groups are similar in terms of BSP, both groups have a high level of immune reaction. A lower degree of immunoreaction was detected in the Px group.
Several studies documented that MEL had been linked to osteoblastic differentiation and bone metabolism [4, 6, 30, 36]. In a previous study, Witt-Enderby et al. (2006) [41] demonstrated that MEL influences bone cell precursors in rat bone marrow which has high concentrations of MEL. Koyama, et al. (2002) [14] showed that the administration of pharmacological doses of MEL during the growth of young rats increased the bone mass through the inhibition of bone resorption. To date, however, the role of MEL replacement has not yet been widely evaluated in adult animals with lower production of endogenous MEL, as did in our functional Px group. In this study, we intended for investigation its role on bone healing; therefore we applied a functional Px rat model to evaluate the effect of exogenous MEL administration histopathologically [23].
Experimental studies revealed that action of exogenous and endogenous MEL upon bone tissue is due to an increased osteoblastic activity, a decreased osteoclastic activity, and an increased osteoclastogenesis inhibitory factor, also known as osteoprotegerin, thus increasing its mass [14, 19, 20, 33, 40]. In addition to its stimulating effect on bone metabolism and production of type I collagen, inhibitory effect on proteasomes has also been suggested [14, 19, 20, 33, 40]. Thus, promotion of bone healing by MEL could be related to one or more of the following possible mechanisms: the promotion of the osteoblast differentiation and/or activity, an increased expression of the osteoprotegerin by osteoblasts, resulting with a decreased differentiation of osteoclasts, and increased scavenging of free radicals which were generated by osteoclasts [31]. Further, and more specifically, it has been speculated that MEL interferes with bone healing in several ways: through modulation of oxidative stress [27, 28, 32], collagen fibril formation [19], differentiation of osteoblasts via PDGF/AKT signalling pathway [9, 25, 26, 35, 43], or activity of osteoblasts and osteoclasts via MEL-MT2 receptor pathway [9, 34, 41] or RANK/NF-kB signalling pathway [14, 26] or Wnt/b-catenin signalling pathway [16, 25, 40]. Moreover, MEL has also been found to stimulate gene expression of BSP and other bone marker proteins including alkaline phosphate, secreted protein, osteocalcin and osteopontin [30].
Despite accumulating evidence about the effects of MEL upon regeneration of the bone tissue during the last decades, its use for cranial defects has received little attention and to date, healing of the bone after application of MEL has been evaluated in few experimental animal studies using model of calvaria [9, 25, 26, 35]. In addition to stimulatory action of MEL related to bone formation, however, possible effects of MEL upon the connective tissue surrounding the cranial defect have been also investigated for the first time in this study. Based on our findings, it is evident that the ossification process is accelerated by the effect of exogenous MEL, while the absence of endogenous MEL also negatively affects ossification.
On the other hand, it has been reported that a lower production of MEL, by some genetic modifications (pineal MEL deficient mice, C57BL/6 and Swiss strains) [12, 34], or various Px techniques (surgical or functional) [21, 22, 24, 37–39] results in alterations in structure, density and mechanical strength of the bone in animals including mice, rat, rabbit, chicken, sheep, and fish [9, 34]. To the best of our knowledge, however, no study was performed to evaluate the healing in endogenous MEL deficiency and exogenous MEL administration, when added to biomaterials or implants [3–6, 36]. Thus, the process of how the absence of endogenous MEL interferes with the bone healing is unclear. Considering this, the purpose of this study was to evaluate the cellular changes that occur in the bone of rats undergoing Px that received or did not receive daily exogenous MEL replacement as a possible therapy for conditions characterised by a decrease in the secretion of this hormone by the pineal gland.
In a previous in vitro study, it has been suggested that exogenous MEL administration has positive effects on both angiogenesis and wound healing [19]. In a recently published paper, Filipowska et al. (2017) [11] also suggested the role of production of vascular endothelial growth factor (VEGF) by either hypertrophic chondrocytes or differentiating mesenchymal cells in bone development and regeneration. In fact, our findings confirm that beneficial effects of MEL on blood vessel invasion for initiation of bone tissue formation, possibly as a result of increased VEGF production by MEL.
Taken together our findings provide an evidence that the absence of MEL delays repair and exogenous MEL replacement regulates this alteration, which would emphasize the role of this neurohormone in bone remodelling, especially in conditions in which a decrease occurs in the circadian release capacity of this hormone by the pineal gland, as did in various clinical disease conditions. Undoubtedly, further understanding of pathological changes in the bone tissue and their reversion is critical for the success of MEL replacement treatment. Two limitations of our study that created an in vivo rat calvaria model are as follows: absence of any analysis for inorganic material content of bone tissue and difference of drilled defects from those in humans in terms of bone remodelling. Also, lack of any imaging study illustrating bone healing and angiogenesis is another limitation of the study. Future studies including imaging are needed for better understanding of modulation of osteoblast and osteoclast function and mineralization with MEL in cases of bone defect that simulates the situation in human.
CONCLUSIONS
Based on the results of this study, we may notice an interaction in cellular responses and protein activity in the absence of MEL, suggesting that when there is a delay in the production of proteins that stimulate bone formation, ossification because of hormone administration increases vascularisation and may result in less scar tissue formation. The MEL replacement, in most parameters evaluated in this study, showed behaviour very close to that of the CO group. Therefore, the absence of the pineal gland impairs the bone repair process during osseointegration; however, the daily exogenous MEL replacement was able to partially restore this response. However, the authors believe that further studies should confirm this information in future.
Acknowledgements
The authors thank to Research Foundation of Aydın Adnan Menderes University (ADU-TPF-15019) for funding of this research. We also thank the anonymous reviewers of the journal “Folia Morphologica” for their constructive comments and help with the manuscript.
