J Clin Periodontol 2015; 42: 96–103 doi: 10.1111/jcpe.12342
Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats F€ ugl A, Gruber R, Agis H, Lzicar H, Keibl C, Schwarze UY, Dvorak G. Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats. J Clin Periodontol 2015; 42: 96–103. doi: 10.1111/jcpe.12342.
Abstract Aim: Vitamin D deficiency is considered to diminish bone regeneration. Yet, raising the serum levels takes months. A topic application of the active vitamin D metabolite, calcitriol, may be an effective approach. Thus, it becomes important to know the effect of vitamin D deficiency and local application on alveolar bone regeneration. Material and Methods: Sixty rats were divided into three groups; two vitamin depletion groups and a control group. Identical single defects (2 mm diameter) were created in the maxilla and mandible treated with calcitriol soaked collagen in one deficiency group while in the other two groups not. Histomorphometric analysis and micro CTs were performed after 1 and 3 weeks. Serum levels of 25 (OH)D3 and PTH were determined. Results: Bone formation rate significantly increased within the observation period in all groups. Bone regeneration was higher in the maxilla than in the mandible. However, bone regeneration was lower in the control group compared to vitamin depletion groups, with no significant effects by local administration of calcitriol (micro CT mandible p = 0.003, maxilla p < 0.001; histomorphometry maxilla p = 0.035, mandible p = 0.18). Conclusion: Vitamin D deficiency not necessarily impairs bone regeneration in the rat jaw and a single local calcitriol application does not enhance healing.
Vitamin D deficiency results from inadequate dietary intake together with insufficient exposure to sunlight or is related to inadequate synthesis by liver and kidney. Although there Conflict of interest and source of funding statement This project was funded by the ITI Research Grant 813_2012. Authors disclose any conflict of interest.
96
is no consensus on optimal levels, a serum level of 25(OH)D (calcidiol) below 20 ng/ml (50 nmol/l) is indicative for vitamin D deficiency, while a level of 30 ng/ml (75 nmol/l) or greater can be considered to indicate sufficient vitamin D and levels in between are considered as insufficiency. An estimated one billion people worldwide have vitamin D deficiency with a prevalence of 40 to 100% of the elderly in the U.S. and
€ gl1,2, Reinhard Alexander Fu Gruber1,2,3, Hermann Agis2,4, Hannah Lzicar2,5, Claudia Keibl2,6, Uwe Yacine Schwarze2,5 and Gabriella Dvorak1,2 1
Department of Oral Surgery, Medical University of Vienna, Vienna, Austria, 2 Austrian Cluster for Tissue Regeneration, Vienna, Austria, 3Laboratory of Oral Biology, School of Dental Medicine, University of Bern, Bern, Switzerland, 4Department of Conservative Dentistry and Periodontology, Medical University of Vienna, Vienna, Austria, 5 Karl Donath Laboratory for Hard Tissue and Biomaterial Research, Medical University of Vienna, Vienna, Austria, 6Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria
Key words: 1,25(OH)D3; bone healing; calcitriol; supplementation; topic administration; vitamin D deficiency Accepted for publication 25 November 2014
Europe (Holick 2007). Without vitamin D, dietary calcium and phosphorus adsorption is severely impaired. Because of the chronic release of calcium and phosphorus, vitamin D deficiency causes a catabolic bone turnover. Vitamin D deficiency has also been linked with impaired fracture healing and osseointegration (Kelly et al. 2009, Dvorak et al. 2012) as vitamin D accumulates in the fracture
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Vitamin D and alveolar bone regeneration callus (Jingushi et al. 1998, Fu et al. 2009). Together, these results support the steroid hormones role in controlling bone regeneration. It is therefore reasonable to suggest that vitamin D deficiency reduces bone regeneration capacity, which can be overcome by vitamin D supplementation (Dvorak et al. 2012, Liu et al. 2014). Only limited data on the effect of vitamin D intake on bone regeneration are available (DelgadoMartinez et al. 1998, Fu et al. 2009). To our knowledge, so far no preclinical study was performed in jawbone. The pleiotropic hormone has endocrine, autocrine and paracrine functions. The classical target tissues are bone, intestine and kidney (Heaney 2005). The active metabolite, calcitriol, stimulates the activity of osteoclasts, and has effects on osteoblasts by increasing the production of extracellular matrix proteins. The vitamin D metabolism is an important intrinsic mechanism for promoting the coupling of bone resorption to bone formation. Cao et al. reported that administration of a vitamin D3 analogue increases the bone mineral content of callus (Cao et al. 2007). Further there is evidence for enhanced bone regeneration by local administration of 1,25(OH)D (Takano-Yamamoto et al. 1992, Kawakami & Takano-Yamamoto 2004). Also scaffolds with incorporated vitamin D were more effective in bone regeneration (Gogolewski et al. 2006, Yoon et al. 2007) by accelerating infiltration of osteoprogenitor cells. Although, these data provide first insights into the potential benefit of vitamin D supplementation it is unknown so far whether there is a similar impact in vitamin D deficiency. In particular, the therapeutic potential of local calcitriol treatment on the process of alveolar bone regeneration under vitamin D deficiency is unclear. Material and Methods Animals
The animals were treated according to the guidelines of animal care. The study protocol was approved by the ethical review board for animal research of the Medical University Vienna (6609/266-II/3b/2011).
Sixty adult male Sprague Dawley rats (Division for Laboratory Animal Science and Genetics Himberg, Austria) were assigned to three groups. Animals in the vitamin D deficient groups (n = 40) were fed a vitamin D deficient diet (Rodent diet TD 89123, ssniff GmbH, Soest, Germany; 0.47% Ca, and 0.3% P). Animals in the control group (n = 20) were fed with the corresponding diet containing 2.400 IU/kg vitamin D and 1.35% Ca, 1.0% P. After 4 weeks of housing a standardized defect in the maxilla and in the mandible was created (Fig. 1). The three groups remained on their diet until they were sacrificed. Body weight was monitored weekly. Surgery
The animals were pre-medicated with ketamine hydrochloride and xylazine (Ketavetâ and Rompunâ, Bayer, Germany). Lidocaine with adrenaline (0.3 ml; Xylocaine, AstraZeneca, London, GB) was used for local anaesthesia of the oral mucosa. A 1.5 cm incision was made in the transitional zone of attached to free oral mucosa, muscle and maxillary periosteum was elevated. The maxilla and zygomatic bone were completely undermined, similarly the diastema in the mandibular bone. A highspeed rosehead burr with constant irrigation was used to create a unilateral bone defect in the left maxillary and mandibular diastema. The dimensions (2 mm in depth, 2 mm in diameter) of the hemispheric defects were controlled with a calliper. Then the drill holes were filled with a 2 9 2 mm native, absorbable collagen fleece (Lyostyptâ B. Braun Melsungen AG, Melsungen, Germany) soaked with either 40 ll dimethyl sulfoxide (DMSO) in control and deficiency group or 40 ll 10 9 mol/l 1,25(OH)2D3 (calcitriol) in DMSO in calcitriol group. Wound edges were sutured with absorbable, synthetic, braided suture (Vicrylâ, Ethicon, Switzerland). Piritramide (Dipidolorâ, Janssen-Cilag Pharma GmbH, Vienna, Austria) was administered for pain relief. Serum chemistry measurement
Approximately, 1–2 ml of blood was extracted at the time of sacrifice by
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
97
intracardial puncture. Serum levels of calcidiol (25(OH)D) and parathyroid hormone (PTH) were determined using a corresponding ELISA kit (25-OH Vitamin D, Immundiagnostik AG, Bensheim, Germany; PTH 1-84 intact rat, Immutopics International, San Clemente, CA, USA) according to the protocol of the manufacturer. Biopsy and Preparation of Histological Sections
The defect bearing maxillae and mandibles were excised after one and 3 weeks, respectively and fixed in 4% neutral-buffered formalin. To prepare undecalcified, thin ground sections the specimens were dehydrated in ascending grades of ethanol and embedded in light-curing resin. Using the cutting-grinding technique by Donath (1988) sections approx. 30 lm thick were produced and stained with Levai Laczko dye. For each trauma one ground section in bucco-lingual direction was available (Fig. 1). The histological slides were scanned using a digital virtual microscopy system (Olympus dotSlide 2.4, Olympus, Tokyo, Japan) resulting in images with a resolution of 0.32 lm per pixel. Histomorphometric analysis
The surgical defect was manually demarcated as the region of interest (ROI) using Adobe Photoshop CS5 Extended (Adobe Systems Inc., San Jose, USA) (Fig. 1). This preselected region was subdivided into homogenous smaller objects by combining neighbouring pixels using multi-resolution segmentation with a histomorphometry software (Definiens Developer XD 2.0, Munich, Germany). These resulting objects were classified, merged and re-segmented in multiple iterations as bone tissue or void, depending on colour, size and relations to neighbouring objects. The classification was manually corrected and followed by area measurements of new bone, void and total defect size. New mineralized Bone Area per Tissue Area (newBAr/TAr) was calculated as the area of newly formed bone divided by the area of the total defect. Additionally, the depth of the resorption spaces was measured to
98
F€ ugl et al.
(a)
(b)
(c)
(d)
Fig. 1. Example of a micro computed tomography with the defect in the mandible (a) and the thin ground section with the new bone in the region of interest (b), the surgical defect area (c) (drawn in red) and a schematic presentation how the measurements of the maximum resorption depth was performed (d).
(a)
(b)
Fig 2. (a) BoxPlot indicating the serum levels of 25(OH)D (calcidiol) in nmol/l after 7 and 21 days in control, calcitriol and vitamin D deficiency group. *p < 0.05. (b) BoxPlot indicating the serum levels of PTH in pg/ml after 7 and 21 days in control, calcitriol and vitamin D deficiency group. *p < 0.05.
evaluate the strength and extend of resorptive processes. For this purpose, the point of deepest resorption extending from the surface of the semicircular defect border into the surrounding autochthonous bone was identified by histologic examination. It could be easily recognized by the presence of osteoclasts and/or Howships lacunae. The shortest
direct distance to the contour of the drill defect was measured in lm using the Olyvia software (Olympus Inc. Tokyo, Japan). Micro computed tomography
For the micro computed tomography data, Mineralized Bone Volume per Tissue Volume (BV/TV) within
the defect was calculated as bone divided by the sum of bone and void. Micro computed tomography (lCT) of each specimen was performed by vivaCT75 (SCANCO Medical AG, Br€ uttisellen, Switzerland) (Fig. 1). Scanning was performed at 70 kV/114 lA with a resolution of 20.5 lm and an integration time of 300 ms. A grey value
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Vitamin D and alveolar bone regeneration threshold of 1077 Hounsfield units was used to characterize bone tissue and to distinguish it from the surrounding soft tissues. After performing this tissue classification, the volume of bone (BV) and the volume of the total defect (TV) were measured in mm3 using the Definiens Developer XD 2.0 software (Definiens, Munich, Germany). Statistics
Sample sized calculations were based on a significance level of 0.05 and a desired power of 80%, assuming standard deviation of eight and a clinically significant group difference of 11. These calculations resulted in a sample size of nine rats, assuming a drop out of one animal in each group we selected a group size of 10 animals. Visual diagnostics suggested assuming normality was not valid. Descriptive Statistics including median and interquartile range (IQR) for all variables are presented in box plots for newBAr/TAr, BV/ TV from CT data and maximum resorption depth (mrd) as well as Vitamin D and PTH serum levels (Figs 2 and 3). Since almost all values for newBAr/TAr (histomorphometry and micro CT data) after 7 days were zero or close to zero, only data resulting from measurements after 21 days were considered for inference. Assuming normality was not valid, a non-parametric approach was chosen. In order to test for differences in treatment groups, Kruskal–Wallis rank sum tests were performed for mandible and maxilla separately. In case of significance multiple Wilcoxon rank sum tests were calculated to determine differences between treatment groups. Spearman’s correlation coefficient between Vitamin D and all of newBAr/TAr and maximum resorption depth was calculated; additionally adjusted percentile bootstrap 95% intervals were given (Davison & Hinkley 1997). Holm’s method was used to adjust for multiple testing, resulting p-values 0.05) (Table 1). Nevertheless, there was a negative correlation between the maximum resorption depth and serum vitamin D levels in the mandible (r = 0.47, CI 0.72; 0.11). (Table 1, Fig 3c). Discussion
Fig. 4. a, b, c The defect area and new bone in control (a), calcitriol (b) and deficiency group (c) after 21 days.
21.7% (IQR 13.0) in the maxilla. Vitamin D deficiency group had a median of 34.4% (IQR 27.0), 55.1% (IQR 26.2) in mandible and 31.4% (IQR 9.3) in the maxilla. New bone volume ratio was significantly higher in vitamin D deficiency groups with and without local calcitriol application in both jaws (Kruskal–Wallis mandible p = 0.003, maxilla p < 0.001) (Table 1, Fig. 3a). Histomorphometry There was no statistically significant difference in defect dimensions between the groups. (Kruskal–Wallis p = 0.51 for the mandible and p = 0.67 for maxilla). Mean tissue area in the control group was 1.31 mm2 in the mandible and 1.79 mm2 in the maxilla respectively, calcitriol group showed 1.68 mm2 in the mandible and 1.29 mm2 in the maxilla, and vitamin D deficiency group 2.02 mm2 in the mandible and 1.46 mm2 in the maxilla.
New mineralized bone area
After 7 days new bone area was negligible in all three groups, while after 21 days control group showed a median percentage of 2.1% (IQR 14.9) with 14.9% (IQR 16.4) in the mandible and 0.5% (IQR 1.8) in the maxilla, vitamin D application group 15.9% (IQR 16.7) with 24.8% (IQR 4.1) in the mandible and 13.9% (IQR 7.6) in the maxilla and vitamin D deficiency group 17.5% (IQR 20.8) with 30.3% (IQR 13.6) in the mandible and 10.5% (IQR 7.5) in the maxilla (Table 1, Fig 3b). There was a significant difference between controls and vitamin D deficiency groups in the maxilla (Kruskal–Wallis p = 0.035). Between control and vitamin D application animals (Wilcoxon p = 0.048) as well as vitamin D deficiency animals without local application (Wilcoxon p = 0.023). While in the mandible the new bone area did not show any difference in between groups (Kruskal–Wallis p = 0.18) (Table 1, Fig 3b).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
The preliminary results indicate no impairment of vitamin D deficiency on alveolar bone regeneration and no significant influence of topic vitamin D application on the early healing process. Surprisingly, vitamin D deficiency showed a beneficial impact on alveolar bone regeneration. Rodent models have been used to investigate the role of bone-inductive agents on alveolar bone regeneration. Nguyen et al. established a critical-sized alveolar defect model in rats and determined the ossification rate at 4 to 12 weeks. The highest rate of ossification occurred after 2 to 4 weeks (Raposo-Amaral et al. 2010), based on the study of Nguyen et al. suggesting that 4 weeks would be a plateau of bone formation (Nguyen et al. 2009). Therefore, a maximum of 4 weeks should be a satisfactory timing to assess the potential of bone-inductive agents. Hence, in this study we examined the early onset of bone regeneration after one and 3 weeks. Nevertheless biomaterials, such as collagen, may delay hard tissue modelling (Lindhe et al. 2013) and probably therefore the data after 7 days showed mainly resorptive activity and not enough bone formation to be sufficiently studied. Further many studies induce vitamin D deficiency state by a longer depletion period of 10– 12 weeks (Anderson et al. 2007, 2010), yet previous studies have
102
F€ ugl et al.
Table 1. Median (IQR) for Vitamin D and PTH, newBAr/TAr of the histomorphometric measurements, BV/TV according to lCT and maximum resorption depth (mrd) 7 days Control Vitamin D (nmol/l) PTH (nmol/l) BV/TV (%) Total Mandible Maxilla newBAr/TAr (%) Total Mandible Maxilla mrd (lm) Total Mandible Maxilla
80.59 39.17 0.5 0.6 0.4
Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats F€ ugl A, Gruber R, Agis H, Lzicar H, Keibl C, Schwarze UY, Dvorak G. Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats. J Clin Periodontol 2015; 42: 96–103. doi: 10.1111/jcpe.12342.
Abstract Aim: Vitamin D deficiency is considered to diminish bone regeneration. Yet, raising the serum levels takes months. A topic application of the active vitamin D metabolite, calcitriol, may be an effective approach. Thus, it becomes important to know the effect of vitamin D deficiency and local application on alveolar bone regeneration. Material and Methods: Sixty rats were divided into three groups; two vitamin depletion groups and a control group. Identical single defects (2 mm diameter) were created in the maxilla and mandible treated with calcitriol soaked collagen in one deficiency group while in the other two groups not. Histomorphometric analysis and micro CTs were performed after 1 and 3 weeks. Serum levels of 25 (OH)D3 and PTH were determined. Results: Bone formation rate significantly increased within the observation period in all groups. Bone regeneration was higher in the maxilla than in the mandible. However, bone regeneration was lower in the control group compared to vitamin depletion groups, with no significant effects by local administration of calcitriol (micro CT mandible p = 0.003, maxilla p < 0.001; histomorphometry maxilla p = 0.035, mandible p = 0.18). Conclusion: Vitamin D deficiency not necessarily impairs bone regeneration in the rat jaw and a single local calcitriol application does not enhance healing.
Vitamin D deficiency results from inadequate dietary intake together with insufficient exposure to sunlight or is related to inadequate synthesis by liver and kidney. Although there Conflict of interest and source of funding statement This project was funded by the ITI Research Grant 813_2012. Authors disclose any conflict of interest.
96
is no consensus on optimal levels, a serum level of 25(OH)D (calcidiol) below 20 ng/ml (50 nmol/l) is indicative for vitamin D deficiency, while a level of 30 ng/ml (75 nmol/l) or greater can be considered to indicate sufficient vitamin D and levels in between are considered as insufficiency. An estimated one billion people worldwide have vitamin D deficiency with a prevalence of 40 to 100% of the elderly in the U.S. and
€ gl1,2, Reinhard Alexander Fu Gruber1,2,3, Hermann Agis2,4, Hannah Lzicar2,5, Claudia Keibl2,6, Uwe Yacine Schwarze2,5 and Gabriella Dvorak1,2 1
Department of Oral Surgery, Medical University of Vienna, Vienna, Austria, 2 Austrian Cluster for Tissue Regeneration, Vienna, Austria, 3Laboratory of Oral Biology, School of Dental Medicine, University of Bern, Bern, Switzerland, 4Department of Conservative Dentistry and Periodontology, Medical University of Vienna, Vienna, Austria, 5 Karl Donath Laboratory for Hard Tissue and Biomaterial Research, Medical University of Vienna, Vienna, Austria, 6Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria
Key words: 1,25(OH)D3; bone healing; calcitriol; supplementation; topic administration; vitamin D deficiency Accepted for publication 25 November 2014
Europe (Holick 2007). Without vitamin D, dietary calcium and phosphorus adsorption is severely impaired. Because of the chronic release of calcium and phosphorus, vitamin D deficiency causes a catabolic bone turnover. Vitamin D deficiency has also been linked with impaired fracture healing and osseointegration (Kelly et al. 2009, Dvorak et al. 2012) as vitamin D accumulates in the fracture
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Vitamin D and alveolar bone regeneration callus (Jingushi et al. 1998, Fu et al. 2009). Together, these results support the steroid hormones role in controlling bone regeneration. It is therefore reasonable to suggest that vitamin D deficiency reduces bone regeneration capacity, which can be overcome by vitamin D supplementation (Dvorak et al. 2012, Liu et al. 2014). Only limited data on the effect of vitamin D intake on bone regeneration are available (DelgadoMartinez et al. 1998, Fu et al. 2009). To our knowledge, so far no preclinical study was performed in jawbone. The pleiotropic hormone has endocrine, autocrine and paracrine functions. The classical target tissues are bone, intestine and kidney (Heaney 2005). The active metabolite, calcitriol, stimulates the activity of osteoclasts, and has effects on osteoblasts by increasing the production of extracellular matrix proteins. The vitamin D metabolism is an important intrinsic mechanism for promoting the coupling of bone resorption to bone formation. Cao et al. reported that administration of a vitamin D3 analogue increases the bone mineral content of callus (Cao et al. 2007). Further there is evidence for enhanced bone regeneration by local administration of 1,25(OH)D (Takano-Yamamoto et al. 1992, Kawakami & Takano-Yamamoto 2004). Also scaffolds with incorporated vitamin D were more effective in bone regeneration (Gogolewski et al. 2006, Yoon et al. 2007) by accelerating infiltration of osteoprogenitor cells. Although, these data provide first insights into the potential benefit of vitamin D supplementation it is unknown so far whether there is a similar impact in vitamin D deficiency. In particular, the therapeutic potential of local calcitriol treatment on the process of alveolar bone regeneration under vitamin D deficiency is unclear. Material and Methods Animals
The animals were treated according to the guidelines of animal care. The study protocol was approved by the ethical review board for animal research of the Medical University Vienna (6609/266-II/3b/2011).
Sixty adult male Sprague Dawley rats (Division for Laboratory Animal Science and Genetics Himberg, Austria) were assigned to three groups. Animals in the vitamin D deficient groups (n = 40) were fed a vitamin D deficient diet (Rodent diet TD 89123, ssniff GmbH, Soest, Germany; 0.47% Ca, and 0.3% P). Animals in the control group (n = 20) were fed with the corresponding diet containing 2.400 IU/kg vitamin D and 1.35% Ca, 1.0% P. After 4 weeks of housing a standardized defect in the maxilla and in the mandible was created (Fig. 1). The three groups remained on their diet until they were sacrificed. Body weight was monitored weekly. Surgery
The animals were pre-medicated with ketamine hydrochloride and xylazine (Ketavetâ and Rompunâ, Bayer, Germany). Lidocaine with adrenaline (0.3 ml; Xylocaine, AstraZeneca, London, GB) was used for local anaesthesia of the oral mucosa. A 1.5 cm incision was made in the transitional zone of attached to free oral mucosa, muscle and maxillary periosteum was elevated. The maxilla and zygomatic bone were completely undermined, similarly the diastema in the mandibular bone. A highspeed rosehead burr with constant irrigation was used to create a unilateral bone defect in the left maxillary and mandibular diastema. The dimensions (2 mm in depth, 2 mm in diameter) of the hemispheric defects were controlled with a calliper. Then the drill holes were filled with a 2 9 2 mm native, absorbable collagen fleece (Lyostyptâ B. Braun Melsungen AG, Melsungen, Germany) soaked with either 40 ll dimethyl sulfoxide (DMSO) in control and deficiency group or 40 ll 10 9 mol/l 1,25(OH)2D3 (calcitriol) in DMSO in calcitriol group. Wound edges were sutured with absorbable, synthetic, braided suture (Vicrylâ, Ethicon, Switzerland). Piritramide (Dipidolorâ, Janssen-Cilag Pharma GmbH, Vienna, Austria) was administered for pain relief. Serum chemistry measurement
Approximately, 1–2 ml of blood was extracted at the time of sacrifice by
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
97
intracardial puncture. Serum levels of calcidiol (25(OH)D) and parathyroid hormone (PTH) were determined using a corresponding ELISA kit (25-OH Vitamin D, Immundiagnostik AG, Bensheim, Germany; PTH 1-84 intact rat, Immutopics International, San Clemente, CA, USA) according to the protocol of the manufacturer. Biopsy and Preparation of Histological Sections
The defect bearing maxillae and mandibles were excised after one and 3 weeks, respectively and fixed in 4% neutral-buffered formalin. To prepare undecalcified, thin ground sections the specimens were dehydrated in ascending grades of ethanol and embedded in light-curing resin. Using the cutting-grinding technique by Donath (1988) sections approx. 30 lm thick were produced and stained with Levai Laczko dye. For each trauma one ground section in bucco-lingual direction was available (Fig. 1). The histological slides were scanned using a digital virtual microscopy system (Olympus dotSlide 2.4, Olympus, Tokyo, Japan) resulting in images with a resolution of 0.32 lm per pixel. Histomorphometric analysis
The surgical defect was manually demarcated as the region of interest (ROI) using Adobe Photoshop CS5 Extended (Adobe Systems Inc., San Jose, USA) (Fig. 1). This preselected region was subdivided into homogenous smaller objects by combining neighbouring pixels using multi-resolution segmentation with a histomorphometry software (Definiens Developer XD 2.0, Munich, Germany). These resulting objects were classified, merged and re-segmented in multiple iterations as bone tissue or void, depending on colour, size and relations to neighbouring objects. The classification was manually corrected and followed by area measurements of new bone, void and total defect size. New mineralized Bone Area per Tissue Area (newBAr/TAr) was calculated as the area of newly formed bone divided by the area of the total defect. Additionally, the depth of the resorption spaces was measured to
98
F€ ugl et al.
(a)
(b)
(c)
(d)
Fig. 1. Example of a micro computed tomography with the defect in the mandible (a) and the thin ground section with the new bone in the region of interest (b), the surgical defect area (c) (drawn in red) and a schematic presentation how the measurements of the maximum resorption depth was performed (d).
(a)
(b)
Fig 2. (a) BoxPlot indicating the serum levels of 25(OH)D (calcidiol) in nmol/l after 7 and 21 days in control, calcitriol and vitamin D deficiency group. *p < 0.05. (b) BoxPlot indicating the serum levels of PTH in pg/ml after 7 and 21 days in control, calcitriol and vitamin D deficiency group. *p < 0.05.
evaluate the strength and extend of resorptive processes. For this purpose, the point of deepest resorption extending from the surface of the semicircular defect border into the surrounding autochthonous bone was identified by histologic examination. It could be easily recognized by the presence of osteoclasts and/or Howships lacunae. The shortest
direct distance to the contour of the drill defect was measured in lm using the Olyvia software (Olympus Inc. Tokyo, Japan). Micro computed tomography
For the micro computed tomography data, Mineralized Bone Volume per Tissue Volume (BV/TV) within
the defect was calculated as bone divided by the sum of bone and void. Micro computed tomography (lCT) of each specimen was performed by vivaCT75 (SCANCO Medical AG, Br€ uttisellen, Switzerland) (Fig. 1). Scanning was performed at 70 kV/114 lA with a resolution of 20.5 lm and an integration time of 300 ms. A grey value
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Vitamin D and alveolar bone regeneration threshold of 1077 Hounsfield units was used to characterize bone tissue and to distinguish it from the surrounding soft tissues. After performing this tissue classification, the volume of bone (BV) and the volume of the total defect (TV) were measured in mm3 using the Definiens Developer XD 2.0 software (Definiens, Munich, Germany). Statistics
Sample sized calculations were based on a significance level of 0.05 and a desired power of 80%, assuming standard deviation of eight and a clinically significant group difference of 11. These calculations resulted in a sample size of nine rats, assuming a drop out of one animal in each group we selected a group size of 10 animals. Visual diagnostics suggested assuming normality was not valid. Descriptive Statistics including median and interquartile range (IQR) for all variables are presented in box plots for newBAr/TAr, BV/ TV from CT data and maximum resorption depth (mrd) as well as Vitamin D and PTH serum levels (Figs 2 and 3). Since almost all values for newBAr/TAr (histomorphometry and micro CT data) after 7 days were zero or close to zero, only data resulting from measurements after 21 days were considered for inference. Assuming normality was not valid, a non-parametric approach was chosen. In order to test for differences in treatment groups, Kruskal–Wallis rank sum tests were performed for mandible and maxilla separately. In case of significance multiple Wilcoxon rank sum tests were calculated to determine differences between treatment groups. Spearman’s correlation coefficient between Vitamin D and all of newBAr/TAr and maximum resorption depth was calculated; additionally adjusted percentile bootstrap 95% intervals were given (Davison & Hinkley 1997). Holm’s method was used to adjust for multiple testing, resulting p-values 0.05) (Table 1). Nevertheless, there was a negative correlation between the maximum resorption depth and serum vitamin D levels in the mandible (r = 0.47, CI 0.72; 0.11). (Table 1, Fig 3c). Discussion
Fig. 4. a, b, c The defect area and new bone in control (a), calcitriol (b) and deficiency group (c) after 21 days.
21.7% (IQR 13.0) in the maxilla. Vitamin D deficiency group had a median of 34.4% (IQR 27.0), 55.1% (IQR 26.2) in mandible and 31.4% (IQR 9.3) in the maxilla. New bone volume ratio was significantly higher in vitamin D deficiency groups with and without local calcitriol application in both jaws (Kruskal–Wallis mandible p = 0.003, maxilla p < 0.001) (Table 1, Fig. 3a). Histomorphometry There was no statistically significant difference in defect dimensions between the groups. (Kruskal–Wallis p = 0.51 for the mandible and p = 0.67 for maxilla). Mean tissue area in the control group was 1.31 mm2 in the mandible and 1.79 mm2 in the maxilla respectively, calcitriol group showed 1.68 mm2 in the mandible and 1.29 mm2 in the maxilla, and vitamin D deficiency group 2.02 mm2 in the mandible and 1.46 mm2 in the maxilla.
New mineralized bone area
After 7 days new bone area was negligible in all three groups, while after 21 days control group showed a median percentage of 2.1% (IQR 14.9) with 14.9% (IQR 16.4) in the mandible and 0.5% (IQR 1.8) in the maxilla, vitamin D application group 15.9% (IQR 16.7) with 24.8% (IQR 4.1) in the mandible and 13.9% (IQR 7.6) in the maxilla and vitamin D deficiency group 17.5% (IQR 20.8) with 30.3% (IQR 13.6) in the mandible and 10.5% (IQR 7.5) in the maxilla (Table 1, Fig 3b). There was a significant difference between controls and vitamin D deficiency groups in the maxilla (Kruskal–Wallis p = 0.035). Between control and vitamin D application animals (Wilcoxon p = 0.048) as well as vitamin D deficiency animals without local application (Wilcoxon p = 0.023). While in the mandible the new bone area did not show any difference in between groups (Kruskal–Wallis p = 0.18) (Table 1, Fig 3b).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
The preliminary results indicate no impairment of vitamin D deficiency on alveolar bone regeneration and no significant influence of topic vitamin D application on the early healing process. Surprisingly, vitamin D deficiency showed a beneficial impact on alveolar bone regeneration. Rodent models have been used to investigate the role of bone-inductive agents on alveolar bone regeneration. Nguyen et al. established a critical-sized alveolar defect model in rats and determined the ossification rate at 4 to 12 weeks. The highest rate of ossification occurred after 2 to 4 weeks (Raposo-Amaral et al. 2010), based on the study of Nguyen et al. suggesting that 4 weeks would be a plateau of bone formation (Nguyen et al. 2009). Therefore, a maximum of 4 weeks should be a satisfactory timing to assess the potential of bone-inductive agents. Hence, in this study we examined the early onset of bone regeneration after one and 3 weeks. Nevertheless biomaterials, such as collagen, may delay hard tissue modelling (Lindhe et al. 2013) and probably therefore the data after 7 days showed mainly resorptive activity and not enough bone formation to be sufficiently studied. Further many studies induce vitamin D deficiency state by a longer depletion period of 10– 12 weeks (Anderson et al. 2007, 2010), yet previous studies have
102
F€ ugl et al.
Table 1. Median (IQR) for Vitamin D and PTH, newBAr/TAr of the histomorphometric measurements, BV/TV according to lCT and maximum resorption depth (mrd) 7 days Control Vitamin D (nmol/l) PTH (nmol/l) BV/TV (%) Total Mandible Maxilla newBAr/TAr (%) Total Mandible Maxilla mrd (lm) Total Mandible Maxilla
80.59 39.17 0.5 0.6 0.4
