The aim of this study was to evaluate the effects of local administration of human amniotic fluid (HAF) on newly formed bone obtained by mandibular distraction osteogenesis (DO) with histomorphometry. A unilateral mandibular osteotomy at the left corpus was performed in 32 adult male rabbits. After a 5-day latency period, the left mandibles were lengthened by mandibular DO over 5 days, at a rate of 1 mm/day, via a custom-made distractor. After the distraction, the rabbits were divided randomly into four groups: 0.3 ml HAF was injected into the distraction gap followed by 21 (group 1) or 45 (group 2) days of consolidation; or 0.3 ml normal saline (NS) was administered followed by 21 (group 3) or 45 (group 4) days of consolidation. Mandibles were removed at the end of the consolidation period and investigated histomorphometrically. The newly formed bone area (NFBA) and number of fibroblasts increased significantly in the HAF groups compared to the NS groups (NFBA: group 1 vs. group 3, P < 0.05; group 2 vs. group 4, P < 0.01; fibroblasts: group 1 vs. group 3, and group 2 vs. group 4, P < 0.05), and also in both 45-day consolidation groups compared to the 21-day consolidation groups (NFBA: group 1 vs. group 2, and group 3 vs. group 4, P < 0.001; fibroblasts: group 1 vs. group 2, and group 3 vs. group 4, P < 0.01). Additionally, the numbers of osteoblasts and capillaries were increased significantly at 45 days of consolidation compared to 21 days in both the HAF and NS groups (osteoblasts: group 1 vs. group 2, P < 0.01; group 3 vs. group 4, P < 0.05; capillaries: group 1 vs. group 2, and group 3 vs. group 4, P < 0.01). Histomorphometric analysis demonstrated that local HAF administration effectively accelerated bone formation. Thus, a HAF injection procedure could improve new bone formation around the bone in maxillofacial operations such as DO.
Distraction osteogenesis (DO), used widely in the treatment of oral and maxillofacial deformities or deficiencies, is a biological procedure that produces new bone formation via gradually separated bone segments, using an external lengthener. Traditional surgical techniques for skeletal expansion include osteotomies, acute movements of variable magnitude, and the necessity for bone grafts. Problems include donor site morbidity, unpredictable graft resorption, and the risk of relapse because of soft tissue resistance to large skeletal movements. Many of these limitations can be avoided with the use of DO to lengthen or expand the skeleton. The benefits of DO include the minimally invasive nature of the procedure, the ability to achieve movements of great magnitude without the need for a bone graft, and the elimination of donor site morbidity. The main advantage of the technique is that the new bone forms together with elongation of the surrounding soft tissue envelope. In addition, concurrent soft tissue histogenesis may decrease the relapse.
Although this technique is used to treat several abnormalities of hard and soft tissues, there are some drawbacks, particularly related to long-term consolidation periods. To accelerate the maturation of the regenerated bone, numerous approaches including growth factors, calcitonin, calcium sulphate, bisphosphonates, and electronic and ultrasonic stimulation have been researched.
Several growth-promoting factors have been identified after the manifestation of bone defects including those caused by injuries, fractures, and DO; these include platelet-derived growth factor, transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), interleukin (IL)-1, and IL-6. FGF has angiogenic properties and mitogenic activity on the osteoblast lineage. A rich content of growth and trophic factors such as epidermal growth factor (EGF), FGF, and insulin-like growth factors I and II (IGF-I and IGF-II), which are critical for development, have been identified in human amniotic fluid (HAF). Additionally, hyaluronic acid (HA), hyaluronic acid stimulating activator (HASA), chondroitin-4- and -6-sulphate, dermatan sulphate, and heparan sulphate have been identified in HAF.
HAF, generally obtained by amniocentesis during the second trimester of gestation, contains high molecular weight HA and HASA in high concentrations. HA has been shown to increase osteoblastic bone formation in vitro, through increased mesenchymal cell differentiation and migration. Also, HASA has been shown to stimulate and increase the production of endogenous HA. Thus, HAF may increase both endogenous and exogenous HA around the region of application.
Several studies have investigated the effect of HAF on cell differentiation. HAF has been reported to enhance new cartilage, bone formation, and nerve and tendon healing. However, new bone regeneration in the distraction gap after DO of the mandible has not been documented, and there are no data in the literature regarding the acceleration of bone regeneration in the distraction gap with the use of HAF. We hypothesized that HAF would have a positive stimulating effect on bone formation after DO. Hence, the aim of this study was to investigate the effects of HAF, collected between weeks 16 and 24 of gestation, on the acceleration of new bone formation in animal subjects undergoing mandibular DO.
Materials and methods
All procedures were performed in the experimental animal breeding and research centre of the military medical academy. Approval was obtained from the institutional ethics committee and local clinical research ethics committee for the animal research and use of HAF.
Subjects and surgery
The study subjects were 32 adult male New Zealand White rabbits of the same age with an average weight of 2.9 kg (range 2.2–3.5 kg). They had free access to a standard pellet diet and tap water and were adapted to a 12:12-h light–dark cycle in separate cages.
All animals were operated on under general anaesthesia. The rabbits were anaesthetized with a combination of xylazine hydrochloride (5 mg/kg) (Alfazyne 2%; Ege Vet, Izmir, Turkey) and ketamine hydrochloride (50 mg/kg) (Alfamine 10%; Ege Vet, Izmir, Turkey) before the application of distractors. Isoflurane inhalation anaesthetic (10 mg/kg) was used before the various procedures.
After sterilization of all surgical equipment, the left mandible was shaved and disinfected with iodine. After surgical preparation of the experimental side of the mandible, a 2–2.5-cm long submandibular skin incision was made. The subcutaneous tissues were exposed by careful dissection down to the periosteum and the bone was exposed with a periosteal incision. Using a reciprocating saw, a vertical corticotomy line was outlined between the premolars and extended to the inferior mandibular border under saline irrigation. The extraoral bone-borne distraction device used consisted of a 7-mm hyrax expansion screw (Dentaurum GmbH & Co., Ispringen, Germany) with three holes; the retention legs were bent for bone attachment. The distractor device was fixed with two posterior (7 mm) and one anterior (9 mm) titanium screws. The bone cut was completed using a thin osteotome through the vertical corticotomy line, and mobilization of the bone fragments was achieved. Activation of the distraction device was tested. The gap between the bone fragments was narrowed by reverse-directed activation of the distractor. The periosteal flaps were repositioned and closed with 4–0 Vicryl sutures ( Fig. 1 ).
After closure of the mandible, the rabbit was awoken from general anaesthesia and allowed to recover. An Elizabethan collar was placed around the rabbit’s neck to protect the device from dislodgement. A subcutaneous bolus of 30 ml normal saline (NS) was given every 8 h during the first 24–48 h depending on the rabbit’s water intake. After the rabbit had recovered fully as per the facility criteria, it was returned to its cage and placed on a soft diet. Prophylactic antibiotics (Colicillin 0.1 ml/kg (100 mg/ml ampicillin + 250,000 IU/ml colistin sulphate); Ege Vet, Izmir, Turkey) were administered prior to surgery to prevent any infection that could have resulted from the trauma caused during surgery, and were continued every 12 h for the next 48 h.
Distraction protocol, administration, and groups
HAF samples were obtained by diagnostic amniocentesis from normal pregnant women in the second trimester of pregnancy, between weeks 16 and 24 of gestation, under sterile conditions. Samples were then centrifuged at high speeds; the serum was collected and used without being stored. Oral consent was obtained from each woman before HAF collection.
After a latency period of 5 days, distraction was started at a rate of 0.5 mm twice a day for 5 days. The animals were divided randomly into four equal groups consisting of eight rabbits per group. The experimental groups (groups 1 and 2) were treated with a single dose of 0.3 ml HAF, and the control groups (groups 3 and 4) were treated with single dose of 0.3 ml NS solution. The HAF or NS was injected into the distraction gap with a micro-syringe (Hamilton Injection Syringe; Hamilton Company, Reno, NV, USA) at the end of the distraction process. The group protocols were as follows: group 1, 0.3 ml local HAF administered and sacrifice at day 21 of consolidation; group 2, 0.3 ml local HAF administered and sacrifice at day 45 of consolidation; group 3, 0.3 ml local NS administered and sacrifice at day 21 of consolidation; and group 4, 0.3 ml local NS administered and sacrifice at day 45 of consolidation. After the consolidation period, all animals were sacrificed by injection of 200 mg/kg sodium thiopental (Pentothal; Abbott Laboratories, Abbott Park, IL, USA). The mandibles were dissected subperiosteally and fixed in a 10% formalin solution for histomorphometric evaluation.
In order to investigate the distraction process and the newly formed bone area (NFBA) at the distraction gap, serial cone beam computed tomography (CBCT) scans were obtained for all rabbits at the time of placement of the distractor, at the end of the distraction period, and at the end of the consolidation period (after 21 or 45 days) postoperatively. A 3D Accuitomo 170 (J. Morita Manufacturing Corp., Kyoto, Japan) was used, with a voxel size of 0.08 mm (field of view (FOV) 170 × 120 mm). The tube settings were set to 65 kV, 2.0 mA, and an exposure time of 30 s. After scanning, the CBCT datasets were transferred to an independent computer workstation with SimPlant–OMS software (Materialise Dental, Leuven, Belgium), and three-dimensional (3D) virtual models were generated for macroscopic comparisons between groups. The same investigator evaluated all CBCT images and generated 3D models at the medical design and manufacturing centre.
The histomorphometric evaluation was carried out in the pathology department. All materials were fixed in 10% buffered paraformaldehyde for 48 h then decalcified in ethylenediaminetetraacetic acid (EDTA) solutions. Tissue specimens were prepared, embedded in paraffin, and sectioned with a microtome. The sections were stained with haematoxylin and eosin. Stained specimens were investigated under a Nikon Eclipse E400 light microscope (Nikon, Tokyo, Japan). For each specimen, the same area was photographed after staining using a Nikon Coolpix 5000 camera attachment (Nikon, Tokyo, Japan). A photograph of the Nikon micrometer microscope slide (MBM11100 Stage Micrometer Type A) was also obtained during the procedure. All photographs were then transferred to a PC and analyzed by Clemex Vision Lite 3.5 image analysis programme (Clemex Technologies Inc., Longueuil, Quebec, Canada). The length was calibrated by comparing the photograph of the specimen with the photograph of the Nikon micrometer microscope slide, which was obtained at the same magnification. A 0.5-mm 2 area was designated using the Clemex Vision Lite 3.5 image analysis programme, and osteoblasts, osteoclasts, fibroblasts, and vessels were marked with the same image analysis programme in an area of 445,928.3 mm 2 . Damaged cells were not evaluated. The marked cells were counted automatically with the same image analysis programme. The NFBA regions per unit area were measured with the same image analysis programme in an area of 445,928.3 mm 2 . The reader was blinded to the origin of the specimen ( Fig. 2 ).
The sample size for each group was calculated and based on the estimated power of the study (=0.90) according to the effect size (=0.65), with a significance level of α = 0.05 and β = 0.20. The sample size calculations showed eight rabbits in each group to be sufficient. So, each group consisted of eight rabbits. All statistical evaluations were performed with SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Results of the evaluation were given as the mean ± standard deviation (SD), median, minimum, and maximum. The Kolmogorov–Smirnov test was used at baseline to determine whether all the values were normally distributed. The NFBA data had a normal distribution; thus the independent samples t -test was used for the comparison of NFBA values between the groups. Differences amongst the four groups with regard to the numbers of osteoblasts, osteoclasts, fibroblasts, and capillaries were evaluated using the Mann–Whitney U -test. A value of P < 0.05 was considered as statistically significant.
During the study period, no serious weight loss was seen in any of the rabbits. No deep mucosal infection, dehiscence, or other adverse effects were observed in most of the animals. However, due to a faulty distractor device and infection, two rabbits in the NS control group and one rabbit in the HAF group were excluded from the study and substituted with new ones.
Bone lengthening in the range of 4–7 mm (mean 4.2 mm) was achieved in the mandible of all animals. Laterognathia and malocclusion were observed in all subjects due to the unilateral lengthening of the mandible. However, this malformation did not seem to affect the rabbits’ nutrition.
A total of 32 distracted hemimandibles were examined. Differences in the gross appearance between the groups were not evident. While all distracted mandibles revealed a fibrous tissue-filled gap and had a similar appearance to the surrounding bone tissue, the same bone tissue formation was observed in the experimental and control group animals ( Fig. 3 ).
The 3D digital models formed by the initial CBCT scans, demonstrated that the distractors were placed successfully ( Fig. 4 A) . At the end of the distraction period, on day 5, gaps were observed in the distraction region of all groups ( Fig. 4 B). At the end of the each consolidation period (21 and 45 days), callus formation had occurred in the distraction gap in all samples. The digital models revealed evidence of ossification, which was increased substantially in the experimental groups when compared with the control groups after the consolidation periods ( Fig. 4 C–F).
Histological findings indicated that bone formation was sparse in all groups and the distraction gaps were filled with intramembranous new bone and fibrovascular tissue. The results showed that HAF (groups 1 and 2) effectively accelerated the new bone formation when compared with the NS control groups ( Fig. 5 ). All parameters were compared by group and consolidation period, separately ( Tables 1 and 2 ).
|21 days||HAF (group 1)||Mean||143,924.23||37.43||2.86||32.29||3.43|
|NS (group 3)||Mean||118,885.38||32.60||2.00||27.60||2.60|
|45 days||HAF (group 2)||Mean||201,511.72||49.71||1.86||40.29||7.00|
|NS (group 4)||Mean||159,124.40||45.83||1.67||34.67||5.50|
|HAF||21 (group 1)||Mean||143,924.23||37.43||2.86||32.29||3.43|
|45 (group 2)||Mean||201,511.72||49.71||1.86||40.29||7.00|
|NS||21 (group 3)||Mean||118,885.38||32.60||2.00||27.60||2.60|
|45 (group 4)||Mean||159,124.40||45.83||1.67||34.67||5.50|