|Year : 2014 | Volume
| Issue : 1 | Page : 22-28
Influence of pre-implant bone augmentation on diving fitness: An in vitro study
Constantin von See1, Marcus Stoetzer1, Andreas Koch2, Martin Ruecker1, Nils-Claudius Gellrich1
1 Department of Oral and Maxillofacial Surgery, Hanover Medical School, Carl Neuberg Strasse 1, 30625 Hanover, Germany
2 Naval Institute of Maritime Medicine, Kiel, Germany
|Date of Web Publication||19-Apr-2014|
Constantin von See
Department of Oral and Maxillofacial Surgery, Hanover Medical School, Carl Neuberg Strasse 1, D-30625 Hanover
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Purpose: Autogenous bone grafts are commonly used for pre-implant bone augmentation or defect coverage in the maxillofacial region. Particulate bone or bone blocks without a vascular pedicle are normally used for this purpose. The objective of this study was to investigate the effects of hyperbaric conditions on the cell activity of augmented bone under pressure changes such as those to which combat divers and mine clearance divers are exposed.
Materials and Methods: Systematic studies were performed using an animal model (isogeneic Lewis rats). We collected bone chips (n = 4) from rat femurs with a Safescraper; and obtained isogeneic bone blocks (n = 4) with a bone rongeur. Bone samples were exposed to three simulated 45-min dives to 5.0 bar using oxygen or compressed air. Bone chips and blocks that were not exposed to pressure served as controls. After 9 and 15 days of in vitro cultivation, osteoblast proliferation rates were assessed in a Neubauer counting chamber and the results were statistically analyzed.
Results: Irrespective of the atmospheric conditions, bone blocks showed significantly higher proliferation rates (P = 0.05) than bone chips. When exposed to compressed air during simulated dives, both groups showed considerably lower proliferation rates than the control groups. When exposed to oxygen, both groups showed significantly higher proliferation rates than the control groups.
Conclusion: Depending on the breathing gas used, changes in atmospheric conditions during simulated dives can lead to a decrease in osteoblast activity. Patients who underwent bone augmentation should therefore be advised not to dive using compressed air until osseointegration is complete.
Keywords: Air pressure, bone chip, diving, hyperbaric, oxygen
|How to cite this article:|
See Cv, Stoetzer M, Koch A, Ruecker M, Gellrich NC. Influence of pre-implant bone augmentation on diving fitness: An in vitro study. J Dent Implant 2014;4:22-8
|How to cite this URL:|
See Cv, Stoetzer M, Koch A, Ruecker M, Gellrich NC. Influence of pre-implant bone augmentation on diving fitness: An in vitro study. J Dent Implant [serial online] 2014 [cited 2020 Feb 20];4:22-8. Available from: http://www.jdionline.org/text.asp?2014/4/1/22/130949
| Introduction|| |
An increase in external pressure during diving leads to a variety of physiological and pathophysiological changes. The partial pressures of gases in tissues change as a result of biochemical and mechanical effects. In addition, barotrauma to air-filled spaces within the body can occur.  This depends in particular on the type of breathing gas used as well as on the depth and duration of diving. Especially in military diving, breathing gas composition is mainly influenced by the depth of diving and the operational scenario.
As a rule, divers should have fixed prostheses that prevent complications in association with mouthpieces.  Fixed prostheses often require the use of dental implants that act as an anchor for restorations. Dental implant procedures require the presence of sufficient bone to hold and support the implant. If there is a lack of underlying bone, bone augmentation procedures are usually performed prior to the placement of an implant. , It is not yet clear whether mechanical loading or breathing gases can influence osseointegration after pre-implant bone augmentation.
The insertion of endosseous implants has become an accepted standard procedure for dental rehabilitation. It involves the placement of a usually cylindrical implant within the bone and requires the presence of adequate bone. Professional societies have developed guidelines according to which implant stability requires the presence of a minimum of 1 mm of bone around the implant and sufficient bone density.  Many patients who sustained a trauma or present with wound healing problems after tooth extraction or alveolar ridge atrophy do not meet these requirements. In these cases, bone augmentation must be performed in order to create sufficient bone volume for implant placement.
A variety of materials are available for bone augmentation  and include autogenous and alloplastic materials. Important aspects are the availability and biocompatibility of the augmentation material as well as functional properties of the material, donor site morbidity and the risk of irreversible damage.  Bone substitutes have the advantages of almost unlimited availability and no donor site morbidity. By contrast, the availability of autogenous bone is limited. In addition, autogenous bone graft procedures can be associated with donor site morbidity that can lead to further complications and co-morbidities. 
Excellent compatibility is a key advantage that autogenous bone grafts have over bone substitutes. Despite enormous advances in the field of the osseointegration of bone substitutes, autogenous bone grafts are still considered to be the gold standard for bone augmentation. 
A variety of sites and procedures for autogenous bone grafting are used. In the majority of cases, bone grafts are obtained from intraoral locations since these sites are associated with low co-morbidity. Cortical, cancellous or corticocancellous bone grafts can be harvested from intraoral locations. Depending on the grafting procedure, particulate bone or bone blocks are distinguished in routine clinical practice. Grafting procedures involving the use of piezosurgery for harvesting block grafts allow surgeons to obtain bone augmentation material that is as well vitalized as bone chips collected with a Safescraper® . 
The vitality of bone structures can be investigated using a variety of methods that assess cell activity, proliferation rates or apoptosis rates in vitro. These methods allow the effects of exogenous factors on bone to be directly compared.
Irrespective of the donor site, free grafts are usually used for bone augmentation at oral sites. This means that the graft is usually incorporated into the recipient site without a vascular supply. In the region of the jaw, bone grafts are transferred by anastomosis only in very rare cases involving large bone defects.  First experimental approaches to the prevascularization of bone grafts for augmentation procedures have provided promising results but are not yet of clinical relevance.
At the time of augmentation, the graft is separated from its supplying vessels. Until neovascularization, the transplanted material obtains oxygen and nutrients through diffusion from surrounding vessels. ,
Neovascularization of the bone augmentation material is essential in order to prevent resorption and to enhance osseous incorporation into the recipient bone. ,, Both the bone graft bed and the surrounding soft tissues are involved in this process.  During the initial phase after bone augmentation, a high functional vessel density in the area around the augmentation material is therefore required to ensure initial supply and revascularization. For this reason, primary wound closure should be achieved without tension in order not to compromise neovascularization. For this reason, primary wound closure should be achieved without tension in order not to compromise vascularization.  Especially when a large amount of augmentation material is required, however, the increase in bone volume can make it impossible for a surgeon to achieve a tension-free soft-tissue coverage of the bone. In any case, this surgical procedure is likely associated with a temporarily reduced supply of nutrients and oxygen to the region of the augmented bone even under physiological atmospheric conditions.
A number of approaches have been used in an attempt to enhance local microcirculation and to prevent graft rejection and resorption. For example, the administration of transmitter proteins such as vascular endothelial growth factor (VEGF) is a method of accelerating vascularization.  Animal experiments have reported promising results. Transmitter proteins, however, are not yet used clinically because they can have undesired side effects on surrounding tissues and they are distributed in an uncontrolled manner. Another approach to improving microcirculation is hyperbaric oxygen therapy, which has been successfully used in patients with osteoradionecrosis or osteomyelitis.  The percentage of oxygen in air and external pressure are increased in an attempt to improve local oxygenation in terminal vessels.  The effectiveness of hyperbaric oxygen therapy, however, is a matter of controversy. , Among the issues discussed is the role of mechanical pressure effects and microthromboses that may adversely affect metabolism.
In the literature, there are no systematic studies addressing possible effects of hyperbaric exposure after bone augmentation and before dental implant placement. The objective of this study was therefore to investigate the effects of compressed air and oxygen on the cellular activity of osteoblasts from bone blocks and bone chips similar to those that are used for pre-implant bone augmentation.
| Materials and Methods|| |
Isolation and cultivation of rat osteoblasts
Bone samples were taken from male Lewis rats (Charles River, Sulzfeld, Germany) weighing approximately 300 grams. The rats were anesthetized with carbon dioxide and killed by cervical dislocation. The experiments were performed in accordance with the pertinent animal protection laws and regulations and had been approved by the responsible local authority.
After the rats were killed, the surface of the skin in the region of the thighs was disinfected using 70% ethanol and a skin incision was made. All thigh muscles were removed and the femurs were exposed. The tendons around the knee and hip joints were divided and the femurs were disarticulated at the hip and knee joints. All soft tissues were removed and the femurs were stored on ice in Hanks' balanced salt solution (HBSS, PAA, Coelbe, Germany) until further processing.
Bone samples were obtained for cultivation using two independent methods:
(a) Collection of bone blocks using a Luer bone rongeur
Rat femurs were cut into small fragments with a length of approximately 2 mm using a Luer bone rongeur (Martin, Solingen, Germany). Bones and bone fragments were kept moist with HBSS. The bone blocks were washed three times with HBSS with a view to preventing contamination by adherent bone marrow and bone marrow cells.
(b) Collection of cortical bone chips using a Safescraper®
Cortical bone chips were harvested using a suitable commercial bone collector (Safescraper® Twist, META, Italy). The Safescraper® was pressed toward the bone surface and pulled backward in order to collect bone [Figure 1]. The bone chips were directly collected in a sterile chamber that is integrated in the handle of the instrument. The harvested bone chips were taken from the chamber and washed three times with HBSS with a view to preventing contamination.
|Figure 1: Collection of bone chips using a Safescraper®. The Safescraper® was pressed toward the bone surface and pulled backward in order to collect bone (arrow). The bone chips were directly collected in a sterile chamber that is integrated in the handle of the instrument (asterisk)|
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The bone blocks and chips were separated from HBSS and their fresh weight was determined using an analytical balance (BP61, Sartorius, Goettingen, Germany) [Table 1].
|Table 1: Wet weight of samples, dry weight of fresh bone samples before testing, and weight of air-dried bone samples after testing (day 15)|
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After weighing, a total of 16 bone fragments with similar weights were placed into groups, each consisting of two samples [Table 2]. The bone fragments were seeded into cell culture flasks with a culture area of 25 c 2 (Nunc, Roskilde, Denmark) in 5.0 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), 1000 IU/ml penicillin and 0.1 mg/ml streptomycin (all obtained from PAA, Coelbe, Germany). Group 1 (bone blocks) and Group 5 (bone chips) served as controls for any effects of transportation to the pressure chamber. These preparations were immediately incubated at 37°C in a humidified atmosphere of 8.5% CO 2 and 91.5% air.
All other samples were transferred to culture flasks that were tightly closed and placed in the pressure chamber, where they were exposed to pressure during simulated 45-min dives to 5.0 bar. Compressed air was used as breathing gas in Group 3 (bone blocks) and Group 7 (bone chips) and oxygen (medical oxygen, Linde, Germany) in Group 4 (bone blocks) and Group 8 (bone chips).
The flasks that contained the preparations were placed in the pressure chamber with the lid opened with a view to achieving rapid pressure and gas equalization [Figure 2]. Similar preparations (identical bone material and weight) were kept at normal pressure for control purposes. Before and after the simulated dives, culture media were changed in order to rule out any effects of pressure on the media. Preparations were then incubated at 37°C for 9 and 15 days in a humidified atmosphere of 8.5% CO 2 and 91.5% air.
|Figure 2: The diving chamber with an open culture fl ask and medium (DMEM) containing 10% fetal calf serum (FCS), 1000 IU/ml penicillin and 0.1 mg/ml streptomycin (all obtained from PAA, Coelbe, Germany)|
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We placed a further culture flask that contained 5 ml of culture medium in the pressure chamber and measured the pH value of the medium at the end of a dive in order to assess whether a simulated dive influences the pH value of the medium. For the duration of the dive, another flask with 5 ml of culture medium was kept outside the pressure chamber and served as a control.
Following observation periods of 9 and 15 days, adherent cells that had grown from the bone fragments were counted for analysis. Cell monolayers were rinsed three times with HBSS. After accutase (PAA, Coelbe, Germany) had been added, the cells were incubated for 15 min at 37°C and detached from the culture flasks. The detached cells were pelleted by centrifugation at 300 x g and resuspended in 0.2 ml of culture medium. The cells in the cell suspensions were counted using a Neubauer counting chamber (four replicates).
Following the tests, bone fragments were completely washed with HBSS using 40 μm sieves and dried in air for 7 days. The exact dry weight of the fragments was measured using an analytical balance (BP61, Sartorius, Goettingen, Germany) [Table 1].
SigmaStat software (SigmaStat Version 10, Jandel Corporation, San Rafael, USA) was used for statistical analysis. Results are expressed as means (MV) ± standard error of the mean (SEM). A test of normality was conducted to confirm a normal distribution. Two groups were then compared using a parametric t-test or a non-parametric Wilcoxon rank-sum test for dependent or independent samples. Differences were considered significant if the probability of error was <0.05.
| Results|| |
Representative samples were obtained for all groups using either a bone rongeur or a Safescraper® [Figure 3].
|Figure 3: Representative samples of bone chips (a) obtained with a Safescraper® and bone blocks (b) immediately after harvesting|
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All samples exhibited measurable proliferation after 9 and 15 days. Comparative analyses of newly proliferated cells and statistical analyses of the various groups were thus possible at both time points.
There were no differences in pH values between pressure-exposed and control groups [Table 3].
|Table 3: pH values of media for control groups and groups subjected to simulated dives. There were no significant differences between the groups (P<0.05)|
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All groups showed higher cell proliferation rates after 15 days than after 9 days [Figure 4].
|Figure 4: Representative bone block samples after exposure to oxygen (a and c) and compressed air (b and d) during simulated dives. Irrespective of the gas used, the samples show lower cell proliferation rates after 9 days (A and B) than after 15 days (c and d)|
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At both time points, there were no significant differences in proliferation rates between the control and transportation groups.
In all control and study groups, bone blocks showed higher proliferation rates than bone chips at both time points.
After 9 and after 15 days, the bone block and bone chip groups that had been exposed to compressed air showed lower rates of proliferation than the control groups. At both time points, however, these differences were not significant for either the bone block groups or the bone chip groups.
By contrast, the bone block groups and bone chip groups that had been exposed to hyperbaric oxygen showed significantly higher proliferation rates at both time points than the control groups and the groups that had been exposed to compressed air.
| Discussion|| |
In the control groups, the proliferation rates of osteoblasts in bone tissue obtained from isogeneic rats were significantly higher in bone blocks than in bone chips. Compared with the control groups, the groups that had been exposed to compressed air during simulated dives showed lower proliferation rates. By contrast, the groups that had been exposed to pure oxygen during simulated dives exhibited significantly higher proliferation rates than the control groups that were not subjected to simulated dives. Irrespective of ambient pressure and partial gas pressure levels, pH values did not change in cell media.
Changes in atmospheric pressure, for example during diving or flying, lead to a variety of physiological and pathophysiological responses within the body. The simulation of dives in a hyperbaric chamber has become a well established method for performing systemic studies under different ambient conditions.  Increases in pressure similar to those seen during diving can be simulated almost realistically by increasing gas pressure in a diving chamber. During diving, pressure increases in all gas-filled spaces that can communicate with the environment. As a result, pressure in the oral cavity during diving is likely to be equivalent to that of the environment. The results of the simulations therefore allow conclusions to be drawn on the effects of atmospheric pressure changes on bone under real conditions.
In the present study, bone samples were obtained from isogeneic rats and pooled in order to minimize interindividual variances.
Osteoblast proliferation rates after simulated dives were analyzed in vitro. This allowed us to systematically analyze various influencing factors. This approach, however, has the disadvantage that metabolic processes involving the entire organism are reflected only to a limited extent. Since bone tissue is separated from its supplying vessels during an augmentation procedure and osseointegration requires revascularization, this disadvantage can be largely ignored in the present study. In addition, influencing factors can be directly analyzed on the basis of a comparison of pressure-exposed and control samples. In vivo studies have shown that osteocyte activity varies considerably. Gundberg et al.  reported that age, gender and other factors appear to play a key role in osseoinductive effects. Systematic tests using an isogeneic animal model are therefore better suited for basic studies than are in vivo tests. The results, however, can be directly transferred to humans only to a limited extent. The present study should provide the basis for clinical trials involving an appropriate group of patients.
In vivo, vessel density in the region above the augmented bone plays a particularly important role in the incorporation of bone grafts without a vascular pedicle. Apart from interindividual differences, vessel density can vary widely. Results of studies on human subjects are therefore difficult to assess and should be interpreted with caution. Vessel density in the periosteum is of particular importance for ensuring the initial provision of nourishment to the bone.  Every surgical procedure that involves subperiosteal exposure compromises periosteal perfusion.  This makes realistic in vivo studies of bones even more difficult. For this reason, the use of animal models is appropriate and indispensable for basic studies that are conducted to systematically assess a variety of influencing factors.
An analysis of pH values in cell media revealed no significant differences between the various groups. This can be explained by the high buffer capacity of the solution that was used in the tests. In clinical in vivo studies, Thorsen et al. too detected no pH changes in the blood or tissue of pressure-exposed subjects. 
From day 9 to day 15, all groups of bone blocks and bone chips showed significant increases in cell proliferation rates. This finding is in line with previous studies and shows that vital bone cells were present in all groups. 
In addition, all bone block groups showed higher proliferation rates than the bone chip groups. This finding has been reported in other systematic studies as well.  This can possibly be explained by the number of cells per sample and the relative surface area. Outer cell layers are assumed to show very low proliferation rates as a result of mechanical damage. This is supported by the results of this study since a direct comparison of all groups showed higher proliferation rates for bone blocks than bone chips.
The effects of hyperbaric oxygen exposure on bone are a matter of controversy in the literature.  A comparison of the control groups that were not subjected to simulated dives and the groups that had been exposed to oxygen reveals that the samples that had been exposed to oxygen during diving had significantly higher proliferation rates than the control samples irrespective of the harvesting method. The effects of exposure to oxygen during simulated dives are similar to those produced by hyperbaric oxygen therapy, which is used, for example, for the treatment of osteomyelitis. The purpose of hyperbaric oxygen therapy is to increase local oxygen levels in the areas of osteomyelitis in order to stimulate proliferation and achieve bone healing. Our results thus support the findings reported by Hsieh et al., who reported an increase in osteoblast activity after hyperbaric oxygenation under in vitro conditions. 
The literature provides no data allowing us to directly compare our results for the effects of compressed air on osteoblasts with the results of other studies. A comparison with the control groups, which had not been subjected to simulated dives, demonstrates lower proliferation rates for the groups that had been exposed to compressed air. Irrespective of the harvesting method used, the gases to which the samples had been exposed during simulated dives directly influenced bone cell proliferation rates in both bone blocks and bone chips.
In summary, our study shows that exposure to pressure alone has no influence on bone cell proliferation rates. By contrast, the breathing gases that are used during diving have a direct effect on cell activity and cell proliferation.
Our results suggest that a bone augmentation procedure does not generally disqualify a patient from diving. The use of compressed air, however, is likely to adversely affect osseointegration.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]