Examination of Local Periosteal Microcirculation After Application of Absorbable and Nonabsorbable Membranes

In medicine and especially in dentoalveolar surgery, membranes are regularly inserted beneath the periosteum to cover native bone or bone whose dimensions have been altered. The objective, first and foremost, is to prevent the undesired ingrowth of soft tissue into the bone defect which is caused by the rapid proliferation of epithelial cells and connective tissue. Membranes are also used as “internal patches” in surgery involving the maxillary sinus or to cover bone augmentation, where they serve as a mechanical buffer to the maxillary sinus or oral cavity. Membranes can serve as placeholders for osteoblasts and osteoclasts, which proliferate slowly. Although many different materials are used, membranes are usually divided into absorbable and nonabsorbable membranes.

Guided bone regeneration (GBR) and guided tissue regeneration (GTR) are currently standard therapeutic procedures in dental surgery.¹  These procedures are based on the isolation of regenerative cell types—such as periodontal fibroblasts and osteoblasts—from rapidly proliferating epithelial and connective tissue cells.^2–4  Over the years, absorbable membranes have become more commonly used.

Nonabsorbable membranes are manufactured from modern synthetic materials or titanium. Filter membranes or polytetrafluorethylene (PTFE) membranes (e-PTFE, GORE-TEX, Gore & Assoc, Newark, Del; n-PTFE, TefGen, Keystone Dental, Inc, Burlington, Mass) or titanium membranes⁵  (FRIOS BoneShield, Dentsply Implants, Mölndal, Sweden) are placed over a cavity in such a way that the bone is completely sealed off and the membrane slightly overlaps the defect at the bone edges.^6–9 

The periosteum covers the surface of the bone and is detached in many types of bone surgery, particularly in dentoalveolar surgery. The objective of this study is to clarify the extent to which the periosteum is compromised by surgical procedures, what impacts standard medical products (such as different membranes) that regularly must be inserted between periosteum and bone, the effect on the biological function of the periosteum, and whether the periosteum suffers less damage when piezo surgical instruments are used.

We studied female Lewis rats from Charles River Laboratories. The experiments were conducted and animals were kept in accordance with the German Animal Protection Act and the Guide for the Care and Use of Laboratory Animals.^10,11  The research applications were reviewed by the animal welfare officers of Hannover Medical School and then submitted for approval to the Animal Protection Division (Dezernat 33 “Tierschutz”) of the Lower Saxony State Office for Consumer Protection and Food Safety. All experiments were performed at the central animal laboratory of Hannover Medical School (CrossBIT).

In this study, we compared a special piezoelectric approach to periosteum preparation with a conventional approach involving a periosteal elevator, focusing on the impact of these methods on the periosteum. The surgical procedure was performed either at the calvarium or intraorally on the maxilla of female Lewis rats.

The animal model of Stuehmer et al¹²  provided the basis for this study and was considerably modified and adapted for this purpose.

To analyze the influence of different biomaterials on microcirculatory processes in the periosteum, we examined and compared absorbable and nonabsorbable membranes. After we prepared the rat calvaria either with a periosteal elevator or by using the new piezoelectric approach, the biomaterials were placed beneath the periosteum on the calvaria. To determine microcirculatory parameters, intravital fluorescence microscopy (IVM) was performed on days 0, 3, 8, and 28. For this examination, we used a cranial window fixed to the head of each rat to avoid reopening the wound. The study involved a total of 32 animals divided into 4 groups.

The groups were as follows:

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  Group 1: n = 8, subperiosteal preparation with periosteal elevator, absorbable membrane, intravital microscopy on days 0, 3, 8, and 28.

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  Group 2: n = 8, subperiosteal preparation with periosteal elevator, nonabsorbable membrane, intravital microscopy on days 0, 3, 8, and 28.

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  Group 3: n = 8, subperiosteal preparation with piezoelectric device, absorbable membrane, intravital microscopy on days 0, 3, 8, and 28.

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  Group 4: n = 8, subperiosteal preparation with piezoelectric device, nonabsorbable membrane, intravital microscopy on days 0, 3, 8, and 28.

The laboratory animals were female isogenic Lewis rats (Charles River Laboratories, Wilmington, Mass) weighing between 300 and 330 g. They were provided by the Central Animal Laboratory and the Institute for Laboratory Animal Science of the Hannover Medical School.

The animals were housed in groups of 5 in group-sized cages. After the surgical procedure, the animals were housed singly. All laboratory animals were exposed to identical conditions. During the entire experiment, the rats were kept in a stable microenvironment and in a normal cycle of 12-hour periods of light and dark.

A window chamber consists of a basic frame, a glass coverslip, two bone screws, and a snap ring. The frame has 2 holes for the 1-mm bone screws as well as lateral notches for the circular fixation of tissue/skin around the frame. In the center, the frame has a round hole with a diameter of 8 mm in which the glass coverslip was placed. The glass coverslip was then fixed to the frame with a snap ring. Intravital fluorescence microscopy was performed through this glass coverslip.

The images analyzed in this study were recorded using intravital microscopy. Intravital fluorescence microscopy (IVM) was performed in accordance with the experimental protocol. During each examination, the animals were anesthetized. We used a modified Zeiss Axiotech microscope (Zeiss, Oberkochen, Germany) for epifluorescence microscopy. To illuminate the examination area, the microscope was equipped with a mercury arc lamp and a blue filter combination (excitation wavelength: 450–490 nm). The microscopic images were recorded using a highly sensitive video camera (FK 6990 IQ-S, Pieper, Schwerte, Germany) and transferred to a DVD system (LQ-MS 800, Panasonic, Hamburg, Germany). To improve the visualization of the structures requiring analysis, the animals were injected with specific fluorescent dyes. The injected dye and the use of the blue filter made the periosteum's vascular structures visible. We placed each animal under the microscope, brought the structures into focus, and analyzed and recorded the periosteum for subsequent evaluation using each filter and magnification for 1 min.

For surgery and the examination, the animals were anesthetized by an intraperitoneal injection of ketamine and xylazine (Ketavet, Pfizer, New York, NY, 75 mg/kg bw; xylazine, Vetpharm, East Rochester, NY, 25 mg/kg bw). One milliliter of contrast agent was injected into 1 of the 4 veins in the tail of each of the anesthetized rats. The agent contained fluorescein isothiocyanate-labeled dextran (FITC-labeled dextran, molecular weight: 150 000 Da; Sigma, Taufkirchen, Germany; 150 mg/ml in 0.9% NaCl solution) in a ratio of 1:1. After disinfection, a scalpel was used to make an incision through the scalp. The cranial bone was exposed through blunt dissection and the covering periosteum was cut unilaterally with a horizontal incision. Depending on the group, we used either a periosteal elevator or a piezoelectric device to expose an area of 1.5 cm × 1 cm of the calvaria below the periosteum. A membrane was then placed over this area, and the cranial chamber with the observation window was fixed with 2 screws to the calvaria. The scalp was then sutured with Ethicon-Vicryl size: 4.0 (Johnson & Johnson, Neuss, Germany) around the cranial chamber. We then recorded microscopic images of the surgical area at magnifications of ×2.5, ×5.0, and ×20 using a blue filter set. The images were recorded on a DVD recorder (LQ-MS 800, Panasonic). Intravital microscopy imaging took an average of 30 minutes per animal. During the entire procedure, the body temperature of the laboratory animals was maintained at +36°C with a heating pad (ThermoLux, Witte+Sutor, Murrhardt, Germany).

Over the course of the experiment, the animals were examined using intravital microscopy on days 3, 8, and 28 after the start of the healing period. The periosteum was once again examined after anesthesia through the observation window with intravital microscopy at magnifications of ×2.5, ×5, ×10, and ×20. An analysis of the images was conducted at a later time to keep the anesthesia duration to a minimum. Immediately after the last microscopic examination, the laboratory animals were euthanized under anesthesia by an overdose of anesthetic.

Microsoft Excel was used to collect and store all data. A statistical analysis was conducted using SPSS (SPSS Statistics 23, IBM GmbH, Ehningen, Germany).

We obtained the following results for functional capillary density when we compared the use of a periosteal elevator and a piezoelectric device together with a titanium membrane as an example of a nonabsorbable membrane (Figure 1, Table 1).

With absorbable membranes, we obtained the following results for functional capillary density (Figure 2, Table 2).

Between the groups with nonabsorbable membranes, there was a significant difference (P = .01) in vessel diameter measurements on day 0 for the group whose periosteum was dissected with a piezoelectric device. On days 3 and 8, there was no statistical significance. Only on day 28 did the P-value almost attain a level of significance (P = .0075). The data for absorbable membranes are totally different: On days 0, 3, and 28, a statistical significance can be seen (P < .05). On day 8, the P-value was almost significant (P = .053).

With regard to blood flow velocity and nonabsorbable membranes, there was a significant difference (P = .02) only on day 0. There was no significance on all other days. With regard to blood flow velocity and absorbable membranes, P-values were statistically significant (P < .05) on days 0, 3, and 28 and almost significant (P = .053) on day 8.

Rats were chosen as biological systems for all experiments in this study. These experiments require intact, healthy organisms that allow us to examine the periosteum and periosteal microcirculatory processes. Previous studies have already demonstrated that rats are good subjects for examining the quantity and quality of periosteal microcirculation.¹²  The results cannot, however, be directly transferred to humans; however, since we compare the use of 2 instruments in the same surgical procedure, the results can be transferred to humans to a limited extent. This has been shown by other studies in which Lewis rats were also used in the first phase to transfer the obtained results to humans.¹³ 

Intravital microscopy was chosen as the key method for examining local periosteal microcirculation. It was used to determine functional capillary density, vessel diameter, and blood flow velocity. Functional capillary density is the most appropriate and informative parameter¹⁴  for determining whether the periosteum was damaged, as it reveals all functioning vessels and thus provides information on the supply and, moreover, the vitality of the periosteum. Vessel diameter and blood flow velocity confirm the validity of functional capillary density by providing information on the condition of the vessels.¹⁵  IVM is an important method of describing the perfusion of the prepared periosteum and observing changes in local periosteal microcirculation over the entire period of study.

The objective of this study was to examine local periosteal microcirculation. While it is possible to conduct a histological examination of the periosteum, this cannot be done in vivo and we would merely obtain a snapshot and not an overview of the entire study period. Therefore, the in vivo observation of microcirculation has proven to be a valuable indicator.^12,14–16  Reduced functional capillary density correlates directly with reduced blood flow in the examined area.¹⁷ 

For vessel diameter measurements, we chose vessels with a diameter between 10 and 30 μm in which the flow of red blood cells was visible. This ensured that we were examining active venules, playing a decisive role in periosteal perfusion, rather than venule fragments that are visible but no longer fulfill any purpose. This method of determining the vessel diameter has been confirmed by other working groups.¹⁸  We drew a line perpendicular to the two vessel walls and calculated the distance between the vessel walls in μm.

The last parameter measured was blood flow velocity. Blood flow—and thus the delivery of oxygen to the organs (in this case, the periosteum)—are regulated by alterations in vascular tone. This parameter provided information on the quality of blood supply, as other studies have shown.¹⁹ 

This measurement was carried out with the computer-assisted line shift diagram method on vessels whose diameter had been measured. We drew a line inside the vessel lumen in the direction of flow and played back a recorded sequence for 10 seconds. We read the gray scale value data for each half-frame along the line and transferred this data to an image memory where it was aligned vertically. With the CapImage program (Image Analysis Systems, Dr. Zeintl, München, Germany) and the appropriate contrast level, we could determine the length and slope of the line. We chose 10 measurement sites that we used to calculate the mean velocity in μm/s on the basis of slope.

In this way, we had three meaningful parameters for our analysis of periosteal microcirculation. All parameters were measured without any difficulty at all measurement times. As already examined and confirmed in several studies, animal data collected by the IVM method can be transferred to humans.^20,21 

With regard to functional capillary density, a significant difference was seen only for the nonabsorbable membranes on day 28 in the piezoelectric device group. Although functional capillary density increased in all groups over the course of the study, there was no significant difference on days 0, 3, and 8. However, functional capillary density was higher at every time point in the piezoelectric device group than in the periosteal elevator group. There is no explanation for the significant difference between the two membrane groups on day 28. This is probably due to the rigidity of the titanium membrane. We cannot rule out that when the membrane was inserted under the periosteum, the latter was damaged and functional capillary density was thus impaired. The values for absorbable membranes indicate a difference between the periosteal elevator and the piezoelectric device. Although this difference is not significant, the values do favor the piezoelectric device. In direct comparison to the periosteal elevator, the piezoelectric device was better for functional capillary density. This became even more apparent toward the end of the study. Similar to a study by Siar et al,²²  our study also provides evidence that periosteal vascularization was higher when absorbable membranes were used.

Over the course of 28 days, vessel diameters in both nonabsorbable membrane groups were similar, with no significant difference observed. This is different from the results obtained for absorbable membranes, as the working groups of Rothamel²³  and Schwarz²⁴  have already shown. In our study, we observed a significant difference in favor of the new piezoelectric instrument. The data clearly show that, over the course of the study, the vessel diameter in the group with a periosteal elevator did not vary to a great extent. Although the same can be seen with the piezoelectric device, vessel diameter values for the periosteal elevator remained markedly below those achieved with the piezoelectric device.

Blood flow velocity values were comparable to those of the vessel diameter. There was no significant difference between the groups with nonabsorbable membranes, but a considerable difference was detected between those groups with absorbable membranes. Blood flow velocity was significantly higher in the absorbable membrane group in which the periosteum was prepared with the piezoelectric approach.

The integrity of the interface between bone and periosteum is a crucial factor in oral and maxillofacial surgery. As part of an animal experiment designed to optimize this interface between periosteum and bone, a total of 32 rats were divided into 4 groups. We examined the extent to which the periosteum was affected by the subperiostal insertion of different medical products and whether 2 different methods of preparing the periosteum offered any advantages. For this purpose, we compared absorbable and nonabsorbable membranes. By determining functional capillary density, vessel diameter, and blood flow velocity, we found that neither method offered any advantages when nonabsorbable membranes were used. We obtained different results for absorbable membranes. In this case, the piezoelectric preparation of the periosteum offered significant advantages. The degradation of absorbable membranes did not impair periosteal microcirculation. Nonabsorbable membranes had no significant impact on local microcirculation, irrespective of which instrument we used.

We can thus conclude that the piezoelectric device allowed us to detach the periosteum from the bone with fewer adverse biological consequences than the manual approach. The periosteum plays a decisive role in bone healing and should be protected. The preparation of the periosteum with the piezoelectric device is thus a positive development in dental as well as in oral and maxillofacial surgery. Further studies must be conducted to determine the extent to which piezoelectric oscillation stimulates and influences bone metabolism.

Abbreviations

Da

Dalton

GBR

guided bone regeneration

GTR

guided tissue regeneration

IVM

intravital fluorescence microscopy

PTFE

polytetrafluorethylene

Marcus Stoetzer contributed to the conception and design of the study as well as to the acquisition, analysis, and interpretation of data. He also drafted and critically revised the manuscript. Björn Rahlf and Nils-Claudius Gellrich contributed to the conception and design of this study and critically revised the manuscript. Anna Joop and Constantin von See contributed to the conception and design of the study as well as to the acquisition and interpretation of data; they also drafted and critically revised the manuscript. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of this study.

We declare that all authors have read the guidelines on ethical considerations. We received no funding and no conflict of interest for this study.