Abstract: This study was designed to determine if vacuum-induced suction increased the number of blood vessels in healthy dog gingiva as a prelude to future studies testing vacuum therapy for improving local blood supply and controlling periodontal disease. Methods. The buccal gingiva of five dogs was treated with subatmospheric pressure for 5 days, with untreated tissues acting as controls. Biopsies were analyzed for vascular endothelial growth factors (VEGF) and blood vessels were counted. Results. VEGF and vessel numbers were elevated in treatment groups compared to controls (P < 0.05). Conclusion. A single daily application of subatmospheric pressure might be beneficial for healing damaged or diseased gingival tissues.

Introduction

  Several epidemiological studies have linked moderate and advanced chronic periodontitis to systemic diseases1–7 and undesirable outcomes in pregnancy.8,9 Periodontitis and associated comorbidities have been shown to increase inflammatory markers, such as C-reactive protein (CRP) and interleukin-6 (IL-6), and to adversely increase systemic complications,10,11 including inflammation and obliteration of peripheral blood vessels.12 Inhibited tissue regeneration, poor nourishment, and delayed healing are a few significant risk factors for reduced blood supply.13 Healthcare professionals are challenged to improve periodontal health and diminish suboptimal outcomes associated with periodontitis. Preventing and reversing systemic negative outcomes14 associated with decreased inflammatory markers after periodontal treatment15 are 2 ways to treat periodontal infections. The formation of new blood supply from existing vasculature is a key mechanism required for tissue regeneration.16 Several studies accentuated the need for improved angiogenic activity to supply the periodontium with a better vascular network through vascular regeneration in patients who are healthy, and in patients with systemic complications.17,18   Previous studies demonstrated the beneficial effect of increased angiogenesis, fibrovascular proliferation, and capillary sprouting induced by increasing cellular tension.19–23 Some investigators used low-intensity ultrasound to stimulate vascular regeneration.24 It was found that elevated osmotic pressure increased angiogenesis in periodontal soft tissues in the presence of periodontal disease in diabetic rats.25,26 Mechanical forces, such as using vacuum (subatmospheric pressure), showed improvement in healing after diabetic foot amputation and in acute wounds.27–29 Additionally, subatmospheric pressure improved growth of granulation tissue, reduced tissue edema,23,27,28,30 increased expression of vascular endothelial growth factors (VEGF) and fibroblast growth factor-2, induced collagen organization and wound maturation,31 and lessened secondary infection resulting in shortened hospital stays.32 Vacuum application after chemotherapy and radiation following resection of musculoskeletal tumors with removal of substantial bone and soft tissues showed reduced flap ischemia in primary closure, lessened wound dehiscence, and decreased incidence of deep tissue infections.33 None of these studies suggested an optimal time or amount of subatmospheric pressure to use. The present study was designed to test the hypothesis that subatmospheric pressure stimulates blood vessels to increase in number within the gingival tissues of a dog model. The purpose of this study was to evaluate the effectiveness of subatmospheric pressure in inducing the appearance of blood vessels, and to identify the optimal amount of time and pressure for vacuum application in healthy dog gingiva. Follow-up studies would test the effects of optimal times and amounts of subatmospheric pressure administration on unhealthy gingiva, and ultimately apply these methods to inflamed human periodontal tissues.

Methods

  Animals. The Institutional Animal Care and Use Committee of the Texas A&M Health Science Center Baylor College of Dentistry (TAMHSC-BCD) approved all animal experiments prior to commencement of the study. Five healthy adult Foxhounds, approximately 2 years old, (Schachtele Kennels Lab, Salisbury, MO) were given complete physicals, and kept in quarantine for 10 days at the Animal Research Unit, TAMHSC-BCD. To diminish the possibility of gingivitis during the study, and to avoid contamination of the surgical site during biopsies, each dog underwent ultrasonic gingival scaling before the study began. After the ultrasonic scaling procedures, the dogs were allowed to rest over the weekend (approx. 48 hours). On the following Monday, the experimental treatments were started and continued for 5 consecutive days. At the beginning of each treatment day, each dog received 2.2 mg/kg ketamine (Bioniche Animal Health USA, Inc. Athens, GA) and 0.22 mg/kg, xylazine (Butler Animal Health Supply Dublin, OH) intramuscularly (IM). Anesthesia was maintained with intravenous (IV) titration. Heart rate, oxygen saturation, respiration rate, and end tidal carbon dioxide levels were monitored throughout the procedures. Weight and behavioral changes were recorded each day.   Treatment procedure. The vacuum machine (Gomco 4010, Doral Medical Equipment, Miami, FL) was calibrated at -150 mmHg for the maxilla and -300 mmHg for the mandible (Figure 1A). A 6 cm x 1 cm suction tip was dipped in a synthetic plastic coating (Plasti-Dip, Plasti-Dip International, Blaine, MN) 3 times to coat and pad the tip before treatment. The suction tip was connected to the vacuum machine to perform the treatments on the right side of the jaw for 2, 3, or 5 minutes (Figure 1B). The same suction tip was used on the left control side at 0 mmHg pressure with the tip resting on the tissue. Treated and control sides were divided into 3 sites, 15 mm apart. After each suction application, the vacuum was turned off in order to remove the hand piece without damaging the tissue. On the first study day, the perimeters of the post-treatment hyperemic borders were outlined using a blue surgical marker (Figure 1C). The location of the suction tip was outlined on the control site. On each of the 4 subsequent days, the size of the post-treatment hyperemic site was measured and photographed before and after each treatment. Any visual abnormalities over the course of treatment were also recorded.   Biopsies. Following 2 days of rest after the last treatment, the dogs were sedated using intramuscular (IM) ketamine and xylazine, 2.2 mg/kg and 0.22 mg/kg, respectively, for biopsy collection. Surgical plane anesthesia was maintained with isoflurane at 1.5% and inhalation of oxygen at 1 L per minute. All dogs were monitored throughout the entire procedure. One centimeter diameter biopsies using a scalpel were performed to the periosteal surface of the bone. Gingival tissue, along with periosteum, was collected from each treatment and control site. At the treatment site, visible erythema was used to identify the exact location for the biopsy. The biopsy area indicated with the surgical marker, was used for control-side sample collection. Once the tissue was removed, it was sectioned into 2 pieces with 1 half placed in a sterile 0.5-mL vial filled with 0.3-mL RNA-Later, immediately placed on ice, and transferred to a freezer at -80˚C. The other half of the tissue was dissected in half again, with 1 part placed in a 0.5-mL vial with 0.3-mL protein extraction lysis buffer, and the other placed in a 0.5-mL vial with 0.3-mL 4% paraformaldehyde/PBS fixative. All biopsy sites were sutured and the dogs were given 0.3 mg buprenorphine and 900,000 international units (IU) of penicillin G benzathine IM. The sutures were evaluated on each dog on the following day. Photographs of the sutured sites were taken for 3 consecutive days.   Protein extraction. The frozen tissue samples were homogenized in protein extraction lysis buffer (RIPA buffer, Sigma, St. Louis, MO), mixed with a phosphatase inhibitor cocktail, and centrifuged at 3000 rpm for 20 minutes at 4˚C. Supernatant fluid was used to quantify proteins using a BCA assay and to analyze VEGF levels by ELISA, as recommended by the manufacturer (R&D Systems, Minneapolis, MN).   Histology. The tissue samples were transferred into larger vials containing 4% paraformaldehyde/ PBS fixative and stored overnight. Samples were dehydrated and embedded in paraffin, and cut into 36, 6-µm thick sections. The 1st, 6th, 12th, 18th, 26th, and 32nd sections were stained with H&E. After staining, the slides were coded and blinded for counting blood vessels. Photomicrographs of the slides were taken at 10x magnification to count large vessels and at 20x to count collateral vessels surrounding larger blood vessels. Two lower magnification photos were selected from each slide, 1 of superficial tissues adjacent to the rete peg ridges, and the other showing the deepest part of the tissue visible on the slide. An experienced, trained technician who was blinded to the study design, counted superficial and deep vessels.   Immunohistochemistry. Histological sections were de-paraffinized and rehydrated in preparation for immunostaining. All slides were incubated at 4˚C with 10% hamster serum (1 mL hamster serum, [Abcam, Cambridge, MA]) plus 9-mL PBS (pH 7.4) for 30 minutes. The primary antibody podoplanin/gp36 (Abcam) was diluted 1:200 in PBS (pH 7.4). Experimental slides were incubated overnight at 4˚C with 300 L of primary antibody, while control slides were incubated in hamster serum alone. The next morning, the slides were washed in the 10% hamster serum for 5 minutes. Experimental and control slides were then incubated with a secondary rabbit anti-hamster polyclonal antibody at 1:300 in PBS (pH 7.4) for 2 hours at room temperature (Santa Cruz Biotechnology, Inc. CA). Thereafter, each slide was washed in 300-µL of PBS (pH 7.4) 3 times for 5 minutes, and incubated in a diaminobenzidine tetrahydrochloride solution for 5 minutes. Counterstaining was done with hematoxylin & eosin (H&E). Photomicrographs were taken and stained vessels counted as before.

Statistical Analysis

  Statistical analysis was conducted using SPSS v.17 (SPSS Inc, Chicago, IL). Univariate ANOVA was used to test the effects of suction (treated vs. control), pressure (-150 mmHg vs. -300 mmHg), time intervals (2, 3, and 5 minutes), day of treatment (days 2, 3, 4, and 5), and time of treatment (pre vs. post), where applicable. The Kolmogorov-Smirnov test was employed to test for normality of the data. Levene’s test was used to confirm equality of variances between the groups.

Results

  Gross morphology and hyperemia. No visible signs of tissue damage were noted in any of the animals in any of the treatment groups. As subatmospheric pressure was applied to each site, blanching of the gingival tissues was noted around the suction tip. This blanching remained for the duration of treatment. The gingival tissue was slightly raised compared to the surrounding tissue immediately post-subatmospheric pressure treatment, and the treated area became reddened (Figure 2). By the following day, the treatment area had returned to a normal appearance.   Hyperemic areas were significantly larger on the fifth day (P ≤ 0.0001) than the earlier time points (Table 1). Hyperemic area measured immediately post treatment was significantly larger (P ≤ 0.0001) than the pre-treatment area. Subatmospheric pressure (-300 mmHg) caused significantly larger hyperemic areas than those seen at 150 mmHg (P ≤ 0.001). After 2 minutes of treatment, the sizes of the hyperemic areas were significantly larger (P ≤ 0.003) than those seen at the 3- and 5-minute treatment intervals. Post-hoc analysis showed that the sizes of the hyperemic areas caused after 2 minutes of -300 mmHg of vacuum treatment were significantly increased compared to those seen at 2 minutes and -150 mmHg (P ≤ 0.0001). No significant differences between the 2 subatmospheric pressure groups were found after 3 minutes and 5 minutes.   VEGF production. Subatmospheric pressure of -150 mmHg and -300 mmHg for 2, 3, and 5 minutes significantly (P ≤ 0.0001) increased VEGF levels (Table 2). There were no differences between subatmospheric pressure treatments or between time intervals, and no interactive effect of pressure and time.   Histology and blood vessels. All control and treatment groups showed large, wavy collagen fibers interspersed with blood vessels (Figure 3). Superficial tissues had numerous small vessels, while larger blood vessels were visible in the deep tissues. No evidence of separation of the subepithelial tissues from the epidermis was noted, and there were no signs of edema, inflammation, or hematoma formation.   No differences in response to treatment were noted between different vessel sizes or between superficial and deep vessels in all groups, and no differences were noted between control groups (Table 3). For further analysis, all control groups were combined, and all sizes of vessels were combined in the control group and in each of the treatment groups. Subatmospheric pressure treatment produced more blood vessels compared to control treatment (P ≤ 0.001), with no differences between treatment groups when time was not factored in. There were no differences between time intervals, although the interactive effect of pressure and time had a significant effect (P ≤ 0.001). Post-hoc analysis of the effect of pressure at different time intervals showed that treatment with -150 mmHg for 2 minutes was not different from control, but both control and -150 mmHg groups had significantly fewer vessels than the -300 mmHg group (P ≤ 0.0001). After 3 minutes of subatmospheric pressure application, the -150 mmHg group (P ≤ 0.001) and the -300 mmHg group (P ≤ 0.028) had significantly more vessels than the control group, but the numbers of vessels in the 2 treated groups did not differ from one another. After 5 minutes, the -150 mmHg group (P < 0.0001) and the -300 mmHg group (P ≤ 0.047) had significantly more vessels than the control, but the 2 treated groups did not differ from one another.   Immunohistochemistry. Many large and small vessels remained unstained with the podoplanin/GP36 antibody (Figure 4). The majority of the stained vessels appeared to be small collateral vessels surrounding larger vessels. Slides incubated with only secondary antibody showed no immunoreactvity. Subatmospheric pressure significantly increased the number of small superficial vessels immunoreactive for podoplanin/GP36 (P ≤ 0.05), and there was no difference between the 2 treatment groups (Table 4). In the deep tissues, no differences in vessel numbers were seen between groups. Although not statistically significant (P ≤ 0.06), subatmospheric pressure appeared to increase the total number of vessels (superficial plus deep) compared to control treatment, but no differences were seen between the two treatment groups.

Discussion

  From 1968–1970, patients with periodontitis were treated using a combination of vacuum-massage and hydro-massage.34,35 Vacuum was created using a dental compressor, and was applied until the patient reported pain or discomfort. The vacuum was not calibrated and pain intensity was not measured. Water pressure for hydro-massage at 4–6 atmospheres (atm) was applied immediately post-vacuum treatment. The investigators reported hematoma formation that resolved within 2 days after the treatments, along with decreased periodontal pocket depth. Decreased gingival bleeding was reported in 97% of the patients; the other 3% reported no change after treatment.34 Investigators using a vacuum compression vibration device (VCVD) demonstrated that patients had decreased gingival bleeding after vacuum application at -140 mmHg and vibration of 25 GHz for 20 minutes each day.35 The investigators reported that after 3–4 procedures, bleeding from the gingiva completely or partially stopped during tooth brushing. In patients who underwent 5–7 procedures, bleeding completely stopped and periodontal pocket depths decreased. Other investigators also recorded that sialic acid and CRP decreased in the patient’s blood, indicating potentially decreased levels of gingival inflammation and vascular wall permeability.36 Resistance to damage was increased for up to 6 months after VCVD application. Between 6–12 months after treatment, patients with initially severe periodontal disease developed recurrence of inflammation and bleeding. However, inflammation was lower than that observed pre-treatment.   A limitation of previous dental studies was the lack of optimized conditions for subatmospheric pressure application and amount of treatment time. While clinical outcomes appeared promising and a decrease in inflammatory markers was seen, no underlying mechanism was identified to explain the improvement in gingival health. The present study tested the hypothesis that subatmospheric pressure increases the number of blood vessels in gingival tissues, and proposed that these vessels can improve blood supply to the periodontium. Results demonstrated that subatmospheric pressure of -150 mmHg and -300 mmHg significantly increased the appearance of blood vessels when applied for 5 consecutive days. While the shortest treatment time did not produce an overall significant increase in numbers of blood vessels, it did produce a significant increase in numbers of small vessels stained with an antibody that optimally identifies new lymphatic vessels. The specificity of this antibody is for staining thin-walled vessels not visible with H&E staining, and significantly increased numbers of small vessels were noted with both treatment pressures. These data are similar to the findings of Scherer et al,37 who showed that vacuum assisted full-thickness wound closure in a diabetic mouse model resulted in a 2-fold increase in vascularity compared to control, as well as an increase in cell proliferation. The idea that increased vascularity results in increased blood flow is supported by the findings of Ichioka et al,38 who showed that subatmospheric pressures similar to those used in this study produced increased blood flow in wound beds. Argenta et al30 also showed a 4-fold increase in blood flow when subatmospheric pressures were applied to wounds. Evidence of increased blood flow at the edges of a wound after subatmospheric pressure treatment provides further support of this idea.39   In association with the increased numbers of blood vessels seen in response to subatmospheric pressures, significantly increased VEGF expression was found. Interestingly, decreasing amounts of VEGF were noted at higher pressures and longer treatment times, paralleling the decreased blood flow seen at high subatmospheric pressures,27,38 and increased VEGF expression was noted in response to intermittent subatmospheric pressure application in cell culture.40 However, in contrast to Scherer et al37 these researchers reported decreased cell proliferation.37 In a rat wound healing model, increased VEGF expression accompanied accelerated wound closure.41 A recent clinical study showed elevated VEGF in wound fluid after vacuum treatment compared to wounds not undergoing vacuum therapy, while serum VEGF levels remained unchanged.42 This implies that the angiogenic effects of subatmospheric pressure remain localized to the wound area. It would be interesting to see if serum markers of inflammation are decreased in response to subatmospheric treatment of inflamed gingival tissues.   A localized hyperemic response was seen in all treatment groups, comparable to that seen by Grubianov et al,35 where some patients had a hyperemic border on the periphery of the area of vacuum compression massage, while other patients developed an anemic or pale border at the treatment site. In both situations, inflammation was observed for 4–6 hours after the procedure.35 In this study, blanching of the treatment site was noted during treatment prior to the appearance of the hyperemic area. A Doppler temperature recorder showed that these blanched areas had temperatures 3˚C–6˚C lower than surrounding tissues for several minutes post-treatment (unpublished data), indicating that subatmospheric pressure treatment may lead to a transient hypoxic event. Reduced expression of hypoxia inducible factor-1 is noted after subatmospheric pressure treatment of irradiated tissue,43 so it would be interesting to determine if this factor changes expression in gingival tissues after subatmospheric pressure treatment. The hyperemic response appeared immediately after each treatment was completed, and was diminished by the next day in all treatment groups.   This study did not examine shorter treatment times and lower treatment pressures. However, given that the shortest treatment time and lowest pressure produced increased numbers of small vessels detectable with immunohistochemistry, it is possible that even shorter treatments or lower pressures might not produce a measurable effect. Future studies will measure the durability of the increased vasculature after treatment is completed, and if subatmospheric pressure decreases gingivitis and/or periodontitis symptoms and severity.

Conclusion

  The present study demonstrated that subatmospheric pressure stimulated the appearance of increased numbers of blood vessels in healthy dog gingiva, associated with increased levels of VEGF expression. Within the test parameters, the shortest treatment time and lower treatment pressure produced both increased numbers of small vessels and increased VEGF expression, and will be used in future studies on animals and humans to evaluate the effects of noncontinuous subatmospheric pressure on periodontal disease.

Acknowledgements

  The authors wish to thank Mr. Gerald Hill for help during clinical procedures, as well as his exceptional supervision and care for the animals during the study. The authors also thank Ms. Claudia Fernandez for her help in counting the vessels, and Dr. Maria Serrano for her technical laboratory support. The authors appreciate the help of Dr. Raghunath Puttaiah in editing the manuscript.

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Lee, BS; Paul C. Dechow, PhD; Kathy K.H. Svoboda, PhD; and Lynne A. Opperman, PhD are from the Department of Biomedical Sciences, Texas A&M Health Science Center, Baylor College of Dentistry, Dallas, TX. Roman A. Budinskiy, BS, BA is from the Departments of Biochemistry and Petroleum Engineering, Texas Tech University, Lubbock, TX. Address correspondence to: Oksana Budinskaya, DDS Diagnostic Sciences Department Texas A&M Health Science Center Baylor College of Dentistry 3302 Gaston Ave. Dallas, TX, 75246 obudinskaya@bcd.tamhsc.edu