- Email: [email protected]
Quantitative sensory testing of intraoral open wounds
Int. J. Oral Maxillofac. Surg. 2013; 42: 401–405 http://dx.doi.org/10.1016/j.ijom.2012.11.010, available online at http://www.sciencedirect.com
Research Paper Wound Healing
Quantitative sensory testing of intraoral open wounds D. A. Ettlin, T. Hitz, C. Ramel, M. L. Meier, M. Roos, L. M. Gallo, P. Svensson, C. H. Ha¨mmerle: Quantitative sensory testing of intraoral open wounds. Int. J. Oral Maxillofac. Surg. 2013; 42: 401–405. # 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Abstract. Wound healing is an important aspect of oral and maxillofacial surgery. Positive sensory signs (allodynia, hyperalgesia) and negative sensory signs (hypoesthesia, hypoalgesia) may be encountered. Quantitative sensory testing (QST) has moved from bench to bedside for the detection, therapy selection and monitoring the recovery of individuals with sensory disturbances. Tracking somatosensory changes during normal and abnormal wound healing has not previously been reported. This report presents data obtained by a novel, automated, non-contact psychophysical method for assessment of wound sensitivity after standardized oral mucosal biopsy. By directing graded air puffs towards palatal biopsy wounds, thresholds for sensory detection, pain detection and pain tolerance were repeatedly assessed across 19 days, demonstrating high reliability. Participants recorded daily spontaneous and chewing-evoked maximum pains. Pain detection and tolerance thresholds increased linearly across time. Comparison between air puff evoked pain detection threshold and chewing-evoked pain demonstrated a strong correlation. Thus, for the first time, this study tracked the time course of somatosensory sensitivity of wounds induced by oral biopsies. The psychophysical data on wound healing obtained by this automated, contact-free stimulation method can be utilized as a surrogate marker for clinical pain improvements and standardized assessment of intraoral pain sensitivity, for example in oral mucositis.
A thorough understanding of the wound healing process is critical in oral and maxillofacial surgery for interpreting the relationship between patient discomfort, wound presentation, and underlying biological events. Knowledge has accumulated regarding cellular and molecular mechanisms responsible for epithelial wound healing.1 Briefly, blood clot formation begins immediately following tissue trauma. At 0901-5027/030401 + 05 $36.00/0
the interface between the clot and surrounding normal tissue, epidermal migration begins within 24 h after injury, yet no tissue cells have invaded the clot before 3 days. After 3 days the clot is enriched by abundant neutrophils secreting growth factors into the wound environment, stimulating further epidermal cell proliferation. Compared to skin wounds, re-epithelialization of oral mucosa occurs more rapidly and
D. A. Ettlin1, T. Hitz2, C. Ramel2, M. L. Meier1, M. Roos3, L. M. Gallo1, P. Svensson4, C. H. Ha¨mmerle2 1 University of Zurich, Center of Dental Medicine, Clinic of Masticatory Dysorders, Removable Prosthodontics, Geriatric and Special Care Dentistry, Zurich, Switzerland; 2 University of Zurich, Center of Dental Medicine, Clinic of Fixed Prosthodontics and Dental Material Science, Zurich, Switzerland; 3 University of Zurich, Division of Biostatistics, Institute of Social and Preventive Medicine, Zurich, Switzerland; 4University of Aarhus, Department of Clinical Oral Physiology, Aarhus University Hospital, Center for Functionally Integrative Neuroscience (CFIN), MindLab, Aarhus, Denmark
Keywords: wound healing; neurophysiology; mouth mucosa; pain threshold; pain perception. Accepted for publication 12 November 2012 Available online 11 December 2012
with minimal scar formation.2 This may in part be attributable to earlier resolution of inflammation and differential regulation of various cellular and molecular mechanism in oral mucosal compared to dermal wounds.3,4 Despite these advances in the understanding of biological processes involved in acute wound healing, surprisingly little is known about the time course of somatosensory changes and relation to
# 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
402
Ettlin et al.
pain evoked by natural function, such as chewing. One possible explanation for the scarce data on extra-oral and intraoral wound sensitivity might be the lack of appropriate stimulus methods because physical contact between the stimulator or stimulus device could impinge natural healing and lead to infection. The authors recently developed a computer-controlled, multi-valve air puff delivery system for quantitative sensory testing (QST) of open wounds.5 This novel approach applies a standardized contactfree stimulus to tissue and it is suggested to improve understanding of somatosensory changes in the healing phase. This new knowledge may lead to better pain management and patient care by oral and maxillofacial surgeons caring for mucosal wounds of traumatic, surgical, cancerous or chemoradiation related origin. Thus, the aims of this study were to examine in an oral mucosal wound healing model: the test–retest reliability of a new air jet tissue stimulation method on palatal wounds; the wound sensitivity in response to standardized 2 s air puff stimuli of quantified flow rates across a 19-day healing period; and to test for correlations between air-puff evoked pain and clinical levels of pain provoked by chewing. Materials and methods
10 healthy, pain-free individuals (aged 22–39 years; 7 females) were recruited for this study. Subjects received detailed information about the experimental procedure and provided written informed consent. Participants consented not to take any analgesic medication (including over the counter medicine) 3 days prior to or during the study. CHF 500 was offered to each subject for study participation. Air puff stimulator
A modified portable version of the air puff delivery system previously described was used for stimulation of the oral mucosa (Fig. 1a).5,6 This system enables application of graded air streams with flow rates starting at 2 l/min (barely noticeable) to 20 l/min which corresponds to an air stream exiting from a triple air syringe commonly used in dental offices. Pilot laboratory observation has shown that on intact oral mucosa, the highest flow rates do not cause any painful sensations. The application of standardized air puffs first requires fabrication of customized intraoral splints. Alginate impressions were taken of the upper (maxillary) dental arch and palate. The clinician performing the
Fig. 1. (a) Air jet delivery system. (b) Delivery tubes in situ (on model).
surgery marked the location of the designated palatal biopsy site on the maxillary cast. A second mark was placed equidistant from the midline to serve as control site. A soft dental acrylic splint was fabricated for placement on the maxillary teeth (Fig. 1b). Two clear polyurethane tubes (Festo, Dietikon, Switzerland) of 4 mm inner diameter for air stimulation were permanently mounted onto this splint with GC acrylic resin (GC Europe, Heverlee, Belgium). The air tubes served to target the air puffs to the biopsy wound as well as the palatal control site. At the oral end of the tubes, 908 angled nozzles of 2 mm inner diameter were inserted with their palatal openings located 3 mm from the indicated stimulation target centre. Surgical biopsy
Seated subjects were requested to rinse their mouth with chlorhexidine 0.2% for 60 s. Photographs of the palatal mucosa were taken and subsequently, the stimulation splint was inserted. On one side of the palate (side selection by simple randomization, i.e. tossing a coin) approximately 10 mm from the palatal gingival margin of the second premolar, a mucosal mark at the opening of the stimulation tube was placed indicating the biopsy site. For local anaesthesia, articaine 4% (1 ml) was infil-
trated submucosally. After 3 min, a 6 mm punch biopsy of 2–3 mm thickness was obtained. Psychophysical assessment
The study protocol (with a focus on the subject’s rating procedure) was explained to participants prior to the first examination. Assessment of intraoral somatosensory sensitivity with the air puff stimulator lasted no longer than 30 min and was always performed in the early mornings (8–9 am) of the following days: prior to surgery (baseline at day 1; always a Monday morning) and on subsequent days 2, 4, 5, 8, 10, 12, 15 and 19. Photographs were taken prior to psychophysical testing. Comfortable seating of stimulation tubes was checked and the palatal mucosa dried with cotton gauze before air stimulation. At baseline (day 1), air-puff stimuli of 2 s duration and randomized interstimulus intervals between 4 and 6 s were applied bilaterally (randomization by Excel 2003, Microsoft Corp., Redmond, USA). The sensory detection threshold (SDT) was defined as the lowest flow rate at which the volunteer sensed an air puff. The SDT was determined, starting at a flow rate of 2 l/min (system inherent lower limit), with subsequent 1 l/min increments. In order to assess the variability between repeated
Quantitative sensory testing of intraoral open wounds determinations, testing was repeated three times for each site. On postoperative days (days 2–19), photographs of the biopsy wounds were taken and air-puff stimuli applied as described above. SDT, pain detection threshold (PDT; the lowest flow rate that was perceived as just painful) and pain tolerance threshold (PTT; the maximum air flow rate that the subject would tolerate) were determined three times, with 4 min pauses between each series. The average of the three determinations was used for statistical analyses and to provide a coefficient of variation (CV). Self-reported pain
During the postoperative period (days 2– 19), subjects were asked to fill in a pain diary for the three main meals (breakfast, lunch, and dinner), and to indicate spontaneous pain before meals as well as maximum chewing-evoked pain during meals. Subjects were free to select their food. Pain diaries consisted of vertical 0–10 numerical rating scales (NRS) with 0 labelled as ‘no pain’ and 10 as ‘worst imaginable pain’. Statistical analysis
Means and standard errors (SEM) of the three repeated measurements for SDT, PDT and PTT were computed. The CV was calculated as a measure of intra-subject variability. Threshold data (SDT, PDT, PPT) were analyzed with Repeated Measures Analyses of Variance (RM ANOVA) with Greenhouse–Geisser correction and polynomial within-subject contrasts to check for linear relationships across days. Within-subject contrasts between specific days against subsequent
days and against the last measurement day (day 19) were used. Additionally, the paired Wilcoxon signed ranks test together with Bonferroni correction was applied for pain level differences between the time points. Given the small sample size, none of the tests was significant after adjusting for multiple testing. Inter-subject variability for thresholds was analyzed by employing a one-way ANOVA. Spearman correlations between psychophysical and self-reported pain were computed. Results
All subjects completed the study without adverse events (excessive bleeding, infection). A typical example of the clinical time course is shown in Fig. 2. Psychophysical assessment
At baseline, SDT was reached with the lowest flow rate of 2 l/min (Fig. 3a). No subject reported any pain when the intact mucosa was stimulated with the highest flow rate (20 l/min). Intra-subject variability across the 19-day measurement period revealed excellent test–retest reliability with CV values of 5.9% for the control side, and 8.1% for the test side. ANOVAs revealed no significant withinsubject differences across time for both sides (p > 0.13). During the first postoperative week, flow rates to reach PDT on the test site ranged widely between 4 and 20 l/min with substantial inter-individual variability on day 2 (p = 0.003), day 4 (p = 0.0004), day 5 (p < 0.0001) and day 8 (p < 0.0001), but no longer on day 10 (p = 0.05) (Fig. 3a). Intra-subject variability for the three repeated measurements was again small
403
with a CV of 8.6%. ANOVA within-subject contrasts revealed an overall significant linear relationship between PDT and postoperative time course (p = 0.005). The test site was most sensitive to air puff pulses at day 2, compared with day 10 and subsequent days (p = 0.03), while by day 4, PDTs reached levels which did not significantly differ from day 10 (p = 0.068). In between these days, PDTs temporarily differed on day 5 (p = 0.049) and day 8 (p = 0.039). When comparing day to day, changes were mainly evident during the early phase (day 2 vs. day 4, p = 0.028) and late phase of wound healing (day 8 vs. day 10, p = 0.039). Flow rates required to reach PTT varied widely between 10 and 20 l/min. PTTs were lowest during the initial phase (day 2 vs. day 10, p = 0.047), but on average, maximum air flows were readily tolerated on subsequent days (Fig. 3a). As for SDT and PDT, intra-subject variability was small (mean CV 3.1%). Inter-subject variability significantly varied on day 2 (p = 0.0001) and day 8 (p < 0.0001) whereas variability decreased on days 4 (p = 0.19) and 5 (p = 0.1). ANOVA within-subject contrasts revealed a trend towards a linear relationship between PTT and postoperative time course (p = 0.054). Self-reported pain
The maximum NRS pain scores before and after the three daily meals were recorded daily. Chewing evoked significantly higher NRS scores of pain than spontaneous pain (p = 0.001), but in general only moderate levels of pain were experienced by the subjects (Fig. 3b). Comparison between pain ratings for spontaneous pain, chewing evoked pain
Fig. 2. Example of wound healing in one subject from day 1 (baseline) through day 19 (d1–d19).
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Fig. 3. (a) Sensitivity to air puff stimuli on test site (red) and control site (green). Mean flow rates required to reach SDT, PDT and PTT (n = 10). I bars represent standard errors. Arrow indicates time of biopsy. n.a., not applicable. (b) Mean NRS scores of spontaneous and chewing pain from day 2 through day 19.
and PDT, revealed the following coefficients of correlation: spontaneous pain vs. chewing-evoked pain, r = 0.83 (p = 0.003); chewing-evoked pain vs. PDT, r = 0.64 (p < 0.0001); spontaneous pain vs. PDT, r = 0.51 (p < 0.0001). Discussion
The key finding of this study is that the novel technique with standardized air puff stimuli5 provides reliable information on postoperative pain sensitivity in an oral mucosal pain model. Somatosensory sensitivity to air puff stimuli gradually subsided during the time course of wound healing and closely correlated to chewing-evoked pain as well as spontaneous pain. In the experimental and clinical pain field, QST has emerged as a reliable method for investigating subjective responses to stimuli applied to intact tissue.7,8 Yet, psychophysical aspects of wound healing have received little attention,9 despite emerging knowledge on molecular aspects of epidermal pain and nociception.10–12 The most likely reason for a lack of QST data on open wounds is that commonly applied test instruments
(thermodes, von Frey filaments, pinprick stimulators, graded tuning forks, and pressure algometers) are unsuitable for wound investigations due to contamination risks and impingement on the wound. Intraoral laser stimuli might also be an option for a non-contact stimulator and could provide additional information on thermal heat pain sensations following injury.13 The wound stimulation instrument used in this study demostrated high reliabilty for repeated measurements, documented by low CVs for SDT, PDT and PTT (all below 10%). Part of the low CV could be due to inherent limitations with SDT for all subjects less than 2 ml/min and PTT greater than 20 ml/min for many subjects. Nevertheless, flow rates to reach SDTs were minimal which may be due to the fine innervation of the hard palate.14 A remarkable variation for PDT and PTT among subjects was observed mainly during the first week of wound healing. Variability of pain complaints is commonly observed during postoperative wound healing,15 possibly due to genetic factors.16–18 Scab coverage of the wound might explain why subjects responded to both experimental (air puffs) and natural stimuli (meals) with a rapid decrease in
pain sensitivity by day 4. Wounds tended to become slightly more sensitive again until day 12 (Fig. 3a). It appears pain caused by palatal punch biopsies lasts up to 14 days. These results reveal that during wound healing across time, pain ‘naturally’ provoked by chewing decreased and, at the same time, the experimental stimulus strength required to evoke pain (PDT) increased, which is reflected by a negative correlation coefficient of r = 0.64. Similarly, the correlation coefficient between spontaneous pain and experimental stimulus strength to evoke pain (PDT) was r = 0.51. Regarding study limitations, alternative statistical methods might also have been appropriate. The authors opted for RM ANOVA as it offers the best description of longitudinal changes of pain levels given the data at hand (n = 10). A key advantage of RM ANOVA is keeping the global alpha-level at 5% and still being able to investigate longitudinal changes of pain levels by applying appropriate contrasts. Also, the study design did not aim to test differential functional recovery of nerve fibre subpopulations. Nonetheless, the observations of recovery of somatosensory sensitivity in mucosal wounds seem to at least partly match reports on nerve function recovery after trigeminal nerve injuries following bilateral sagittal split operations.19 These earlier studies have shown that detection of brush stroke direction occurred in the majority of patients after 2 weeks. On the other hand, recovery of other somatosensory modalities such as thermal heat pain, cold pain and mechanical pain requires up to 3 months and recovery of warm sensation up to 1 year.19 Future studies may address these latter aspects. In conclusion, use of a new automated contact-free air puff stimulation method enabled reliable measurements for sensory and pain detection thresholds as well as for pain tolerance thresholds during a 19 day wound healing period. Experimental quantitative data correlated well with scores from chewing and spontaneous pain. This new psychophysical method shows promise for standardized assessment of sensitivity of intraoral mucosal or facial skin wounds and may allow comparison of the efficacy of wound healing interventions supplemental to other measures.20 Funding
The authors appreciate the financial support for the development of the stimulation device by GlaxoSmithKline (GSK),
Quantitative sensory testing of intraoral open wounds Consumer Healthcare, Weybride, London. GSK had no role in the study design; collection, analysis and interpretation of data; manuscript writing; decision to submit the manuscript for publication. Competing interests
The authors declare no financial relationship that might pose a conflict of interest. Ethical approval
The study was approved by local ethics committee (StV07/12) and conducted according to the guidelines of the Declaration of Helsinki for treatment of experimental human subjects. References 1. Watkins SA, Zippin JH. When wound healing goes awry. A review of normal and abnormal wound healing, scar pathophysiology, and therapeutics. J Drugs Dermatol 2008;7:997–1005. 2. Szpaderska AM, Zuckerman JD, DiPietro LA. Differential injury responses in oral mucosal and cutaneous wounds. J Dent Res 2003;82:621–6. 3. Enoch S, Peake MA, Wall I, Davies L, Farrier J, Giles P, et al. ‘Young’ oral fibroblasts are geno/phenotypically distinct. J Dent Res 2010;89:1407–13. 4. Mak K, Manji A, Gallant-Behm C, Wiebe C, Hart DA, Larjava H, et al. Scarless healing of oral mucosa is characterized by faster resolution of inflammation and control of myofibroblast action compared to skin wounds in the red Duroc pig model. J Dermatol Sci 2009;56:168–80.
5. Megias-Alguacil D, Keller T, Lutz K, Barlow AP, Ettlin DA. Design and construction of a magnetic resonance compatible multiinjector gas jet delivery system. Rev Sci Instrum 2008;79:014301. 6. Thoma DS, Sancho-Puchades M, Ettlin DA, Hammerle CH, Jung RE. Impact of a collagen matrix on early healing, aesthetics and patient morbidity in oral mucosal wounds – a randomized study in humans. J Clin Periodontol 2012;39:157–65. 7. Haanpaa ML, Backonja MM, Bennett MI, Bouhassira D, Cruccu G, Hansson PT, et al. Assessment of neuropathic pain in primary care. Am J Med 2009;122:S13–21. 8. Pigg M, Baad-Hansen L, Svensson P, Drangsholt M, List T. Reliability of intraoral quantitative sensory testing (QST). Pain 2009; 148:220–6. 9. Wright C, Goudas LC, Bentch A, Mehdi M, Perry PP, Carr DB. Hyperalgesia in outpatients with dermal injury: quantitative sensory testing versus a novel simple technique. Pain Med 2004;5:162–7. 10. Chen L, Arbieva ZH, Guo S, Marucha PT, Mustoe TA, DiPietro LA. Positional differences in the wound transcriptome of skin and oral mucosa. BMC Genomics 2010;11:471. 11. Huang SM, Lee H, Chung MK, Park U, Yu YY, Bradshaw HB, et al. Overexpressed transient receptor potential vanilloid 3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2. J Neurosci 2008;28:13727–37. 12. Khodorova A, Montmayeur JP, Strichartz G. Endothelin receptors and pain. J Pain 2009;10:4–28. 13. Svensson P, Bjerring P, Rendt-Nielsen L, Kaaber S. Sensory and pain thresholds to orofacial argon laser stimulation in patients with chronic burning mouth syndrome. Clin J Pain 1993;9:207–15.
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14. Dixon AD. Sensory nerve termination in the oral mucosa. Arch Oral Biol 1961;5:105–14. 15. Joly V, Richebe P, Guignard B, Fletcher D, Maurette P, Sessler DI, et al. Remifentanilinduced postoperative hyperalgesia and its prevention with small-dose ketamine. Anesthesiology 2005;103:147–55. 16. Costigan M, Belfer I, Griffin RS, Dai F, Barrett LB, Coppola G, et al. Multiple chronic pain states are associated with a common amino acid-changing allele in KCNS1. Brain 2010;133:2519–27. 17. Shi Q, Cleeland CS, Klepstad P, Miaskowski C, Pedersen NL. Biological pathways and genetic variables involved in pain. Qual Life Res 2010;19:1407–17. 18. Waxman SG. Polymorphisms in ion channel genes: emerging roles in pain. Brain 2010;133:2515–8. 19. Van Boven RW, Johnson KO. A psychophysical study of the mechanisms of sensory recovery following nerve injury in humans. Brain 1994;117:149–67. 20. Schultz G, Mozingo D, Romanelli M, Claxton K. Wound healing and TIME; new concepts and scientific applications. Wound Repair Regen 2005;13:S1–1.
Address: Dominik A. Ettlin University of Zurich Center of Dental Medicine Clinic of Masticatory Dysorders Removable Prosthodontics Geriatric and Special Care Dentistry Plattenstrasse 11 CH-8032 Zurich Switzerland Tel.: +41 44 634 32 31 fax: +41 44 634 43 02 E-mail: [email protected]
Research Paper Wound Healing
Quantitative sensory testing of intraoral open wounds D. A. Ettlin, T. Hitz, C. Ramel, M. L. Meier, M. Roos, L. M. Gallo, P. Svensson, C. H. Ha¨mmerle: Quantitative sensory testing of intraoral open wounds. Int. J. Oral Maxillofac. Surg. 2013; 42: 401–405. # 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Abstract. Wound healing is an important aspect of oral and maxillofacial surgery. Positive sensory signs (allodynia, hyperalgesia) and negative sensory signs (hypoesthesia, hypoalgesia) may be encountered. Quantitative sensory testing (QST) has moved from bench to bedside for the detection, therapy selection and monitoring the recovery of individuals with sensory disturbances. Tracking somatosensory changes during normal and abnormal wound healing has not previously been reported. This report presents data obtained by a novel, automated, non-contact psychophysical method for assessment of wound sensitivity after standardized oral mucosal biopsy. By directing graded air puffs towards palatal biopsy wounds, thresholds for sensory detection, pain detection and pain tolerance were repeatedly assessed across 19 days, demonstrating high reliability. Participants recorded daily spontaneous and chewing-evoked maximum pains. Pain detection and tolerance thresholds increased linearly across time. Comparison between air puff evoked pain detection threshold and chewing-evoked pain demonstrated a strong correlation. Thus, for the first time, this study tracked the time course of somatosensory sensitivity of wounds induced by oral biopsies. The psychophysical data on wound healing obtained by this automated, contact-free stimulation method can be utilized as a surrogate marker for clinical pain improvements and standardized assessment of intraoral pain sensitivity, for example in oral mucositis.
A thorough understanding of the wound healing process is critical in oral and maxillofacial surgery for interpreting the relationship between patient discomfort, wound presentation, and underlying biological events. Knowledge has accumulated regarding cellular and molecular mechanisms responsible for epithelial wound healing.1 Briefly, blood clot formation begins immediately following tissue trauma. At 0901-5027/030401 + 05 $36.00/0
the interface between the clot and surrounding normal tissue, epidermal migration begins within 24 h after injury, yet no tissue cells have invaded the clot before 3 days. After 3 days the clot is enriched by abundant neutrophils secreting growth factors into the wound environment, stimulating further epidermal cell proliferation. Compared to skin wounds, re-epithelialization of oral mucosa occurs more rapidly and
D. A. Ettlin1, T. Hitz2, C. Ramel2, M. L. Meier1, M. Roos3, L. M. Gallo1, P. Svensson4, C. H. Ha¨mmerle2 1 University of Zurich, Center of Dental Medicine, Clinic of Masticatory Dysorders, Removable Prosthodontics, Geriatric and Special Care Dentistry, Zurich, Switzerland; 2 University of Zurich, Center of Dental Medicine, Clinic of Fixed Prosthodontics and Dental Material Science, Zurich, Switzerland; 3 University of Zurich, Division of Biostatistics, Institute of Social and Preventive Medicine, Zurich, Switzerland; 4University of Aarhus, Department of Clinical Oral Physiology, Aarhus University Hospital, Center for Functionally Integrative Neuroscience (CFIN), MindLab, Aarhus, Denmark
Keywords: wound healing; neurophysiology; mouth mucosa; pain threshold; pain perception. Accepted for publication 12 November 2012 Available online 11 December 2012
with minimal scar formation.2 This may in part be attributable to earlier resolution of inflammation and differential regulation of various cellular and molecular mechanism in oral mucosal compared to dermal wounds.3,4 Despite these advances in the understanding of biological processes involved in acute wound healing, surprisingly little is known about the time course of somatosensory changes and relation to
# 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
402
Ettlin et al.
pain evoked by natural function, such as chewing. One possible explanation for the scarce data on extra-oral and intraoral wound sensitivity might be the lack of appropriate stimulus methods because physical contact between the stimulator or stimulus device could impinge natural healing and lead to infection. The authors recently developed a computer-controlled, multi-valve air puff delivery system for quantitative sensory testing (QST) of open wounds.5 This novel approach applies a standardized contactfree stimulus to tissue and it is suggested to improve understanding of somatosensory changes in the healing phase. This new knowledge may lead to better pain management and patient care by oral and maxillofacial surgeons caring for mucosal wounds of traumatic, surgical, cancerous or chemoradiation related origin. Thus, the aims of this study were to examine in an oral mucosal wound healing model: the test–retest reliability of a new air jet tissue stimulation method on palatal wounds; the wound sensitivity in response to standardized 2 s air puff stimuli of quantified flow rates across a 19-day healing period; and to test for correlations between air-puff evoked pain and clinical levels of pain provoked by chewing. Materials and methods
10 healthy, pain-free individuals (aged 22–39 years; 7 females) were recruited for this study. Subjects received detailed information about the experimental procedure and provided written informed consent. Participants consented not to take any analgesic medication (including over the counter medicine) 3 days prior to or during the study. CHF 500 was offered to each subject for study participation. Air puff stimulator
A modified portable version of the air puff delivery system previously described was used for stimulation of the oral mucosa (Fig. 1a).5,6 This system enables application of graded air streams with flow rates starting at 2 l/min (barely noticeable) to 20 l/min which corresponds to an air stream exiting from a triple air syringe commonly used in dental offices. Pilot laboratory observation has shown that on intact oral mucosa, the highest flow rates do not cause any painful sensations. The application of standardized air puffs first requires fabrication of customized intraoral splints. Alginate impressions were taken of the upper (maxillary) dental arch and palate. The clinician performing the
Fig. 1. (a) Air jet delivery system. (b) Delivery tubes in situ (on model).
surgery marked the location of the designated palatal biopsy site on the maxillary cast. A second mark was placed equidistant from the midline to serve as control site. A soft dental acrylic splint was fabricated for placement on the maxillary teeth (Fig. 1b). Two clear polyurethane tubes (Festo, Dietikon, Switzerland) of 4 mm inner diameter for air stimulation were permanently mounted onto this splint with GC acrylic resin (GC Europe, Heverlee, Belgium). The air tubes served to target the air puffs to the biopsy wound as well as the palatal control site. At the oral end of the tubes, 908 angled nozzles of 2 mm inner diameter were inserted with their palatal openings located 3 mm from the indicated stimulation target centre. Surgical biopsy
Seated subjects were requested to rinse their mouth with chlorhexidine 0.2% for 60 s. Photographs of the palatal mucosa were taken and subsequently, the stimulation splint was inserted. On one side of the palate (side selection by simple randomization, i.e. tossing a coin) approximately 10 mm from the palatal gingival margin of the second premolar, a mucosal mark at the opening of the stimulation tube was placed indicating the biopsy site. For local anaesthesia, articaine 4% (1 ml) was infil-
trated submucosally. After 3 min, a 6 mm punch biopsy of 2–3 mm thickness was obtained. Psychophysical assessment
The study protocol (with a focus on the subject’s rating procedure) was explained to participants prior to the first examination. Assessment of intraoral somatosensory sensitivity with the air puff stimulator lasted no longer than 30 min and was always performed in the early mornings (8–9 am) of the following days: prior to surgery (baseline at day 1; always a Monday morning) and on subsequent days 2, 4, 5, 8, 10, 12, 15 and 19. Photographs were taken prior to psychophysical testing. Comfortable seating of stimulation tubes was checked and the palatal mucosa dried with cotton gauze before air stimulation. At baseline (day 1), air-puff stimuli of 2 s duration and randomized interstimulus intervals between 4 and 6 s were applied bilaterally (randomization by Excel 2003, Microsoft Corp., Redmond, USA). The sensory detection threshold (SDT) was defined as the lowest flow rate at which the volunteer sensed an air puff. The SDT was determined, starting at a flow rate of 2 l/min (system inherent lower limit), with subsequent 1 l/min increments. In order to assess the variability between repeated
Quantitative sensory testing of intraoral open wounds determinations, testing was repeated three times for each site. On postoperative days (days 2–19), photographs of the biopsy wounds were taken and air-puff stimuli applied as described above. SDT, pain detection threshold (PDT; the lowest flow rate that was perceived as just painful) and pain tolerance threshold (PTT; the maximum air flow rate that the subject would tolerate) were determined three times, with 4 min pauses between each series. The average of the three determinations was used for statistical analyses and to provide a coefficient of variation (CV). Self-reported pain
During the postoperative period (days 2– 19), subjects were asked to fill in a pain diary for the three main meals (breakfast, lunch, and dinner), and to indicate spontaneous pain before meals as well as maximum chewing-evoked pain during meals. Subjects were free to select their food. Pain diaries consisted of vertical 0–10 numerical rating scales (NRS) with 0 labelled as ‘no pain’ and 10 as ‘worst imaginable pain’. Statistical analysis
Means and standard errors (SEM) of the three repeated measurements for SDT, PDT and PTT were computed. The CV was calculated as a measure of intra-subject variability. Threshold data (SDT, PDT, PPT) were analyzed with Repeated Measures Analyses of Variance (RM ANOVA) with Greenhouse–Geisser correction and polynomial within-subject contrasts to check for linear relationships across days. Within-subject contrasts between specific days against subsequent
days and against the last measurement day (day 19) were used. Additionally, the paired Wilcoxon signed ranks test together with Bonferroni correction was applied for pain level differences between the time points. Given the small sample size, none of the tests was significant after adjusting for multiple testing. Inter-subject variability for thresholds was analyzed by employing a one-way ANOVA. Spearman correlations between psychophysical and self-reported pain were computed. Results
All subjects completed the study without adverse events (excessive bleeding, infection). A typical example of the clinical time course is shown in Fig. 2. Psychophysical assessment
At baseline, SDT was reached with the lowest flow rate of 2 l/min (Fig. 3a). No subject reported any pain when the intact mucosa was stimulated with the highest flow rate (20 l/min). Intra-subject variability across the 19-day measurement period revealed excellent test–retest reliability with CV values of 5.9% for the control side, and 8.1% for the test side. ANOVAs revealed no significant withinsubject differences across time for both sides (p > 0.13). During the first postoperative week, flow rates to reach PDT on the test site ranged widely between 4 and 20 l/min with substantial inter-individual variability on day 2 (p = 0.003), day 4 (p = 0.0004), day 5 (p < 0.0001) and day 8 (p < 0.0001), but no longer on day 10 (p = 0.05) (Fig. 3a). Intra-subject variability for the three repeated measurements was again small
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with a CV of 8.6%. ANOVA within-subject contrasts revealed an overall significant linear relationship between PDT and postoperative time course (p = 0.005). The test site was most sensitive to air puff pulses at day 2, compared with day 10 and subsequent days (p = 0.03), while by day 4, PDTs reached levels which did not significantly differ from day 10 (p = 0.068). In between these days, PDTs temporarily differed on day 5 (p = 0.049) and day 8 (p = 0.039). When comparing day to day, changes were mainly evident during the early phase (day 2 vs. day 4, p = 0.028) and late phase of wound healing (day 8 vs. day 10, p = 0.039). Flow rates required to reach PTT varied widely between 10 and 20 l/min. PTTs were lowest during the initial phase (day 2 vs. day 10, p = 0.047), but on average, maximum air flows were readily tolerated on subsequent days (Fig. 3a). As for SDT and PDT, intra-subject variability was small (mean CV 3.1%). Inter-subject variability significantly varied on day 2 (p = 0.0001) and day 8 (p < 0.0001) whereas variability decreased on days 4 (p = 0.19) and 5 (p = 0.1). ANOVA within-subject contrasts revealed a trend towards a linear relationship between PTT and postoperative time course (p = 0.054). Self-reported pain
The maximum NRS pain scores before and after the three daily meals were recorded daily. Chewing evoked significantly higher NRS scores of pain than spontaneous pain (p = 0.001), but in general only moderate levels of pain were experienced by the subjects (Fig. 3b). Comparison between pain ratings for spontaneous pain, chewing evoked pain
Fig. 2. Example of wound healing in one subject from day 1 (baseline) through day 19 (d1–d19).
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Fig. 3. (a) Sensitivity to air puff stimuli on test site (red) and control site (green). Mean flow rates required to reach SDT, PDT and PTT (n = 10). I bars represent standard errors. Arrow indicates time of biopsy. n.a., not applicable. (b) Mean NRS scores of spontaneous and chewing pain from day 2 through day 19.
and PDT, revealed the following coefficients of correlation: spontaneous pain vs. chewing-evoked pain, r = 0.83 (p = 0.003); chewing-evoked pain vs. PDT, r = 0.64 (p < 0.0001); spontaneous pain vs. PDT, r = 0.51 (p < 0.0001). Discussion
The key finding of this study is that the novel technique with standardized air puff stimuli5 provides reliable information on postoperative pain sensitivity in an oral mucosal pain model. Somatosensory sensitivity to air puff stimuli gradually subsided during the time course of wound healing and closely correlated to chewing-evoked pain as well as spontaneous pain. In the experimental and clinical pain field, QST has emerged as a reliable method for investigating subjective responses to stimuli applied to intact tissue.7,8 Yet, psychophysical aspects of wound healing have received little attention,9 despite emerging knowledge on molecular aspects of epidermal pain and nociception.10–12 The most likely reason for a lack of QST data on open wounds is that commonly applied test instruments
(thermodes, von Frey filaments, pinprick stimulators, graded tuning forks, and pressure algometers) are unsuitable for wound investigations due to contamination risks and impingement on the wound. Intraoral laser stimuli might also be an option for a non-contact stimulator and could provide additional information on thermal heat pain sensations following injury.13 The wound stimulation instrument used in this study demostrated high reliabilty for repeated measurements, documented by low CVs for SDT, PDT and PTT (all below 10%). Part of the low CV could be due to inherent limitations with SDT for all subjects less than 2 ml/min and PTT greater than 20 ml/min for many subjects. Nevertheless, flow rates to reach SDTs were minimal which may be due to the fine innervation of the hard palate.14 A remarkable variation for PDT and PTT among subjects was observed mainly during the first week of wound healing. Variability of pain complaints is commonly observed during postoperative wound healing,15 possibly due to genetic factors.16–18 Scab coverage of the wound might explain why subjects responded to both experimental (air puffs) and natural stimuli (meals) with a rapid decrease in
pain sensitivity by day 4. Wounds tended to become slightly more sensitive again until day 12 (Fig. 3a). It appears pain caused by palatal punch biopsies lasts up to 14 days. These results reveal that during wound healing across time, pain ‘naturally’ provoked by chewing decreased and, at the same time, the experimental stimulus strength required to evoke pain (PDT) increased, which is reflected by a negative correlation coefficient of r = 0.64. Similarly, the correlation coefficient between spontaneous pain and experimental stimulus strength to evoke pain (PDT) was r = 0.51. Regarding study limitations, alternative statistical methods might also have been appropriate. The authors opted for RM ANOVA as it offers the best description of longitudinal changes of pain levels given the data at hand (n = 10). A key advantage of RM ANOVA is keeping the global alpha-level at 5% and still being able to investigate longitudinal changes of pain levels by applying appropriate contrasts. Also, the study design did not aim to test differential functional recovery of nerve fibre subpopulations. Nonetheless, the observations of recovery of somatosensory sensitivity in mucosal wounds seem to at least partly match reports on nerve function recovery after trigeminal nerve injuries following bilateral sagittal split operations.19 These earlier studies have shown that detection of brush stroke direction occurred in the majority of patients after 2 weeks. On the other hand, recovery of other somatosensory modalities such as thermal heat pain, cold pain and mechanical pain requires up to 3 months and recovery of warm sensation up to 1 year.19 Future studies may address these latter aspects. In conclusion, use of a new automated contact-free air puff stimulation method enabled reliable measurements for sensory and pain detection thresholds as well as for pain tolerance thresholds during a 19 day wound healing period. Experimental quantitative data correlated well with scores from chewing and spontaneous pain. This new psychophysical method shows promise for standardized assessment of sensitivity of intraoral mucosal or facial skin wounds and may allow comparison of the efficacy of wound healing interventions supplemental to other measures.20 Funding
The authors appreciate the financial support for the development of the stimulation device by GlaxoSmithKline (GSK),
Quantitative sensory testing of intraoral open wounds Consumer Healthcare, Weybride, London. GSK had no role in the study design; collection, analysis and interpretation of data; manuscript writing; decision to submit the manuscript for publication. Competing interests
The authors declare no financial relationship that might pose a conflict of interest. Ethical approval
The study was approved by local ethics committee (StV07/12) and conducted according to the guidelines of the Declaration of Helsinki for treatment of experimental human subjects. References 1. Watkins SA, Zippin JH. When wound healing goes awry. A review of normal and abnormal wound healing, scar pathophysiology, and therapeutics. J Drugs Dermatol 2008;7:997–1005. 2. Szpaderska AM, Zuckerman JD, DiPietro LA. Differential injury responses in oral mucosal and cutaneous wounds. J Dent Res 2003;82:621–6. 3. Enoch S, Peake MA, Wall I, Davies L, Farrier J, Giles P, et al. ‘Young’ oral fibroblasts are geno/phenotypically distinct. J Dent Res 2010;89:1407–13. 4. Mak K, Manji A, Gallant-Behm C, Wiebe C, Hart DA, Larjava H, et al. Scarless healing of oral mucosa is characterized by faster resolution of inflammation and control of myofibroblast action compared to skin wounds in the red Duroc pig model. J Dermatol Sci 2009;56:168–80.
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Address: Dominik A. Ettlin University of Zurich Center of Dental Medicine Clinic of Masticatory Dysorders Removable Prosthodontics Geriatric and Special Care Dentistry Plattenstrasse 11 CH-8032 Zurich Switzerland Tel.: +41 44 634 32 31 fax: +41 44 634 43 02 E-mail: [email protected]