Regional associations between inspiratory tongue dilatory movement and genioglossus activity during wakefulness in people with obstructive sleep apnoea.

Inspiratory tongue dilatory movement is believed to be mediated via changes in neural drive to genioglossus. However, this has not been studied during quiet breathing in humans. Therefore, this study investigated this relationship and its potential role in obstructive sleep apnoea (OSA). During awake supine quiet nasal breathing, inspiratory tongue dilatory movement, quantified with tagged magnetic resonance imaging, and inspiratory phasic genioglossus EMG normalised to maximum EMG were measured in nine controls [apnoea-hypopnea index (AHI) ≤5 events/h] and 37 people with untreated OSA (AHI >5 events/h). Measurements were obtained for 156 neuromuscular compartments (85%). Analysis was adjusted for nadir epiglottic pressure during inspiration. Only for 106 compartments (68%) was a larger anterior (dilatory) movement associated with a higher phasic EMG [mixed linear regression, beta = 0.089, 95% CI [0.000, 0.178], t(99) = 1.995, P = 0.049, hereafter EMG↗/mvt↗]. For the remaining 50 (32%) compartments, a larger dilatory movement was associated with a lower phasic EMG [mixed linear regression, beta = -0.123, 95% CI [-0.224, -0.022], t(43) = -2.458, P = 0.018, hereafter EMG↘/mvt↗]. OSA participants had a higher odds of having at least one decoupled EMG↘/mvt↗ compartment (binary logistic regression, odds ratio [95% CI]: 7.53 [1.19, 47.47] (P = 0.032). Dilatory tongue movement was minimal (>1 mm) in nearly all participants with only EMG↗/mvt↗ compartments (86%, 18/21). These results demonstrate that upper airway dilatory mechanics cannot be predicted from genioglossus EMG, particularly in people with OSA. Tongue movement associated with minimal genioglossus activity suggests co-activation of other airway dilator muscles. KEY POINTS: Inspiratory tongue movement is thought to be mediated through changes in genioglossus activity. However, it is unknown if this relationship is altered by obstructive sleep apnoea (OSA). During awake supine quiet nasal breathing, inspiratory tongue movement, quantified with tagged magnetic resonance imaging (MRI), and inspiratory phasic genioglossus EMG normalised to maximum EMG were measured in four tongue compartments of people with and without OSA. Larger tongue anterior (dilatory) movement was associated with higher phasic genioglossus EMG for 68% of compartments. OSA participants had an ∼7-times higher odds of having at least one compartment for which a larger anterior tongue movement was not associated with a higher phasic EMG than controls. Therefore, higher genioglossus phasic EMG does not consistently translate into tongue dilatory movement, particularly in people with OSA. Large dilatory tongue movements can occur despite minimal genioglossus inspiratory activity, suggesting co-activation of other pharyngeal muscles.

Task-dependent neural control of regions within human genioglossus.

Anatomical and imaging evidence suggests neural control of oblique and horizontal compartments of the genioglossus differs. However, neurophysiological evidence for differential control remains elusive. This study aimed to determine whether there are differences in neural drive to the oblique and horizontal regions of the genioglossus during swallowing and tongue protrusion. Adult participants (n = 63; 48 M) were recruited from a sleep clinic; 41 had obstructive sleep apnea (OSA: 34 M, 8 F). Electromyographic (EMG) was recorded at rest (awake, supine) using four intramuscular fine-wire electrodes inserted percutaneously into the anterior oblique, posterior oblique, anterior horizontal, and posterior horizontal genioglossus. Epiglottic pressure and nasal airflow were also measured. During swallowing, two distinct EMG patterns were observed - a monophasic response (single EMG peak) and a biphasic response (2 bursts of EMG). Peak EMG and timing of the peak relative to epiglottic pressure were significantly different between patterns (linear mixed models, P < 0.001). Monophasic activation was more likely in the horizontal than oblique region during swallowing (OR = 6.83, CI = 3.46-13.53, P < 0.001). In contrast, during tongue protrusion, activation patterns and EMG magnitude were not different between regions. There were no systematic differences in EMG patterns during swallowing or tongue protrusion between OSA and non-OSA groups. These findings provide evidence for functional differences in the motoneuronal output to the oblique and horizontal compartments, enabling differential task-specific drive. Given this, it is important to identify the compartment from which EMG is acquired. We propose that the EMG patterns during swallowing may be used to identify the compartment where a recording electrode is located.NEW & NOTEWORTHY During swallowing, we observed two distinct, stereotyped muscle activation patterns that define the horizontal (monophasic, maximal EMG) and oblique (biphasic, submaximal EMG) neuromuscular compartments of genioglossus. In contrast, volitional tongue protrusions produced uniform activation across compartments. This provides evidence for task-dependent, functionally discrete neuromuscular control of the oblique and horizontal compartments of genioglossus. The magnitude and temporal patterning of genioglossus EMG during swallowing may help guide electrode placement in tongue EMG studies.

Effect of upper airway fat on tongue dilation during inspiration in awake people with obstructive sleep apnea.

STUDY OBJECTIVES: To investigate the effect of upper airway fat composition on tongue inspiratory movement and obstructive sleep apnea (OSA). METHODS: Participants without or with untreated OSA underwent a 3T magnetic resonance imaging (MRI) scan. Anatomical measurements were obtained from T2-weighted images. Mid-sagittal inspiratory tongue movements were imaged using tagged MRI during wakefulness. Tissue volumes and percentages of fat were quantified using an mDIXON scan. RESULTS: Forty predominantly overweight participants with OSA were compared to 10 predominantly normal weight controls. After adjusting for age, BMI, and gender, the percentage of fat in the tongue was not different between groups (analysis of covariance [ANCOVA], p = 0.45), but apnoeic patients had a greater tongue volume (ANCOVA, p = 0.025). After adjusting for age, BMI, and gender, higher OSA severity was associated with larger whole tongue volume (r = 0.51, p < 0.001), and greater dilatory motion of the anterior horizontal tongue compartment (r = -0.33, p = 0.023), but not with upper airway fat percentage. Higher tongue fat percentage was associated with higher BMI and older age (Spearman r = 0.43, p = 0.002, and r =0.44, p = 0.001, respectively), but not with inspiratory tongue movements. Greater inspiratory tongue movement was associated with larger tongue volume (e.g. horizontal posterior compartment, r = -0.44, p = 0.002) and smaller nasopharyngeal airway (e.g. oblique compartment, r = 0.29, p = 0.040). CONCLUSIONS: Larger tongue volume and a smaller nasopharynx are associated with increased inspiratory tongue dilation during wakefulness in people with and without OSA. This compensatory response was not influenced by higher tongue fat content. Whether this is also true in more obese patient populations requires further investigation.

Treatment usage patterns of oral appliances for obstructive sleep apnea over the first 60 days: a cluster analysis.

STUDY OBJECTIVES: Oral appliance (OA) therapy usage can be objectively measured through temperature-sensing data chips embedded in the appliance. Initial reports of group data for short-term treatment usage suggest good nightly hours of usage. However, individual variability in treatment usage patterns has not been assessed. We aimed to identify OA treatment usage subtypes in the first 60 days and the earliest predictors of these usage patterns. METHODS: OSA patients were recruited for a study of OA therapy with an embedded compliance chip (DentiTrac, Braebon, Canada). Fifty-eight participants with 60 days of downloadable treatment usage data (5-minute readings) were analyzed. A hierarchical cluster analysis was used to group participants with similar usage patterns. A random forest classification model was used to identify the minimum number of days to predict usage subtype. RESULTS: Three user groups were identified and named: "Consistent Users" (48.3%), "Inconsistent Users," (32.8%) and "Non-Users" (19.0%). The first 20 days provided optimal data to predict the treatment usage group a patient would belong to at 60 days (90% accuracy). The strongest predictors of user group were downloaded usage data, average wear time, and number of days missed. CONCLUSIONS: Granular analysis of OA usage data suggests the existence of treatment user subtypes (Consistent, Inconsistent, and Non-Users). Our data suggest that 60-day usage patterns can be identified in the first 20 days of treatment using downloaded treatment usage data. Understanding initial treatment usage patterns provide an opportunity for early intervention to improve long-term usage and outcomes. CITATION: Sutherland K, Almeida FR, Kim T, et al. Treatment usage patterns of oral appliances for obstructive sleep apnea over the first 60 days: a cluster analysis. J Clin Sleep Med. 2021;17(9):1785-1792.

Influence of mandibular advancement on tongue dilatory movement during wakefulness and how this is related to oral appliance therapy outcome for obstructive sleep apnea.

STUDY OBJECTIVES: To characterize how mandibular advancement splint (MAS) alters inspiratory tongue movement in people with obstructive sleep apnea (OSA) during wakefulness and whether this is associated with MAS treatment outcome. METHODS: A total of 87 untreated OSA participants (20 women, apnea-hypopnea index (AHI) 7-102 events/h, aged 19-76 years) underwent a 3T MRI with a MAS in situ. Mid-sagittal tagged images quantified inspiratory tongue movement with the mandible in a neutral position and advanced to 70% of the maximum. Movement was quantified with harmonic phase methods. Treatment outcome was determined after at least 9 weeks of therapy. RESULTS: A total of 72 participants completed the study: 34 were responders (AHI < 5 or AHI ≤ 10events/h with >50% reduction in AHI), 9 were partial responders (>50% reduction in AHI but AHI > 10 events/h), and 29 nonresponders (change in AHI <50% and AHI ≥ 10 events/h). About 62% (45/72) of participants had minimal inspiratory tongue movement (<1 mm) in the neutral position, and this increased to 72% (52/72) after advancing the mandible. Mandibular advancement altered inspiratory tongue movement pattern for 40% (29/72) of participants. When tongue dilatory patterns altered with advancement, 80% (4/5) of those who changed to a counterproductive movement pattern (posterior movement >1 mm) were nonresponders and 71% (5/7) of those who changed to beneficial (anterior movement >1 mm) were partial or complete responders. CONCLUSIONS: The mandibular advancement action on upper airway dilator muscles differs between individuals. When mandibular advancement alters inspiratory tongue movement, therapeutic response to MAS therapy was more common among those who convert to a beneficial movement pattern.

Mandibular advancement splint response is associated with the pterygomandibular raphe.

STUDY OBJECTIVES: To investigate whether the presence of tendinous PMR could predict treatment outcome and how it affects lateral wall mechanical properties. Mandibular advancement increases the lateral dimensions of the nasopharyngeal airway via a direct connection from the airway to the ramus of the mandible. The anatomical structure in this region is the pterygomandibular raphe (PMR), but a tendinous component is not always present. Whether tendon presence influences treatment outcome is unknown. METHODS: In total, 105 participants with obstructive sleep apnea completed detailed anatomical magnetic resonance imaging with and without mandibular advancement. The study design was case-control. Variables were compared between participants with and without the tendon present. RESULTS: The amount of maximum mandibular advancement decreased when pterygomandibular tendon was present (4.0 ± 1.2 mm present versus 4.6 ± 1.4 mm absent, p = 0.04). PMR tendon-absent participants had a lower posttreatment apnea hypopnea index (16 ± 12 events/hour tendon present versus 9 ± 9 events/hour absent, p = 0.007) and were more likely to have complete response (63% versus 36%, p = 0.02). However, tendon-absent participants were more likely to not complete the study (χ 2 (3) = 10.578, p = 0.014). Tendon-absent participants had a greater increase in midline anteroposterior airway diameter (1.6 ± 1.7 mm versus 0.6 ± 2.3 mm, p = 0.04). CONCLUSION: When PMR tendon is absent, treatment response and amount of maximum advancement improve, possibly at the expense of reduced splint tolerability. Tendon presence may help predict a group less likely to respond to mandibular advancement splint therapy.

Toward a rapid method for the study of biodiversity in cold environments: the characterization of psychrophilic yeasts by MALDI-TOF mass spectrometry.

To investigate the potential of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) as a platform to support biodiversity and phylogenetic studies of psychrophilic yeasts in cold environments, the technique was employed to rapidly characterize and distinguish three psychrophilic yeasts (Rhodotorula mucilaginosa, Naganishia vishniacii, and Dioszegia cryoxerica) from three mesophilic counterparts (Saccharomyces cerevisiae Cry Havoc, S. cerevisiae California V Ale, and S. pastorianus). A detailed workflow for providing reproducible mass spectral fingerprints of low molecular weight protein/peptide features specific to the organisms studied is presented. The potential of this approach as a tool in the study of biodiversity, systematics, and phylogeny of psychrophilic microorganisms is highlighted.

Effect of head and jaw position on respiratory-related motion of the genioglossus.

Head and jaw position influence upper airway patency and electromyographic (EMG) activity of the main upper airway dilator muscle, the genioglossus. However, it is not known whether changes in genioglossus EMG activity translate into altered muscle movement during respiration. The aim of this study was to determine the influence of head and jaw position on dilatory motion of the genioglossus in healthy adult men during quiet breathing by measuring the displacement of the posterior tongue in six positions--neutral, head extension, head rotation, head flexion, mouth opening, and mandibular advancement. Respiratory-related motion of the genioglossus was imaged with spatial modulation of magnetization (SPAMM) in 12 awake male participants. Tissue displacement was quantified with harmonic phase (HARP) analysis. The genioglossus moved anteriorly beginning immediately before or during inspiration, and there was greater movement in the oropharynx than in the velopharynx in all positions. Anterior displacements of the oropharyngeal tongue varied between neutral head position (0.81 ± 0.41 mm), head flexion (0.62 ± 0.45 mm), extension (0.39 ± 0.19 mm), axial rotation (0.39 ± 0.2 mm), mouth open (1.24 ± 0.72 mm), and mandibular advancement (1.08 ± 0.65 mm). Anteroposterior displacement increased in the mouth-open position and decreased in the rotated position relative to cross-sectional area (CSA) (P = 0.002 and 0.02, respectively), but CSA did not independently predict anteroposterior movement overall (P = 0.057). The findings of this study suggest that head position influences airway dilation during inspiration and may contribute to variation in airway patency in different head positions.

Tongue stiffness is lower in patients with obstructive sleep apnea during wakefulness compared with matched control subjects.

STUDY OBJECTIVES: This study aimed to determine whether tongue stiffness (shear modulus) in patients with obstructive sleep apnea (OSA) is different for controls matched for age, sex, and body mass index (BMI), and to investigate the effect of continuous positive airway pressure (CPAP) on stiffness. DESIGN: Controlled experimental study. SETTING: Medical research institute. PARTICIPANTS: Patients with OSA and age-, sex-, and BMI-matched healthy controls. MEASUREMENTS: Magnetic resonance elastography was performed in nine patients with OSA (apnea-hypopnea index (AHI) > 15 events/h) and seven controls (AHI < 10 events/h) matched for age, sex, and BMI. Six of these OSA subjects were also scanned while 10 cmH2O CPAP was applied. Mean isotropic shear modulus and anisotropic shear moduli parallel and perpendicular to the muscle fascicles in the tongue were calculated. RESULTS: Tongue shear modulus in patients with OSA was lower than that in matched controls (2.68 ± 0.35 (mean ± standard deviation) kPa versus 2.98 ± 0.44 kPa, P < 0.001). Shear modulus decreased with increasing AHI (R = -0.496, P = 0.043), but not age, BMI, or percentage tongue fat. Anisotropic analysis revealed that reduction in stiffness was greatest parallel to the muscle fibers. CPAP had no significant effect on tongue shear modulus. CONCLUSIONS: In awake subjects with obstructive sleep apnea, the tongue is less stiff than in similar healthy subjects and this difference occurs in the muscle fiber direction. CPAP did not significantly reduce tongue stiffness. Thus, any change in neural drive to genioglossus during wakefulness is insufficient to restore normal tongue stiffness.

Healthy humans with a narrow upper airway maintain patency during quiet breathing by dilating the airway during inspiration.

A patent upper airway is essential for survival. Increased age, obesity and some upper airway anatomical features are associated with failure to maintain upper airway patency during sleep, leading to obstructive sleep apnoea. However, many healthy subjects with these risk factors do not develop this condition. The aim of this study was to determine how anatomical factors and active dilator muscle contraction contribute to upper airway patency in healthy volunteers across a broad range of age and body mass index (BMI). A 'tagged' magnetic resonance imaging technique quantified respiratory-related motion of the anterior and lateral walls of the upper airway during quiet breathing in the supine position. Fifty-two subjects aged 22-68 years with BMI from 17.5 to 40.1 kg m(-2) were studied. Higher BMI was associated with smaller airway cross-sectional area at the level of soft palate (P < 0.05). The genioglossus moved anteriorly to dilate the upper airway during inspiration. This movement increased with increasing BMI, increasing age, a smaller airway area, and steeper tongue-base angle (all P < 0.05). Motion of the lateral upper airway at the soft-palate level was variable and less strongly linked to anatomical features of the upper airway. Multiple regression indicated that anterior genioglossus motion decreased with increasing airway area (P = 0.03) and with increasing tongue-base angle (P = 0.02). These data suggest that healthy humans, including those whose anatomy places them at increased risk of airway closure, can maintain upper airway patency by dynamically dilating the airway during inspiration.

Respiratory Movement of Upper Airway Tissue in Obstructive Sleep Apnea.

STUDY OBJECTIVES: To measure real-time movement of the tongue and lateral upper airway tissues in obstructive sleep apnea (OSA) subjects during wakefulness using tagged magnetic resonance imaging. DESIGN: Comparison of the dynamic imaging of three groups of increasing severity OSA and a control group approximately matched for age and body mass index (BMI). SETTING: Not-for-profit research institute. PARTICIPANTS: 24 subjects (apnea hypopnea index [AHI] range 2-84 events/h, 6 with AHI < 5 events/h). METHODS: The upper airway was imaged awake in two planes using SPAtial Modulation of Magnetization (SPAMM). Tissue displacements were quantified with harmonic phase analysis. MEASUREMENTS AND RESULTS: All subjects had dynamic airway opening in the sagittal plane associated with inspiration. In the nasopharynx, the increase in airway cross-sectional area during inspiration correlated with minimal cross-sectional area of the airway (R = 0.900, P < 0.001). AHI correlated negatively with movement of the nasopharyngeal lateral walls (R = - 0.542, P = 0.006). Four movement patterns were observed during inspiration: "en bloc" anterior movement of the whole posterior tongue; movement of only the oropharyngeal posterior tongue; bidirectional movement; or minimal movement. Some subjects showed different inspiratory movement patterns with different breaths. A low AHI (< 5) was associated with en bloc movement (P = 0.002). CONCLUSIONS: Inspiratory movement of the tongue varied between and within subjects, likely as a result of local and neural factors. However, in severe OSA inspiratory movement was minimal. CITATION: Brown EC; Cheng S; McKenzie DK; Butler JE; Gandevia SC; Bilston LE. Respiratory movement of upper airway tissue in obstructive sleep apnea. SLEEP 2013;36(7):1069-1076.

Tongue and lateral upper airway movement with mandibular advancement.

STUDY OBJECTIVES: To characterize tongue and lateral upper airway movement and to image tongue deformation during mandibular advancement. DESIGN: Dynamic imaging study of a wide range of apnea hypopnea index (AHI), body mass index (BMI) subjects. SETTING: Not-for-profit research institute. PARTICIPANTS: 30 subjects (aged 31-69 y, AHI 0-75 events/h, BMI 17-39 kg/m(2)). INTERVENTIONS: Subjects were imaged using dynamic tagged magnetic resonance imaging during mandibular advancement. Tissue displacements were quantified with the harmonic phase technique. MEASUREMENTS AND RESULTS: Mean mandibular advancement was 5.6 ± 1.8 mm (mean ± standard deviation). This produced movement through a connection from the ramus of the mandible to the pharyngeal lateral walls in all subjects. In the sagittal plane, 3 patterns of posterior tongue deformation were seen with mandibular advancement-(A) en bloc anterior movement, (B) anterior movement of the oropharyngeal region, and (C) minimal anterior movement. Subjects with lower AHI were more likely to have en bloc movement (P = 0.04) than minimal movement. Antero-posterior elongation of the tongue increased with AHI (R = 0.461, P = 0.01). Mean anterior displacements of the posterior nasopharyngeal and oropharyngeal regions of the tongue were 20% ± 13% and 31% ± 17% of mandibular advancement. The posterior tongue compressed 1.1 ± 2.2 mm supero-inferiorly. CONCLUSIONS: Mandibular advancement has two mechanisms of action which increase airway size. In subjects with low AHI, the entire tongue moves forward. Mandibular advancement also produces lateral airway expansion via a direct connection between the lateral walls and the ramus of the mandible. CITATION: Brown EC; Cheng S; McKenzie DK; Butler JE; Gandevia SC; Bilston LE. Tongue and lateral upper airway movement with mandibular advancement. SLEEP 2013;36(3):397-404.

Single motor unit recordings in human geniohyoid reveal minimal respiratory activity during quiet breathing.

Maintenance of airway patency during breathing involves complex interactions between pharyngeal dilator muscles. The few previous studies of geniohyoid activity using multiunit electromyography (EMG) have suggested that geniohyoid shows predominantly inspiratory phasic activity. This study aimed to quantify geniohyoid respiration-related activity with single motor unit (SMU) EMG recordings. Six healthy subjects of normal body mass index were studied. Intramuscular EMG recordings of geniohyoid activity were made with a monopolar needle with subjects in supine and seated positions. The depth of the geniohyoid was identified by ultrasound, and the electrode position was confirmed with maneuvers to isolate activity in geniohyoid and genioglossus. Activity was recorded at 85 sites in the geniohyoid during quiet breathing (45 supine and 40 seated). When subjects were supine, 33 sites (73%) showed no activity during breathing and 10 (22%) showed tonic activity. In addition, one site showed a tonic SMU with increased expiratory discharge, and one site in another subject had one unit with expiratory phasic activity. When subjects were seated, 27 sites (68%) in the geniohyoid showed no activity, 12 sites (30%) showed tonic activity that was not respiration related, and one unit at one site showed phasic expiratory activity. The average peak discharge frequency of geniohyoid motor units was 16.2 ± 3.1 impulses/s during the "geniohyoid maneuver," which was the first part of a swallow. In contrast to previous findings, the geniohyoid shows some tonic activity but minimal respiration-related activity in healthy subjects in quiet breathing. The geniohyoid has little active role in airway stability under these conditions.

Communication challenges of HPV vaccination.

BACKGROUND: The UK has introduced a national human papillomavirus (HPV) immunisation programme for girls aged 12-13 from autumn 2008. Previous research has explored public understanding of the causes of cervical cancer and found little awareness of the role of HPV. OBJECTIVE: To explore GPs' and practice nurses' views of HPV vaccination, prior to implementation of the national immunisation programme, with a focus on their role and anticipated difficulties. METHODS: Qualitative semi-structured interviews were conducted with 17 GPs and practice nurses from practices in Hampshire and Wiltshire, in March 2008. RESULTS: The prevailing theme of new communication challenges was identified and these were connected to the activities of (i) explaining, (ii) consenting and (iii) managing conflicts between parents and their children with respect to vaccination decisions. The importance of decisions being fully informed was emphasized and concern was expressed about the adequacy of the information provided to girls and their parents in schools. Whether consent would be granted by the parent or by the child and how potential disagreements should be managed remained ambiguous. Participants considered it appropriate to offer the vaccine without parental consent if other criteria, such as an assessment of competency, were met. CONCLUSIONS: Success of the national immunization programme will depend on overcoming the challenges of providing explanations to ensure that individuals understand the potential benefits of HPV vaccination. Primary care can play an important role, but this study suggests there are fundamental issues that need further clarification.

Interleukin-1 and tumor necrosis factor antagonists attenuate ethanol-induced inhibition of bone formation in a rat model of distraction osteogenesis.

Chronic ethanol exposure inhibits rapid bone formation during distraction osteogenesis (DO; fracture and limb lengthening) and decreases volumetric bone mineral density (BMD) in a model of intragastric dietary infusion [total enteral nutrition (TEN)] in the rat. The hypothesis tested herein was that overexpression of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha mediates these deleterious effects of ethanol on the rat skeleton. Two studies (study 1, female rats; study 2, male rats) were performed to test the potential protective effects of the IL-1 and TNF antagonists: IL-1 receptor antagonist (IL-1ra) and 30-kDa polyethylene glycol-conjugated soluble TNF receptor type 1 (sTNFR1). All rats were infused with a liquid diet +/- ethanol (EtOH) and underwent tibial fractures and DO. During distraction, the animals received a combination of IL-1ra (1.8-2.0 mg/kg/day) and sTNFR1 (2.0 mg/kg/2 days) or vehicle. A comparison of distracted tibial histological sections demonstrated 1) significant antagonist-related increases in bone column formation over the EtOH controls (studies 1 and 2), and 2) restoration of new bone equivalent to that of the TEN controls (study 2). In contrast, examination of intact proximal tibial metaphyses by peripheral quantitative computerized tomography revealed decreases in volumetric BMD of both EtOH control and EtOH antagonist groups (study 2). These results demonstrate that short-term systemic administration of IL-1 and TNF antagonists together protect rapid bone formation during DO from the deleterious effects of chronic ethanol but are ineffective in regard to intact bone homeostasis.

Skeletal toxicity associated with chronic ethanol exposure in a rat model using total enteral nutrition.

Chronic alcohol abuse decreases bone mass, inhibits osteoblast differentiation and function, increases fracture incidence, and delays fracture healing. Four studies were designed to use intragastric ethanol delivery as part of a total enteral nutrition (TEN) system to determine the negative systemic effects of chronic ethanol on 1) the rat skeleton and 2) local rapid bone formation during limb lengthening (distraction osteogenesis, DO). In study 1, three-point bending tests demonstrated that after 75 days of ethanol exposure, the tibiae had significantly lower load to failure versus control diet (p = 0.0006) or ad libitum chow-fed rats (p = 0.0029). Study 2 examined alcohol's effects on the density and cross-sectional area of the proximal tibial metaphysis using peripheral quantitative computed tomography and found that after 25 days of ethanol exposure the trabecular volumetric bone mineral density (p = 0.011) and cortical cross-sectional area (p = 0.011) were lower compared with controls. In study 3, a comparison of distracted tibial radiographs and histological sections demonstrated ethanol-related decreases in both gap mineralization (p = 0.03) and bone column formation (p = 0.01). Histological comparisons in study 4 reproduced the ethanol-related deficits in new bone formation during DO (p = 0.001). These results indicate that the TEN system is a viable model to study ethanol's effects on the skeleton and that chronic ethanol delivery via TEN decreases trabecular bone density, cortical area, and mature bone strength. Also, the DO studies demonstrate, for the first time, that chronic ethanol inhibits rapid bone formation during limb lengthening.

Immunohistochemical study of osteopontin expression during distraction osteogenesis in the rat.

Distraction osteogenesis (DO) is a limb-lengthening procedure that combines mechanical tension stress with fracture healing to provide a unique opportunity for detailed histological examination of bone formation. Osteopontin (OPN) is a multifunctional matricellular protein believed to play a key role in wound healing and cellular response to mechanical stress. We studied the expression of OPN during DO using standard immunohistochemical (IHC) staining techniques. In addition, we compared the expression of OPN to proliferation (PCNA-positive cells) in the DO gap. After 14 days of distraction in the rat, these stains revealed variations in OPN expression and its relationship to proliferation according to the cell type, tissue type, and mode of ossification examined. Fibroblast-like cells within the central fibrous area exhibited intermittent low levels of OPN, but no relationship was observed between OPN and proliferation. In areas of transchondral ossification, OPN expression was very high in the morphologically intermediate oval cells. During intramembranous ossification, osteoblasts appeared to exhibit a bimodal expression of OPN. Specifically, proliferating pre-osteoblasts expressed osteopontin, but OPN was not detected in the post-proliferative pre-osteoblasts/osteoblasts that border the new bone columns. Finally, intracellular OPN was detected in virtually all of the mature osteoblasts/osteocytes within the new bone columns, while detection of OPN in the matrix of the developing bone columns may increase with the maturity of the new bone. These results imply that the expression of OPN during DO may be more similar to that seen during embryogenesis than would be expected from other studies. Furthermore, the biphasic expression of OPN during intramembranous ossification may exemplify the protein's multi-functional role. Early expression may facilitate pre-osteoblastic proliferation and migration, while the latter downregulation may be necessary for hydroxyapatite crystal formation.