In situ label‐free cell viability assessment of nucleus pulposus tissue

Regenerative medicine approaches aiming at treating degenerating intervertebral discs, a major cause of back pain, are increasingly tested in ex‐vivo disc explant models mimicking in‐vivo conditions. For assessing the efficacy of regenerative therapies, cell viability is commonly measured requiring specific labels to stain cells. Here, we demonstrate and evaluate how cellular auto‐fluorescence can be utilized to non‐invasively assess viability in disc tissue in‐situ using label‐free two‐photon microscopy. Live and dead bovine disc cells (0% and 100% cell viability) from the nucleus pulposus were seeded into collagen gels and auto‐fluorescence was characterized. Subsequently, nucleus pulposus explants were cultured for 6 days in media with different glucose supplementation (0, 0.25, 0.5, and 1 g/L) to induce different degrees of cell death. Then, samples were split and viability was assessed using label‐free two‐photon microscopy and conventional staining. Results show that live and dead nucleus pulposus cells systematically emit auto‐fluorescent light with distinct characteristics. Cell viability values obtained with label‐free microscopy did not significantly differ from those acquired with staining. In summary, monitoring auto‐fluorescence facilitates accurate cell viability assessment in nucleus tissue requiring no additional dyes. Thus, this technique may be suitable for pre‐clinical testing of regenerative therapies in nucleus pulposus cultures. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:545–550, 2014.     

An Evaluation of the Accuracy of Labeling of Percent Sodium Hypochlorite on Various Commercial and Professional Sources: Is Sodium Hypochlorite from These Sources Equally Suitable for Endodontic Irrigation?

Introduction

The shelf life of sodium hypochlorite (NaOCl) is limited, and a previous article showed that there can be a discrepancy between the expected concentration of free available chlorine (FAC) and the actual FAC concentration in NaOCl solutions intended for endodontic irrigation. The current study investigates the FAC content of domestic and professional NaOCls and evaluates the influences of dilution and storage on FAC concentration.

Methods

First, domestic and professional NaOCls not obtained from manufacturers were iodometrically titrated. Then, NaOCls were diluted with demineralized water or tap water and stored at 4°C or 18°C and analyzed at baseline and 2 and 22 weeks. Statistical analyses included paired samples, independent samples t tests and repeated multivariate analysis of variance. Correlations were calculated with the Pearson or Spearman rank correlation test. A P < .05 was considered significant.

Results

Label specifications of domestic NaOCl were very imprecise (ie, <5% NaOCl). Domestic NaOCl contained 1.8%–3.5% NaOCl (w/v). Professional NaOCl varied from 14.3% relative less FAC than specified on the label to 23.5% relative more FAC than specified. After 22 weeks, the relative average loss of FAC in all conditions was 5.4% FAC (P = .002). Dilution, diluents, or storage temperature had no effect on the decline of FAC caused by aging.

Conclusions

There is a great variation in NaOCl concentrations, with domestic NaOCl being the least accurate. NaOCl can be stored up to 5 months. The FAC concentration of domestic NaOCl is unpredictable, and, therefore, it appears less suitable for clinical application as root canal irrigant.     

The effect of early detection of anterolateral thigh free flap crisis on the salvage success rate,based on 10 years of experience and 1072 flaps

The aim of this study was to assess the effectiveness of early exploration of anterolateral thigh (ALT) free flap compromise in head and neck reconstruction and to correlate this with the salvage success rate. The perioperative data of 1051 patients with 1072 ALT flap reconstructions were reviewed retrospectively for the period January 2002 to December 2012. Outcome measures included ethnicity, defect type, incidence and timing of flap compromise, type of flap compromise, causes of vascular occlusion, and salvage rate. The success rate of free flap reconstruction was 97.3% (1043/1072). Of the 29 failures, 21 were complete and eight were partial failures (10–40% of the flap). Venous occlusions occurred in 39 flaps (83.0%) and arterial occlusions in five flaps (17.0%). Six cases were detected within 8 h postoperatively, 13 at 8–16 h postoperatively, seven at 16–24 h postoperatively, and 18 at 24–48 h postoperatively, with respective salvage rates of 66.7%, 61.5%, 28.6%, and 22.2%; three cases detected after 48 h failed. The salvage rate at ≤16 h (62.2%) was much higher than that at >16 h (21.4%, P = 0.0039). Early detection, re-exploration, and effective handling of the flap crisis increases the rate of flap salvage tremendously.     

颏下岛状皮瓣与前臂皮瓣修复口腔鳞癌术后缺损的比较

目的：比较颏下岛状皮瓣与游离前臂皮瓣修复口腔鳞癌术后缺损的结果。方法：2009年5月~2011年8月,69例口腔鳞癌术后缺损采用颏下岛状皮瓣或游离前臂皮瓣修复,比较2种修复方法的皮瓣大小、手术时间、住院时间、并发症、口腔功能恢复及复发情况。结果：69例患者中,采用颏下岛状皮瓣修复32例,游离前臂皮瓣修复37例。颏下岛状皮瓣组与游离前臂皮瓣组在平均皮瓣大小（32.78cm2 VS 48.27cm2）、平均手术时间（351min VS 508min）、平均住院日（13.06dVS 17.48d）等均有统计学差异（P〈0.001）;两组患者总的并发症发生相似,但颏下岛状皮瓣组发生颌下区积液的比例较高（P〈0.05）;两组患者在术后口腔功能恢复及肿瘤复发方面无差异。结论：颏下岛状皮瓣修复可明显缩短手术时间及患者住院时间,可作为口腔鳞癌术后中小型缺损修复的首选。     

Effects of geometry and chemistry on hydrophobic solvation

Inserting an uncharged van der Waals (vdw) cavity into water disrupts the distribution of water and creates attractive dispersion interactions between the solvent and solute. This free-energy change is the hydrophobic solvation energy (ΔG_vdw). Frequently, it is assumed to be linear in the solvent-accessible surface area, with a positive surface tension (γ) that is independent of the properties of the molecule. However, we found that γ for a set of alkanes differed from that for four configurations of decaalanine, and γ = −5 was negative for the decaalanines. These findings conflict with the notion that ΔG_vdw favors smaller A. We broke ΔG_vdw into the free energy required to exclude water from the vdw cavity (ΔG_rep) and the free energy of forming the attractive interactions between the solute and solvent (ΔG_att) and found that γ < 0 for the decaalanines because −γ_att > γ_rep and γ_att < 0. Additionally, γ_att and γ_rep for the alkanes differed from those for the decaalanines, implying that none of ΔG_att, ΔG_rep, and ΔG_vdw can be computed with a constant surface tension. We also showed that ΔG_att could not be computed from either the initial or final water distributions, implying that this quantity is more difficult to compute than is sometimes assumed. Finally, we showed that each atom’s contribution to γ_rep depended on multibody interactions with its surrounding atoms, implying that these contributions are not additive. These findings call into question some hydrophobic models.Many techniques in computational biophysics require the computation of free-energy differences. However, directly computing these free-energy differences with quantum or classical molecular dynamics (MD) methods has proven challenging because doing so requires long simulation times. To make such computations more tractable, techniques have been developed based on the observation that these free-energy changes could be computed by combining estimates of the changes in vacuum, where they are usually easier to compute, with estimates of the solvation energies (ΔG) of the initial and final states (1, 2).One common technique for computing ΔG is to break it into electrostatic (ΔG_el) and hydrophobic (ΔG_vdw) terms (3, 4). In this breakdown, ΔG_vdw is the free energy required to place an uncharged van der Waals (vdw) cavity into solution, and ΔG_el is the free energy of charging the cavity once it has been placed into solution. Several techniques, including the Poisson–Boltzmann equation (5), the generalized Born model (6), structured continuum approaches (7–9), and integral equation methods (10), have been developed to compute ΔG_el. In the present study, we continue a study of ΔG_vdw begun previously (11–13).Many models have been constructed to compute ΔG_vdw. The first of these models, and perhaps the most widespread, isΔG_vdw = γ_vdwA + b, [1]where γ_vdw is a positive constant independent of the properties of the molecule and b is the energy of solvating a point-like solute (2, 14–17). This model follows from assuming that ΔG_vdw for microscopic cavities should scale with A in the same manner as for macroscopic cavities (18). Alternatively, several studies have pointed out that experimental measurements often are not consistent with Eq. 1 (19–27). These studies have argued that ΔG_vdw should be split into purely repulsive (ΔG_rep) and attractive (ΔG_att) parts:ΔG_vdw = ΔG_rep + ΔG_att.[2]Often, this breakdown follows that of Weeks et al. (19) and Chandler et al. (20). These studies usually then proceed to assume that ΔG_rep is a linear function of A or the molecular volume (V),ΔG_rep = γ_repA + b, ?or[3]ΔG_rep = ρ_repV + b, ?or[4]ΔG_rep = γ_repA + ρ_repV + b, [5]where γ_rep and ρ_rep are positive constants independent of the properties of the molecule. Studies (28–31) have argued that ΔG_rep should obey Eq. 3 for large solute molecules, Eq. 4 for small solutes, and Eq. 5 in a cross-over regime. Alternatively, one could construct other functional forms that would approach Eqs. 3 and 4 in the appropriate limits, such asΔG_rep = 1/((1/γ_repA) + (1/ρ_repV)).[6]Some researchers (23, 24, 27) have then estimated ΔG_att by integrating over approximate solvent densities:ΔGatt=〈Uatt(1)〉ρ¯,[7]where this average was taken over ρ¯, an approximate solvent distribution, and U_att was the attractive dispersive interaction energy between the solute and solvent, as described in Materials and Methods.In three previous studies, we obtained computational results that appeared to contradict some common expectations of ΔG_vdw (11–13). For example, the idea that ΔG_vdw drives the initial collapse during protein folding by opposing the formation of cavities in the solvent with an energy penalty that increases with A has frequently been discussed (2, 32, 33). However, in the first of our studies (11), where we computed ΔG_vdw for a series of glycine peptides ranging in length from 1 to 5 monomers, we found that although ΔG_vdw was linear in the number of monomers, as would be expected if it scaled with A, ΔG_vdw was negative and decreased with the number of monomers, seemingly implying that γ_vdw was negative. In the second of our studies (12), we computed ΔG_vdw for a set of decaalanine conformations and found that ΔG_vdw was negative for these peptides as well. Finally, in the third study (13), we computed ΔG_vdw for a series of alanine peptides ranging in length from 1 to 10 monomers, with the peptides either held in fixed, extended conformations or allowed to assume a normal distribution of conformations. We once again found that ΔG_vdw was negative, and it decreased with the number of monomers when the peptides were held in extended conformations but increased with the number of monomers when the peptides were allowed to adopt a normal distribution of conformations.To better understand these findings, here we computed with free-energy perturbation (FEP) (34, 35) not just ΔG_vdw but also ΔG_rep and ΔG_att for four conformations of decaalanine from a previous study (12) and a series of alkanes that has been examined in the literature (22). As discussed below, none of Eqs. 1 and 3–7 are consistent with our data. Each atom’s contributions to ΔG_rep, ΔG_att, and ΔG_vdw appear to depend on their chemical surroundings and the geometry of the molecular surface. We do not know of a theory that can explain the calculations presented here.     

VitC与不同抗氧化剂联用辅治病毒性心肌炎疗效观察

目的比较不同氧自由基清除剂配伍治疗病毒性心肌炎疗效，以探讨最佳治疗方案。方法对确诊为病毒性心肌炎的患儿随机分成４组（治疗组），并设对照组。采用ＶｉｔＣ与不同氧自由基清除剂配伍治疗，比较治疗效果。结果治疗组各方案疗效均明显优于对照组（χ２＝２６．２７，Ｐ＜０．０１），而治疗组各组疗效并非随抗氧化剂种类增多而增强（χ２＝１．８１，Ｐ＞０．０５）。结论病毒性心肌炎在常规治疗基础上，加用ＶｉｔＣ静脉推注足以发挥明显的抗氧化作用。