Methods to reduce the contraction of tissue-engineered buccal mucosa for use in substitution urethroplasty

Background

We previously described the production and clinical outcomes of tissue-engineered buccal mucosa (TEBM) used to treat recurrent urethral strictures. In this study, two patients developed a recurrent stricture and there was also evidence of graft contraction.

Objective

Assess possible preclinical methods to reduce contraction of TEBM.

Design, setting and participants

Using the model of TEBM in use clinically (ie, oral keratinocytes and fibroblasts cultured on de-epidermised acellular dermal scaffold), three methods of reducing TEBM contraction were investigated in vitro.

Interventions

The techniques assessed were pretreatment of de-epidermised dermis (DED) with glutaraldehyde, culture with β-aminopropionitrile (β-APN; a lysyl oxidase inhibitor), and physical restraint of TEBM grafts during culture.

Measurements

Contraction was assessed using serial digital image analysis. The cytotoxicity of the pharmacologic manipulations was assessed using monolayer cultures of oral mucosa cells.

Results and limitations

Control TEBM lost a mean of 45.4% of its original surface area over 28 d of culture. Treating TEBM with glutaraldehyde, β-APN, or mechanical restraint during culture all significantly inhibited graft contraction. Glutaraldehyde treatment was most effective (only 5.5% loss of area with 0.1% glutaraldehyde), followed by mechanical restraint for at least 7 d (21.4% loss of area), and then β-APN (28.7% loss of area). None of the treatments had any significant effect on cell viability. This in vitro study identifies solutions for graft contracture to explore in the clinic.

Conclusions

Glutaraldehyde pretreatment and restraint of TEBM grafts during culture both reduce graft contraction.     

戊二醛联合丹宁酸处理增加去细胞牛心包的组织稳定性的实验研究

目的初步观察戊二醛联合丹宁酸处理对去细胞后牛心包组织稳定性的影响。方法新鲜牛心包切割成同样大小的试片数块,分为4组,A组：新鲜牛心包;B组：新鲜牛心包去细胞处理;C组：牛心包去细胞后丹宁酸处理;D组：牛心包去细胞后戊二醛联合丹宁酸处理。通过测定各组试片的厚度、热皱缩温度、抗拉负荷、抗张强度、断裂伸长率及抗胶原蛋白酶降解试验来比较各组的组织稳定性并行形态学和组织学观察。结果 D组在厚度、热皱缩温度、抗拉负荷、抗张强度及抗胶原纤维酶降解能力方面均高于B组（P〈0.05）,组织学发现D组纤维排列较B组更加紧凑。结论戊二醛联合丹宁酸处理能够增加去细胞后牛心包的组织稳定性。     

基于膨胀实验和逆向建模技术评估戊二醛对兔角膜生物力学性能的影响

目的基于膨胀实验和逆向建模技术评估戊二醛对角膜生物力学性能的影响。方法实验研究。选取10只日本大耳白兔,处死摘取眼球。实验开始前测量角膜的厚度与直径。用随机数字表法选取每只白兔其中一眼进入实验组,另一眼进入对照组,分别浸入包含和不包含4%戊二醛的PBS磷酸盐缓冲液30 min。处理完毕后分离2组眼球制成附带巩膜环的完整角膜试件,固定于自制前房模拟装置,于角膜后表面施加液压,加压范围为2.1~45.0 mmHg,记录角膜前顶点的前突位移值;采用逆向建模技术计算角膜的应力、应变和正切模量值。数据采用配对t检验和配对Hotelling T2检验进行分析。结果戊二醛处理前实验组角膜的厚度、直径和对照组差异无统计学意义,戊二醛处理后角膜厚度下降约1/4。实验组测得的角膜正切模量值明显大于对照组;随着应力增加,相比于对照组,实验组角膜正切模量的增加比例逐渐减少（从199.5%下降到59.7%）。结论戊二醛处理后角膜正切模量明显增大,戊二醛促使胶原纤维间产生交联而增强角膜硬度。     

成人Still病的观察与护理

目的 探讨强氧化离子水与传统的戊二醛溶液对呼吸机管道消毒效果的差异。方法对连续使用24h的呼吸机管道60组随机分为实验组和对照组各30组。实验组采用离子水浸泡进行呼吸机管道的消毒；对照组采用戊二醛溶液浸泡消毒，溶液均没过管道。消毒前后采样进行细菌培养及细菌菌落计数，对2组的消毒效果进行比较。结果消毒前所有样本细菌培养阳性率为100％，强氧化离子水与戊二醛溶液消毒后灭菌率分别为：1min83％及17％；5min94％及33％；30min100％及90％。浸泡后1，5min时2组杀灭率比较，差异有统计学意义（P〈0．01），而浸泡30min后杀灭率比较差异无统计学意义（P〉0．05）。结论对呼吸机管道消毒，强氧化离子水效果优于传统的戊二醛溶液。     

多聚环氧化合物预处理同种异体静脉重建犬股动脉的实验研究

目的探索新型交联材料多聚环氧化合物(PolyepoxyCompound,PC)预处理同种异体静脉的实验应用。方法将多聚环氧化合物、戊二醛(Glutaraldehyde,GA)交联后的同种异体犬脉以及自体犬静脉(Fresh)进行30只杂种犬双侧股动脉移植 ,在术后3d、7d、14d、30d、60d、90d各个时间点以多普勒超声检测或DSA造影观测血管通畅情况 ,并取血管进行射电镜观察。结果①在移植后的早期(<14d内) ,PC组、GA组和Fresh组累积通畅率分别为64.20%、42.86%和90.00%。但从移植后30d起 ,GA组累积通畅率急剧下降到6.12% ,而PC组在术后30d、60d和90d的累积通畅率分别为48.15%、21.88 %和13.13% ,在各个观察时间期都较GA组高 ,两组之间累积通畅率曲线的分布有统计学的差异(P<0.05)。Fresh组各时间段的累积通畅率也较GA组高 ,两组之间有统计学上的显著差异(P<0.01)。而PC组和Fresh组累积通畅率曲线的分布无统计学差异(P>0.05) ;②PC组在7d时可见内皮细胞从受体动脉向静脉爬行 ,90d时新生内皮细胞已覆盖静脉表面。GA组14d吻合口处内皮细胞开始爬行 ,但爬行距离较PC组短 ,90d可见内膜面有局灶状分布的新生内皮细胞。Fresh组7d内膜面见残存的内皮细胞 ,30d内皮细胞已重新覆盖内膜面。结论PC处理静脉毒性小于GA处理静脉 ,更有利于内皮细胞生长。     

An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method

Controlled and efficient conjugation of synthetic peptides to proteins, for use in immunization or in assay procedures, is a prerequisite for the immunological applications of synthetic peptides. This study describes a new method of conjugating synthetic peptides to proteins in such a way that no homopolymers of synthetic peptides or proteins occur. To achieve this, the protein is first activated with glutaraldehyde and subsequently excess glutaraldehyde is removed. Then coupling of the synthetic peptide to the activated protein occurs while subsequetly the surplus reactive glutaraldehyde groups on the protein are blocked with lysine. Excess free peptide and lysine is then removed by dialysis. This improvement not only results in better defined conjugates when compared to classical glutaraldehyde coupling, but also in the consumption of smaller amounts of synthetic peptide during conjugate formation. When used for immunization we obtained similar and sometimes even better responses with the glutaraldehyde based conjugates than with succinimidyl (MBS) conjugates of the same peptides. The performance of the modified conjugates in ELISA procedures, immunization and immunocytochemistry suggests that they are superior to conjugates formed by classical glutaraldehyde coupling.     

Prevention of Calcification of Tissue Valves

Abstract: In this study an attempt was made to find an optimum method of chemical treatment to prevent the calcification of bioprosthetic heart valves. Bovine pericardium was washed in a 5% sodium chloride solution followed by trypsin (Tr) treatment and was kept in 0. 1% glutaraldehyde (GA) with a gradual increase in concentration up to 0. 25% GA and finally posttreated with a 4% chitosan (Ch) solution. Fresh, 0. 2% GA, 0. 625% GA, and sodium chloride–Tr–GA treated pericardial samples were taken for comparative study. Tensile testing showed comparable strength and elongation at the breaking point for all groups. The thermal shrinkage studies indicated merit of the proposed treatment (5% sodium chloride–trypsinglutaraldehyde treated pericardia with chitosan and without chitosan posttreatment). Collagenase assay showed that all differently treated (GA) materials were equally resistant to collagenase. All samples were implanted subcutaneously in rats for 2, 4, 8, or 12 weeks for calcification study. Morphological and mineral analyses showed complete prevention of calcification in sodium chloride–trypsin–GA–chitosan treated pericardium (Ca was 1. 1 ± 0. 27 mg/g, von Kossa 0) at the 12th week of implantation.     

Effects of glutaraldehyde fixation on renal tubular function

Using the in vitro microperfusion technique on isolated rat papillary collecting duct (PCD), we examined whether the glutaraldehyde-fixation method can be also applied to the mammalian collecting duct for preservation of the vasopressin-stimulated water and urea transport. Arginine vasopressin (AVP) at 10^–9 mol/l increased diffusional water permeability (P _dw) from 101.9±10.76 to 283.3±16.67×10^–7 cm² s^–1 (n=8,P<0.01) and urea permeability (P _urea) from 30.3±2.24 to 83.5±7.80×10^–7 cm² s^–1 (n=8,P<0.01). Both parameters remained elevated after fixation with 0.1 mol/l glutaraldehyde even in the absence of AVP, with the values being 265.0±14.47 and 74.5±7.15×10^–7 cm² s^–1, respectively. Glutaraldehyde fixation did not affect the basal levels ofP _dw orP _urea. Phloretin at 2.5×10^–4 mol/l decreased glutaraldehyde-fixed AVP-stimulatedP _urea from 79.0±7.96 to 29.7±3.66×10^–7 cm² s^–1 (n=4,P<0.01) and from 73.2±7.05 to 38.7±3.53×10^–7 cm² s^–1 (n=4,P<0.01) when the drug was added to the lumen or to the bath, respectively. Phloretin also decreased glutaraldehyde-fixed non-stimulatedP _urea by 25–40%. However, this drug did not affect glutaraldehyde-fixedP _dw. These findings indicate that the glutaraldehyde fixation method can be applied to mammalian collecting tubules for studying vasopressin stimulatedP _dw andP _urea.P _urea fixed by glutaraldehyde is functionally flexible and may be distinct from the water pathway.     

Biochemical differences between dystrophic calcification of cross-linked collagen implants and mineralization during bone induction

Summary Ectopic calcification of diseased tissues or around prosthetic implants can lead to serious disability. Therefore, calcification of implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen was compared with mineralization induced by demineralized bone matrix (DBM). Whereas implants of DBM accumulated large amounts of calcium and a bone-specific γ-carboxyglutamic acid protein (BGP or osteocalcin) following implantation in both young and older rats, implants of cross-linked pericardium calcified with only traces of BGP. Glutaraldehyde-cross-linked DBM failed to calcify after implantation in 8-month-old rats for 2–16 weeks. Implants of cross-linked type I collagen exhibited small calcific deposits 2 weeks postimplantation but calcium content eventually dropped to levels equal to those of soft tissues as the implants were resorbed. The calcium content of DBM implanted in 1- and 8-month-old rats reached comparable levels after 4 weeks, but the BGP content was approximately twice as high in the younger animals than in the older ones. Glutaraldehyde-cross-linked implants of DBM, tendon, and cartilage calcified significantly in young but not in old animals. This form of dystrophic calcification was associated with only trace amounts of BGP. Alkaline phosphatase activity was high in implants of DBM and undetectable in implants of cross-linked collagenous tissues. These results show that implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen calcify to different extents depending upon their origin and the age of the host, and that the mechanism of dystrophic calcification differs significantly from the process of mineralization associated with bone induction as reflected by alkaline phosphatase activity and BGP accumulation.     

Residual glutaraldehyde levels in fiberoptic endoscopes: measurement and implications for patient toxicity

Most gastroenterology societies recommend glutaraldehyde for fiberoptic endoscope disinfection. However, glutaraldehyde toxicity has been suspected in patients examined with endoscopes disinfected with this compound. The aim of our study was to determine the residual levels of glutaraldehyde in fiberoptic endoscopes after either manual or automatic disinfection and to evaluate the extent of toxicity. Furthermore, the procedures for disinfection currently performed by the department were compared with the new French guidelines. We used both manual and automatic disinfection procedures and flushed sterile distilled water through the lumens of endoscopes before use. Residual glutaraldehyde levels were determined using liquid chromatography coupled to spectrophotometric detection. In a total of 92 measurements it was found that residual glutaraldehyde levels were higher and more variable after manual disinfection (< 0.2-159.5 mg/L) than after automatic disinfection (< 0.2-6.3 mg/L). We conclude that local procedures for disinfection need to be improved to conform to the new French guidelines. Since thresholds for the toxic dose of glutaraldehyde and international norms for levels of residual glutaraldehyde in equipment have not been defined, additional studies combining accurate measurements in fiberoptic endoscopes and clinical observations of endoscopy patients will be required to draw more definitive conclusions.