Mandibular reconstruction using an axially vascularized tissue-engineered construct

Background

Current reconstructive techniques for continuity defects of the mandible include the use of free flaps, bone grafts, and alloplastic materials. New methods of regenerative medicine designed to restore tissues depend mainly on the so-called extrinsic neovascularization, where the neovascular bed originates from the periphery of the construct. This method is not applicable for large defects in irradiated fields.

Methods

We are introducing a new animal model for mandibular reconstruction using intrinsic axial vascularization by the Arterio-Venous (AV) loop. In order to test this model, we made cadaveric, mechanical loading, and surgical pilot studies on adult male goats. The cadaveric study aimed at defining the best vascular axis to be used in creating the AV loop in the mandibular region. Mechanical loading studies (3 points bending test) were done to ensure that the mechanical properties of the mandible were significantly affected by the designed defect, and to put a base line for further mechanical testing after bone regeneration. A pilot surgical study was done to ensure smooth operative and post operative procedures.

Results

The best vascular axis to reconstruct defects in the posterior half of the mandible is the facial artery (average length 32.5 ± 1.9 mm, caliber 2.5 mm), and facial vein (average length 33.3 ± 1.8 mm, caliber 2.6 mm). Defects in the anterior half require an additional venous graft. The defect was shown to be significantly affecting the mechanical properties of the mandible (P value 0.0204). The animal was able to feed on soft diet from the 3^rd postoperative day and returned to normal diet within a week. The mandible did not break during the period of follow up (2 months).

Conclusions

Our model introduces the concept of axial vascularization of mandibular constructs. This model can be used to assess bone regeneration for large bony defects in irradiated fields. This is the first study to introduce the concept of axial vascularization using the AV loop for angiogenesis in the mandibular region. Moreover, this is the first study aiming at axial vascularization of synthetic tissue engineering constructs at the site of the defect without any need for tissue transfer (in contrast to what was done previously in prefabricated flaps).

     

Dynamic deformational loading results in selective application of mechanical stimulation in a layered, tissue-engineered cartilage construct

The application of dynamic physiologic loading to a bilayered chondrocyte-seeded agarose construct with a 2% (wt/vol) top layer and 3% (wt/vol) bottom layer was hypothesized to (1) improve overall construct properties and (2) result in a tissue that mimics the mechanical inhomogeneity of native cartilage. Dynamic loading over the 28 day culture period was found to significantly increase bulk mechanical and biochemical properties versus free-swelling culture. The initial depth-distribution of the compressive Young's modulus (EY) reflected the intrinsic properties of the gel in each layer and a similar trend to the native tissue, with a softer 2% gel layer and a much stiffer 3% gel layer. After 28 days in culture, free-swelling conditions maintained this general trend while loaded constructs possessed a reverse profile, with significant increases in EY observed only in the 2% gel. Histological analysis revealed preferential matrix formation in the 2% agarose layer, with matrix localized more pericellularly in the 3% agarose layer. Finite element modeling revealed that, prior to significant matrix elaboration, the 2% layer experiences increased mechanical stimuli (fluid flow and compressive strain) during loading that may enhance chondrocyte stimulation and nutrient transport in that layer, consistent with experimental observations. From these results, we conclude that due to the limitations in 3% agarose, the use of this type of bilayered construct to construct depth-dependent inhomogeneity similar to the native tissue is not likely to be successful under long-term culture conditions. Our study underscores the importance of other physical properties of the scaffold that may have a greater influence on interconnected tissue formation than intrinsic scaffold stiffness.     

蛋白质的玻璃化

在成玻载体(糖类)和保护剂(分子伴侣和脱水蛋白)存在下,蛋白质类生物活性物质可在室温下被玻璃化.这类玻璃态产品在室温下极其稳定,已被用于可在室温下长期储、运的新一代体外诊断试剂盒的生产.有迹象表明,植物种子的休眠即是由于种子内含有全套成玻物和保护剂,使其所含活性分子能在自然条件下玻璃化而高度稳定.     

Nucleation and Crystal Growth in a Vitrification Solution Tested for Organ Cryopreservation by Vitrification

Nucleation and crystal growth are investigated for vitrification solution VS41A (dimethyl sulfoxide, formamide, and 1,2-propanediol) in an aqueous carrier solution giving, when added to these three cryoprotectants, a concentration of other solutes in the whole solution the same as that in Euro-Collins, with a 55% (w/v) cryoprotectant concentration. This concentration is assumed to achieve physical properties under 1 atmosphere similar to those of solution VS4 used under 1000 atmospheres. The thermal range and the kinetics of nucleation and crystal growth are investigated by DSC through different thermal treatments. It is found that the nucleation thermal range is below -90 degrees C and that of crystal growth is above -85 degrees C for a relatively long experimental time. The nucleation density is also studied through direct observations by cryomicroscopy and is related to the amount of crystallization calorimetrically recorded. The effect of storage below the glass transition shows the possibility of a slow increase in nucleation below the glass transition, as already observed by other authors for different aqueous solutions. Isothermal crystallization is analyzed within the Johnson-Mehl-Avrami model for temperatures above -75 degrees C. The corresponding samples have been cooled and warmed at the same rate of 40 degrees C/min and calculations give, at constant nuclei numbers, an activation energy of 9.3 +/- 0.3 kcal/mol and the Avrami exponent n = 2.2 +/- 0.05. This shows a two-dimensional crystal growth as observed by cryomicroscopy. The estimated critical warming rate relevant to the preservation of rabbit kidneys by vitrification is 270 degrees C/min with or without an increase in the nucleus density during storage. The present results support the possibility of using VS4 solution for vitrification of rabbit kidneys if pressure is not a limiting factor. Copyright 1993, 1999 Academic Press.     

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer

We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blastocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated.     

Digital Microfluidic Processing of Mammalian Embryos for Vitrification

Cryopreservation is a key technology in biology and clinical practice. This paper presents a digital microfluidic device that automates sample preparation for mammalian embryo vitrification. Individual micro droplets manipulated on the microfluidic device were used as micro-vessels to transport a single mouse embryo through a complete vitrification procedure. Advantages of this approach, compared to manual operation and channel-based microfluidic vitrification, include automated operation, cryoprotectant concentration gradient generation, and feasibility of loading and retrieval of embryos.     

大苞鞘石斛原球茎玻璃化超低温保存技术的研究

本文研究建立了大苞鞘石斛(Dendrobium wardianum Warner)原球茎玻璃化法超低温保存的技术体系。结果发现,预处理和玻璃化溶液(plant vitrification solution 2,PVS2)装载脱水是影响大苞鞘石斛原球茎相对存活率的两个关键步骤,高渗与低温-高渗两种预处理方法测定的相对存活率具有显著性差异;玻璃化溶液的种类以及脱水时间对冻后存活率具有重要的影响。基于此,建立了大苞鞘石斛原球茎的超低温保存体系,即:以继代培养60 d的大苞鞘石斛原球茎为材料,1/2MS+0.8 mol/L蔗糖的培养基上4℃低温预处理6 d后,转至1/2 MS+2 mol/L甘油+0.4 mol/L蔗糖的装载液中室温下装载40 min,在0℃下装载PVS2脱水40 min,然后转入装有新鲜PVS2冷冻管中并迅速投入液氮。在液氮保存1 h后放在40℃水浴中快速解冻1 min,利用含1.2 mol/L蔗糖的1/2MS培养液洗涤3次,每次间隔10 min;待恢复培养30 d后统计存活率,可使大苞鞘石斛原球茎超低温保存后存活率达到20.0%。     

循证护理在骨科护理质量管理中的应用

目的探讨循证护理在骨科护理质量管理中的应用。方法抽取2013年8月至2014年8月台州市第一人民医院骨科收治的84例患者作为研究对象,将其随机分为对照组和循证组,对照组采用常规护理,循证组在常规护理的基础上实施循证护理措施,分别评价两组患者对护士服务态度、操作水平、沟通能力及患者满意度的差异。结果循证组患者对护士服务态度、操作水平、沟通能力评价得分分别为4.15±0.67、4.57±0.32和4.70±0.57,与对照组相比显著提高;循证组患者满意度由对照组的66.7%提高至85.7%,两组患者各项目比较差异有统计学意义(P0.05)。结论循证护理以护理研究为依据,在临床实践中为患者提供最新、最科学、最有效的护理方案。循证护理促进了和谐护患关系的建立,对提高护理质量管理具有重要意义。     

Poliovirus retention in 75-cm soil cores after sewage and rainwater application.

The adsorption rate of a guanidine-resistant strain of poliovirus LSc 2ab was measured in Long Island soils with in situ field cores (10.1 by 75 cm). The test virus was chosen because it exhibited soil adsorption and elution characteristics of a number of non-polioviruses. After the inoculation of cores with seeded sewage effluent at a 1-cm/h infiltration rate, cores were extracted, fractionated, and analyzed for total plaque-forming units per each 5-cm fraction. The results showed that 77% of the viruses were adsorbed in the first 5 cm of soil. An additional 11% were found in the 5- to 10-cm fraction, and a total of 96% of the viruses were adsorbed by 25 cm. The remaining 4% were uniformly distributed over the next 50 cm of soil, with a minimum of 0.23% in each soil section. Few viruses (< 0.22%) were observed in core filtrates. Analysis of the viral distribution pattern in seeded cores, after an application of a single rinse of either sewage effluent or rainwater, indicated that large-scale viral mobilization was absent. However, localized areas of viral movement were noted in both of the rinsed cores, with the rainwater-rinsed cores exhibiting more expensive movement. All mobilized viruses were resorbed at lower core depths.