Tissue engineering of bone: the reconstructive surgeon's point of view

Bone defects represent a medical and socioeconomic challenge. Different types of biomaterials are applied for reconstructive indications and receive rising interest. However, autologous bone grafts are still considered as the gold standard for reconstruction of extended bone defects. The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Tissue engineering is, according to its historic definition, an "interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function". It is based on the understanding of tissue formation and regeneration and aims to rather grow new functional tissues than to build new spare parts. While reconstruction of small to moderate sized bone defects using engineered bone tissues is technically feasible, and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions, the reconstruction of large-volume defects remains challenging. Therefore vascularization concepts gain on interest and the combination of tissue engineering approaches with flap prefabrication techniques may eventually allow application of bone-tissue substitutes grown in vivo with the advantage of minimal donor site morbidity as compared to conventional vascularized bone grafts. The scope of this review is the introduction of basic principles and different components of engineered bioartificial bone tissues with a strong focus on clinical applications in reconstructive surgery. Concepts for the induction of axial vascularization in engineered bone tissues as well as potential clinical applications are discussed in detail.     

Natural secretory products of human neural and microvessel endothelial cells

Neurons, glia, and endothelial cells of the cerebral microvasculature co-exist in intimate proximity in nervous tissues, and their homeostatic interactions in health, as well as coordinated response to injury, have led to the concept that they form the basic elements of a functional neurovascular unit. During the course of normal cellular metabolism, growth, and development, each of these brain cell types secrete various species of potentially neurotoxic peptides and factors, events that increase in magnitude as brain cells age. This article reviews contemporary research on the secretory products of the three primary cell types that constitute the neurovascular unit in deep brain regions. We provide some novel in vitro data that illustrate potentially pathogenic paracrine effects within primary cells of the neurovascular unit. For example, the pro-inflammatory cytokine interleukin (IL)-1β was found to stimulate amyloid-β (Aβ) peptide release from human neural cells, and human brain microvessel endothelial cells exposed to transient hypoxia were found to secrete IL-1β at concentrations known to induce Aβ42 peptide release from human neural cells. Hypoxia and excessive IL-1β and Aβ42 abundance are typical pathogenic stress factors implicated in the initiation and development of common, chronic neurological disorders such as Alzheimer's disease. These data support the hypothesis that paracrine effects of stressed constituent cells of the neurovascular unit may contribute to “spreading effects” characteristic of progressive neurodegenerative disorders.     

Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways

Flap endonuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease that plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair, resolution of DNA secondary structures and stalled DNA replication forks, and apoptotic DNA fragmentation. How FEN-1 is involved in multiple pathways, of which some are seemingly contradictory, is of considerable interest. To date, at least 20 proteins are known to interact with FEN-1; some form distinct complexes that affect one or more FEN-1 activities presumably to direct FEN-1 to a particular DNA metabolic pathway. FEN-1 consists of a nuclease core domain and a C-terminal extension. While the core domain harbors the nuclease activity, the C-terminal extension may be important for protein-protein interactions. Here, we have truncated or mutated the C-terminus of FEN-1 to identify amino acid residues that are critical for interaction with five proteins representing roles in different DNA replication and repair pathways. We found with all five proteins that the C-terminus is important for binding and that each protein uses a subset of amino acid residues. Replacement of one or more residues with an alanine in many cases leads to the complete loss of interaction, which may consequently lead to severe biological defects in mammals.     

即时扩展型背阔肌肌皮瓣乳房再造在保留乳头乳晕复合体乳癌术后的运用分析

目的：探讨分析即时扩展型背阔肌肌皮瓣乳房再造在保留乳头乳晕复合体乳癌术后的运用。方法：回顾性分析我院2008年2月-2012年4月收治的乳腺癌术后患者106例,采用乳癌术即时扩展型背阔肌肌皮瓣乳房再造保留乳头乳晕复合体,观察手术效果以及满意度。结果：术后患者乳房美容优良率为88．68％明显大于对照组的47．17％,并且术后6个月治疗组生活质量评价总分明显大于对照组总分术后6个月患者生活质量评价总分明显大于术前评价总分,差异具有条件下意义（P〈0．05）,差异均具有统计学意义（P〈0．05）。结论：即时扩展型背阔肌肌皮瓣乳房再造在保留乳头乳晕复合体乳癌术后患者乳房美容效果较好,提高患者生活质量高,值得在临床上推广,但在手术后需积极处理可能存在的并发症情况。     

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5′-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.     

Crystal structures of the structure‐selective nuclease Mus81‐Eme1 bound to flap DNA substrates

The Mus81‐Eme1 complex is a structure‐selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross‐links, replication fork collapse, or double‐strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81‐Eme1, we determined crystal structures of human Mus81‐Eme1 bound to various flap DNA substrates. Mus81‐Eme1 undergoes gross substrate‐induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre‐ and post‐nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post‐nick DNA. These features are crucial for comprehensive protein‐DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81‐Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81‐Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends.     

游离皮瓣移植术后血运监测的研究进展

游离皮瓣移植已经成为修复重建外科常用的修复手段之一,其成功率已经超过95%,但微循环障碍的风险仍然存在,临床上皮瓣救助率与缺血时间呈负相关,因此游离皮瓣移植术后的血运监测至关重要。作者以"皮瓣"和"监测"为检索词在PubMed数据库检索出2000年1月到2014年1月期间关于游离皮瓣移植术后监测的相关文献,纳入标准为至少5篇以上的相关报道,通过分析相关文献概述当前主要的监测方法,从临床实用性、可靠性等方面分析其优缺点。     

新型小鼠跨区供血耳瓣模型的构建

目的：建立一个实时活体观察血管形态学变化小鼠跨区供血耳瓣模型。方法体重25～30 g清洁级ICR小鼠30只,双耳脱毛后,观察其血管分布情况。小鼠麻醉后,用眼科剪从尾侧向头侧剪断鼠耳基底部尾侧2/3,保留头侧1/3,形成耳前血管蒂跨三个血管体、二个choke区的耳瓣模型。将小鼠侧卧置于二维图像采集系统的动物承载台上,调节体视显微镜物镜并固定为25倍,设置步进参数,“弓”型路线渐次、局部采集造模后0,1,2,3,5,7,10,14,21,30 d的时间点图像,合成鼠耳全景图。重点观察皮瓣的坏死率、皮瓣内choke血管的形态学变化。结果 ICR小鼠耳有三个恒定的血管体来供养,从内到外依次为头侧血管体、中间血管体及尾侧血管体。术后5 d,耳瓣坏死面积趋于稳定,坏死率为（15±7）％。内侧血管体与中间血管体之间的choke动静脉的管径出现快速扩增,两者都在第10天左右达最大,choke静脉管径最高峰可达到原来的（3.9±0.5）倍,choke动脉管径最高峰可达到原来的（3.5±0.7）倍。10 d后,choke静脉管径开始减小,21 d后逐渐平稳,而choke动脉管径于术后10 d左右开始平稳,之后无明显减小。结论①跨区皮瓣切取后,静脉扩张是被动扩张,而动脉扩张是主动增值;②跨区皮瓣切取后血流动力学供区与潜力供区之间的choke区参与扩张的choke血管数量及扩张度均小于解剖供区与血流动力学供区之间的choke血管;③小鼠耳瓣模型为研究血管扩张机制及遴选促皮瓣存活药物的理想动物模型。