从皮肤的细胞外基质（NECM）中制取可注射胶原

常规制取注射性胶原的方法是用酶降解法，经反复酸提、盐析、离心、交联等步骤，进行纯化，该法将胶原降解，酶提取分子片断，用交联剂组成可注射胶原。本法是从ＮＥＣＭ开始制取，ＮＥＣＭ已为胶原框架，因此步骤简化很多，仅需解裂胶原网状结构，酶解末端蛋白以消除特异性，最后降解成３００ｎｍ左右胶原。本法不用交联剂和稳定剂。生物原材料来源广泛，有利临床推广应用。经半年动物实验证明，本法制取的可注射胶原无排斥反应和炎     

从NECM制取的可注射性胶原的生物学评价

从ＮＥＣＭ制取可注射胶原是从大分子降解为胶原分子，因此不需要交联剂和稳定剂。溶液中仅有胶原分子、无交联剂。本实验是将可注射胶原液注入大鼠尾部皮下组织内，共注射３０只，分三批杀死（１．５个月，３．０个月和半年），取注射部位的皮下组织作组织切片，染色（ＨＥ）、光镜观察（包括偏光显微镜）。     

从NECM制取的可注射性胶原的生物学评价

从ＮＥＣＭ制取可注射胶原是从大分子降解为胶原分子，因此不需要交联剂和稳定剂。溶液中仅有胶原分子，无交联剂。本实验是将可注射胶原液（１２０ｍｇ／ｍｌ）１ｍｌ注入大鼠尾部皮下组织内，共注射３０只，分三批杀死（１．５个月，３．０个月和半年），取注射部位的皮下组织作组织切片、染色（ＨＥ）、光镜观察（包括偏光显微镜）。发现三组在注射部位皮下组织内均有胶原分散存在。半年时胶原量少而且排列有序，未见包裹现象。除１．５个月少数动物皮下组织内有轻度炎性反应外，其余均未见到炎性反应。无排斥反应。局部组织无坏死现象。实验认为：本法所制可注射胶原液在半年内未发现特殊的生物学反应，胶原未完全吸收。     

组织工程学研究中偏光显微镜的应用

偏光显微镜是地质界用来观察晶体矿物的重要仪器，其理论基础是结晶光学，特点是即可观察结构，又可根据光学性质（折光率值不同）来判断成分，通过在研究ＮＥＣＭ和注射性胶原制作的过程中，用偏光显微镜和光镜观察同一部位的组织片，加以对比分析。因为胶原是生物体内的特殊“晶体”，故认为偏光显微镜应用在组织工程研究中，不仅开阔了观察领域，而且是重要的观测手段和方法。用偏光显微镜观察了猪骨ＥＣＭ胶原框架，以及注射性胶     

组织工程学研究中偏光显微镜的应用

偏光显微镜是地质界用来观察晶体矿物的重要仪器。其理论基础是结晶光学，特点是即可观察结构，又可根据光学性质（折光率值不同）来判断成分。通过在研究ＮＥＣＭ和注射性胶原制作的过程中，用偏光显微镜和光镜观察同一部位的组织片，加以对比分析。因为胶原是生物体内的特殊“晶体”，故认为偏光显微镜应用在组织工程研究中，不仅开阔了观察领域，而且是重要的观测手段和方法。用偏光显微镜观察了猪骨ＥＣＭ胶原框架，以及注射性胶原制作过程中胶原变化特点。认为本方法简单不需染色，并可特异的观察到胶原的分布和走行。     

动物NECM的稳定性评价

“动物ＮＥＣＭ的制作”文章发表近半年，已制作好的动物皮肤、肌腱、软骨和骨的ＮＥＣＭ已在保存液中保存了１３个月。为了观察ＮＥＣＭ和保存液的稳定性，本文做了如下几个方面的研究：①保存液：未放置ＮＥＣＭ新配制的和已放置１年的保存液；放置ＮＥＣＭ的保存液（１９９７年２月）。上述保存液均做细菌培养、镜下观察和成分分析。②电镜和组织学光镜观察：１９９７年２月制作的ＮＥＣＭ的光镜观察；放置１３个月后的ＮＥＣＭ电镜和光镜观察（包括偏光显微镜）；新鲜的正常相应组织的电镜和光镜观察。实验结果证明：猪皮肤和牛肌腱的ＮＥＣＭ已无细胞成分，基质为平行排列的胶原纤维丝或松散的胶原纤维网。而正常组织中细胞完整，胶原纤维呈致密交叉的网状编织结构。保存液清晰，无细菌生长，成分未变。     

肾小球硬化及老年肾细胞外基质的变化研究

本文通过一侧肾切加重复阿霉素注射制作加速肾小球硬化大鼠模型，应用酶联免疫吸附试验（ＥＬＩＳＡ）对分离的肾小球内细胞外基质［胶原Ⅲ、胶原Ⅳ、层粘连蛋白（ＬＮ）和纤维连结蛋白（ＦＮ）］进行了硬化过程动态的定量研究，并将正常鼠（３月）和老年鼠（２６月）作了比较。结果显示，胶原Ⅲ在正常鼠肾小球内用ＥＬＪＳＡ方法未检测到；疾病所致的肾小球硬化过程胶原Ⅲ持续增加，弥漫性肾小球硬化期，较正常肾增加１１．８倍。胶原Ⅳ、ＬＮ和ＦＮ为正常肾小球内存在的细胞外基质（ＥＣＭ），在肾小球硬化的早、中期明显增加；弥漫性肾小球硬化期分别为正常肾的１２．３、１６．３和５．１倍。老年鼠与正常鼠相比，胶原Ⅲ无显著性变化（Ｐ＞０．０５），胶原Ⅳ、ＬＮ和ＦＮ分别为正常鼠的９．１、１０．０和２．８倍。本研究证实，ＥＣＭ的积聚是肾小球硬化过程中重要的病理特征，胶原Ⅲ（一种间质胶原）似与疾病所致的肾小球硬化发展有更密切和直接的关系。     

动物NECM的稳定性评价

“动物ＮＥＣＭ的制作”文章发表近半年，已制作好的动物皮肤、肌腱、软骨和骨的ＮＥＣＭ已在保存液中保存了１３个月，为了观察ＮＥＣＭ和保存液的稳定性，本文做了如下几个方面的研究：（１）保存液：未放置ＮＥＣＭ新配制的和已放置１年的保存液；放置ＮＥＣＭ的保存液（１９９７年２月）。上述保存液均细菌培养，镜下观察和成分分析。（２）电镜和组织学光镜观察；１９９７年和２月制作的ＮＥＣＭ的光镜观察；放置１３个月后的Ｎ     

免疫电镜技术研究肾小球硬化过程中细胞外基质的变化

为探讨肾小球硬化机制，应用胶体金免疫电镜技术对重复阿霉累注射加一侧肾切除所诱发的进行性肾小球硬化模型中细胞外基质（ＥＣＭ）成份－Ⅲ型、Ⅳ型胶原、层粘连蛋白（ＬＮ）和纤维连结蛋白（ＦＮ）进行了定位、定量、定性的研究，并对肾小球基底膜（ＧＢＭ）和系膜区上述成份的正常分布及硬化过程中的变化作了比较。结果显示：正常鼠ＬＮ、ＦＮ在系膜区和ＧＢＭ中的含量有显著性差异（Ｐ＜０．０１），而Ⅳ型胶原则基本相同，这三种成份在ＧＢＭ分布有一定的规律。肾小球硬化过程中ＬＮ、ＦＮ、Ⅳ型胶原明显积聚，弥漫性肾小球硬化期与正常鼠相比，增加幅度为１．０～３．２倍（Ｐ＜０．０１）；肾小球硬化后期ＧＢＭ内不再显著增加，而系膜区内则持续增加．正常肾小球内不存在的Ⅲ型胶原在硬化早期无明显积聚，其后急聚增多至弥漫性肾小球硬化期。与对照组相比，ＧＢＭ与系膜区Ⅲ型胶原分别增加了１３．９及３２．３倍。我们用该技术证实了ＥＣＭ成份积聚是肾小球硬化的基本病理特征。Ⅲ型间质胶原从无至有，增加幅度最大，似提示与肾小球硬化有更直接、密切的关系。     

同种异体兔耳软骨ECM复制兔耳软骨的实验研究──软骨组织工程的实验研究预试验报告

我们这项研究旨在寻求一种能够避免组织移植和人工材料的缺点，又能获得自体组织移植相同的效果的移植材料。选用１０只纯种新西兰大耳白兔，异体兔耳软骨细胞外基质（ＥＣＭ）。兔耳软骨缺损分别以下列四组方式修复：①兔耳软骨ＥＣＭ＋兔耳软骨骨膜，②兔耳软骨ＥＣＭ，③兔耳软骨膜，④空白。术后６、１２、２４、４８周分别切取修复区域标本，做ＨＥ、奥新蓝两种染色。结果显示，ＥＣＭ植入体内后６周有轻度的炎症反应，术后１２周炎症异物反应消失，ＥＣＭ周边无包裹现象，术后１２周骨膜细胞长入ＥＣＭ，但细胞排列不规则。术后２４周、４８周ＥＣＭ仍然存在，无吸收现象，软骨膜与ＥＣＭ覆着处细胞长入ＥＣＭ，排列较１２周时规则，呈奥新蓝反应强阳性。结论：①ＮＥＣＭ可以作为组织充填物而长期存在并有较好的组织亲和性和相容性，因此有希望成为新一代的组织替代物。②ＮＥＣＭ可以诱导软骨膜细胞向其中生长而重建软骨，因此软骨ＮＥＣＭ有可能是复合生长因子。③软骨膜可以作为软骨ＮＥＣＭ复制软骨组织的细胞来源。④根据本实验观察一年的结果证明应用兔耳软骨ＥＣＭ，可以复制出新的软骨。     

人发制备软组织填充材料的微观结构及皮内填充的实验观察

目的对人发材料制备中微观结构变化及皮内填充效果进行初步观察，为临床修复体表凹陷寻求良好的软组织填充材料。方法经漂白和机械加工制备可注射性人发材料，扫描电镜观察材料的微观结构。将制备的材料注射到小香猪皮内，分别在1周、4周、12周和24周取材，观察材料在体内的变化。结果经过漂白后的人发较好地保留了人发的结构，可注射性人发材料以角蛋白纤维束存在。材料填充后无局部及全身不良反应，24周时填充部位材料大部分被吸收，代之以自体成纤维细胞及其分泌的自身胶原，材料周围无明显纤维包膜形成。12周和24周时，填充部位真皮较对照组增厚，差别有统计学意义（P〈0.05）。结论人发填充材料具有良好的体内生物相容性，并能促进局部真皮增厚；具有良好的填充效果，可能成为一种新型的填充材料。     

Injectable autogenous corium: technique development in the rat model

Difficult facial rhytides have been treated with injectable bovine collagen. To our knowledge, there has been no reported use of injectable autogenous collagen. We studied several techniques using homogenization of dermis in the rat model. Commercial tissue homogenizers produced a fine emulsification, which serial clinical and histological examinations showed to be largely resorbed. A household blender produced coarse homogenates, which persisted 5 to 6 months; however, most of the implants eventually were resorbed. We conclude that coarse homogenates of dermis persist longer as implants than do fine homogenates in the rat model.     

激光等物理治疗对置人的聚甲基丙烯酸甲酯微球结构影响的观察

目的 评估注射皮肤填充剂爱贝芙（Artecoll）后,再联合使用激光等其他美容手段后的有效性及安全性。方法 体外实验：直接对爱贝芙进行点阵激光照射后,观察其微球结构的完整性与表面的光滑程度等变化。动物实验：分别用猪及豚鼠建立相应的短期和中长期填充剂动物模型,并进行后续的激光、射频等治疗,光镜、电镜观察比较激光治疗前后的微球结构及组织的炎症程度变化等。结果 体外研究结果,未发现经多次激光照射后爱贝芙中的聚甲基丙烯酸甲酯（polymethyl-methacrylate,PMMA）微球的形状和结构发生变形或破坏等情况,微球表面仍光滑如初。动物实验表明,爱贝芙较长期置入动物后,PMMA微球仍能保持良好的结构和光滑的表面,未见微球被吞噬破坏。在多次激光等其他手段治疗后,微球置人部位炎症细胞浸润程度与未经激光治疗的对照组比较无差异。结论 爱贝芙是一种安全有效的永久性皮肤填充剂,临床使用某些激光、射频的治疗不会影响其安全性。     

Bulking agents: a urogynecology perspective

A bulking agent is a material injected into the wall of the urethra to improve urethral coaptation in women suffering from stress incontinence. The concept was initially described in the 1930s when sodium morrhuate and paraffin were used to augment urethral resistance. Sclerosing agents were also used for inducing permanent urethral scaring to improve urinary leakage. Eventually, collagen and autologous fat were found to be efficacious, and only collagen demonstrated proven safety and endured extensive testing, becoming the gold standard for injectable agents. Since then, multiple other products have been developed by the industry, each with its particular success rates and complications.     

Collagen fillers

This article reviews the currently available collagen injectable fillers and describes injection techniques. Collagen was one of the first fillers for aesthetic enhancement and has been in use for more than 20 years. In recent years there has been a surge of new injectable fillers, with more anticipated in the near future. Despite the large number of new products for augmentation and wrinkle filling, there still remains a role for injectable collagen--alone and in combination with other wrinkle fillers.     

Symptomatic calcification of subureteral collagen ten years after injection

Bovine glutaraldehyde cross-linked collagen is an injectable bulking agent used to treat urinary incontinence and vesicoureteral reflux. A 16-year-old girl had previously undergone left subureteral injection of collagen at the age of 6 years. She presented to our clinic after passing a 7-mm calcification with a history of hematuria and intermittent back pain. Computed tomography showed two hyperdense foci measuring 2 mm each in the region of the left ureterovesical junction that did not change on follow-up imaging. On cystoscopy, two submucosal calcifications were located medially and inferiorly to the left ureteral orifice and were removed endoscopically.     

Injectable biomaterials for the treatment of stress urinary incontinence: their potential and pitfalls as urethral bulking agents

Injectable urethral bulking agents composed of synthetic and biological biomaterials are minimally invasive treatment options for stress urinary incontinence (SUI). The development of an ideal urethral bulking agent remains challenging because of clinical concerns over biocompatibility and durability. Herein, the mechanical and biological features of injectable urethral biomaterials are investigated, with particular emphasis on their future potential as primary and secondary treatment options for SUI. A literature search for English language publications using the two online databases was performed. Keywords included “stress urinary incontinence”, “urethral bulking agent” and “injectable biomaterial”. A total of 98 articles were analysed, of which 45 were suitable for review based on clinical relevance and importance of content. Injectable biomaterials are associated with a lower cure rate and fewer postoperative complications than open surgery for SUI. They are frequently reserved as secondary treatment options for patients unwilling or medically unfit to undergo surgery. Glutaraldehyde cross-linked bovine collagen remains the most commonly injected biomaterial and has a cure rate of up to 53 %. Important clinical features of an injectable biomaterial are durability, biocompatibility and ease of administration, but achieving these requirements is challenging. In carefully selected patients, injectable biomaterials are feasible alternatives to open surgical procedures as primary and secondary treatment options for SUI. In future, higher cure rates may be feasible as researchers investigate alternative biomaterials and more targeted injection techniques for treating SUI.     

The augmentation of soft tissue with injectable collagen

Clinical experience has demonstrated the value of injectable collagen in treating small soft-tissue defects. Among the lesions that respond best to injectable collagen therapy are nasolabial and glabellar lines as well as areas of atrophy and soft scars. This material is a useful adjunct to surgical procedures and can correct contour irregularities that may follow rhinoplasty or rhytidectomy. The safety of injectable collagen has also been well demonstrated. The nature and incidence of treatment reactions (less than 3 per cent) remains unchanged since the close of clinical trials. Reactions have been localized, and although some have been cosmetically undesirable, none has been health-threatening. All ultimately resolve without therapeutic intervention. Immunologic studies have confirmed the benign nature and specificity of reactions to injectable collagen. The importance of proper injection technique cannot be overstated. Immediately upon injection, tissue blanching followed by whealing and overcorrection should be evident. These indicate the desired superficial placement of the material. The overcorrection helps to compensate for the loss of carrier saline, and it quickly dissipates. When properly injected, injectable collagen can be used safely and effectively to correct an array of soft-tissue contour irregularities.     

Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives

Whereas xenogeneic collagen has provided a safe and effective biomaterial for numerous medical applications, there are few instances in which data permit the correlation of the immunologic profile of well-defined devices with their clinical sequelae. A major exception is the use of injectable bovine dermal collagen for soft-tissue contour correction. The low incidence of hypersensitivity has been studied in the context of clinical efficacy and safety with several devices. The findings indicate that such immunity usually results in the manifestation of local symptoms of dermal inflammation at sites of treatment that resolve as the implant is resorbed by the host. In contrast, more immunogenic hemostatic agents may elicit a more frequent or vigorous immune response that is not clinically visible or relevant in that application. Recent experiences with collagen-based devices for the repair and regeneration of bone have also demonstrated that the presence of immunity to their collagenous or non-collagenous components does not necessarily predict adverse clinical sequelae. Indeed, numerous specific data indicate that this immunity can exist as an epiphenomenon with no effect on osteogenesis. To get a true composite picture of biocompatibility, significant steps must be taken to characterize biomaterials properly and to ensure that immunologic, clinical, histologic, and other pertinent laboratory data are viewed in relation to one another and not in isolation.