研究背景：

近几十年来，人们对面部年轻化的方法发生了观念上的转变，从减法手术逐渐转向了加法手术。而面部注射填充术因其微创、操作方便而成为最常用的面部年轻化手段。目前，临床上常用的软组织填充剂种类繁多，其中以透明质酸相关制剂应用最为广泛。透明质酸作为人体广泛存在的一种糖胺聚糖，拥有可降解、生物相容性高等优点。然而，其在体内降解速率较快，不易存留，无法获得稳定的疗效，需要通过交联剂对其进行改构从而发挥更好的疗效。这些交联剂对机体的影响有待于更多的研究追踪与验证，且反复注射存在交联剂的聚集可能。近年来，可以刺激组织新新生的可吸收微球类产品的应用逐渐增多，如羟基磷灰石微球、聚左旋乳酸微球等。与传统的软组织填充剂相比，这类材料除了单纯为组织提供容积以外，还可以通过免疫炎症反应更有效地修复组织缺损，增加组织容积，提供更为持久的疗效，并减少了反复注射所引起的并发症。此外，随着组织工程和再生医学的不断发展，一些含有生物活性成分的新型复合材料如细胞外基质、富血小板血浆等也逐渐走进了研究者们的视野并引起了极大的关注。此类材料可以通过其所含的细胞成分或生物因子发挥作用刺激组织生成，是一类理想的软组织填充材料，具有良好的应用前景。

尽管这些材料在临床应用前均做过相关的动物研究，验证了其安全性和有效性，但这些研究可能存在观察时间较短或检测手段较为单一等局限性，需要进一步研究验证。在实际临床应用时，我们观察到不同的软组织填充材料之间在性质和引起的组织学反应上存在明显差异，各有优缺点。如果应用不当，则可能会导致患者在接受注射治疗后仍无法达到预期效果，甚至于产生一些不良反应。因此，本实验选用了透明质酸（Hyaluronic Acid, HA）、羟基磷灰石（Calcium Hydroxylapatite, CaHA）和细胞外基质（Extracellular Matrix, ECM）作为上述三种具有代表性的软组织填充材料进行了详细对比研究，以全面地了解了它们的基本特性，引起免疫炎症反应的强度以及刺激组织新生的能力。拟为整形外科医生在临床上合理选择软组织填充材料提供更为确切的理论依据并为未来软组织填充剂的发展提供更多参考。

研究目的：

1.以生理盐水作为对照组比较ECM凝胶、HA凝胶和CaHA微球的性质，观察三种材料在注射后局部的外观及质地变化。测量计算并对比不同软组织填充材料在注射后即刻、第1周、第4周、第8周、第16周和第24周时移植物的体积保留率。

2.观察三种软组织填充材料在注射后1周、4周、8周和16周，24周在周围组织中所引起的炎症细胞数量和种类的变化，并对产生的免疫炎症反应进行组织学评分，了解免疫炎症反应的变化趋势。同时，从基因和蛋白两个层面，对各组软组织填充材料附近的炎症因子水平进行定量分析，观察其变化趋势。

3.从胶原生成和血管新生两方面对三种软组织填充材料刺激周围组织新生的能力进行定量分析和对比研究，并观察其变化趋势。

研究方法：

第一部分：本部分建立了C57BL/6N小鼠的注射模型。总共分为四组:生理盐水对照组、HA凝胶组、CaHA微球组和ECM凝胶组。使用6周龄的雌性C57BL/6N小鼠，在IVC环境中适应一周后开始实验。将四组填充材料按0.1 ml的体积按顺时针方向皮下注射至40只小鼠的背部，每只小鼠四个位点。观察并记录注射后即刻，1周、4周、8周和16周，24周等各个时间点注射部位的大体形态和质地，以及注射部位局部反应，用游标卡尺测量各组所形成的皮丘的长宽高，并通过半椭球体体积公式计算出每个时间点的残留体积和体积保留率。

第二部分：本部分实验评估了各组材料注射后局部组织的免疫炎症反应。在注射后第1周、第4周、第8周、第16周和第24周处死小鼠，将注射部位包含注射物、周边包膜及附近的全层皮肤完全切除。进行HE染色，观察注射材料周围炎症细胞数量和种类的变化，邀请两位病理学家用Duranti分级对各组组织学反应讨论后进行评分，并测量第8周时各组所形成的纤维囊壁厚度。然后通过PCR和ELISA检测TNF-α、IL-1β、IL-6三种炎症因子在各时间点基因和蛋白的表达水平，进行定量和对比分析，观察其变化趋势。

第三部分：本部分通过胶原生成和血管新生两个方面评估了HA凝胶组、CaHA微球组和ECM凝胶组对周围组织新生的作用。将取材后的标本进行Masson三色染色（胶原）、CD31（新生血管）免疫组化染色和Ⅰ型胶原蛋白及Ⅲ型胶原蛋白的免疫荧光双染，观察胶原生成和血管新生的情况，并测量注射物中心部位皮肤的真皮厚度及真皮内胶原纤维的密度。然后通过PCR和ELISA检测Ⅰ型胶原蛋白、Ⅲ型胶原蛋白及VEGF在注射物周围组织内的表达水平，进行定量分析和对比分析。

研究结果：

第一部分：ECM凝胶、HA凝胶和CaHA微球均具有较好的生物相容性，不会在注射局部引起明显的不良反应。三组中，以ECM凝胶的流动性最好，易于推注，因不易形成包膜包裹，难以维持原始形状。ECM注射后质地最软，与软组织最相像，HA凝胶次之，CaHA微球最硬。HA凝胶和CaHA微球均可在体内存留24周以上，且这两种材料周围可形成较薄的纤维包膜，能长时间地维持一定的体积和形态。HA凝胶的填充效果较为稳定，体积下降速度基本保持恒定。CaHA微球早期易出现快速的体积的丢失，但其长期的体积保留率优于HA凝胶。

第二部分：实验过程中三组材料的免疫炎症反应均在正常的生理反应范围内，均未出现异物肉芽肿、组织坏死等不良反应。其中，HA和ECM两种材料所引起的免疫炎症反应较轻，且很快消退。CaHA微球所引起的免疫炎症反应较上述两组重，但随着注射时间增加，其组织学评分呈逐渐降低的趋势。HA凝胶和ECM凝胶在注射后4周内可短暂的刺激周围组织炎症因子的分泌，而CaHA在16周内均较对照组的炎症因子水平升高。三组实验组其炎症因子水平随注射时间呈逐渐下降的趋势，最终都恢复至正常水平。

第三部分：HA凝胶、ECM凝胶和CaHA微球均能显著改善注射局部皮肤的质量，增加其真皮的厚度和真皮内胶原纤维的密度。三组实验材料均能持续性刺激附近组织中胶原的生成和局部血管的新生，且随着刺激时间呈累加效果。产生的胶原主要以III型胶原为主。总体来说，三组中引起免疫炎症反应最轻的ECM对周围组织新生的影响最小，而CaHA虽然引起的免疫炎症反应最重，但其刺激周围组织新生的效果也最好。这提示免疫炎症反应的刺激可能在软组织填充材料诱导周围组织新生起着决定性的作用。

结论：

本研究综合比较了临床上上关注度较高的三种软组织材料，为临床应用提供了理论基础。所有实验材料都被证明是生物相容性和组织学安全的，且三种填料各有优缺点。ECM柔韧，与天然软组织高度相似，免疫反应反应最小。但其注射后很难保持原来的体积和位置，填充效果及长期体积保持率较差，且促进组织新生能力最弱。HA具有良好的生物相容性，填充效果较稳定，但其长期的体积保留率低于CaHA。CaHA引起的免疫炎症反应最重，短期存在快速的体积丢失，但长期的填充效果较好，且其诱导胶原蛋白生成和血管生成的能力最强。研究中发现注射材料引起的免疫炎症反应在一定范围内与其促进促进组织新生的作用呈正相关。因此，精准地操纵和控制填充材料周围的免疫炎症反应有助于软组织填充取得更好的疗效。

Background:

In recent decades, a conceptual shift in the approach to facial rejuvenation has occurred, from subtraction surgery to additive surgery. The facial injection has become the most commonly used facial rejuvenation surgery because of its minimally invasive and convenient operation. At present, there are many kinds of soft tissue fillers in the market, among which hyaluronic acid-related preparations are the most widely used. As a kind of glycosaminoglycan widely existing in the human body, hyaluronic acid has the advantages of biodegradability and high biocompatibility. However, its degradation rate in vivo is fast, not easy to retain, and can not obtain a stable efficiency. So, it needs to be modified by a cross-linking agent to play a better therapeutic effect. The effects of these cross-linking agents on the human body need to be tracked and verified by more research. And the repeated injection of the fillers may lead to the accumulation of cross-linking agents. In recent years, the application of absorbable microsphere products that can stimulate tissue regeneration has gradually increased, such as hydroxyapatite microspheres, poly-L-lactic acid microspheres, and so on. Compared with traditional soft tissue fillers, this kind of material can not only provide volume but also repair tissue defects through immuno-inflammatory reactions. It can provide a more prolonged effect and reduce the complications caused by repeated injections. In addition, with the continuous development of tissue engineering and regenerative medicine, some new composites containing bioactive components, such as extracellular matrix and platelet-rich plasma, have gradually come into the view of researchers and aroused great attention. These materials can stimulate tissue regeneration through their cellular components or biological factors, so they are the ideal materials for soft tissue fillers and has a good application prospect in the future.

Although these materials have done relevant animal studies before clinical application to verify their safety and effectiveness, they may have limitations, such as short observation time or single detection methods, which need further verification. In clinical application, we have observed significant differences in properties and histological reactions among different soft tissue fillers, each of which has its own advantages and disadvantages. If the surgeons appropriately use them, the patients may be unable to achieve their expected effect after receiving injection treatment and even have some adverse reactions. Therefore,  in this experiment, we selected hyaluronic acid, calcium hydroxyapatite microsphere, and extracellular matrix as three representative soft tissue fillers to conduct a detailed comparative study in order to fully understand their basic characteristics, the intensity of the immuno-inflammatory response and the ability to stimulate tissue regeneration. It is intended to provide a theoretical basis for plastic surgeons to select soft tissue fillers in the clinic reasonably and provide a reference for the development of soft tissue fillers in the future.

Objectives:

1. With normal saline as the control group, compare the properties of ECM gel, HA gel, and CaHA microspheres. And observe the local appearance and texture changes of the three materials after injection. The volume retention rates of different soft tissue filling materials were measured and compared at immediate time, 1 week, 4 weeks, 8 weeks, 16 weeks, and 24 weeks after injection.

2. In this study, the changes in the number and types of the inflammatory cells around the soft tissue fillers were observed at 1 week, 4 weeks, 8 weeks, 16 weeks, and 24 weeks after injection, and the immuno-inflammatory reaction was scored to understand the changing trend of the immuno-inflammatory response At the same time, the levels of inflammatory factors in the vicinity of soft tissue fillers in each group were quantitatively analyzed from the gene and protein levels, and the changing trend was observed.

3. The stimulation effects of three kinds of soft tissue fillers on the regeneration of surrounding tissues were quantitatively analyzed and compared from the aspects of collagen generation and angiogenesis, and the changing trend was observed.

Methods:

Part I: The injection model of C57BL/6N mice was established in this part. The injection was divided into four groups: control group(normal saline), ECM gel group, HA gel group, and CaHA microsphere group. Six-week-old female C57BL/6N mice were used in the experiment after a week of adaptation in the IVC laboratory. To reduce the difference caused by different injection sites, the four groups of fillers were injected into the subcutaneous back of 32 mice in a clockwise direction according to the volume of 0.1ml. The general shape and texture of the injection site and the local reaction of the injection site were recorded at immediate time, 1 week, 4 weeks, 8 weeks, 16 weeks, and 24 weeks after injection. The vernier caliper was used to measure the fillers’ length, width, and height. And the volume formula for hemiellipse was used to calculate the volume and the retention rate of the mass.

Part Ⅱ: In this part of the study, we evaluated the immuno-inflammatory reaction of the surrounding tissues after the injection of fillers in each group. The mice were killed at 1 week, 4 weeks, 8 weeks, 16 weeks and 24 weeks after injection. And the test site was removed completely for further experiments, including the implant, the capsule, and the whole skin. HE staining was performed to observe the changes in the number and types of inflammatory cells around the injection material. Two pathologists were invited to blindly evaluate the local foreign-body response of all slides according to the Duranti histologic score. And the thickness of the fibrous capsule wall in each group was measured at 8 weeks. Then, PCR and ELISA were used to detect the gene and protein expression levels of TNF-α, IL-1β, and IL-6 at each time point for quantitative and comparative analysis. The expression trend of inflammatory factors was monitored.

Part Ⅲ: In this part, the effects of the HA gel group, CaHA microsphere group, and ECM gel group on peripheral tissue regeneration were evaluated through collagen generation and angiogenesis. The specimens were stained with Masson trichrome (for collagen) and CD31 (for neovascularization) staining according to standard immunohistochemistry protocols. Also, the double immunofluorescence staining of type Ⅰ collagen and type Ⅲ collagen was performed. Collagen production and angiogenesis were observed, and the thickness of the dermis and the density of collagen fibers in the dermis were measured. Then, the expression levels of type Ⅰ collagen, type Ⅲ collagen, and VEGF in the surrounding tissues were detected by PCR and ELISA for quantitative and comparative analysis.

Results:

Part I: ECM gel, HA gel, and CaHA microspheres have good biocompatibility and will not cause obvious adverse reactions in the injection site. Among the three groups, ECM gel has the best fluidity. It is easy to push during the injection. And it is hard to maintain the original shape due to the ECM gel could hardly be encapsulated after injection. After ECM gel was injected, the texture of the injection site was the softest and most similar to the soft tissue, followed by the HA group, and the CaHA group was the hardest. Both HA gel and CaHA microspheres can remain in vivo for more than 24 weeks, and a thin fiber capsule can be formed around the two materials. They can maintain a certain volume and shape for a long time. The volume of the HA gel groups maintained a relatively stable decline from the beginning to the end. The CaHA microsphere groups experienced a rapid volume loss in the early stage, but their long-term volume retention is better than that of HA gel.

Part Ⅱ: During the experiment, the immune inflammatory reactions of the three groups of materials were all within the range of normal physiological responses. There were no adverse reactions, such as foreign body granuloma and tissue necrosis. Among them, the immuno-inflammatory response caused by HA and ECM was slight and disappeared quickly. The immuno-inflammatory reaction induced by CaHA microspheres was more severe than that in the above two groups, but the Duranti score decreased gradually with the increase of injection time. HA gel and ECM gel could temporarily stimulate the secretion of inflammatory factors in the surrounding tissue within 4 weeks after injection, while the level of inflammatory factors in CaHA groups was higher than that in the control group within 16 weeks. Finally, the expression level of inflammatory factors in the three experimental groups decreased gradually with the injection time and eventually returned to the average level.

Part Ⅲ: HA gel, ECM gel, and CaHA microspheres can significantly improve the quality of local skin and increase the thickness and the density of collagen fibers of the dermis. All three experimental groups could continuously stimulate collagen production and local angiogenesis in nearby tissues and showed a cumulative effect with the stimulation time. The new collagen was mainly type Ⅲ collagen. Generally speaking, the ECM, which causes the most minor immuno-inflammatory response, has the least efficiency on the regeneration of surrounding tissue among the three experimental groups. Although CAHA causes the most severe immuno-inflammatory reaction, it is the most effective in stimulating surrounding tissue regenerations. It is suggested that the stimulation of immuno-inflammatory response may play a decisive role in the tissue regeneration induced by soft tissue fillers.

Conclusions:

This study comprehensively compares three kinds of soft tissue fillers with great attention in the market, providing a theoretical basis for clinical application. All the experimental fillers have been proven to be biocompatible and histologically safe and have their own advantages and disadvantages. The ECM gel is flexible, highly similar to natural soft tissue, and has the least immune response. However, it is difficult to maintain the original volume and position after injection, and the ability to promote tissue regeneration is the weakest. HA has good biocompatibility and stable degradation and absorption, but its long-term volume retention rate is lower than CaHA. The immuno-inflammatory reaction caused by CaHA is the most severe, and there is a rapid volume loss in the short term. However, the filling efficiency of CaHA over a long time is better, and its ability to induce collagen production and angiogenesis is the strongest. In this study, it was found that the materials with severe immuno-inflammatory responses were more efficient in promoting tissue regeneration. Therefore, accurately manipulating the immuno-inflammatory response around the fillers may lead to better results of soft tissue augmentation.