Background

The present study evaluated the cellular tissue reaction of two equine-derived collagen hemostatic sponges (E-CHS), which differed in thickness after pressing, over 30 days in vivo. The inflammatory response during physiological wound healing in sham-operated animals was used as control group.

Material and methods

First, the E-CHS was pressed by applying constant pressure (6.47 ± 0.85 N) for 2 min using a sterile stainless-steel cylinder until the material was uniformly flattened. Consequently, the original (E-CHS), the pressed (P-E-CHS), as well as the control group (CG; sham operation) were studied independently. The 3 groups were evaluated in vivo after subcutaneous implantation in Wistar rats during 3, 15, and 30 days. Histochemical and immunohistochemical methods provided observations of biomaterial degradation rate, cellular inflammatory response, and vascularization pattern. A derivative of human blood known as platelet-rich fibrin (PRF) was used as an ex vivo model to simulate the initial biomaterial-cell interaction. Segments of E-CHS and P-E-CHS were cultivated for 3 and 6 days with PRF, and the release of pro-inflammatory proteins was measured using ELISA. PRF cultivated alone was used as a control group.

Results

At day 3, the CG induced a statistically significant higher presence of monocytes/macrophages (CD68+), pro-inflammatory macrophages (M1; CCR7+), and pro-wound healing macrophages (M2; CD206+) compared to E-CHS and P-E-CHS. At the same time point, P-E-CHS induced a statistically significant higher presence of CD68+ cells compared to E-CHS. After 15 days, E-CHS was invaded by cells and vessels and showed a faster disintegration rate compared to P-E-CHS. On the contrary, cells and vessels were located only in the outer region of P-E-CHS and the biomaterial did not lose its structure and accordingly did not undergo disintegration. The experimental groups induced similar inflammatory reaction primarily with positive pro-inflammatory CD68+/CCR7+ macrophages and a low presence of multinucleated giant cells (MNGCs). At this time point, significantly lower CD68+/CCR7+ macrophages and no MNGCs were detected within the CG when compared to the experimental groups (P < 0.05). After 30 days, E-CHS and P-E-CHS were fully degraded. All groups showed similar inflammatory reaction shifted to a higher presence CD206+ macrophages. A low number of CCR7+ MNGCs were still observable in the implantation bed of both experimental groups. In the ex vivo model, the cells and fibrin from PRF penetrated E-CHS. However, in the case of P-E-CHS, the cells and fibrin stayed on the surface and did not penetrate towards materials central regions. The cultivation of P-E-CHS with PRF induced a statically significant higher release of pro-inflammatory proteins compared to the CG and E-CHS after 3 days.

Conclusion

Altering the original presentation of a hemostatic sponge biomaterial by pressing modified the initial biomaterial-cell interaction, delayed the early biomaterial’s degradation rate, and altered the vascularization pattern. A pressed biomaterial seems to induce a higher inflammatory reaction at early time points. However, altering the biomaterial did not modify the polarization pattern of macrophages compared to physiologic wound healing. The ex vivo model using PRF was shown to be an effective model to simulate the initial biomaterial-cell interaction in vivo.

Clinical relevance

A pressed hemostatic sponge could be applied for guided tissue regeneration and guided bone regeneration. In that sense, within the limitations of this study, the results show that the same biomaterial may have two specific clinical indications.

中文翻译：

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一项基于胶原蛋白的海绵的修饰可以诱导早期炎症反应的上调和由M1巨噬细胞导致的慢性炎症反应的升高：一项体内研究。

背景

本研究评估了两种马来源的胶原蛋白止血海绵（E-CHS）在30天内的厚度变化后的细胞组织反应。假手术动物在生理伤口愈合过程中的炎症反应用作对照组。

材料与方法

首先，通过使用无菌不锈钢圆筒施加恒定压力（6.47±0.85 N）2分钟来压制E-CHS，直到材料被均匀压平为止。因此，独立地研究了原稿（E-CHS），压制（PE-CHS）以及对照组（CG;假手术）。在3、15和30天内，将Wistar大鼠皮下植入后，对3组进行了体内评估。组织化学和免疫组织化学方法提供了生物材料降解率，细胞炎症反应和血管形成模式的观察。人类血液的一种衍生物，称为富血小板纤维蛋白（PRF），被用作离体模型来模拟初始生物材料与细胞之间的相互作用。用PRF将E-CHS和PE-CHS的片段培养3天和6天，用ELISA法测定促炎蛋白的释放。单独培养的PRF用作对照组。

结果

在第3天，与E-CHS和PE-CHS相比，CG诱导了统计上显着更高的单核细胞/巨噬细胞（CD68 +），促炎性巨噬细胞（M1; CCR7 +）和伤口愈合的巨噬细胞（M2; CD206 +）存在。 。在同一时间点，与E-CHS相比，PE-CHS诱导CD68 +细胞存在统计学上显着更高的存在。15天后，E-CHS被细胞和血管侵入，与PE-CHS相比，崩解速度更快。相反，细胞和血管仅位于PE-CHS的外部区域，生物材料不会丢失其结构，因此不会发生崩解。实验组主要通过阳性的促炎性CD68 + / CCR7 +巨噬细胞和少量存在的多核巨细胞（MNGC）诱导了类似的炎症反应。此时，P  <0.05）。30天后，E-CHS和PE-CHS完全降解。所有组均显示相似的炎症反应，转移至较高存在的CD206 +巨噬细胞。在两个实验组的植入床中仍观察到少量的CCR7 + MNGC。在离体模型中，来自PRF的细胞和纤维蛋白穿透了E-CHS。但是，在PE-CHS的情况下，细胞和纤维蛋白停留在表面上，并没有渗透到材料的中心区域。3天后，与CG和E-CHS相比，用PRF培养PE-CHS诱导了促炎性蛋白的静态显着更高的释放。

结论

通过按压改变止血海绵生物材料的原始外观，可以改变初始生物材料与细胞之间的相互作用，延缓早期生物材料的降解速度，并改变血管化方式。受压的生物材料似乎在早期时间点会引起更高的炎症反应。但是，与生理伤口愈合相比，改变生物材料不会改变巨噬细胞的极化模式。使用PRF的离体模型被证明是模拟体内初始生物材料-细胞相互作用的有效模型。

临床相关性

压制的止血海绵可用于引导组织再生和引导骨再生。从这个意义上说，在本研究的范围内，结果表明同一生物材料可能具有两种特定的临床适应症。