Department of Orthopedic Surgery, Key Laboratory of Musculoskeletal Trauma &war Injuries PLA, Beijing Key Laboratory of Regenerative Medicine in Orthopedics, Chinese PLA General Hospital, Beijing 100853, People's Republic of China.
Department of orthopedics, The First Hospital of Shanxi Medical University YanHu District Branch, Yuncheng, Shanxi 044000, People's Republic of China.
Int J Nanomedicine. 2020 Sep 29;15:7155-7171. doi: 10.2147/IJN.S238399. eCollection 2020.
Tissue engineering approaches seem to be an attractive therapy for tendon rupture. Novel injectable porous gelatin microcryogels (GMs) can promote cell attachment and proliferation, thus facilitating the repair potential for target tissue regeneration. The research objectives of this study were to assess the efficacy of tissue-like microunits constructed by multiple GMs laden with adipose-derived mesenchymal stem cells (ASCs) in accelerated tendon regeneration in a rat model.
Through a series of experiments, such as isolation and identification of ASCs, scanning electron microscopy, mercury intrusion porosimetry (MIP), laser scanning confocal microscopy and the CCK-8 test, the biocompatibility of GMs was evaluated. In an in vivo study, 64 rat right transected Achilles tendons were randomly divided into four groups: the ASCs+GMs group (microunits aggregated by multiple ASC-laden GMs injected into the gap), the ASCs group ( injected into the gap), the GMs group (GMs injected into the gap) and the blank defect group (non-treated). At 2 and 4 weeks postoperatively, the healing tissue was harvested to evaluate the gross observation and scoring, biomechanical testing, histological staining and quantitative scoring. Gait analysis was performed over time. The 64 rats were randomly assigned into 4 groups: (1) micro-unit group (ASCs+GMs) containing ASC (10)-loaded 120 GMs in 60 μL DMEM; (2) cell control group (ASCs) containing 10 ASCs in 60 μL DMEM; (3) GM control group (GMs) containing 120 blank GMs in 60 μL DMEM; (4) blank defect group (Defect) containing 60 μL DMEM, which were injected into the defect sites. All animals were sacrificed at 2 and 4 weeks postsurgery (Table 1).
In an in vitro study, GMs (from 126 μm to 348 μm) showed good porosities and a three-dimensional void structure with a good interpore connectivity of the micropores and exhibited excellent biocompatibility with ASCs. As the culture time elapsed, the extracellular matrix (ECM) secreted by ASCs encased the GMs, bound multiple microspheres together, and then formed active tendon tissue-engineering microunits. In animal experiments, the ASCs+GMs group and the ASCs group showed stimulatory effects on Achilles tendon healing. Moreover, the ASCs+GMs group was the best at improving the macroscopic appearance, histological morphology, Achilles functional index (AFI), and biomechanical properties of repair tissue without causing adverse immune reactions.
Porous GMs were conducive to promoting cell proliferation and facilitating ECM secretion. The ASCs-GMs matrices showed an obvious therapeutic efficiency for Achilles tendon rupture in rats.
组织工程方法似乎是一种有吸引力的治疗肌腱断裂的方法。新型可注射多孔明胶微球（GMs）可促进细胞附着和增殖，从而促进目标组织再生的修复潜力。本研究的目的是评估由负载脂肪间充质干细胞（ASCs）的多个 GMs 构建的类组织微单位在加速大鼠模型中的肌腱再生中的疗效。
通过一系列实验，如 ASCs 的分离和鉴定、扫描电子显微镜、压汞法（MIP）、激光共聚焦显微镜和 CCK-8 试验，评估 GMs 的生物相容性。在体内研究中，将 64 只大鼠右侧横断的跟腱随机分为四组：ASCs+GMs 组（多个负载 ASC 的 GMs 聚集注入间隙）、ASCs 组（注入间隙）、GMs 组（注入间隙）和空白缺损组（未处理）。术后 2 周和 4 周时，采集愈合组织进行大体观察和评分、生物力学测试、组织学染色和定量评分。随着时间的推移进行步态分析。将 64 只大鼠随机分为 4 组：（1）微单位组（ASCs+GMs），含 120 个 ASC（10）负载的 GMs 在 60 μL DMEM 中；（2）细胞对照组（ASCs），含 10 个 ASCs 在 60 μL DMEM 中；（3）GMs 对照组（GMs），含 120 个空白 GMs 在 60 μL DMEM 中；（4）空白缺陷组（缺陷），含 60 μL DMEM，注入缺陷部位。所有动物均在术后 2 周和 4 周时处死（表 1）。
在体外研究中，GMs（126 μm 至 348 μm）表现出良好的孔隙率和三维空隙结构，微孔之间具有良好的孔间连通性，与 ASCs 具有极好的生物相容性。随着培养时间的延长，ASCs 分泌的细胞外基质（ECM）包裹 GMs，将多个微球结合在一起，然后形成活跃的肌腱组织工程微单位。在动物实验中，ASCs+GMs 组和 ASCs 组对跟腱愈合有刺激作用。此外，ASCs+GMs 组在改善宏观外观、组织形态学、跟腱功能指数（AFI）和修复组织的生物力学性能方面效果最佳，且无不良反应免疫反应。
多孔 GMs 有利于促进细胞增殖并促进 ECM 分泌。ASCs-GMs 基质对大鼠跟腱断裂有明显的治疗效果。
