Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables the bone to regenerate with capsule maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC are derived from odontogenic epithelium remnants at the embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization. Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues was analyzed by morphology, surface marker, and multi-differentiation assays. Comparison of proliferation ability between MSCs isolated from DC capsules before (Bm-DCSCs) and after (Am-DCSCs) marsupialization was evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F), and 5′-ethynyl-2′-deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice was performed to detect their bone regeneration and bone defect repairability. Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony-forming cells expressed MSC markers (CD44+, CD90+, CD31−, CD34−, CD45−), and they could differentiate into osteoblast-, adipocyte-, and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed a greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. There are MSCs residing in capsules of DC, and the cell viability as well as the osteogenic capacity of them is largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.

中文翻译：

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间充质干细胞的活化促进了齿状囊肿内新骨的形成

齿囊肿（DC）是一种骨破坏性疾病，仍然是临床医生面临的挑战。有袋化使骨骼在保持胶囊的情况下得以再生，使其成为邻近重要解剖结构的DC的首选治疗手段。鉴于DC胶囊在胚胎期源自牙源性上皮残余物，我们调查了DC胶囊中是否存在间充质干细胞（MSC）以及它们在有袋化后在骨再生中所起的作用。有袋化之前和之后获得的样品用于组织学检测和细胞培养。从新鲜组织分离的细胞的干细胞通过形态，表面标记和多分化分析进行了分析。通过Cell Counting Kit-8（CCK-8），成纤维细胞集落形成单位（CFU-F）和5'-乙炔基-2'-脱氧尿苷（EdU）分析。通过碱性磷酸酶（ALP）和茜素红染色（ARS）结合实时聚合酶链反应（RT-PCR）和免疫荧光（IF）染色检测其体外成骨能力。进行裸鼠皮下异位成骨以及颅骨缺损模型以检测其骨再生和骨缺损可修复性。有袋化后在DC的胶囊中检测到骨组织和强大的ALP活性。从（Bm-DCSCs）和（Am-DCSCs）有袋化之前和之后，从DC的纤维囊中分离出两种类型的MSC。这些成纤维细胞样的集落形成细胞表达MSC标记（CD44 +，CD90 +，CD31-，CD34-，CD45-），并且在诱导下可以分化为成骨细胞，脂肪细胞和软骨细胞样细胞。值得注意的是，Am-DCSC在细胞增殖和自我更新方面表现更好。而且，与Bm-DCSCs相比，Am-DCSCs在体外和体内均显示出更大的成骨能力。DC胶囊中存在MSC，有袋化后其细胞活力以及成骨能力大大增强。我们的发现表明，MSCs在有袋化后可能在DC的愈合过程中起关键作用，从而通过促进胶囊内MSCs的成骨分化为DC的治疗提供了新的见识。CD45-），在诱导下它们可以分化为成骨细胞，脂肪细胞和软骨细胞样细胞。值得注意的是，Am-DCSC在细胞增殖和自我更新方面表现更好。而且，与Bm-DCSCs相比，Am-DCSCs在体外和体内均显示出更大的成骨能力。DC胶囊中存在MSC，有袋化后其细胞活力以及成骨能力大大增强。我们的发现表明，MSCs在有袋化后可能在DC的愈合过程中起关键作用，从而通过促进胶囊内MSCs的成骨分化为DC的治疗提供了新的见识。CD45-），在诱导下它们可以分化为成骨细胞，脂肪细胞和软骨细胞样细胞。值得注意的是，Am-DCSC在细胞增殖和自我更新方面表现更好。而且，与Bm-DCSCs相比，Am-DCSCs在体外和体内均显示出更大的成骨能力。DC胶囊中存在MSC，有袋化后其细胞活力以及成骨能力大大增强。我们的发现表明，MSCs在有袋化后可能在DC的愈合过程中起关键作用，从而通过促进胶囊内MSCs的成骨分化为DC的治疗提供了新的见识。Am-DCSC在细胞增殖和自我更新方面表现更好。而且，与Bm-DCSCs相比，Am-DCSCs在体外和体内均显示出更大的成骨能力。DC胶囊中存在MSC，有袋化后其细胞活力以及成骨能力大大增强。我们的发现表明，MSCs在有袋化后可能在DC的愈合过程中起关键作用，从而通过促进胶囊内MSCs的成骨分化为DC的治疗提供了新的见识。Am-DCSC在细胞增殖和自我更新方面表现更好。而且，与Bm-DCSCs相比，Am-DCSCs在体外和体内均显示出更大的成骨能力。DC胶囊中存在MSC，有袋化后其细胞活力以及成骨能力大大增强。我们的发现表明，MSCs在有袋化后可能在DC的愈合过程中起关键作用，从而通过促进胶囊内MSCs的成骨分化为DC的治疗提供了新的见识。