Ieva Gendviliene^*; Milda Alksne; Povilas Barasa; Milda Vitosyte; Egle Marija Jonaityte; Egidijus Simoliunas; Rokas Borusevicius; Virginija Bukelskiene; Vygandas Rutkunas; Algirdas Kaupinis

Vilnius University, Vilnius, Lithuania

Background: To date, almost all bone augmentations in clinical practice are performed using autogenous, allogeneic, or xenogeneic bone grafts because no osteoconductive 3D scaffolds meet the ideal bone substitute requirements. Scaffold decoration with extracellular matrix (ECM) is one of the most progressive osteoinductive bone tissue engineering treatment strategy. However, ECM structure and functions depend on the type of stem cells used for its production.

Aim/Hypothesis: This study aimed to evaluate dental pulp stem cells (DPSC) secreted ECM impact on new bone formation in vitro and in vivo.

Material and Methods: Polylactic acid (PLA) scaffolds were created using the FDM 3D printer in 3D structured threads rotated at an angle of 60°. First, DPSC were isolated from the incisors of adult Wistar rats. Then DPSC were seeded and grown on PLA scaffolds for 7 days. Afterwards, decellularisation was done, and the remaining ECMs detail proteomic analysis was performed. For the in vitro studies the DPSC were seeded on decellularized scaffolds again. Then DPSC behaviour, spontaneous osteogenic differentiation (alkaline phosphatase (ALP) assay, determination of collagen amount, ECM mineralisation assay, quantitative PCR) and cytokine evaluation in DPSC secretome were carried out. Finally, bone regeneration was evaluated using a critical-size Wistar rat's calvarial defect model with micro-computer tomography (µCT) and histology 8 weeks after surgery. Approval of the Ethics Committee and permission for the experimentation were received from Lithuania's State Food and Veterinary Service, No G2-40, 2016-03-18.

Results: Detailed composition of proteins retained on PLA scaffolds after DPSC decellularisation showed that left ECM contributes to biological processes associated with bone/cartilage formation, angiogenesis, ECM formation, immune response, protein processing, and membrane transport. In vitro results showed significantly higher numbers of focal adhesions within the cells (p < 0.001), better cell proliferation (p < 0.05), ALP activity (p < 0.05), ECM mineralisation in the PLA ECM group compared to pure PLA samples. In addition, DPSC showed significantly better proliferative activity on PLA ECM scaffolds. In vivo results obtained by quantitative histology and µCT showed a statistically significant difference between PLA and PLA ECM groups (p < 0.05) when males and females were counted together. Some qualitative histology sections of PLA ECM scaffolds showed the new bone islands formed separately from the newly formed bone at the defect edges.

Conclusion and Clinical implications: The DPSC-produced ECM enhanced cell attachment, migration, proliferation, new bone formation and angiogenesis both in vitro and in vivo. Therefore, DPSC-secreted ECM could be a promising osteoinductive technique to decorate various osteoconductive materials for bone tissue engineering. However, further research is needed to analyse the effect of ECM on composite biomaterials.

Disclosure of Interest: None Declared.

Keywords: animal study, biomaterial, bone regeneration

Ieva Gendviliene ^* ; 米尔达·阿尔克斯尼；波维拉斯巴拉萨；米尔达维托赛特；Egle Marija Jonaityte; 埃吉迪尤斯·西莫利乌纳斯；罗卡斯·博鲁塞维修斯；维尔吉尼娅·布克尔斯基恩；维干达斯·鲁特库纳斯；阿尔吉尔达斯·考皮尼斯

维尔纽斯大学，立陶宛维尔纽斯

背景：迄今为止，临床实践中几乎所有的骨增强都是使用自体、同种异体或异种骨移植物进行的，因为没有骨传导性 3D 支架满足理想的骨替代要求。带有细胞外基质 (ECM) 的支架装饰是最先进的骨诱导骨组织工程治疗策略之一。然而，ECM 的结构和功能取决于用于其生产的干细胞类型。

目的/假设：本研究旨在评估牙髓干细胞 (DPSC) 分泌的 ECM在体外和体内对新骨形成的影响。

材料和方法：聚乳酸 (PLA) 支架是使用 FDM 3D 打印机在以 60° 角旋转的 3D 结构螺纹中创建的。首先，从成年 Wistar 大鼠的门牙中分离出 DPSC。然后在 PLA 支架上接种 DPSC 并生长 7 天。之后，进行去细胞化，并对剩余的 ECM 进行详细的蛋白质组学分析。对于体外研究将 DPSC 再次接种在脱细胞支架上。然后进行DPSC行为，自发成骨分化（碱性磷酸酶（ALP）测定，胶原蛋白量测定，ECM矿化测定，定量PCR）和DPSC分泌组中的细胞因子评估。最后，在手术后 8 周，使用具有微计算机断层扫描 (μCT) 和组织学的临界尺寸 Wistar 大鼠颅骨缺损模型评估骨再生。立陶宛国家食品和兽医服务处 (No G2-40, 2016-03-18) 获得了伦理委员会的批准和实验许可。

结果： DPSC 脱细胞后保留在 PLA 支架上的蛋白质的详细组成表明，左 ECM 有助于与骨/软骨形成、血管生成、ECM 形成、免疫反应、蛋白质加工和膜转运相关的生物过程。体外结果显示，与纯 PLA 样品相比，PLA ECM 组中细胞内粘着斑的数量显着更多 ( p <0.001)、更好的细胞增殖 ( p <0.05)、ALP 活性 ( p <0.05)、ECM 矿化。此外，DPSC 在 PLA ECM 支架上显示出明显更好的增殖活性。体内通过定量组织学和 μCT 获得的结果显示，当男性和女性一起计数时，PLA 和 PLA ECM 组之间存在统计学显着差异 ( p <0.05)。PLA ECM 支架的一些定性组织学切片显示新骨岛与缺损边缘处新形成的骨分开形成。

结论和临床意义： DPSC 产生的 ECM 增强了体外和体内的细胞附着、迁移、增殖、新骨形成和血管生成。因此，DPSC 分泌的 ECM 可能是一种很有前途的骨诱导技术，用于装饰用于骨组织工程的各种骨传导材料。然而，需要进一步的研究来分析 ECM 对复合生物材料的影响。

利益披露：无声明。

关键词: 动物研究，生物材料，骨再生