State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, 3rd Sec., Ren Min Nan Road, Chengdu 610041, Sichuan Province, PR China; National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, No. 14, 3rd Sec., Ren Min Nan Road, Chengdu 610041, Sichuan Province, PR China; Department of Oral and Maxillofacial Surgery, West China School of Stomatology, Sichuan University, No. 14, 3rd Sec., Ren Min Nan Road, Chengdu 610041, Sichuan Province, PR China.
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, 3rd Sec., Ren Min Nan Road, Chengdu 610041, Sichuan Province, PR China; National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, No. 14, 3rd Sec., Ren Min Nan Road, Chengdu 610041, Sichuan Province, PR China.
Biomaterials. 2014 Jun;35(18):4929-39. doi: 10.1016/j.biomaterials.2014.03.016. Epub 2014 Mar 27.
Cryopreservation has been identified as an efficient approach to preserve tissue engineered products for a long term. Our prior studies have suggested that the treated dentin matrix (TDM) could be an ideal bioactive scaffold for dental tissue regeneration. In this study, we hypothesize that the cryopreservation could effectively maintain the survival and viability of dentinogenesis-related proteins of TDM and the cryopreserved dentin matrix (CDM) would provide the suitable biological scaffold and inductive microenvironment for the regeneration of dentin-pulp like tissue. CDM-3 and CDM-6 were prepared by cryopreserving TDM in liquid nitrogen (-196 °C) with cryoprotectant for 3 months and 6 months, respectively. Various biological characteristics of CDM, including mechanical properties, cell proliferation, and odontogenesis ability, were investigated. To further evaluate the inductive capacity of CDM, human dental follicle cells were encapsulated within CDM, and implanted the scaffold into a mouse model for 8 weeks, and the grafts were harvested and assessed histologically. The CDM showed superior mechanical properties than TDM. Compared to TDM, CDM can release more dentinogenesis-related proteins due to the larger pore diameter. Cell proliferation with the addition of CDM extract liquid was similar to that of TDM in the first five days. Human dental follicle cells, under the effect of CDM extract liquid, highly expressed bone sialoprotein, collagen-1, alkaline phosphatase, indicating that CDM, regarded as the inductive microenvironment, plays an important role in odontogenesis. Most importantly, in vivo, CDM could induce dental follicle cells to regenerate new dentin-pulp like tissues, such as dentinal tubules, predentin, collagen fibers, nerves, and blood vessels which were positive for dentin sialophosphoprotein, dental matrix protein-1, Tubulin, and collagen-1. In conclusion, CDM is an ideal biological scaffold material for human dentin-pulp like tissue regeneration. These findings indicated that TDM could be preserved as the tissue engineering scaffold that is readily available for patient treatments. Furthermore, the success of cryopreservation of TDM may also provide an insight into preserving other bioactive scaffold materials of tissue engineering.
冷冻保存已被确定为长期保存组织工程产品的有效方法。我们之前的研究表明，处理牙本质基质（TDM）可以成为牙齿组织再生的理想生物活性支架。在这项研究中，我们假设冷冻保存可以有效地维持 TDM 中牙发生相关蛋白的存活和活力，并且冷冻保存的牙本质基质（CDM）将为牙本质 - 牙髓样组织的再生提供合适的生物支架和诱导微环境。通过在液氮（-196°C）中用冷冻保护剂冷冻保存 TDM 3 个月和 6 个月分别制备了 CDM-3 和 CDM-6。研究了 CDM 的各种生物学特性，包括机械性能、细胞增殖和牙发生能力。为了进一步评估 CDM 的诱导能力，将人牙滤泡细胞包封在 CDM 中，并将支架植入小鼠模型 8 周，收获移植物并进行组织学评估。与 TDM 相比，CDM 表现出更好的机械性能，因为其具有更大的孔径。与 TDM 相比，CDM 可以释放更多的牙发生相关蛋白。在最初的五天内，添加 CDM 提取物的细胞增殖与 TDM 相似。在 CDM 提取物的作用下，人牙滤泡细胞高度表达骨唾液蛋白、胶原蛋白-1、碱性磷酸酶，表明 CDM 作为诱导微环境在牙发生中起重要作用。最重要的是，在体内，CDM 可以诱导牙滤泡细胞再生新的牙本质 - 牙髓样组织，例如牙本质小管、前期牙本质、胶原纤维、神经和血管，这些组织对牙本质涎磷蛋白、牙基质蛋白-1、微管蛋白和胶原蛋白-1呈阳性。总之，CDM 是人类牙本质 - 牙髓样组织再生的理想生物支架材料。这些发现表明 TDM 可以作为组织工程支架保存，以备患者治疗使用。此外，TDM 的冷冻保存成功也为保存其他组织工程生物活性支架材料提供了思路。
