94 / 2018-08-04 01:32:49

Evaluation of decellularization process to obtain matrices for tissue regeneration approaches

Tissue engineering,decellularized matrices,Matrices,adipose derived stem cells

全体主题 > vi. BAMOS (Biomaterials and Additive Manufacturing: Osteochondral Scaffold innovation)

Introduction
Different strategies focus on Tissue Engineering approaches have been pursued to tackle osteoarthritis. In fact, the incredible progress achieved, resulted in the capability to modulate cells’ behaviour by changing biomaterial features, allowing its application into different final applications [1]. Decellularized extracellular matrices (dECM) emerged as a promising biomaterial since it can naturally emulate the native microenvironment. In fact, dECM preserves specific properties, such as the composition, the biomechanical properties and the structure, which can direct cells and promote tissue regeneration. Nevertheless, the method to obtain the dECM is critical since it should remove the cellular content while maintain the minimal disruption of the matrix [2]. With this in mind, we aimed to develop a method to obtain dECM with different characteristics that could be used in a wide range of applications. We hypothesized that human adipose derived stem cells (hASCs) could be used to produce such matrices, which upon specific induction would develop different characteristics of native tissues, such as mineralization deposits typical of bone tissue. Such matrices could be used to provide an environment that emulates more closely the native tissue, enhancing tissue regeneration.
Materials and methods
hASCs were cultured under basal conditions (BM, αMEM supplemented with 10% FBS and 1% antibiotic/antimycotic) and osteogenic conditions (OM, BM supplemented with 100 nM dexamethasone, 10 mM β-glycerophosphate and 0.05 mM ascorbic acid) during 6 weeks. Upon this time of culture, BM and OM matrices were submitted to decellularization process. For that, they were incubated in 2% Triton X-100 for 24h, followed by 0.1% sodium dodecyl sulfate (SDS) for 48h. Then, microtissues were sonicated and treated with 0.0025% DNase I for 23h. Finally, the resulted matrices were lyophilized and used to evaluate the decellularization process. Matrices that were not submitted to the decellularization process were used as control (BM CTRL and OM CTRL). The efficiency of the decellularization process was validated using Quant-IT PicoGreen dsDNA Assay Kit, for DNA quantification, and H&E staining, for nucleus observation. Additionally, the presence and preservation of collagen within the matrices was assessed by Masson's trichrome staining. Finally, Alizarin Red staining was used to observe the presence of mineralization deposits.
Results
The DNA quantification and H&E staining showed the successful reduction of nuclear content present in each matrix (BM and OM) after decellularization process as compared with controls (CTRL OM and CTRL BM). Moreover, Masson’s trichrome staining showed no visible effect of decellularization process on collagen content on treated matrices. Finally, Alizarin Red staining conformed the deposition of mineralization along the OM matrices.
Conclusions
This study showed the effective production of decellularized hASCs-derived matrices with different features, such as mineral deposits, typical of bone tissue matrix. Overall, this promising results showed a new class of raw material with improved features, which could be used to address a variety of tissue engineering applications, including patient-specific scaffolds and bioinks.
References
1. Khademhosseini, A. and Langer, A. (2016) A decade of progress in tissue engineering. Nature Protocols. 11: p. 1775.
2. Crapo, P.M., Gilbert, T.W. and Badylak, S.F. (2011) An overview of tissue and whole organ decellularization processes. Biomaterials. 32: p. 3233-43.