A simple method for decellularizing a cell-derived matrix for bone cell cultivation and differentiation

Abstract

The extracellular matrix regulates cell survival, proliferation, and differentiation. In vitro two-dimensional cell experiments are typically performed on a plastic plate or a substrate of a single extracellular matrix constituent such as collagen or calcium phosphate. As these approaches do not include extracellular matrix proteins or growth factors, they fail to mimic a complex cell microenvironment. The cell-derived matrix is an alternative platform for better representing the in vivo microenvironment in vitro. Standard decellularization of a cell-derived matrix is achieved by combining chemical and physical methods. In this study, we compared the decellularization efficacy of several methods: ammonium hydroxide, sodium dodecyl sulfate (SDS), or Triton X-100 with cold or heat treatment on a matrix of Saos-2 cells. We found that the protocols containing SDS were cytotoxic during recellularization. Heat treatment at 47 °C was not cytotoxic, removed cellular constituents, inactivated alkaline phosphatase activity, and maintained the levels of calcium deposition. Subsequently, we investigated the differentiation efficiency of a direct bone coculture system in the established decellularized Saos-2 matrix, an inorganic matrix of calcium phosphate, and a plastic plate as a control. We found that the decellularized Saos-2 cell matrix obtained by heat treatment at 47 °C enhanced osteoclast differentiation and matrix mineralization better than the inorganic matrix and the control. This simple and low-cost method allows us to create a Saos-2 decellularized matrix that can be used as an in vivo-like support for the growth and differentiation of bone cells.

Maqui Berry and Ginseng Extracts Reduce Cigarette Smoke-Induced Cell Injury in a 3D Bone Co-Culture Model

“…As well as cell viability, osteoclast function (CAII and TRAP activity) was measured in the co-culture systems. CAII and TRAP activities are early and late indicators of osteoclast differentiation, respectively [ 46 ]. Our CAII and TRAP activity results ( Figure 1 c,d) showed that osteoclast function was markedly increased in co-cultures exposed to 5% CSE, contributing to the development of an osteoporotic bone environment in vitro.…”

“…Experimental values were corrected to background control (scaffold without cells). After that, data were normalized to viable cell numbers by resazurin conversion as previously described [ 46 ].…”

Preparation and Use of Decellularized Extracellular Matrix for Tissue Engineering

“…In a study by Kawasaki T et al (2015) comparing SDS with another detergent, SDS was found to damage the ECM microstructure, destroy the ECM laminar array, remove most of the sulfated glycosaminoglycans (sGAG), and affect the growth factors and cytokines [ 149 ]. Weng et al (2021) reported that cell-cultured bone ECM decellularized using SDS was cytotoxic to cells during recellularization [ 103 ]. Furthermore, Uhl FE et al (2020) demonstrated that the decellularization of lungs using SDS resulted in high losses of GAGs, and the remaining GAGs were dysfunctional and unable to bind key matrix-associated growth factors [ 150 ].…”

“…The essential steps are providing a substrate for cells to grow, followed by providing the necessary nutrients and physiological conditions, and the the cells will produce the ECM that can be decellularized and used. This can achieve simple sheets of dECM that can be used for other experiments or procedures [ 101 , 103 , 126 , 209 , 247 , 248 , 249 , 250 , 251 , 252 , 253 , 254 , 255 , 256 , 257 ], the modification of existing dECM to include tissue-specific ECM [ 258 ], or the modification of the surface of synthetic substrates [ 84 , 204 , 205 , 259 , 260 , 261 ]. Such a technique can be used in more complex situations, such as the surgical implantation of a scaffold for autologous cells to produce the ECM, followed by explanting then decellularizing the resulting scaffold, and finally implanting it in the orthotopic location [ 262 ].…”

“…Stem cells have the unique ability to differentiate into different tissue types, but they generally require specialized treatments to induce this differentiation into a particular cell type. The specific make-up of the ECM is unique to each tissue type, and the ECM has been shown to have the potential to induce and enhance the differentiation of stem cells towards the phenotype associated with the tissue source of the ECM [ 14 , 56 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 ].…”