Modified enzymatic collagen digestion-mediated isolation of osteocytes

Wenjuan Xu,Xiaolin Tu,Guangming Dai,Yining Zhang,Yifei Lyu

doi:10.32604/biocell.2022.017505

Abstract

This study established a method for isolating large numbers of high-purity osteocytes from high-density bone. Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times, and the digested cells and bone chips (BC) were cultured, digested, and passaged when cells were fully grown. The types of cells obtained were identified by morphology, viable cell counts, alkaline phosphatase staining, and biochemical activity analyses, and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction. Our results showed that among the cells obtained from the third digestion (fractions 7–9) of wild mice tibias and femurs and the remaining BCs, 85%–90% of the cells were osteocytes. Moreover, their morphology was approximately one-tenth to one-fifth the size of osteoblasts, star-shaped or polygonal, with a dendritic structure, negative for alkaline phosphatase staining, and showed a high expression of dmp1 and sclerostin. Ninety percent of the cells in fractions 1–3 were osteoblasts, and were fusiform or polygonal shape. The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction, while the expression of osteocyte-specific dmp1 and sclerostin was not detected. In the second portion (fractions 4–6), a large number were osteoblasts, mixed with a small number of osteocytes, and had high alkaline phosphatase activity and osteocyte mRNA levels, a specific level of the osteocyte marker dmp1, and no sclerostin was detected. Osteocytes in daβcatot mice were also successfully isolated by this method, and we found that Wnt signaling increased the proliferation of these osteocytes. The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone, which provides an avenue for the study of osteocyte function in vitro.

Highlights

Osteocytes are the most abundant cells in bones, accounting for more than 95% of all bone cells
Establishment of isolation and culture methods for osteocytes By changing the concentration, digestion time, and application sequence of digestive enzymes, osteoblasts, and osteocytes of high purity were extracted from dense bone tissue
The results showed that the morphological characteristics of osteocytes in the control group and daβcatot mice were still smaller than that of osteoblasts, about 5 times smaller than osteoblasts, and the morphology was still negative for alkaline phosphatase staining outside the cell body with multiple synapses (Fig. 3C)

Summary

Introduction

Osteocytes are the most abundant cells in bones, accounting for more than 95% of all bone cells. Osteocytes have previously been considered to be static and non-functional cells because they are embedded in the bone matrix (Schaffler et al, 2014). The osteocytes embedded in the matrix connect themselves with abundant synaptic structures to form a special crisscross network structure (Bonewald, 2011), which makes them. The isolation and culture of osteocytes originated from the work of Bonewald and coresearchers. They were the first to obtain an immortalized MLOY4 osteocyte-like cell line (Bonewald, 2011; Kato et al, 1997) for studying the communication between osteocytes and

Methods

Results

Conclusion