The Osteocyte Stimulated by Wnt Agonist SKL2001 Is a Safe Osteogenic Niche Improving Bioactivities in a Polycaprolactone and Cell Integrated 3D Module.

Yangxi Liu,Jie Chen,Jun Li,Xiaojie Ruan,Pengtao Wang,Bo Wang,Xiaolin Tu,Xiaofang Wang

doi:10.3390/cells11050831

Abstract

Finding and constructing an osteogenic microenvironment similar to natural bone tissue has always been a frontier topic in orthopedics. We found that osteocytes are targeting cells controlling bone anabolism produced by PTH (JBMR 2017, PMID: 27704638), and osteocytes with activated Wnt signaling orchestrate bone formation and resorption (PNAS 2015, PMID: 25605937). However, methods for taking advantage of the leading role of osteocytes in bone regeneration remain unexplored. Herein, we found that the osteocytes with SKL2001-activated Wnt signaling could be an osteogenic microenvironment (SOOME) which upregulates the expression of bone transcription factor Runx2 and Bglap and promotes the differentiation of bone marrow stromal cell ST2 into osteoblasts. Interestingly, 60 μM SKL2001 treatment of osteocytic MLO-Y4 for 24 h maintained Wnt signaling activation for three days after removal, which was sufficient to induce osteoblast differentiation. Triptonide, a Wnt inhibitor, could eliminate this differentiation. Moreover, on day 5, the Wnt signaling naturally decreased to the level of the control group, indicating that this method of Wnt-signaling induction is safe to use. We quickly verified in vivo function of SOOME to a good proximation in 3D bioprinted modules composed of reciprocally printed polycaprolactone bundles (for support) and cell bundles (for bioactivity). In the cell bundles, SOOME stably supported the growth and development of ST2 cells, the 7-day survival rate was as high as 91.6%, and proliferation ability increased linearly. Similarly, SOOME greatly promoted ST2 differentiation and mineralization for 28 days. In addition, SOOME upregulated the expression of angiopoietin 1, promoted endothelial cell migration and angiogenesis, and increased node number and total length of tubes and branches. Finally, we found that the function of SOOME could be realized through the paracrine pathway. This study reveals that osteocytes with Wnt signaling activated by SKL2001 are a safe osteogenic microenvironment. Both SOOME itself and its cell-free culture supernatant can improve bioactivity for osteoblast differentiation, with composite scaffolds especially bearing application value.

Highlights

There are several sources of bone defects, including infection, tumor resection, complex trauma, revisional surgery, etc. [1]
This study reveals that osteocytes with Wnt signaling activated by SKL2001 are a safe osteogenic microenvironment
As we further examined the osteogenic factor of signaling could be an osteogenic microenvironment (SOOME), we found that the expression of the PGE2 receptor EP4 was upregulated both in SOOME and the co-culture of SOOME and ST2 cells (Figure S3D,E)

Summary

Introduction

There are several sources of bone defects, including infection, tumor resection, complex trauma, revisional surgery, etc. [1]. The global orthopedic implant market grows at 4.7% yearly and will reach USD 60 billion by 2025 [3]; the current products still face two challenges: anatomic feature mismatching and lack of bioactivity, resulting in instability, looseness and need for renovation, which is a headache for surgeons. The 3D printing of hard materials has emerged as a attractive candidate for reconstruction of personalized bone implants due to its capacity to use computed tomography (CT)-derived images or computer-aided design (CAD) models [4–6], but more attention needs to be given to improving material bioactivity in order to regenerate the patient’s own bone. Doctors make great efforts to use autogenous and allergenic bone for transplantation. The use of autogenous bone remains the so-called “gold standard”

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