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

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.

A novel decellularized matrix of Wnt signaling-activated osteocytes accelerates the repair of critical-sized parietal bone defects with osteoclastogenesis, angiogenesis, and neurogenesis

“…Cell viability was assessed by LIVE/DEAD™ cell viability assay as reported [ 39 ] in the cell culture on DM modified modules for 24 h. In short, the modules were stained by LIVE/DEAD staining mixture containing calcein AM (0.5 μL/mL) and ethidium-1 (EthD-1, 2 μL/mL) at room temperature for 1 h. Then, the modules were observed and taken pictures under fluorescence microscope (Leica, Wetzlar, Germany). Live and dead cells were exhibited green and red fluorescence under the microscope and calculated with ImageJ software (NIH, Bethesda, USA).…”

“…Cell proliferation activity was measured by cell counting kit-8 assay on day 1, 4, and 7 after BMSCs seeded on the modules as reported [ 39 ]. The modules were washed with PBS and placed in a 96-well plate.…”

“…AP staining was performed per instruction of the BCIP/NBT AP color development kit as reported with modification [ 39 ]. Briefly, after culture, the modules were washed with PBS, and fixed in 3.7% formaldehyde for 5 min at room temperature, followed by staining with the color solution of the kit for 30 min.…”

“…AP biochemical activity assay was carried out as previously described [ 39 ]. Briefly, 200 μL Tris-HCL (50 mmol/L, pH = 7.4) was added to each module.…”

“…In addition, we proved that osteocytes are the target cells of osteogenic parathyroid hormone(PTH)because mice lacking PTH receptors in osteocytes lose their osteogenic response to PTH intermittent therapy [ 38 ]. Furthermore, we found that Wnt agonist activated osteocytic cell line MLO-Y4 promotes osteogenic differentiation and angiogenesis [ 39 ], and primary osteocytes with dominant active Wnt signaling can significantly promote the differentiation of BMSCs torwards osteoblast and induces natural osteogenesis in a 3D-printed PCL module with osteoclastogenesis and neurovascularization (submitted for publication). Therefore, we hypothesized that osteocyes, which account for more than 90% of the total number of bone cells, produces osteogenic microenvironment for bone development after Wnt signaling activation, and as the ideal cell type to biofabricate osteogenic DM to repair defects as the development of nature bone.…”