MC3T3-E1

Simple Preparation and Bone Regeneration Effects of Poly(vinyl alcohol)–Resveratrol Self-Cross-Linked Hydrogels

ACS Omega | DOI | Paper 2024 | By Pengyin Li, Shaoqing Chen, Yanyan Meng, Cheli Wang, Xinye Ni

Background

This study developed poly(vinyl alcohol)–resveratrol (PVA–Res) self-cross-linked hydrogels through a simple, green freeze–thawing process. Resveratrol served as a biological cross-linker via strong hydrogen bonds, enhancing mechanical and viscoelastic properties. The 1.2% PVA–Res hydrogel exhibited the highest viscoelastic modulus, while the 0.4% hydrogel promoted MC3T3-E1 proliferation, alkaline phosphatase activity, mineral deposition, and osteogenic gene expression. It also induced macrophage M2 polarization and reduced M1 markers, indicating immunomodulatory and bone-regenerative potential for bone repair applications.

Key Findings

PVA–Res hydrogels were prepared by mixing PVA with 0.4, 1.2, or 2.0 wt% resveratrol, heating, cooling, and freeze–thawing. FTIR and XRD confirmed hydrogen-bond cross-linking and reduced crystallinity. Rheology showed the 1.2% hydrogel had a viscoelastic modulus of 2299 Pa, 8.5-fold higher than pure PVA, and improved compressive properties. However, excessive resveratrol (2.0%) reduced stability. The 0.4% hydrogel showed sustained resveratrol release over 240 h and best cell compatibility. It enhanced MC3T3-E1 proliferation, ALP activity, Alizarin Red mineralization, and upregulated osteogenic genes BMP-9, OCN, and ALP. In LPS-stimulated Raw 264.7 macrophages, the 0.4% hydrogel downregulated M1 markers iNOS and TNF-α and upregulated M2 markers Arg-1 and CD206, confirmed by flow cytometry. Thus, PVA–Res hydrogels, especially 0.4%, show promise for bone repair by promoting osteogenesis and modulating macrophage polarization.

Contribution

Creative Bioarray supplied the mouse preosteoblastic MC3T3-E1 and mouse monocyte/macrophage leukemia Raw 264.7 cell lines used in this study. These cells were essential for evaluating osteogenic differentiation, mineralization, and macrophage polarization induced by the PVA–Res hydrogels, supporting the key in vitro bone regeneration and immunomodulation findings.

MC3T3-E1 Cell Death Associated with the Accumulation of TAGE

Diabetes is a known risk factor for osteoporotic fractures, and it has been suggested that the hyperglycemia-induced formation of advanced glycation end-products (AGEs) plays a causative role. The AGEs, which are cytotoxic, can also inhibit osteoblast function. Sakasai-Sakai et al. investigated the cytotoxic effect of intracellular glyceraldehyde (GA) and GA-derived toxic AGEs (TAGE) in the MC3T3-E1 osteoblast cell line, and the mechanisms by which GA and TAGE affect osteoblasts.

They first tested the effect of GA on mouse osteoblastic MC3T3-E1 cells using a cell viability assay, as osteoblast death is a key cause of osteoporosis. Cell viability decreased in a dose-dependent manner with increasing GA concentration from 1 mM GA (Fig. 1A). The GA reacts with proteins and forms TAGE that can damage cell functions. Next, they checked the accumulation of TAGE in GA-treated MC3T3-E1 cells by slot blot analysis with an anti-TAGE antibody. TAGE level significantly increased with 2 mM GA (Fig. 1B). Western blot analysis revealed that 2 mM GA increased several TAGE-modified protein bands (Fig. 1C). They used AG, an inhibitor of AGE formation that can react with aldehydes. AG pretreatment decreased GA-induced TAGE accumulation (Fig. 1B) and reduced TAGE band intensities (Fig. 1C). In addition, AG pretreatment inhibited GA-induced cell death (Fig. 1D), which suggests that TAGE accumulation is correlated with MC3T3-E1 cell death.

Fig. 2. Cell death and TAGE accumulation were induced in MC3T3-E1 cells treated with GA (Sakasai-Sakai A, Takata T, et al., 2022).

Apoptosis-Promoting Properties of miR-3074-5p in MC3T3-E1 Cells under Iron Overload Conditions

Iron overload contributes to osteoporosis by triggering apoptosis in osteoblasts. However, the regulatory mechanism of miRNA on osteoblast apoptosis under iron overload is unclear. Using next-generation sequencing analysis to examine the miRNA expression profiles of iron-overloaded MC3T3-E1 cells, Feng's team investigated the mechanism by which miR-3074-5p regulates apoptosis in MC3T3-E1 cells under iron overload.

Differentially expressed miRNAs in MC3T3-E1 cells treated with 1.8 mM FAC for 24 h were identified by miRNA next-generation sequencing (Fig. 2a). The expression of miR-3074-5p was significantly increased in iron-overloaded MC3T3-E1 cells. qRT-PCR results showed that the expression of miR-3074-5p was significantly higher in FAC-treated cells (Fig. 2b). Transfection of a miR-3074-5p inhibitor rescued FAC-induced upregulation (Fig. 2c). These results indicated the potential effect of miR-3074-5p under iron overload. MC3T3-E1 cells were transfected with a miR-3074-5p mimic or inhibitor to perform gain- and loss-of-function experiments. CCK-8 assays and Annexin V-FITC/PI staining confirmed that the miR-3074-5p mimic significantly increased the cell inhibition and apoptosis rates of MC3T3-E1 cells under iron overload, while the inhibitor significantly decreased the cell inhibition and apoptosis rates (Fig. 3a, 3b). Thus, miR-3074-5p promotes the apoptosis of iron-overloaded MC3T3-E1 cells.

Fig. 3. miR-3074-5p expression was increased in MC3T3-E1 cells under iron overload conditions (Feng Y, He P Y, et al., 2021).

Fig. 4. miR-3074-5p mediated the apoptosis of MC3T3-E1 cells under iron overload conditions (Feng Y, He P Y, et al., 2021).