Free glutaraldehyde gelatin microsphere loaded mesenchymal stem cells alleviate osteoarthritis by promoting Ext1 expression

Abstract

Rationale: Osteoarthritis (OA) is a chronic joint disorder with limited treatment efficacy, necessitating innovative therapeutic strategies. This study explores one-pot-synthesized gelatin microspheres devoid of glutaraldehyde as a novel biomaterial for OA management. Focusing on the Ext1 gene, critical for cartilage development and downregulated in OA, we investigated its restoration and immune regulation using gelatin microspheres cultured with mesenchymal stem cells (MSCs). Methods: OA patients undergoing knee replacement surgery have their lateral compartment (remote zone) and medial compartment (lesion zone) cartilage collected for transcriptomic testing. The differential gene Ext1 is identified, and the expression of immune regulatory genes is examined. MSCs were cultured with gelatin microspheres to evaluate their compatibility and ability to promote cell attachment. The effects of the gelatin microspheres on Ext1 gene overexpression, immune regulation, and OA symptom mitigation were investigated through in vitro and in vivo experiments. Results: OA patients exhibit decreased expression of the Ext1 gene in the medial compartment (lesion zone) cartilage area, accompanied by abnormal expression of immune regulatory genes. The study demonstrated that the gelatin microspheres exhibited excellent compatibility with MSCs and facilitated their attachment. Culturing MSCs with the microspheres led to enhanced overexpression of the Ext1 gene, which is crucial for cartilage growth and development. Additionally, the microspheres regulated immune responses, contributing to a reduction in OA symptoms. Conclusion: This study introduces an innovative therapeutic strategy for osteoarthritis using gelatin microspheres cultured with MSCs. By promoting Ext1 gene overexpression and regulating immune responses, these microspheres effectively mitigate OA symptoms. The findings highlight the potential of this biomaterial as a promising treatment option for OA.

Keywords: Ext1; Gelatin microsphere; Immune response; Mesenchymal stem cells; Osteoarthritis.

Figure 1

Gelatin microsphere shows promise in treating osteoarthritis by enhancing Ext1 gene expression and modulating immune responses through mesenchymal stem cell adhesion. This novel approach offers an innovative therapeutic strategy for OA management.

Figure 2

Osteoarthritis patients exhibit abnormal expression of the Ext1 gene and immune response. (A) Representative X-ray image of a knee with osteoarthritis in a patient. (B) Cartilage tissue samples were procured from the medial (Control group) and lateral (cartilage degeneration) compartments of the knee during surgical procedures in patients with knee osteoarthritis. RNA was subsequently extracted from these samples for transcriptomic analysis. (C) Heatmap illustrating the differential expression patterns of the transcriptome in knee joint cartilage tissue. (D) Volcano plot depicting the significant changes in gene expression levels in the knee joint cartilage tissue transcriptome. (E) Table summarizing the KEGG pathways enriched in the transcriptome of knee joint cartilage tissue, with associated gene counts and significance levels. (F) Further selection of immune regulatory genes previously studied and believed to be associated with the pathogenesis of osteoarthritis was conducted to create a correlation heatmap.

Figure 3

Preparation of Gelatin Microsphere and Three-Dimensional culture of MSCs. (A) Gelatin microsphere encapsulating MSCs. Schematic illustration of the gelatin composite preparation process. (B) Particle size detection of gelatin microsphere. (C) Statistical analysis of gelatin microsphere particle size distribution. (D) Scanning electron microscopy image of a gelatin microsphere. (E) MSCs were cultured with gelatin microsphere for 24h. Phalloidin staining of gelatin microsphere loaded with MSCs, highlighting the cytoskeleton. (F) Calcein AM and Propidium Iodide (PI) staining of microsphere loaded with MSCs, serving as the control group. (G) Two-dimensional fluorescence microscopy image of calcein AM and PI-stained microsphere loaded with MSCs. (H) Two-dimensional fluorescence overlay microscopy image of calcein AM and PI-stained microsphere loaded with MSCs. (I) Three-dimensional fluorescence microscopy image of calcein AM and PI-stained microsphere loaded with MSCs, demonstrating the three-dimensional distribution of MSCs within the microsphere.

Figure 4

Enhanced Ext1 Gene Expression in MSCs via Three-Dimensional Culture with Gelatin Microsphere. (A) The control group consists of MSCs cultured in traditional two-dimensional cell culture dishes, while the experimental group involves MSCs loaded onto gelatin microsphere for three-dimensional cell culture. After 48 h of culturing, RNA was extracted from both groups for transcriptomic analysis. Heatmap illustrating the transcriptomic analysis of MSCs cultured in three dimensions with gelatin microsphere. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment plot of the transcriptomic data. (C) Plot of differentially expressed genes identified in the transcriptomic analysis. (D) GO (Gene Ontology) enrichment plot derived from the transcriptomic data. (E) KEGG pathway maps highlighting the biological pathways affected in the transcriptome of MSCs cultured with gelatin microsphere. BP: Biological Process. MF: Molecular Function. CC: Cellular Component.

Figure 5

IFN-γ Enhances Immunomodulatory Properties of MSCs was cultured with Microsphere. (A) The control group consists of MSCs, while the experimental group involves cultured gelatin microsphere with MSCs. After 24 h of culturing, IFN-γ was added to both groups. Following a 24 h intervention, the supernatant was collected from the cells and subjected to Luminex liquid suspension chip immune cytokine analysis. Schematic representation of IFN-γ treatment on MSCs. (B) Heatmap depicting the analysis of cytokine expression. (C) Principal Component Analysis (PCA) results of the cytokine expression analysis. (D) Changes in inflammatory factor levels as determined by cytokine analysis.

Figure 6

Cultured MSCs and gelatin microsphere (GM) is beneficial to alleviate Osteoarthritis (OA) in vivo. (A) Timeline of in vivo experiments. Osteoarthritis rat models were made by anterior cruciate ligament transected. Day 1 was defined by 8 weeks after rat OA model established. Gelatin microsphere loaded with MSCs were intra-articular injected at Day1 (1d). Von Frey test measures knee joint pain sensitivity by applying filaments of different sizes to gauge mechanical pain thresholds. (B) Following intra-articular injection of gelatin microsphere loaded with MSCs. In vivo imaging was measured by 1^st day (1d), 10^th day (10d) and 21^th day (21d). Living cells were labeled by DiR Iodide. The labeled cells appear to be a darker color in the color scheme. (C) Von Frey test of rat knee joint was measured by 1^st day (1d), 6^th day (6d), 12^th day (12d), 18^th day (18d) and 24^th day (24d). Von Frey test was used to assess joint pain by applying varying forces to the skin around the joint to determine pain threshold sensitivity. Lower tactile allodynia means more painful. (D) Rats were sacrificed by 24^th day. Representative H&E staining image of knee joints for Control, OA and OA+GM group.