Multiple Intra-Articular Injections of Adipose-Derived Mesenchymal Stem Cells for Canine Osteoarthritis Treatment

Abstract

Osteoarthritis (OA) is one of the most common degenerative diseases in dogs and humans, which can lead to articular cartilage deterioration, chronic pain, and decreased quality of life. The anti-inflammatory, anti-fibrotic, analgesic, and cartilage regeneration properties of mesenchymal stem cell (MSC) therapy provide a new direction for the treatment development of OA in the future. Currently, MSC therapy lacks confirmed ideal sources, dosages, formulations, and specific characteristics. In this study, we evaluated the efficacy of multiple canine adipose-derived mesenchymal stem cell (ADSC) injections on anti-inflammation and joint cartilage damage in a canine OA model. Considering animal ethics, we simulated the effects of inflammation and cartilage repair during treatment through a mouse OA model. In the mouse OA model, through the detection of cartilage repair and inflammation-related key factors via histology and molecular biology, it was found that MSC therapy has a certain repair effect on cartilage, but the anti-inflammatory effect is time-dependent. In the canine OA model, we verified the feasibility of multiple injections of ADSCs. Compared with the control group, the cartilage repair effect of the treatment group was obvious, and the inflammatory factors decreased, showing an obvious therapeutic effect. This study demonstrates that multiple intra-articular injections of canine ADSCs could be effective in treating OA symptoms.

Keywords: MSC therapy; cartilage repair; mesenchymal stem cells; osteoarthritis.

Figure 1

The isolation, culture, and identification of canine ADSCs. (A) The morphologies of different passages (P0–P4) of canine ADSCs. (B) The cell growth curve of canine ADSCs, (C) Analysis of the differentiation capacity of canine ADSCs through lipogenic, osteogenic, and chondrogenic differentiation. (D) The immunofluorescence identification of canine ADSC surface markers: CD34, CD45, CD90, and CD105.

Figure 2

Intra-articular injection of ADSCs to repair damaged cartilage in DMM mice. (A) The modeling process of the DMM mice model, with the meniscus indicated by arrows. (B) Schematic diagram of injection treatment time schedule. (C) Changes in the body weight of mice during the course of the treatment period. (D) Changes in thickness and width of the knee joints during the course of treatment of the mice. (E) HE staining of articular cartilage. (F) Tomato red-O-solid green staining of articular cartilage. * meanings SHAM compared DMM, & meaning SHAM compared DMM + ADSCs, # meanings SHAM compared DMM. * ^{# &} p ≤ 0.05. ^##. p ≤ 0.01. *** ^{### &&&} p ≤ 0.001.

Figure 3

Analysis of inflammation and cartilage repair-related indexes during treatment in DMM mice. (A) Serum IL-6 levels during treatment. (B) Real-time PCR analysis of the expression of TNF-α and IL-6 in cartilage samples during treatment. (C) Real-time PCR analysis of cartilage samples for chondrogenesis-related genes ADAMTS-5 and MMP-13 during treatment, as well as Aggrecan and Collagen-II mRNA expression. * p ≤ 0.05. ** p ≤ 0.01. *** p ≤ 0.001.

Figure 4

Intra-articular injection of ADSCs to repair damaged cartilage in KOA canines. (A) Modeling process of KOA canine model. (B) Gross observation of articular cartilage, where A1 is the ADSC treatment group, B1 is the control group, and the abrasion point is indicated by an arrow. (C) X-ray examination of the affected limb before and after modeling of the canines. (D) X-ray examination of the affected limb before and after treatment of the canines. Joints are highlighted in the red box (E) HE articular cartilage staining. (F) Toluidine blue articular cartilage staining. (G) Tomatoxylin-O-fixed green articular cartilage staining.

Figure 5

Changes in inflammation and cartilage repair-related indexes during treatment in KOA dogs. (A) Real-time PCR analysis of mRNA expression of inflammatory factors IL-1β and IL-6 in cartilage samples during treatment. (B) Real-time PCR analysis of mRNA expression of chondrogenesis-related genes ADAMTS-5 and MMP-13 in cartilage samples during treatment. (C) Real-time PCR analysis of mRNA expression of chondrogenesis-related genes Collagen-II and Aggrecan in cartilage samples during treatment. (D) Chondrocyte morphology. (E) Immunofluorescence identification of canine chondrocytes’ Col-II. (F) Chondrocyte toluidine blue staining. (G) CCK-8 detection of value-added canine chondrocyte cells. (H) Scratch assay for detection of canine chondrocyte cell migration ability. NS meaning no significance.* p ≤ 0.05. ** p ≤ 0.01. *** p ≤ 0.001.