Attenuation of osteoarthritis progression through intra-articular injection of a combination of synovial membrane-derived MSCs (SMMSCs), platelet-rich plasma (PRP) and conditioned medium (secretome)

Abstract

Purpose: Osteoarthritis (OA) as a progressive destructive disease of articular cartilage is the most common joint disease characterized by reduction of joint cartilage thickness, demolition of cartilage surface and new bone formation. To overcome these problems, the purpose of the current research was to evaluate and compare the in vivo effects of synovial membrane-derived mesenchymal stem cell (SMMSCs), platelet-rich plasma (PRP) and conditioned medium (secretome) on collagenase II-induced rat knee osteoarthritis (KOA) remedy.

Methods: For the first step, SMMSCs were isolated and characterized. Also, secretome was collected from SMMSCs culture. Furthermore, PRP was collect from the rat heart venous blood. Second, two injection of collagenase II with an interval of 3 days was performed in the knee intra-articular space to induce osteoarthritis. Two weeks later, animals were randomly divided into 6 groups. Control group without treatment, positive group: taken an intra-articular sodium hyaluronate injection (0.1 ml), treatment groups taken an intra-articular injection of; treatment 1: SMMSCs (5 × 10⁶), treatment 2: SMMSCs (5 × 10⁶)/secretome (50 µl), treatment 3: SMMSCs (5 × 10⁶)/PRP (50 µl), and treatment 4: SMMSCs (5 × 10⁶)/ secretome (50 µl)/ PRP (50 µl). Three months later, rats were killed and the following assessments were executed: radiography, histopathology, and immunohistochemistry.

Results: Our findings represented that a combination of the SMMSCs/secretome/PRP had a considerable effect on glycosaminoglycans (GAGs) and collagen II contents, articular cartilage preservation, compared with other groups. In addition, combination of the SMMSCs with PRP and secretome showed the lowest expression of mmp3, while SOX9 had the highest expression in comparison with other groups. Also, SMMSCs-injected groups demonstrated better results compared with positive and control groups.

Conclusions: Injecting a combination of the SMMSCs/secretome/PRP resulted in better efficacy in terms of joint space width, articular cartilage surface continuity and integrity, sub-chondral bone and ECM constituents such as collagen II. Indeed, transplantation of this combination could be considered as a preliminary therapy for clinical trial study in the future.

Keywords: Mesenchymal stem cells; Osteoarthritis; PRP; Rat; Secretome.

Fig.1

Morphology (a) (×10), differentiation capacity and cell surface markers of SMMSCs. For SMMSCs, adipogenic differentiation was shown by oil red staining (b), while osteogenic differentiation was demonstrated by calcium deposition in Alizarin red staining (c); ×10 magnification. Flow cytometry study findings exhibited that SMMSCs were negative for hematopoietic markers CD34 (d) and CD45 (e) and also positive for MSC markers CD44 (f) and CD90 (g)

Fig.2

Different stains for knee articular cartilage structure and constituents. H and E staining showed better articular cartilage surface continuity (black arrow) in treatment groups (c1, d1, e1 and f1). Masson’s trichrome staining revealed more collagen type II (black arrow) (c2, d2, e2 and f2). Toluidine blue staining exhibited better extracellular matrix preservation (black arrow) in treatment groups (c3, d3 and f3). (The yellow scale bar shows 100 μm)

Fig. 3

Special staining of knee articular cartilage. Safranin O/ alcian blue staining showed more GAGs (black arrow) in treatment groups c1, e1 and f1. Immunohistochemistry for Col2 (black arrow) revealed better expression in treatment groups (c2 and f2)

Fig. 4

IHC evaluation of MMP3 in OA-induced knee articular cartilage. Compared with other groups, IHC revealed low expression of MMP3 in treatment group 4 (×10)

Fig. 5

IHC evaluation of SOX9 in OA-induced knee articular cartilage. IHC revealed higher expression of SOX9 in treatment groups (2, 3 and 4) (×10)

Fig. 6

Radiography Study. Joint space narrowing (green circle), subchondral sclerosis (red arrow) and osteophyte formation (blue arrow) were observed in OA and Sodium hyaluronate groups