In vitro differentiation capacities and characterization of rat SMMSCs
Figure 1a shows the spindle-shaped morphology of SMMSCs at passage 3 that acquired from the synovial membrane of male Sprague Dawley rat. Oil red-O staining and alizarin red staining are used to demonstrate the differentiation capacities of SMMSCs to adipocyte and osteocyte, respectively. Oil Red-O staining showed of lipid-rich vacuoles after 2 weeks (Fig. 1b). In addition, Alizarin Red staining revealed the formation of mineralized nodules after 2 weeks (Fig. 1c).
To characterize the phenotypes of SMMSCs, flow cytometry was executed to evaluate the surface markers of MSCs. Cells were labeled with FITC, perCP, and PE-conjugated antibodies and investigated by flow cytometry. Cells were stained with CD34, CD45 (hematopoietic cell markers) and CD44, CD90 (mesenchymal stem-cell markers). Based on our findings, the SMMSCs were negative for cell markers CD34, and CD45, while positive for MSC marker CD44 and CD90 (Fig. 1d-g).
As revealed by the H and E staining, treatment groups (c1, d1, e1 and f1) had better articular cartilage surface continuity and integrity. More cell distribution was seen in, treatment 4 group (f1), also, treatment 1, 2 and 3 groups (c1, d1 and e1) showed better cell distribution compared to OA (a1) and Hyalgan (b1) groups. In addition, treatment groups (c1, d1, e1 and f1) demonstrated better cartilage mineralization compared to OA (a1) and Hyalgan (b1) groups. Moreover, better subchondral bone was seen in treatment groups (c1, d1, and e1) especially in treatment 4 (f1). Besides, more cell population was observed in treatment groups (c1, d1, e1 and f1) especially in treatment 2 and 4 (d1 and f1). A regular and intact subchondral bone with normal interstitial space and lamellar orientations is seen in treatment groups (c1, d1 and f1) whereas treatment 3 (e1), OA (a1) and Hyalgan (b1) groups showed destruction of trabecular bone.
Those groups were received treatment groups (c2, d2, e2 and f2) exhibited more collagen type Ⅱ using Masson’s trichrome staining. A potent orientation of collagen is seen at superficial layer of treatment 4 (f2), and deep layer of treatment 2 (d2). In addition, those groups receiving stem cells demonstrated regular surfaces, while loss of superficial cell layers and surface irregularities are observed in OA (a2) and Hyalgan (b2) groups. Articular cartilage fraction and roughness were observed in some parts of e and f groups.
Following toluidine blue staining, better extracellular matrix and orientation of chondrocytes were observed in those animals administrated by treatment groups (c3, d3 and f3). The cartilage layer in treatment groups (c3, d3 and f3) was heavily stained in comparision with OA (a3).
Based on safranin O/ alcian blue staining, similar sulfated glycosaminoglycans of proteoglycan contents (red/orange) were observed in treatment groups (c1, e1 and f1) as the best findings. Likewise, treatment 2 (d1) and Hyalgan (b1) groups were the same in GAGs contents. Generally, all groups showed better results compared to OA group.
Based on IHC, better expression of Col2 was observed in treatment groups c2 and f2. Besides, Similar Col2-positive cells was seen in treatment 2 (d2) and Hyalgan (b2) groups.
Lower expression of MMP3, as a proteolytic enzyme which is well known in the development and spread of inflammatory diseases such as osteoarthritis, was especially observed in treatment group 4 using IHC staining (Fig. 4). However, the level of MMP3 in OA and Hyalgan groups was significantly increased. Treatment groups 1, 2 and 3 exhibited less expression of MMP3 compared with untreated groups (Fig. 4).
To survey the potential effects of SMMSCs, secretome and PRP on OA-induced knee articular cartilage, IHC staining for SOX9 was done. Results showed higher expression of SOX9 in treatment groups 2, 3, and 4 (Fig. 5). Likewise, SOX9 -positive cells were more observed in superficial layer of articular cartilage in treatment group 4 (Fig. 5).
Joint space narrowing, subchondral sclerosis and osteophyte were not observed in treatment group 4. In treatment 1 and 2 groups, findings were normal but osteophyte was seen in early stage. A light Joint space narrowing and osteophytes in early stage were observed in treatment 3 group, while Joint space was normal. Joint space narrowing, moderate subchondral sclerosis and osteophyte were detected in OA and Hyalgan groups, while osteophyte formation was more severe in OA group.