The global incidence of chronic joint diseases such as OA is steadily increasing due to an aging population and rising obesity worldwide (40). The inherent poor ability of articular cartilage to repair, regenerate, and remodel is one of the factors believed to facilitate the development of OA (41–43). There are currently no effective pharmacological treatments for OA, and in many advanced cases joint arthroplasty is the inevitable treatment for patients who have not responded to any other recommended therapies (44, 45). Consequently, there is an urgent medical need for the development of new therapies for OA such as stem cell therapies (46, 47), using different MSCs sources as well as using different sources of MSC, as well as with different biomaterials to be used as scaffolds (48).
The majority cell-based cartilage repair models available on the market have high costs and relatively short lifespan in terms of efficacy (49). Furthermore, to the best of our knowledge, there are no studies comparing the chondrogenic differentiation process of hAD-MSC with that of hAF-MSC. Therefore, in the present study, we assessed the potential of hAF-MSC and hAD-MSC for chondrogenic differentiation directly into chitosan-xanthan gum scaffolds designed for cartilage tissue engineering, under TGF-β3 stimulation, analysing the suitability of a seldom used but abundant and easily obtained source of MSCs, combined with a biocompatible material easily produced, economically accessible and that does not induce inflammatory response, comparing several aspects to establish the most adequate cell source for clinical application. While hAF-MSC are younger cells embryologically closer to embryonic stem cells (ESCs), hAD-MSC are adult stem cells. In recent years, hAF-MSC has been considered a promising option to obtain MSCs, owing to the positive aspects that stimulate its application in clinical practice, such as ethical concerns, unlike the controversial use of embryonic stem cells, hAF-MSC, but also because they are easy to obtain, easy to grow and exhibit no tumorigenicity when compared to adult stem cells (50, 51).
A short time after harvesting the MSCs from both sources, the cells showed rapid adhesion and proliferation, with fibroblastic formimng compatible with the characterization standards of stem cells required by the International Society for Cell Therapy (39) (Fig. 1a and d). In the flow cytometry analysis, hematopoietic lineage markers such as CD34, CD45 and CD19 were present at levels above 2%, differently from the consensus established by Dominici (39). However, as observed previously by our group and by others, CD34 expression was positive in up to 50% of cells (52, 53). hAF-MSC contains several cell types derived from the development of the fetus and, as these cells express different markers according to their lineage and gestation time, it is believed that this peculiarity is responsible for the findings in cytometry. Therefore, it is possible to assume that the minimum criterion proposed by the International Society for Cell Therapy.
Considering that hAF-MSC are less mature and more variable and that this could negatively interfere with cell differentiation, immunomagnetic separation was performed after the second passage of cell expansion. A subpopulation of cells positive for CD117 (50) was selected, representing approximately 1% of the cultured cells (29). After this additional selection step, flow cytometry analysis showed high levels of SSEA-4 and other markers of chondrogenic potential (Fig. 1c and f).
After the fourth passage of the SCs from both sources, it was possible to demonstrate the potential of these cells for differentiation into three mesenchymal lineages: adipogenic, chondrogenic and osteogenic, according to the pre-established consensus (39). The cells showed high ECM production in the differentiation process into three mesenchymal lineages. In the analysis of adipogenic differentiation, we observed the formation of lipid droplets within the newly differentiated adipocytes, in addition to ECM stained in different shades of orange by Oil Red staining. When evaluating chondrogenic differentiation, the intense production of ECM was visualised by staining with Alcian Blue, confirming the presence of proteoglycans (PGs). For osteogenesis analysis, the calcified ECM formed was stained red by Alizarin Red staining, showing the presence of calcification in significant quantities (Fig. 1b and e).
After characterization, MSCs from both sources were seeded into the scaffold. Interestingly, it was observed that, although cells from both sources differentiated into chondrocytes, cells from hAF-MSC adapted better to the scaffold than those from hAD-MSC. This lower affinity of hAD-MSC cells to the scaffold was observed by SEM, with a dispersed cell population, with fewer cells distributed over the scaffold and formation of looser fibers. Regarding TGF-β3 stimulation, in stimulated cells, both from hAF-MSC and hAD-MSC, after 21 days of culture it was possible to observe chondrogenic differentiation, with intense cell condensation and ECM production. In contrast, in groups without stimulation, there was little ECM formation (Fig. 2).
Histological analysis showed very similar results that confirmed the chondrogenic differentiation of MSCs from both sources, because in addition to intense cell condensation and ECM production, the stains used indicated the presence of collagen and proteoglycan. The greatest difference that could be observed in the histology results was in relation to the group stimulated with TGF-β3 and the group without stimulation (Fig. 3). These data enable confirming the influence of the stimulation strategy used (31, 54). The differentiation even without TGF-β3 stimulation can be explained by the high concentration of cells injected together into the scaffold, mimicking the condensation of chondrogenic differentiation that occurs in embryonic development (55, 56). In such conditions, hAD-MSC may recapitule favorable cellular microenvironment. The analysis of the ECM formed in the cell-scaffold set by immunohistochemistry (IH) showed the presence of type II collagen and aggrecan, thus confirming chondrogenic differentiation from the SCs from both sources (Fig. 4). The results of the IH analysis of hAF-MSC showed greater cell condensation and greater ECM production compared to the IH of hAD-MSC SCs, which confirms the lower affinity of hAD-MSC to the CX scaffold, corroborating the results obtained in the other analyses, as well as the immunofluorescence results (Fig. 5).
Despite this study does not include quantitative analysis of cell differentiation, as well as a functional analysis of the set consisting of scaffold, cells and TGF-β3 stimulation, it is possible to infer that hAF-MSC have a favorable potential for use in clinical practice in the treatment of chondral lesions, in association with a scaffold with greater accessibility. Further studies should be carried out to confirm our findings.