The role of bioengineered scaffolds for the induction of osteochondrogenic differentiation of human adipose-derived stem cells

Osteochondral lesions may be due to trauma or congenital conditions; in both cases, modern medicine is limited because of the difficulty of tissue repair. Tissue engineering is proposed as a potential solution in regenerative medicine. Tissue engineering relies on specific mechanical attributes of scaffolds like porosity, mechanical strength, biocompatibility, immunogenicity, and degradability. Based on the assumption that at different stiffness levels mesenchymal stem cells can have different commitment, we decided to study three different scaffolds: a semisynthetic animal-derived hydrogel GelMA, a biopolymer PEGDA, and a decellularized plant-based scaffold. The designated scaffolds have different structural, geometric, and material characteristics and were used to investigate the role of different biomechanical stimuli and microenvironments in the osteochondral differentiation of human adipose-derived stem cells (hASCs). The latter are considered very promising in regenerative medicine because they are abundant, easy to extract, and have excellent differentiation capabilities. In addition, in our previous studies, we have shown two subpopulations of hASCs are able to selectively differentiate toward bone or cartilage. Methods: For the biomechanical analysis of the scaffolds, a Zwick Roell machine for the unconfined compression test was used. To check the swelling, the samples were immersed in water and the weight growth was measured with a precision scale. The porosity analysis was conducted using a HITACHI TM 4000 scanning electron microscope. It was necessary to use different methods to evaluate the degradability of the scaffolds due to their different nature. In particular, for GelMA hydrogel, collagenase type I was used. For PEGDA and the plant-based scaffold, strong acid and base (HCl and NaOH) were used instead. hASCs were isolated according to our lab protocol, consisting in a 0.1% collagenase type I digestion for 45 minutes at 37°C in a shaking bath. Digestion was subsequently filtered and finally seeded on a plastic support for cell culture. Cell viability was measured by CCK8 assay and Dead/live staining. For the assessment of hASC osteochondral differentiation, histochemistry (Alcian blue and Alizarin Red) and immunofluorescence for chondrogenic and osteogenic markers (COL2A1 and OCN, respectively) were used. Images were analyzed by using a confocal and light microscope. Results: In this study, we demonstrated that our scaffolds support cell viability and differentiation toward a spontaneous osteochondral commitment, exploiting the specific intrinsic characteristics of hASCs. Differentiation data obtained are in line with current literature that affirms that the higher the stiffness, the higher the tendency to osteogenic commitment. The obtained biological data highlight the crucial role of the biomaterial structure and the microenvironment in inducing spontaneous cell differentiation without the use of exogenous factors, which makes the approach safer for potential clinical trials. Conclusion: In this study, we intentionally used the heterogeneous unsorted hASC population to exploit their different intrinsic properties in response to various microenvironment stimuli. On the contrary, lower stiffness corresponds to a higher tendency to chondrogenesis. Our next step will be the use of a specific sorted cell population to optimize a specific hASC commitment with the best performing scaffold. Further studies are required to study in vivo tissue regeneration and vascularization in an extended timeframe.