Clinical therapies have traditionally been developed using two-dimensional (2D) cell culture systems, which fail to accurately capture tissue complexity. Therefore, three-dimensional (3D) cell cultures are more attractive platforms to integrate multiple cues that arise from the extracellular matrix and cells, closer to an in vivo scenario. Here we report the development of a 3D cellular model for the in vitro assessment of the outcome of oxygen- and drug-dependent therapies, exemplified by photodynamic therapy (PDT). Using a synthetic self-assembling peptide as a cellular scaffold (RAD16-I), we were able to recreate the in vivo limitation of oxygen and drug diffusion and its biological effect, which is the development of cellular resistance to therapy. For the first time, the production and decay of the cytotoxic species singlet oxygen could be observed in a 3D cell culture. Results revealed that the intrinsic mechanism of action is maintained in both systems and, hence, the dynamic mass transfer effects accounted for the major differences in efficacy between the 2D and 3D models. We propose that this methodological approach will help to improve the efficacy of future oxygen- and drug-dependent therapies such as PDT.

Hair follicle dermal papilla cells (HFDPC) are a specialized cell population located in the bulge of the hair follicle with unique characteristics such as aggregative behavior and the ability to induce new hair follicle formation. However, when expanded in conventional 2D monolayer culture, their hair inductive potency is rapidly lost. Different 3D culture techniques, including cell spheroid formation, have been described to restore, at least partially, their original phenotype, and therefore, their hair inductive ability once transplanted into a recipient skin. Moreover, hair follicle dermal papilla cells have been shown to differentiate into all mesenchymal lineages, but their differentiation potential has only been tested in 2D cultures. In the present work, we have cultured HFDPC in the 3D self-assembling peptide scaffold RAD16-I to test two different tissue engineering scenarios: restoration of HFDPC original phenotype after cell expansion and osteogenic and adipogenic differentiation. Experimental results showed that the 3D environment provided by RAD16-I allowed the restoration of HFDPC signature markers such as alkaline phosphatase, versican and corin. Moreover, RAD16-I supported, in the presence of chemical inductors, three-dimensional osteogenic and adipogenic differentiation. Altogether, this study suggests a potential 3D culture platform based on RAD16-I suitable for the culture, original phenotype recovery and differentiation of HFDPC.

This work describes the development of a three-dimensional (3D) model of osteogenesis using mouse preosteoblastic MC3T3-E1 cells and a soft synthetic matrix made out of self-assembling peptide nanofibers. By adjusting the matrix stiffness to very low values (around 120 Pa), cells were found to migrate within the matrix, interact forming a cell–cell network, and create a contracted and stiffer structure. Interestingly, during this process, cells spontaneously upregulate the expression of bone-related proteins such as collagen type I, bone sialoprotein, and osteocalcin, indicating that the 3D environment enhances their osteogenic potential. However, unlike MC3T3-E1 cultures in 2D, the addition of dexamethasone is required to acquire a final mature phenotype characterized by features such as matrix mineralization. Moreover, a slight increase in the hydrogel stiffness (threefold) or the addition of a cell contractility inhibitor (Rho kinase inhibitor) abrogates cell elongation, migration, and 3D culture contraction. However, this mechanical inhibition does not seem to noticeably affect the osteogenic process, at least at early culture times. This 3D bone model intends to emphasize cell–cell interactions, which have a critical role during tissue formation, by using a compliant unrestricted synthetic matrix.

Cartilage injury and degenerative tissue progression remain poorly understood by the medical community. Therefore, various tissue engineering strategies aim to recover areas of damaged cartilage by using non-traditional approaches. To this end, the use of biomimetic scaffolds for recreating the complex in vivo cartilage microenvironment has become of increasing interest in the field. In the present study, we report the development of two novel biomaterials for cartilage tissue engineering (CTE) with bioactive motifs, aiming to emulate the native cartilage extracellular matrix (ECM). We employed a simple mixture of the self-assembling peptide RAD16-I with either Chondroitin Sulfate (CS) or Decorin molecules, taking advantage of the versatility of RAD16-I. After evaluating the structural stability of the bi-component scaffolds at a physiological pH, we characterized these materials using two different in vitro assessments: re-differentiation of human articular chondrocytes (AC) and induction of human adipose derived stem cells (ADSC) to a chondrogenic commitment. Interestingly, differences in cellular morphology and viability were observed between cell types and culture conditions (control and chondrogenic). In addition, both cell types underwent a chondrogenic commitment under inductive media conditions, and this did not occur under control conditions. Remarkably, the synthesis of important ECM constituents of mature cartilage, such as type II collagen and proteoglycans, was confirmed by gene and protein expression analyses and toluidine blue staining. Furthermore, the viscoelastic behavior of ADSC constructs after 4 weeks of culture was more similar to that of native articular cartilage than to that of AC constructs. Altogether, this comparative study between two cell types demonstrates the versatility of our novel biomaterials and suggests a potential 3D culture system suitable for promoting chondrogenic differentiation.