rhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals.

BACKGROUND: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro. METHODS: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes. RESULTS: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo. CONCLUSIONS: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Polycaprolactone with multiscale porosity and patterned surface topography prepared using sacrificial 3D printed moulds: Towards tailor-made scaffolds.

Biocompatible three-dimensional porous scaffolds are widely used in multiple biomedical applications. However, the fabrication of tailor-made 3D structures with controlled and combined multiscale macroscopic-microscopic, surface and inner porosities in a straightforward manner is still a current challenge. Herein, we use multimaterial fused deposition modeling (FDM) to generate poly (vinyl alcohol) (PVA) sacrificial moulds filled with poly (Ɛ-caprolactone) (PCL) to generate well defined PCL 3D objects. Further on, the supercritical CO2 (SCCO2) technique, as well as the breath figures mechanism (BFs), were additionally employed to fabricate specific porous structures at the core and surfaces of the 3D PCL object, respectively. The biocompatibility of the resulting multiporous 3D structures was tested in vitro and in vivo, and the versatility of the approach was assessed by generating a vertebra model fully tunable at multiple pore size levels. In sum, the combinatorial strategy to generate porous scaffolds offers unique possibilities to fabricate intricate structures by combining the advantages of additive manufacturing (AM), which provides flexibility and versatility to generate large sized 3D structures, with advantages of the SCCO2 and BFs techniques, which allow to finely tune the macro and micro porosity at material surface and material core levels.

The transcription factor DDIT3 is a potential driver of dyserythropoiesis in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.

Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds.

The extracellular matrix (ECM) is a complex network with multiple functions, including specific functions during tissue regeneration. Precisely, the properties of the ECM have been thoroughly used in tissue engineering and regenerative medicine research, aiming to restore the function of damaged or dysfunctional tissues. Tissue decellularization is gaining momentum as a technique to obtain potentially implantable decellularized extracellular matrix (dECM) with well-preserved key components. Interestingly, the tissue-specific dECM is becoming a feasible option to carry out regenerative medicine research, with multiple advantages compared to other approaches. This review provides an overview of the most common methods used to obtain the dECM and summarizes the strategies adopted to decellularize specific tissues, aiming to provide a helpful guide for future research development.