Biotechnological production of reduced and oxidized NAD+ precursors.

Dysregulation of nicotinamide adenine dinucleotide (NAD+) homeostasis by increased activity of NAD+ consumers or reduced NAD+ biosynthesis plays an important role in the onset of prevalent, often age-related, diseases, such as diabetes, neuropathies or nephropathies. To counteract such dysregulation, NAD+ replenishment strategies can be used. Among these, administration of vitamin B3 derivatives (NAD+ precursors) has garnered attention in recent years. However, the high market price of these compounds and their limited availability, pose important limitations to their use in nutritional or biomedical applications. To overcome these limitations, we have designed an enzymatic method for the synthesis and purification of (1) the oxidized NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), (2) their reduced forms NMNH and NRH, and (3) their deaminated forms nicotinic acid mononucleotide (NaMN) and nicotinic acid riboside (NaR). Starting from NAD+ or NADH as substrates, we use a combination of three highly overexpressed soluble recombinant enzymes; (a) a NAD+ pyrophosphatase, (b) an NMN deamidase, and (c) a 5'-nucleotidase, to produce these six precursors. Finally, we validate the activity of the enzymatically produced molecules as NAD+ enhancers in cell culture.

In vivo partial reprogramming of myofibers promotes muscle regeneration by remodeling the stem cell niche.

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.

High temperature CaSiO3-Ca3(PO4)2 ceramic promotes osteogenic differentiation in adult human mesenchymal stem cells.

Silicophosphate calcium ceramics are widely used in orthopedic and oral surgery applications because of their properties for stimulating bone formation and bone bonding. These bioceramics, together with multipotent undifferentiated adult human mesenchymal stem cells, are serious candidates in the field of bone tissue engineering and regenerative medicine. For this reason, the influence of a novel 30 wt%CaSiO3 - 70 wt%Ca3(PO4)2 ceramic over a primary adult human mesenchymal stem cells culture has been investigated in this study, observing a total colonization of the biomaterial by cells at 21 days. The osteoinductive capacity of the materials was also studied: alkaline phosphatase activity, gene quantification of osteoblastic genes and calcium deposits stained by Alizarin Red test, showed evidences of osteogenic differentiation of adult human mesenchymal stem cells seeded with this bioceramic both in growth medium and osteogenic medium. Therefore, the 30 wt%CaSiO3 - 70 wt%Ca3(PO4)2 bioceramic represents a potential scaffold which could be used in the field of biomaterials for bone tissue engineering, allowing cell adhesion, proliferation and promoting osteogenic differentiation of adult human mesenchymal stem cells.

Nurse's A-Phase-Silicocarnotite Ceramic-Bone Tissue Interaction in a Rabbit Tibia Defect Model.

Calcium phosphate materials are widely used as bone substitutes due to their bioactive and biodegradable properties. Also, the presence of silicon in their composition seems to improve the bioactivity of the implant and promote bone tissue repair. The aim of this study was to develop a novel ceramic scaffold by partial solid-state sintering method with a composition lying in the field of the Nurse's A-phase-silicocarnotite, in the tricalcium phosphate-dicalcium silicate (TCP-C2S) binary system. Also, we evaluated its osteogenic and osteoconductive properties after being implanted into tibia defects in New Zealand rabbits. X-ray, microcomputer tomography, and histomorphometry studies demonstrated that this porous ceramic is highly biocompatible and it has excellent osteointegration. The material was being progressively reabsorbed throughout the study and there was no unspecified local or systemic inflammatory response observed. These results suggest that ceramic imitates the physicochemical characteristics of bone substitutes used in bone reconstruction.

Nurse's A-Phase Material Enhance Adhesion, Growth and Differentiation of Human Bone Marrow-Derived Stromal Mesenchymal Stem Cells.

The purpose of this study was to evaluate the bioactivity and cell response of a well-characterized Nurse's A-phase (7CaO·P2O5·2SiO2) ceramic and its effect compared to a control (tissue culture polystyrene-TCPS) on the adhesion, viability, proliferation, and osteogenic differentiation of ahMSCs in vitro. Cell proliferation (Alamar Blue Assay), Alizarin Red-S (AR-s) staining, alkaline phosphatase (ALP) activity, osteocalcin (OCN), and collagen I (Col I) were evaluated. Also, field emission scanning electron microscopy (FESEM) images were acquired in order to visualise the cells and the topography of the material. The proliferation of cells growing in a direct contact with the material was slower at early stages of the study because of the new environmental conditions. However, the entire surface was colonized after 28 days of culture in growth medium (GM). Osteoblastic differentiation markers were significantly enhanced in cells growing on Nurse's A phase ceramic and cultured with osteogenic medium (OM), probably due to the role of silica to stimulate the differentiation of ahMSCs. Moreover, calcium nodules were formed under the influence of ceramic material. Therefore, it is predicted that Nurse's A-phase ceramic would present high biocompatibility and osteoinductive properties and would be a good candidate to be used as a biomaterial for bone tissue engineering.

Morphological and Structural Study of a Novel Porous Nurse's A Ceramic with Osteoconductive Properties for Tissue Engineering.

The characterization process of a new porous Nurse's A ceramic and the physico chemical nature of the remodeled interface between the implant and the surrounding bone were studied after in vivo implantation. Scaffolds were prepared by a solid-state reaction and implanted in New Zealand rabbits. Animals were sacrificed on days 15, 30, and 60. The porous biomaterial displayed biocompatible, bioresorbable, and osteoconductive capacity. The degradation processes of implants also encouraged osseous tissue ingrowths into the material's pores, and drastically changed the macro- and microstructure of the implants. After 60 healing days, the resorption rates were 52.62% ± 1.12% for the ceramic and 47.38% ± 1.24% for the residual biomaterial. The elemental analysis showed a gradual diffusion of the Ca and Si ions from the materials into the newly forming bone during the biomaterial's resorption process. The energy dispersive spectroscopy (EDS) analysis of the residual ceramic revealed some particle categories with different mean Ca/P ratios according to size, and indicated various resorption process stages. Since osteoconductive capacity was indicated for this material and bone ingrowth was possible, it could be applied to progressively substitute an implant.

Assessment of Effects of Si-Ca-P Biphasic Ceramic on the Osteogenic Differentiation of a Population of Multipotent Adult Human Stem Cells.

A new type of bioceramic with osteogenic properties, suitable for hard tissue regeneration, was synthesised. The ceramic was designed and obtained in the Nurse's A-phase-silicocarnotite subsystem. The selected composition was that corresponding to the eutectoid 28.39 wt % Nurse's A-phase-71.61 wt % silicocarnotite invariant point. We report the effect of Nurse's A-phase-silicocarnotite ceramic on the capacity of multipotent adult human mesenchymal stem cells (ahMSCs) cultured under experimental conditions, known to adhere, proliferate and differentiate into osteoblast lineage cells. The results at long-term culture (28 days) on the material confirmed that the undifferentiated ahMSCs cultured and in contact with the material surface adhered, spread, proliferated, and produced a mineralised extracellular matrix on the studied ceramic, and finally acquired an osteoblastic phenotype. These findings indicate that it underwent an osteoblast differentiation process. All these findings were more significant than when cells were grown on plastic, in the presence and absence of this osteogenic supplement, and were more evident when this supplement was present in the growth medium (GM). The ceramic evaluated herein was bioactive, cytocompatible and capable of promoting the proliferation and differentiation of undifferentiated ahMSCs into osteoblasts, which may be important for bone integration into the clinical setting.