Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions. These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Ulvan, a bioactive natural sulfated polysaccharide, and gelatin, a collagen-derived biopolymer, have attracted interest for the preparation of biomaterials for different biomedical applications, due to their demonstrated compatibility for cell attachment and proliferation. Both ulvan and gelatin have exhibited osteoinductive potential, either alone or in combination with other materials. In the current work, a series of novel hybrid scaffolds based on crosslinked ulvan and gelatin was designed, prepared and characterized. Their mechanical performance, thermal stability, porosity, water-uptake and in vitro degradation ability were assessed, while their morphology was analyzed through scanning electron microscopy. The prepared hybrid ulvan/gelatin scaffolds were characterized by a highly porous and interconnected structure. Human adipose-derived mesenchymal stem cells (hADMSCs) were seeded in selected ulvan/gelatin hybrid scaffolds and their adhesion, survival, proliferation, and osteogenic differentiation efficiency was evaluated. Overall, it was found that the prepared hybrid sponge-like scaffolds could efficiently support mesenchymal stem cells’ adhesion and proliferation, suggesting that such scaffolds could have potential uses in bone tissue engineering.

The aim of the study is to determine the effectiveness of stem cells in scaffolds in the treatment of bone deficits, in regard of bone regeneration, safety, rehabilitation and quality of life in humans. The systematic review was conducted in accordance with PRISMA 2020. A systematic search was conducted in three search engines and two registries lastly in 29-9-2022.for studies of the last 15 years. The risk of bias was assessed with RoB-2, ROBINS- I and NIH Quality of Before-After (Pre-Post) Studies with no Control group. The certainty of the results was assessed with the GRADE assessment tool. Due to heterogeneity, the results were reported in tables, graphs and narratively. The study protocol was published in PROSPERO with registration number CRD42022359049. Of the 10,091 studies retrieved, 14 were meeting the inclusion criteria, and were qualitatively analyzed. 138 patients were treated with mesenchymal stem cells in scaffolds, showing bone healing in all cases, and even with better results than the standard care. The adverse events were mild in most cases and in accordance with the surgery received. When assessed, there was a rehabilitation of the deficit and a gain in quality of life was detected. Although the heterogeneity between the studies and the small number of patients, the administration of mesenchymal stem cells in scaffolds seems safe and effective in the regeneration of bone defects. These results pave the way for the conduction of more clinical trials, with greater number of participants, with more standardized procedures. Graphical Abstract

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.

Articular cartilage can be easily damaged from human's daily activities, leading to inflammation and to osteoarthritis, a situation that can diminish the patients' quality of life. For larger cartilage defects, scaffolds are employed to provide cells the appropriate three-dimensional environment to proliferate and differentiate into healthy cartilage tissue. Natural biomaterials used as scaffolds, attract researchers' interest because of their relative nontoxic nature, their abundance as natural products, their easy combination with other materials, and the relative easiness to establish Marketing Authorization. The last 15 years were chosen to review, document, and elucidate the developments on cell-seeded natural biomaterials for articular cartilage treatment in vivo . The parameters of the experimental designs and their results were all documented and presented. Considerations about the newly formed cartilage and the treatment of cartilage defects were discussed, along with difficulties arising when applying natural materials, research limitations, and tissue engineering approaches for hyaline cartilage regeneration. Impact statement: This review includes information from the literature and has never been complied before. The last 15 years were chosen to document and elucidate the developments on in vivo , articular cartilage treatment with cell-seeded natural biomaterials. The parameters of the experimental designs and their results were all documented and presented thoroughly. Natural materials overall seem to have a positive role in the recovery of cartilage defects. We expect that the knowledge from this review will be very useful to researchers organizing experimental protocols on critical analysis of advanced therapy medicinal products for human use.

In this study, we investigated the effect of oxygen tension on the expansion of ADMSCs and on their differentiation toward their chondrocytic phenotype, regenerating a lab-based cartilaginous tissue with superior characteristics. Controversial results with reference to MSCs that were cultured under different hypoxic levels, mainly in 2D culturing settings combined with or without other biochemical stimulus factors, prompted our team to study the role of hypoxia on MSCs chondrogenic differentiation within an absolute 3D environment. Specifically, we used 3D-printed honeycomb-like PCL matrices seeded with ADMSCs in the presence or absence of TGF and cultured with a prototype 3D cell culture device, which was previously shown to favor nutrient/oxygen supply, cell adhesion, and infiltration within scaffolds. These conditions resulted in high-quality hyaline cartilage that was distributed uniformly within scaffolds. The presence of the TGF medium was necessary to successfully produce cartilaginous tissues with superior molecular and increased biomechanical properties. Despite hypoxia’s beneficial effect, it was overall not enough to fully differentiate ADMSCs or even promote cell expansion within 3D scaffolds alone.