Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

Bone regenerative medicine is a clinical approach combining live osteoblast progenitors, such as mesenchymal stromal cells (MSCs), with a biocompatible scaffold that can integrate into host bone tissue and restore its structural integrity. Over the last few years, many tissue engineering strategies have been developed and thoroughly investigated; however, limited approaches have been translated to clinical application. Consequently, the development and clinical validation of regenerative approaches remain a centerpiece of investigational efforts towards the clinical translation of advanced bioengineered scaffolds. The aim of this review was to identify the latest clinical trials related to the use of scaffolds with or without MSCs to regenerate bone defects. A revision of the literature was performed in PubMed, Embase, and Clinicaltrials.gov from 2018 up to 2023. Nine clinical trials were analyzed according to the inclusion criteria: six presented in the literature and three reported in Clinicaltrials.gov. Data were extracted covering background trial information. Six of the clinical trials added cells to scaffolds, while three used scaffolds alone. The majority of scaffolds were composed of calcium phosphate ceramic alone, such as β-tricalcium phosphate (TCP) (two clinical trials), biphasic calcium phosphate bioceramic granules (three clinical trials), and anorganic bovine bone (two clinical trials), while bone marrow was the primary source of the MSCs (five clinical trials). The MSC expansion was performed in GMP facilities, using human platelet lysate (PL) as a supplement without osteogenic factors. Only one trial reported minor adverse events. Overall, these findings highlight the importance and efficacy of cell–scaffold constructs in regenerative medicine under different conditions. Despite the encouraging clinical results obtained, further studies are needed to assess their clinical efficacy in treating bone diseases to optimize their application.

Scaffolds associated with mesenchymal stem cell (MSC) derivatives, such as extracellular vesicles (EVs), represent interesting carriers for bone regeneration. This systematic review aims to analyze in vitro and in vivo studies that report the effects of EVs combined with scaffolds in bone regeneration. A methodical review of the literature was performed from PubMed and Embase from 2012 to 2020. Sixteen papers were analyzed; of these, one study was in vitro, eleven were in vivo, and four were both in vitro and in vivo studies. This analysis shows a growing interest in this upcoming field, with overall positive results. In vitro results were demonstrated as both an effect on bone mineralization and proangiogenic ability. The interesting in vitro outcomes were confirmed in vivo. Particularly, these studies showed positive effects on bone regeneration and mineralization, activation of the pathway for bone regeneration, induction of vascularization, and modulation of inflammation. However, several aspects remain to be elucidated, such as the concentration of EVs to use in clinic for bone-related applications and the definition of the real advantages.

A Deep Molecular Response (DMR), defined as a BCR::ABL1 transcript at levels ≤ 0.01% by RT-qPCR, is the prerequisite for the successful interruption of treatment among patients with Chronic Myeloid Leukemia (CML). However, approximately 50% of patients in Treatment-Free Remission (TFR) studies had to resume therapy after their BCR::ABL1 transcript levels rose above the 0.1% threshold. To improve transcript detection sensitivity and accuracy, transcript levels can be analyzed using digital PCR (dPCR). dPCR increases BCR::ABL1 transcript detection sensitivity 10–100 fold; however, its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapses may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally silent. Flow cytometry can be used to identify and quantify circulating bone marrow Ph+ LSCs CD34+/CD38− co-expressing CD26 (dipeptidylpeptidase-IV). To date, the significance of circulating Ph+ LSCs in TFR is unclear. The aim of this work is to compare and examine the values obtained using the three different methods of detecting minimal residual disease (MRD) in CML at RNA (RT-qPCR and dPCR) and LSC (flowcytometry) levels among patients in TFR or exhibiting a DMR. The twenty-seven patients enrolled received treatment with either imatinib (12), dasatinib (6), nilotinib (7), bosutinib (1), or interferon (1). Twelve patients were in TFR, while the rest exhibited a DMR. The TFR patients had stopped therapy for less than 1 year (3), <3 years (2), 6 years (6), and 17 years (1). Blood samples were collected and tested using the three methods at the same time. Both d-PCR and LSCs showed higher sensitivity than RT-qPCR, exhibiting positive results in samples that were undetectable using RT-qPCR (17/27). None of the patients tested negative with d-PCR; however, 23/27 were under the threshold of 0.468 copies/μL, corresponding to a stable DMR. The results were divided into quartiles, and the lowest quartiles defined the lowest MRD. These data were strongly correlated in 15/27 patients, corresponding to almost half of the TFR patients. Indeed, the TFR patients, some lasting up to 17 years, corresponded to the lowest detectable DMR categories. To the best of our knowledge, this is the first attempt to analyze and compare DMRs in a CML population using standard (RT-qPCR) and highly sensitive (dPCR and LSCs) methods.

One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and −9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system.