Mesenchymal stem cells (MSCs) isolated from peripheral blood (PB) are gaining increasing attention among both researchers and clinicians, representing a promising alternative to traditional stem cell sources such as bone marrow and adipose tissue. Their therapeutic use offers key advantages like high accessibility, minimally invasive collection and autologous nature of the graft. Together with the ability to differentiate into multiple cell types, PB-MSC features make them ideal candidates for a wide range of clinical applications in the fields of regenerative medicine, tissue engineering and immunotherapy. While the therapeutic potential of PB-derived MSCs is more and more acknowledged, several challenges still need to be overcome regarding, for example, their isolation, expansion, and differentiation efficiency. For clinical translation, PB-MSC administration may be specifically indicated when minimally invasive autologous cell therapies are required, especially when bone marrow harvest is not recommended due to age, comorbidity, or prior surgeries. Conversely, PB-MSC auto-transplant should be avoided in patients suffering from hematological malignancies, active infections, or bone marrow disorders, where circulating stem cell populations may be altered, or their isolation poses safety risks. In this Perspective, we highlight recent advances in PB-MSC isolation and characterization, discuss their bioengineering integration into osteochondral repair strategies, and examine their immunomodulatory potential in osteoarthritis (OA). We propose that PB-MSCs integrate regenerative and immunomodulatory properties, positioning them as a promising, autologous cell source for next-generation personalized regenerative therapies. However, their clinical translation critically depends on the development of reproducible, GMP-compliant expansion protocols and validated potency assays.

IntroductionHemophilic arthropathy (HA) is a major complication of hemophilia, being caused by recurrent joint bleeding (hemarthrosis) which leads to iron accumulation in joints and subsequent damage to the articular cartilage (AC) and subchondral bone. Current treatments slow osteochondral degradation but do not promote cartilage regeneration. Tissue engineering offers a promising alternative for addressing AC damage in HA.MethodsThis study developed bio-hybrid scaffolds based on polyvinyl alcohol (PVA) combined with decellularized human AC to enhance bioactivity and mechanical support. AC was harvested from cadaveric donors and decellularized via a detergent-enzymatic protocol, with efficacy confirmed through DNA quantification, histomorphometric analyses and Scanning Electron Microsopy (SEM). The resulting decellularized AC matrix was then homogenized in acetic acid for combination with PVA. Bio-hybrid scaffolds were produced using two fabrication methods: a) cross-linking of a freeze-dried AC layer onto the PVA hydrogel, forming a double-layer structure and b) mechanical incorporation of homogenized decellularized AC into the PVA matrix. Bio-hybrid scaffold morphology, porosity, and mechanical properties were analyzed, and cytocompatibility was evaluated by seeding HM1-SV40 human mesenchymal stem cells (MSC) and assessing cell adhesion and growth by MTT assays and SEM after 7 and 14 days.ResultsQuality control studies on decellularized AC confirmed efficient cell/DNA removal and correct preservation of ECM components. Regarding the bio-hybrid scaffolds, SEM ultrastructural analysis revealed distinct surface roughness and porosity depending on the fabrication method. Compressive tests showed increased stiffness with higher PVA concentrations, while the addition of AC resulted in stiffness reduction, especially in the bilayer configuration. In parallel, consolidation tests revealed that PVA/AC_blend scaffolds showed similar short-term behavior to non-composite materials, while PVA/AC_bilayer exhibited a larger initial force drop, eventually aligning with PVA scaffolds at equilibrium. Cytocompatibility tests demonstrated that acellular AC matrix enhances PVA bioactivity, with PVA/AC_bilayer scaffolds better supporting cell adhesion and growth compared to PVA/AC_blend scaffolds.DiscussionThe findings underscore the potential of PVA/AC bio-hybrid scaffolds for cartilage regeneration in hemophilic patients affected by HA. These scaffolds combine mechanical integrity with enhanced cytocompatibility, representing a novel strategy in the context of tissue-engineered therapies for joint repair.

Hydrogel-based delivery systems have emerged as a promising strategy to enhance the therapeutic efficacy of mesenchymal stromal cells (MSCs) in regenerative medicine. These biomimetic platforms provide a three-dimensional microenvironment that recapitulates key features of native extracellular matrix, supporting MSC viability, retention, and function upon transplantation. Beyond acting as passive carriers, hydrogels can be engineered with tunable biochemical and mechanical properties to modulate MSC behavior, including their differentiation potential, immunomodulatory activity, and paracrine signaling. Recent advances include the development of “smart” hydrogels responsive to physiological stimuli, enabling controlled release of encapsulated cells or bioactive molecules in response to local cues. Preclinical studies have demonstrated enhanced tissue repair in diverse pathological contexts, including musculoskeletal, cardiovascular, gastrointestinal, dermal, and neural injuries. Importantly, translation to clinical settings is being facilitated by the use of xeno-free, good manufacturing practices (GMP)-compliant components such as platelet derivatives and synthetic polymers. Selected early-phase clinical trials support the feasibility, safety, and therapeutic potential of MSC-laden hydrogels, although further studies are required to optimize delivery parameters and regulatory compliance. This review summarizes current progress in hydrogel-MSC systems across application areas, emphasizing design principles, preclinical outcomes, and translational challenges, with the aim of guiding future developments in stem cell-based tissue regeneration.

Abstract Background COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) represents the biggest global health emergency in recent decades. The host immune response to SARS-CoV-2 seems to play a key role in disease pathogenesis and clinical manifestations, with Natural Killer (NK) lymphocytes being among the targets of virus-induced regulation. Methods This study performed a single-cell multi-omics analysis of transcripts and proteins of NK lymphocytes in COVID-19 patients, for the characterization of the innate immunological response to infection. NK cells were isolated from peripheral blood samples collected from adult subjects divided into 3 study groups: (1) non-infected subjects (Naïve group, n = 3), (2) post COVID-19 convalescent subjects (Healed group, n = 3) and (3) patients that were vaccinated against SARS-CoV-2 (Vaccine group, n = 3). Cells were then analysed by the BD Rhapsody System for the single-cell multi-omics investigation of transcriptome and membrane proteins. Results The bioinformatic analysis identified 5 cell clusters which differentially expressed gene/protein markers, defining NK cell subsets as “Active NK cells” and “Mature NK cells”. Calculating the relative proportion of each cluster within patient groups, more than 40% of the Naïve group cell population was found to belong to Mature NKs, whereas more than 75% of the Vaccine group cell population belonged to the cluster of Active NKs. Regarding the Healed group, it seemed to show intermediate phenotype between Active and Mature NK cells. Differential expression of specific genes, proteins and signaling pathways was detected comparing the profile of the 3 experimental groups, revealing a more activated NK cell phenotype in vaccinated patients versus recovered individuals. Conclusions The present study detected differential expression of NK cell markers in relation to SARS-CoV-2 infection and vaccine administration, suggesting the possibility to identify key molecular targets for clinical-diagnostic use of the individual response to viral infection and/or re-infection.

EDITORIAL article Front. Bioeng. Biotechnol., 30 November 2023Sec. Preclinical Cell and Gene Therapy Volume 11 - 2023 | https://doi.org/10.3389/fbioe.2023.1333281

Severe tracheal injuries that cannot be managed by mobilization and end-to-end anastomosis represent an unmet clinical need and an urgent challenge to face in surgical practice; within this scenario, decellularized scaffolds (eventually bioengineered) are currently a tempting option among tissue engineered substitutes. The success of a decellularized trachea is expression of a balanced approach in cells removal while preserving the extracellular matrix (ECM) architecture/mechanical properties. Revising the literature, many Authors report about different methods for acellular tracheal ECMs development; however, only few of them verified the devices effectiveness by an orthotopic implant in animal models of disease. To support translational medicine in this field, here we provide a systematic review on studies recurring to decellularized/bioengineered tracheas implantation. After describing the specific methodological aspects, orthotopic implant results are verified. Furtherly, the only three clinical cases of compassionate use of tissue engineered tracheas are reported with a focus on outcomes.

In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.

BACKGROUND:Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. Adult stem cells isolated from various sources (i.e., cord blood, bone marrow, adipose tissue) are being considered as a realistic option due to their well-documented therapeutic potentials. Our previous studies standardized a method to isolate circulating multipotent cells (CMCs) that are able to sustain long term in vitro culture and differentiate towards mesodermal lineages.METHODS:In this work, long-term cultures of CMCs were stimulated to study in vitro neuronal and myogenic differentiation. After induction, cells were analysed at different time points. Morphological studies were performed by scanning electron microscopy and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. For myogenic plasticity study, CMCs were transplanted into in vivo model of chemically-induced muscle damage.RESULTS:After neurogenic induction, CMCs showed characteristic dendrite-like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of CMCs under stimulation with potassium chloride and the secretion of noradrenalin confirmed their ability to acquire a functional phenotype. In parallel, the myogenic potential of CMCs was confirmed by their ability to form syncytium-like structures in vitro and express myogenic markers both at early and late phases of differentiation. Interestingly, in a rat model of bupivacaine-induced muscle damage, CMCs integrated within the host tissue taking part in tissue repair.CONCLUSION:Overall, collected data demonstrated long-term cultured CMCs retain proliferative and differentiative potentials suggesting to be a good candidate for cell therapy.

S100B is a calcium-binding protein mainly expressed by astrocytes, but also localized in other definite neural and extra-neural cell types. While its presence in biological fluids is widely recognized as a reliable biomarker of active injury, growing evidence now indicates that high levels of S100B are suggestive of pathogenic processes in different neural, but also extra-neural, disorders. Indeed, modulation of S100B levels correlates with the occurrence of clinical and/or toxic parameters in experimental models of diseases such as Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, muscular dystrophy, multiple sclerosis, acute neural injury, inflammatory bowel disease, uveal and retinal disorders, obesity, diabetes and cancer, thus directly linking the levels of S100B to pathogenic mechanisms. In general, deletion/inactivation of the protein causes the improvement of the disease, whereas its over-expression/administration induces a worse clinical presentation. This scenario reasonably proposes S100B as a common therapeutic target for several different disorders, also offering new clues to individuate possible unexpected connections among these diseases.

OBJECTIVE:S100 proteins are demonstrated to exert a protective role in the gastrointestinal tract. In the present study, we investigated whether S100B protein, that is typically expressed by enteroglial cells, is detectable in feces and could be a useful noninvasive indicator of gut chronic inflammation.PATIENTS AND METHODS:This clinical prospective study included n=48 patients suffering Crohn's disease (CD) or ulcerative colitis (UC) and non IBD-controls. The clinical disease activity was evaluated using Harvey-Bradshaw or Mayo Score Index while the diagnosis of IBD was defined based on standard endoscopic and histological criteria. S100B and calprotectin were extracted and analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits.RESULTS:Unlike calprotectin, S100B was significantly decreased in both CD and UC compared to non IBD-patients. The strongest quantitative alterations of S100B were detected concomitantly with signs of active or quiescent disease, including high/normal expression of fecal calprotectin, mucosal damage/cryptitis, mucin depletion and inflammatory infiltrate, as defined by endoscopic evaluation and histological analysis. At the onset of disease and under no Infliximab-based therapy, the lowest was detected suggesting that S100B in feces could have a potential diagnostic value for IBD.CONCLUSIONS:Testing for S100B and calprotectin could be a useful screening tool to better predict IBD activity.

SARS-CoV-2 infection shows a wide-ranging clinical severity, requiring prognostic markers. We focused on S100B, a calcium-binding protein present in biological fluids, being a reliable biomarker in disorders having inflammatory processes as common basis and RAGE as main receptor. Since Covid-19 is characterized by a potent inflammatory response also involving RAGE, we tested if S100B serum levels were related to disease severity. Serum samples (n = 74) were collected from hospitalized SARS-CoV-2 positive patients admitted to Covid center. Illness severity was established by admission clinical criteria and Covid risk score. Treatment protocols followed WHO guidelines available at the time. Circulating S100B was determined by ELISA assay. Statistical analysis used Pearson's χ2 test, t-Test, and ANOVA, ANCOVA, Linear Regression. S100B was detected in serum from Covid-19 patients, significantly correlating with disease severity as shown both by the level of intensity of care (p < 0.006) as well by the value of Covid score (Multiple R-squared: 0.3751); the correlation between Covid-Score and S100B was 0.61 (p < 0.01). S100B concentration was associated with inflammation markers (Ferritin, C-Reactive Protein, Procalcitonin), and organ damage markers (Alanine Aminotransferase, Creatinine). Serum S100B plays a role in Covid-19 and can represent a marker of clinical severity in Sars-CoV-2 infected patients.

The crosstalk between human gut microbiota and intestinal wall is essential for the organ's homeostasis and immune tolerance. The gut microbiota plays a role in healthy and pathological conditions mediated by inflammatory processes or by the gut-brain axes, both involving a possible role for S100B protein as a diffusible cytokine present not only in intestinal mucosa but also in faeces. In order to identify target proteins for a putative interaction between S100B and the microbiota proteome, we developed a bioinformatics workflow by integrating the interaction features of known domains with the proteomics data derived from metataxonomic studies of the gut microbiota from healthy and inflammatory bowel disease (IBD) subjects. On the basis of the microbiota composition, proteins putatively interacting with S100B domains were in fact found, both in healthy subjects and IBD patients, in a reduced number in the latter samples, also exhibiting differences in interacting domains occurrence between the two groups. In addition, differences between ulcerative colitis and Crohn disease samples were observed. These results offer the conceptual framework for where to investigate the role of S100B as a candidate signalling molecule in the microbiota/gut communication machinery, on the basis of interactions differently conditioned by healthy or pathological microbiota.

Nerve wrapping improves neurorrhaphy outcomes in case of peripheral nerve injuries (PNIs). The aim of this preclinical study was to assess the efficacy of two novel biodegradable wraps made of a synthetic 1% oxidized polyvinyl alcohol (OxPVA) and a natural leukocyte-fibrin-platelet membrane (LFPm) versus the commercial product NeuraWrap. After rats sciatic nerve transection and neurorrhaphy, the wraps were implanted and compared for functional outcome, by sciatic function index assessment; structural characteristics, by histological/immunohistochemical analysis; ultrastructural features, by transmission electron microscopy. Moreover, a morphometric study was also performed and collagen distribution was observed by Second Harmonic Generation microscopy. After 12 weeks from implantation, all wraps assured nerve function recovery; no scar tissue/neuromas were visible at dissection. LFPm wraps were completely resorbed, while residues of OxPVA and NeuraWrap were observed. In all groups, biocompatibility was confirmed by the absence of significant inflammatory infiltrate. According to histological/immunohistochemical analysis and morphometric findings, OxPVA and LFPm wraps were both effective in preserving nerve integrity. These results assess that bioengineered OxPVA and LFPm wraps successfully guarantee favorable lesion recovery after PNI/neurorrhaphy and, in future, may be considered an interesting alternative to the commercial NeuraWrap.

Nowadays, research in Tissue Engineering and Regenerative Medicine is focusing on the identification of instructive scaffolds to address the requirements of both clinicians and patients to achieve prompt and adequate healing in case of injury. Among biomaterials, hemocomponents, and in particular Platelet-rich Fibrin matrices, have aroused widespread interest, acting as delivery platforms for growth factors, cytokines and immune/stem-like cells for immunomodulation; their autologous origin and ready availability are also noteworthy aspects, as safety- and cost-related factors and practical aspects make it possible to shorten surgical interventions. In fact, several authors have focused on the use of Platelet-rich Fibrin in cartilage and tendon tissue engineering, reporting an increasing number of in vitro, pre-clinical and clinical studies. This narrative review attempts to compare the relevant advances in the field, with particular reference being made to the regenerative role of platelet-derived growth factors, as well as the main pre-clinical and clinical research on Platelet-rich Fibrin in chondrogenesis and tenogenesis, thereby providing a basis for critical revision of the topic.

Functionalized synthetic conduits represent a promising strategy to enhance peripheral nerve regeneration by guiding axon growth while delivering therapeutic neurotrophic factors. In this work, hollow nerve conduits made of polyvinyl alcohol partially oxidized with bromine (OxPVA_Br2) and potassium permanganate (OxPVA_KMnO4) were investigated for their structural/biological properties and ability to absorb/release the ciliary neurotrophic factor (CNTF). Chemical oxidation enhanced water uptake capacity of the polymer, with maximum swelling index of 60.5% ± 2.5%, 71.3% ± 3.6% and 19.5% ± 4.0% for OxPVA_Br2, OxPVA_KMnO4 and PVA, respectively. Accordingly, hydrogel porosity increased from 15.27% ± 1.16% (PVA) to 62.71% ± 8.63% (OxPVA_Br2) or 77.50% ± 3.39% (OxPVA_KMnO4) after oxidation. Besides proving that oxidized PVA conduits exhibited mechanical resistance and a suture holding ability, they did not exert a cytotoxic effect on SH-SY5Y and Schwann cells and biodegraded over time when subjected to enzymatic digestion, functionalization with CNTF was performed. Interestingly, higher amounts of neurotrophic factor were detected in the lumen of OxPVA_Br2 (0.22 ± 0.029 µg) and OxPVA_KMnO4 (0.29 ± 0.033 µg) guides rather than PVA (0.11 ± 0.021 µg) tubular scaffolds. In conclusion, we defined a promising technology to obtain drug delivery conduits based on functionalizable oxidized PVA hydrogels.

The wound healing is a complex process wherein inflammation, proliferation and regeneration evolve according to a spatio‐temporal pattern from the activation of coagulation cascade to the formation of a plug clot including fibrin matrix, blood‐borne cells and cytokines/growth factors. Creating environments conducive to tissue repair, the haemoderivatives are commonly proposed for the treatment of hard‐to‐heal wounds. Here, we explored in vitro the intrinsic regenerative potentialities of a leucocyte‐ and platelet‐rich fibrin product, known as CPL ‐ MB , defining the stemness grade of cells sprouting from the haemoderivative. Using highly concentrated serum‐based medium to simulate wound conditions, we isolated fibroblast‐like cells ( CPL ‐ CMC s) adhering to plastic and showing stable in vitro propagation, heterogeneous stem cell expression pattern, endothelial adhesive properties and immunomodulatory profile. Due to their blood derivation and expression of CXCR 4, CPL ‐ CMC s have been suggested to be immature cells circulating in peripheral blood at quiescent state until activation by both coagulation event and inflammatory stimuli such as stromal‐derived factor 1/ SDF 1. Expressing integrins ( CD 49f, CD 103), vascular adhesion molecules ( CD 106, CD 166), endoglin ( CD 105) and remodelling matrix enzymes ( MMP 2, MMP 9, MMP 13), they showed a transendothelial migratory potential besides multipotency. Taken together, our data suggested that a standardized, reliable and economically feasible blood product such as CPL ‐ MB functions as an artificial stem cell niche that, under permissive conditions, originate ex vivo immature cells that could be useful for autologous stem cell‐based therapies.

Autologous platelet-rich hemocomponents have emerged as potential biologic tools for regenerative purpose, but their therapeutic efficacy still remains controversial. This work represents the characterization study of an innovative autologous leukocyte-fibrin-platelet membrane (LFPm), which we prepared according to a novel protocol involving multiple cycles of apheresis. The high content in fibrinogen gave to our hemocomponent the appearance of a manipulable and suturable membrane with high elasticity and deformation capacity. Moreover, being highly enriched with platelets, leukocytes, and monocytes/macrophages, the LFPm sustained the local release of bioactive molecules (platelet derived growth factor, vascular endothelial growth factor, interleukin-10, and tumour necrosis factor alpha). In parallel, the evaluation of stemness potential highlighted also that the LFPm contained cells expressing pluripotency and multipotency markers both at the messenger ribonucleic acid (NANOG, SOX2, THY1, NT5E, and ENG) and surface-protein level (CD44high /CD73+ /CD34+ /CD117+ /CD31+ ). Finally, biodegradation analysis interestingly showed a good stability of the membrane for at least 3 weeks in vitro and 1 week in vivo. In both cases, biodegradation was associated with progressive exposure of fibrin scaffold, loss/migration of cellular elements, and release of growth factors. Overall, collected evidence could shed some light on the regenerative effect that LFPms may exert after the autologous implant on a defect site.

In the last decades, the use of multipotent adult stem cells has paved way for the identifica- tion of new therapeutic approaches for the treatment of acquired or degenerative pathologies. Monogenic diseases such as Haemophilia A are also ideal candidates for the treatment by these emerging therapies, which may success in correcting alterated protein expression resulting from genetic mutation. Recently, adipose tissue has proven to serve as an accessible and rich source of adult stem cells with multidifferentiative properties, suitable for regenerative medical appli- cations. Although they have already been studied for these purposes, adipose-derived stem cells are still poorly considered for Haemophilia A cell therapy and few research work have focused on their capacity to produce coagulation factor VIII when properly stimulated. This work has studied the in vitro differentiation of an adipose-derived stem cell line towards the endothelial lineage, considered to be responsible for coagulation factor production. At this end, adipose-derived stem cells were cultured into differentiation medium enriched with endothe- lial growth factors up to 21 days. After the induction treatment, changes in cell morphology, migratory capacity and specific gene/protein expression were evaluated by optical microsco- py, transwell migration assay and qPCR/Dot Blot analysis, respectively. Our data highlighted that, already after 7 days of induction treatment, cell cultures showed to change their fibro- blastoid morphology, starting to form capillary-like structures. They significantly responded to the chemotactic stimuli of endothelial growth factors, and up-regulated the expression of spe- cific endothelial markers (CD34, PDGFRα, VE-cadherin, CD31, vWF) as the time of induction increased. Most important, we gave the first evidence that adipose stem cells are capable of secreting factor VIII after specific endothelial stimulation and without any need for gene trans- fection. On the base of that, stem cells from adipose tissue seem to be promising candidate for Haemophila A cell therapy.

Pediatric Short Bowel Syndrome is a rare malabsorption disease occurring because of massive surgical resections of the small intestine. To date, the issues related to current strategies including intestinal transplantation prompted the attention towards tissue engineering (TE). This work aimed to develop and compare two composite scaffolds for intestinal TE consisting of a novel hydrogel, that is, oxidized polyvinyl alcohol (OxPVA), cross-linked with decellularized intestinal wall as a whole (wW/OxPVA) or homogenized (hW/OxPVA). A characterization of the supports was performed by histology and Scanning Electron Microscopy and their interaction with adipose mesenchymal stem cells occurred by MTT assay. Finally, the scaffolds were implanted in the omentum of Sprague Dawley rats for 4 weeks prior to being processed by histology and immunohistochemistry (CD3; F4/80; Ki-67; desmin; α-SMA; MNF116). In vitro studies proved the effectiveness of the decellularization, highlighting the features of the matrices; moreover, both supports promoted cell adhesion/proliferation even if the wW/OxPVA ones were more effective (p < 0.01). Analysis of explants showed a continuous and relatively organized tissue wall around the supports with a connective appearance, such as myofibroblastic features, smooth muscle, and epithelial cells. Both scaffolds, albeit with some difference, were promising; nevertheless, further analysis will be necessary.