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In this study, zeolitic imidazolate framework 8 (ZIF-8) was coated on porous Ti6Al4V scaffolds, either bare or previously modified using hydroxyapatite (HA) or HA and gelatin (HAgel), via a growing single-step method in aqueous media using two contact times at 6 h and 24 h. The coated scaffolds termed ZIF-8@Ti, ZIF-8@HA/Ti, and ZIF-8@HAgel/Ti were characterized via scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), and molecular plasma-atomic emission spectroscopy (MP-AES). In order to assess the cell proliferation rate, the cytocompatibility of the scaffolds was evaluated in primary osteoblasts (hOBs) using alamarBlue assay, while the osteoconductivity was analyzed in hOBs using a real-time approach, evaluating the expression of secreted phosphoprotein 1 (SPP1). Osteopontin, which is the protein encoded by this gene, represents the major non-collagenous bone protein that binds tightly to HA. The scaffolds were shown to be non-cytotoxic based on hOB proliferation at all time points of analysis (24 h and 72 h). In hOB cultures, the scaffolds induced the upregulation of SPP1 with different fold changes. Some selected scaffolds were assayed in vitro for their antibacterial potential against Staphylococcus epidermidis; the scaffolds coated with ZIF-8 crystals, regardless of the presence of HA and gelatin, strongly inhibited bacterial adhesion to the materials and reduced bacterial proliferation in the culture medium, demonstrating the suitable release of ZIF-8 in a bioactive form. These experiments suggest that the innovative scaffolds, tested herein, provide a good microenvironment for hOB adhesion, viability, and osteoconduction with effective prevention of S. epidermidis adhesion.

Abstract Intercellular adhesion is a key function for epithelial cells. The fundamental mechanisms relying on epithelial cell adhesion have been partially uncovered. Hsa-microRNA-1249-3p (hsa-miR-1249-3p) plays a role in the epithelial mesenchymal transition in carcinoma cells, but its physiological function in epithelial cells is unknown. We aimed to investigate the role and molecular mechanisms of hsa-miR-1249-3p on epithelial cell functions. Hsa-miR-1249-3p was overexpressed in human epithelial cells and uterine cervical tissues, compared to cervical carcinoma cells and precancerous tissues, respectively. Hsa-miR-1249-3p was analyzed to verify its regulatory function on Homeobox A13 (HOXA13) target gene and its downstream cell adhesion gene β-catenin. Functional experiments indicated that hsa-miR-1249-3p inhibition prompted the mRNA and protein overexpression of HOXA13 which, in turn, led to the β-catenin protein expression. Moreover, hsa-miR-1249-3p inhibition induced a strong colony forming ability in epithelial cells, suggesting the miR involvement in cell adhesion machinery. These data indicate that hsa-miR-1249-3p regulates the expression of HOXA13 and its downstream cell adhesion gene β-catenin, possible resulting in cell adhesion modification in epithelial cells. This study will allow the set-up of further investigations aimed at exploring the relationship between the hsa-miR-1249-3p/HOXA13 axis and downstream cell adhesion genes.

IntroductionHuman polyomaviruses (HPyVs) cause persistent/latent infections in a large fraction of the population. HPyV infections may cause severe diseases in immunocompromised patients. Malawi polyomavirus (MWPyV) is the 10th discovered human polyomavirus (HPyV 10). MWPyV was found in stool samples of healthy children. So far, the few investigations carried out on HPyV 10 did not find an association with human disease.MethodsIn this study, to verify the putative association between MWPyV and human diseases, MWPyV seroprevalence was investigated in patients affected by i) lymphoproliferative disorders (LPDs) and ii) immune system disorders, i.e., autoimmune diseases (ADs), and in iii) healthy subjects. An indirect ELISA, employing virus-like particles (VLPs) to detect serum IgG antibodies against MWPyV/HPyV 10, was carried out. The study also revealed the prevalence of another polyomavirus, Merkel cell polyomavirus (MCPyV).ResultsSera from patients with distinct autoimmune diseases (n = 44; mean age 20 years) had a prevalence of MWPyV antibodies of 68%, while in patients with lymphoproliferative disorders (n = 15; mean age 14 years), subjected to bone marrow transplantation, the prevalence was 47%. In healthy subjects (n = 66; mean age 13 years), the prevalence of MWPyV antibodies was 67%. Our immunological investigation indicates that MWPyV/HPyV 10 seroconversion occurs early in life and MWPyV/HPyV 10 appears to be another polyomavirus ubiquitous in the human population. A significantly lower MWPyV antibody reactivity together with a lower immunological profile was detected in the sera of LPD patients compared with HS2 (*p < 0.05) (Fisher’s exact test). LPD and AD patients have a similar MCPyV seroprevalence compared with healthy subjects.DiscussionMWPyV seroprevalence indicates that this HPyV is not associated with lymphoproliferative and autoimmune diseases. However, the ability to produce high levels of antibodies against MWPyV appears to be impaired in patients with lymphoproliferative disorders. Immunological investigations indicate that MWPyV seroconversion occurs early in life. MCPyV appears to be a ubiquitous polyomavirus, like other HPyVs, in the human population.

Abstract Background Blood transfusions represent common medical procedures, which provide essential supportive therapy. However, these procedures are notoriously expensive for healthcare services and not without risk. The potential threat of transfusion-related complications, such as the development of pathogenic infections and the occurring of alloimmunization events, alongside the donor’s dependence, strongly limits the availability of transfusion units and represents significant concerns in transfusion medicine. Moreover, a further increase in the demand for donated blood and blood transfusion, combined with a reduction in blood donors, is expected as a consequence of the decrease in birth rates and increase in life expectancy in industrialized countries. Main body An emerging and alternative strategy preferred over blood transfusion is the in vitro production of blood cells from immortalized erythroid cells. The high survival capacity alongside the stable and longest proliferation time of immortalized erythroid cells could allow the generation of a large number of cells over time, which are able to differentiate into blood cells. However, a large-scale, cost-effective production of blood cells is not yet a routine clinical procedure, as being dependent on the optimization of culture conditions of immortalized erythroid cells. Conclusion In our review, we provide an overview of the most recent erythroid cell immortalization approaches, while also describing and discussing related advancements of establishing immortalized erythroid cell lines.

Abstract The epigenetic role of microRNAs is established at both physiological and pathological levels. Dysregulated miRNAs and their targets appear to be a promising approach for innovative anticancer therapies. In our previous study, circulating miR-197-3p tested dysregulated in workers ex-exposed to asbestos (WEA). Herein, an epigenetic investigation on this circulating miRNA was carried out in sera from malignant pleural mesothelioma (MPM) patients. MiR-197-3p was quantified in MPM (n = 75) sera and comparatively analyzed to WEA (n = 75) and healthy subject (n = 75) sera, using ddPCR and RT-qPCR techniques. Clinicopathological characteristics, occupational, non-occupational information and overall survival (OS) were evaluated in correlation studies. MiR-197-3p levels, analyzed by ddPCR, were significantly higher in MPM than in WEA cohort, with a mean copies/µl of 981.7 and 525.01, respectively. Consistently, RT-qPCR showed higher miR-197-3p levels in sera from MPM with a mean copies/µl of 603.7, compared to WEA with 336.1 copies/µl. OS data were significantly associated with histologic subtype and pleurectomy. Circulating miR-197-3p is proposed as a new potential biomarker for an early diagnosis of the MPM onset. Indeed, miR-197-3p epigenetic investigations along with chest X-ray, computed tomography scan and spirometry could provide relevant information useful to reach an early and effective diagnosis for MPM.

The aim of this study was to investigate whether the presence of Staphylococcus aureus (SA) producing the Panton–Valentine leukocidin (PVL) affects the outcome of Prosthetic Joint Infection (PJI). Patients with acute and chronic PJI sustained by SA were prospectively enrolled at the orthopedic unit of “Casa di Cura Santa Maria Maddalena”, from January 2019 to October 2021. PJI diagnosis was reached according to the diagnostic criteria of the International Consensus Meeting on PJI of Philadelphia. Synovial fluid obtained via joint aspirations was collected in order to isolate SA. The detection of PVL was performed via real-time quantitative PCR (RT-qPCR). The outcome assessment was performed using the criteria of the Delphi-based International Multidisciplinary Consensus. Twelve cases of PJI caused by SA were included. Nine (75%) cases were acute PJI treated using debridement, antibiotic and implant retention (DAIR); the remaining three (25%) were chronic PJI treated using two-stage (n = 2) and one-stage revision (n = 1), respectively. The SA strains that tested positive for PVL genes were 5/12 (41.6%,). Treatment failure was documented in three cases of acute PJI treated using DAIR, all supported by SA–PVL strains (p < 0.045). The remaining two cases were chronic PJI treated with a revision arthroplasty (one and two stage, respectively), with a 100% eradication rate in a medium follow-up of 24 months. Although a small case series, our study showed a 100% failure rate in acute PJI, probably caused by SA PVL-producing strains treated conservatively (p < 0.04). In this setting, toxin research should guide radical surgical treatment and targeted antibiotic therapy.

Bone defects in maxillofacial regions lead to noticeable deformity and dysfunctions. Therefore, the use of biomaterials/scaffolds for maxillofacial bone regrowth has been attracting great interest from many surgical specialties and experts. Many approaches have been devised in order to create an optimal bone scaffold capable of achieving desirable degrees of bone integration and osteogenesis. Osteogenesis represents a complex physiological process involving multiple cooperating systems. A tight relationship between the immune and skeletal systems has lately been established using the concept of “osteoimmunology,” since various molecules, particularly those regulating immunological and inflammatory processes, are shared. Inflammatory mediators are now being implicated in bone remodeling, according to new scientific data. In this study, a profiler PCR array was employed to evaluate the expression of cytokines and chemokines in human adipose derived-mesenchymal stem cells (hASCs) cultured on porous hydroxylapatite (HA)/Collagen derived Bio-Oss®/Avitene scaffolds, up to day 21. In hASCs grown on the Bio-Oss®/Avitene biomaterial, 12 differentially expressed genes (DEGs) were found to be up-regulated, together with 12 DEG down-regulated. Chemokine CCL2, which affects bone metabolism, tested down-regulated. Interestingly, the Bio-Oss®/Avitene induced the down-regulation of pro-inflammatory inter-leukin IL-6. In conclusion, our investigation carried out on the Bio-Oss®/Avitene scaffold indicates that it could be successfully employed in maxillofacial surgery. Indeed, this composite material has the advantage of being customized on the basis of the individual patients favoring a novel personalized medicine approach.

Abstract Tissue engineering‐based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite‐collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3‐year follow‐up. Among DEGs, osteogenesis‐related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in “in vitro” experiments with hASCs.

Multiple sclerosis (MS) is a neurological disease characterized by immune dysregulations. Different viruses may act as MS triggering agents. MS patients respond differently to distinct viruses. The aim of our study is to verify the association between the polyomavirus BKPyV and MS, together with other neurological diseases, through the investigation of serum IgG antibodies against the virus. Sera were from patients affected by MS and other neurologic diseases, both inflammatory (OIND) and noninflammatory (NIND). Control sera were from healthy subjects (HS). Samples were analyzed for IgG antibodies against BKPyV with an indirect ELISA with synthetic peptides mimicking the viral capsid protein 1 (VP1) antigens. As control, ELISAs were carried out to verify the immune response against the Epstein-Barr virus (EBV) of patients and controls. In addition, we assessed values for total IgG in each experimental groups. A significant lower prevalence of IgG antibodies against BKPyV VP 1 epitopes, together with a low titer, was detected in sera from MS patients and other inflammatory neurologic diseases than HS. In MS patients and OIND and NIND groups, the EBV-antibody values and total IgG did not differ from HS. Experimental data indicate that patients affected by neurological diseases, including MS, are poor responders to BKPyV VP 1 antigens, thus suggesting specific immunologic dysfunctions for this polyomavirus. Our findings are relevant in understanding the immune reactions implicated in neurological disorders.

Merkel cell polyomavirus (MCPyV), a small DNA tumor virus, has been detected in Merkel cell carcinoma (MCC) and in normal tissues. Since MCPyV infection occurs in both MCC-affected patients and healthy subjects (HS), innovative immunoassays for detecting antibodies (abs) against MCPyV are required. Herein, sera from HS were analyzed with a novel indirect ELISA using two synthetic peptides mimicking MCPyV capsid protein epitopes of VP1 and VP2. Synthetic peptides were designed to recognize IgGs against MCPyV VP mimotopes using a computer-assisted approach. The assay was set up evaluating its performance in detecting IgGs anti-MCPyV on MCPyV-positive (n=65) and -negative (n=67) control sera. Then, the ELISA was extended to sera (n=548) from HS aged 18-65 yrs old. Age-specific MCPyV-seroprevalence was investigated. Performance evaluation indicated that the assay showed 80% sensitivity, 91% specificity and 83.9% accuracy, with positive and negative predictive values of 94.3% and 71%, respectively. The ratio expected/obtained data agreement was 86%, with a Cohen’s kappa of 0.72. Receiver-operating characteristic (ROC) curves analysis indicated that the areas under the curves (AUCs) for the two peptides were 0.82 and 0.74, respectively. Intra-/inter-run variations were below 9%. The overall prevalence of serum IgGs anti-MCPyV in HS was 62.9% (345/548). Age-specific MCPyV-seroprevalence was 63.1% (82/130), 56.7% (68/120), 64.5% (91/141), and 66.2% (104/157) in HS aged 18-30, 31-40, 41-50 and 51-65 yrs old, respectively (p>0.05). Performance evaluation suggests that our indirect ELISA is reliable in detecting IgGs anti-MCPyV. Our immunological data indicate that MCPyV infection occurs asymptomatically, at a relatively high prevalence, in humans.

Over the past decades, age-related pathologies have increased abreast the aging population worldwide. The increased age of the population indicates that new tools, such as biomaterials/scaffolds for damaged tissues, which display high efficiency, effectively and in a limited period of time, for the regeneration of the body's tissue are needed. Indeed, scaffolds can be used as templates for three-dimensional tissue growth in order to promote the tissue healing stimulating the body's own regenerative mechanisms. In tissue engineering, several types of biomaterials are employed, such as bioceramics including calcium phosphates, bioactive glasses, and glass–ceramics. These scaffolds seem to have a high potential as biomaterials in regenerative medicine. In addition, in conjunction with other materials, such as polymers, ceramic scaffolds may be used to manufacture composite scaffolds characterized by high biocompatibility, mechanical efficiency and load-bearing capabilities that render these biomaterials suitable for regenerative medicine applications. Usually, bioceramics have been used to repair hard tissues, such as bone and dental defects. More recently, in the field of soft tissue engineering, this form of scaffold has also shown promising applications. Indeed, soft tissues are continuously exposed to damages, such as burns or mechanical traumas, tumors and degenerative pathology, and, thereby, thousands of people need remedial interventions such as biomaterials-based therapies. It is known that scaffolds can affect the ability to bind, proliferate and differentiate cells similar to those of autologous tissues. Therefore, it is important to investigate the interaction between bioceramics and somatic/stem cells derived from soft tissues in order to promote tissue healing. Biomimetic scaffolds are frequently employed as drug-delivery system using several therapeutic molecules to increase their biological performance, leading to ultimate products with innovative functionalities. This review provides an overview of essential requirements for soft tissue engineering biomaterials. Data on recent progresses of porous bioceramics and composites for tissue repair are also presented.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) and their derivative enhanced green fluorescent protein (eGFP)-hBMSCs were employed to evaluate an innovative hybrid scaffold composed of granular hydroxylapatite and collagen hemostat (Coll/HA). The cellular morphology/cytoskeleton organization and cell viability were investigated by immunohistochemistry (IHC) and AlamarBlue metabolic assay, respectively. The expression of osteopontin and osteocalcin proteins was analyzed by IHC and ELISA, whereas osteogenic genes were investigated by quantitative PCR (Q-PCR). Cell morphology of eGFP-hBMSCs was indistinguishable from that of parental hBMSCs. The cytoskeleton architecture of hBMSCs grown on the scaffold appeared to be well organized, whereas its integrity remained uninfluenced by the scaffold during the time course. Metabolic activity measured in hBMSCs grown on a biomaterial was increased during the experiments, up to day 21 (p < 0.05). The biomaterial induced the matrix mineralization in hBMSCs. The scaffold favored the expression of osteogenic proteins, such as osteocalcin and osteopontin. In hBMSC cultures, the scaffold induced up-regulation in specific genes that are involved in ossification process (BMP2/3, SPP1, SMAD3, and SP7), whereas they showed an up-regulation of MMP9 and MMP10, which play a central role during the skeletal development. hBMSCs were induced to chondrogenic differentiation through up-regulation of COL2A1 gene. Our experiments suggest that the innovative scaffold tested herein provides a good microenvironment for hBMSC adhesion, viability, and osteoinduction. hBMSCs are an excellent in vitro cellular model to assay scaffolds, which can be employed for bone repair and bone tissue engineering.

Human papillomaviruses (HPVs) are small DNA tumor viruses that mainly infect mucosal epithelia of anogenital and upper respiratory tracts. There has been progressive demand for more analytical assays for HPV DNA quantification. A novel droplet digital PCR (ddPCR) method was developed to simultaneously detect and quantify HPV DNA from different HPV types. DdPCR was initially tested for assay sensitivity, accuracy, specificity as well as intra- and inter-run assay variation employing four recombinant plasmids containing HPV16, HPV18, HPV11, and HPV45 DNAs. The assay was extended to investigate/quantify HPV DNA in Cervical Intraepithelial Neoplasia (CIN, n = 45) specimens and human cell lines (n = 4). DdPCR and qPCR data from clinical samples were compared. The assay showed high accuracy, sensitivity and specificity, with low intra-/inter- run variations, in detecting/quantifying HPV16/18/11/45 DNAs. HPV DNA was detected in 51.1% (23/45) CIN DNA samples by ddPCR, whereas 40% (18/45) CIN tested HPV-positive by qPCR. Five CIN, tested positive by ddPCR, were found to be negative by qPCR. In CIN specimens, the mean HPV DNA loads determined by ddPCR were 3.81 copy/cell (range 0.002–51.02 copy/cell), whereas 8.04 copy/cell (range 0.003–78.73 copy/cell) by qPCR. DdPCR and qPCR concordantly detected HPV DNA in SiHa, CaSki and Hela cells, whereas HaCaT tested HPV-negative. The correlation between HPV DNA loads simultaneously detected by ddPCR/qPCR in CINs/cell lines was good (R2 = 0.9706, p < 0.0001). Our data indicate that ddPCR is a valuable technique in quantifying HPV DNA load in CIN specimens and human cell lines, thereby improving clinical applications, such as patient management after primary diagnosis of HPV-related lesions with HPV-type specific assays.

In the present work, hydroxyapatite (HA) nanoparticles doped with Mg2+, Sr2+, and Zn2+ ions are developed by wet neutralization method and then sintered at 1,250°C to obtain bulk consolidated materials. Physicochemical and microstructural analyses show that the presence of doping ions in the HA structure induced the formation of βTCP as secondary phase, during the sintering process, and we found that this effect is depending on the stability of the various doping ions in the hydroxyapatite lattice itself. We also found that the formation of βTCP as secondary phase, in turn, confines the grain growth of HA induced by the high-temperature sintering process, thus leading to a strong increase of the flexural strength of the bulk materials, according to Hall-Petch-like law. Furthermore, we found that the doping ions enter also in the structure of the βTCP phase; besides the grain growth confinement, also the solubility and ion release ability of the final materials were enhanced. In addition to ameliorate the mechanical performance, the described phenomena also activate multiple biofunctionalities: (i) ability to upregulate various genes involved in the osteogenesis, as obtained by human adipose stem cells culture and evaluated by array technology; (ii) enhanced resistance to the adhesion and proliferation of Gram+ and Gram– bacterial strains. Hence, our results open a perspective for the use of sintered multiple ion-doped HA to develop ceramic biodevices, such as plates, screws, or other osteosynthesis media, with enhanced strength, osteointegrability, and the ability to prevent post-surgical infections.

The regeneration of bone fractures, resulting from trauma, osteoporosis or tumors, is a major problem in our super-aging society. Bone regeneration is one of the main topics of concern in regenerative medicine. In recent years, stem cells have been employed in regenerative medicine with interesting results due to their self-renewal and differentiation capacity. Moreover, stem cells are able to secrete bioactive molecules and regulate the behavior of other cells in different host tissues. Bone regeneration process may improve effectively and rapidly when stem cells are used. To this purpose, stem cells are often employed with biomaterials/scaffolds and growth factors to accelerate bone healing at the fracture site. Briefly, this review will describe bone structure and the osteogenic differentiation of stem cells. In addition, the role of mesenchymal stem cells for bone repair/regrowth in the tissue engineering field and their recent progress in clinical applications will be discussed.