Cellular responses to deproteinized bovine bone mineral biofunctionalized with bone-conditioned medium.

OBJECTIVES The aim of the study was to investigate whether the osteoinductive properties of bone-conditioned medium (BCM) harvested from cortical bone chips within a clinically relevant short-term period can enhance the biologic characteristics of deproteinized bovine bone mineral (DBBM) in vitro. MATERIALS AND METHODS To assess the biofunctionalization of DBBM, the adhesive, proliferative, and differentiation properties of mesenchymal stromal ST2, pre-osteoblastic MC3T3-E1, and primary bone-derived cells grown on BCM-coated DBBM were examined by crystal violet staining of adherent cells, BrdU ELISA, and qRT-PCR, respectively. RESULTS BCM extracted within 20 min or 24 h in either Ringer's solution (BCM-RS) or RS mixed with autologous serum (BCM-RS + S) increased the adhesive properties of all three cell types seeded on DBBM. The 20-min BCM-RS preparation appeared more potent than the 24-h preparation. BCM-RS made within 20 min or 24 h had strong pro-proliferative effects on all cell types grown on DBBM. RS + S alone exhibited a considerable pro-proliferative effect, suggesting an impact of the serum on cellular growth. DBBM coated with BCM-RS or BCM-RS + S, made within 20 min or 24 h each, caused a significant induction of osteogenic differentiation marker expression with a higher potency of the BCM-RS + S. Finally, a strong additive effect of fresh bone chips combined with BCM-coated DBBM on the osteogenic differentiation of the three cell types was observed. CONCLUSIONS Altogether, the data strongly support the biofunctionalization of DBBM with BCM extracted within a clinically relevant time window of 20 min. CLINICAL RELEVANCE Pre-activation of non-osteoinductive biomaterials with BCM, prepared from autologous bone chips during a guided bone regeneration (GBR) procedure, bears the potential of an optimal treatment modality for bone defects in daily practice

Preparation of hybrid samples for scanning electron microscopy (SEM) coupled to focused ion beam (FIB) analysis: A new way to study cell adhesion to titanium implant surfaces.

The study of the intimate connection occurring at the interface between cells and titanium implant surfaces is a major challenge for dental materials scientists. Indeed, several imaging techniques have been developed and optimized in the last decades, but an optimal method has not been described yet. The combination of the scanning electron microscopy (SEM) with a focused ion beam (FIB), represents a pioneering and interesting tool to allow the investigation of the relationship occurring at the interface between cells and biomaterials, including titanium. However, major caveats concerning the nature of the biological structures, which are not conductive materials, and the physico-chemical properties of titanium (i.e. color, surface topography), require a fine and accurate preparation of the sample before its imaging. Hence, the aim of the present work is to provide a suitable protocol for cell-titanium sample preparation before imaging by SEM-FIB. The concepts presented in this paper are also transferrable to other fields of biomaterials research

Discovery and characterization of heterogeneous and multipotent fibroblast populations isolated from excised cleft lip tissue.

BACKGROUND Regularly discarded lip tissue obtained from corrective surgeries to close the cleft lip represents an easily accessible and rich source for the isolation of primary fibroblasts. Primary fibroblasts have been described to show compelling similarities to mesenchymal stem cells (MSCs). Hence, cleft lip and palate (CLP) lip-derived fibroblasts could be thought as an intriguing cell source for personalized regenerative therapies in CLP-affected patients. METHODS Initially, we thoroughly characterized the fibroblastic nature of the lip-derived mesenchymal outgrowths by molecular and functional assays. Next, we compared their phenotype and genotype to that of bone marrow-mesenchymal stem cells (BM-MSCs) and of human lung-derived fibroblasts WI38, by assessing their morphology, surface marker expression, trilineage differentiation potential, colony-forming (CFU) capacity, and immunomodulation property. Finally, to better decipher the heterogeneity of our CLP cultures, we performed a single cell clonal analysis and tested expanded clones for surface marker expression, as well as osteogenic and CFU potential. RESULTS We identified intriguingly similar phenotypic and genotypic properties between CLP lip fibroblasts and BM-MSCs, which makes them distinct from WI38. Furthermore, our own data in combination with the complex anatomy of the lip tissue indicated heterogeneity in our CLP cultures. Using a clonal analysis, we discovered single cell-derived clones with increased levels of the MSC markers CD106 and CD146 and clones with variabilities in their commitment to differentiate into bone-forming cells and in their potential to form single cell-derived colonies. However, we were not able to gain clones possessing superior MSC-like capacities when compared to the heterogeneous parental CLP population. Additionally, all clones could still generate contractile forces and retained robust levels of the fibroblast specific marker FSP1, which was not detectable in BM-MSCs. CONCLUSIONS Our results suggest that we isolate heterogeneous populations of fibroblasts from discarded CLP lip tissue, which show a prominently multipotent character in their entirety avoiding the need for elaborate subpopulation selections in vitro. These findings suggest that CLP lip fibroblasts might be a novel potential cell source for personalized regenerative medicine of clinical benefit for CLP patients

Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes.

Variants within the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic Cleft Lip/Palate (CLP) cases. IRF6 plays a vital role in the regulation of the proliferation/differentiation balance in keratinocytes and is involved in wound healing and migration. Since a fraction of CLP patients undergoing corrective cleft surgery experience wound healing complications, IRF6 represents an interesting candidate gene linking the two processes. However, Irf6 function has been mainly studied in mice and knowledge on IRF6 in human cells remains sparse. Here, we aimed to elucidate the role of IRF6 in human postnatal skin- and oral mucosa-derived keratinocytes. To do so, we applied CRISPR/Cas9 to ablate IRF6 in two TERT-immortalized keratinocyte cultures, which we used as model cell lines. We show that IRF6 controls the appearance of single cells and colonies, with the latter being less cohesive in its absence. Consequently, IRF6 knockout keratinocytes often moved as single cells instead of a collective epithelial sheet migration but maintained their epithelial character. Lack of IRF6 triggered severe keratinocyte differentiation defects, which were already apparent in the stratum spinosum and extended to the stratum corneum in 3D organotypic skin cultures, while it did not alter their growth rate. Finally, proteomics revealed that most of the differentially expressed proteins in the absence of IRF6 could be associated with differentiation, cell-cell adhesion as well as immune response. Our data expand the knowledge on IRF6 in human postnatal keratinocytes, which will help to better understand IRF6-related pathologies

Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro.

BACKGROUND/PURPOSE Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. MATERIALS AND METHODS We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. RESULTS Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. CONCLUSION These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application

Aptamer-Mediated Selective Protein Affinity to Improve Scaffold Biocompatibility

Protein adsorption on surfaces occurs shortly after scaffold insertion. This process is of pivotal importance to achieve therapeutic success in tissue engineering (TE), and favorable proteins should be adsorbed at the interface without unfolding to preserve their structure and function. Protein misfolding at the interface is a common phenomenon, which can impair cell adhesion and scaffold colonization. Many efforts have been done to improve scaffold biocompatibility by ameliorating protein adsorption, but with poor results. In the present chapter, we propose the use of a novel class of molecules, aptamers, to improve scaffold biocompatibility. Aptamers are small, single stranded oligonucleotides, which specifically bind to a target molecule: they work as antibodies, but without many of the drawbacks associated to the use of antibodies. We propose to immobilize aptamers on scaffolds to retain specific proteins, acting as docking points to guide cell activity. In particular, we show the results obtained by enriching different polymeric scaffolds with aptamers against human fibronectin, a naturally abundant protein in tissues, which plays a pivotal role in cell adhesion. We demonstrate that scaffold enrichment with aptamers lead to a better colonization of the substrate from cells. The results we obtained pave the way to the possibility of further investigating the role of aptamers as useful molecules to improve scaffold biocompatibility in the contest of tissue engineering

Role of HBcAb Positivity in Increase of HIV-RNA Detectability after Switching to a Two-Drug Regimen Lamivudine-Based (2DR-3TC-Based) Treatment: Months 48 Results of a Multicenter Italian Cohort

: The aim of this study was to evaluate whether the presence of anti-hepatitis B (HBV) c antibodies (HBcAb positivity) could influence the control of HIV viremia in patients living with HIV (PLWH) who switch to two-drug antiretroviral therapy (2DR) containing lamivudine (3TC) (2DR-3TC-based). A retrospective multicentre observational study was conducted on 160 PLWH switching to the 2DR-3TC-based regimen: 51 HBcAb-positive and 109 HBcAb-negative patients. The HBcAb-positive PLWH group demonstrated a significantly lower percentage of subjects with HIV viral suppression with target not detected (TND) at all time points after switching (24th month: 64.7% vs. 87.8%, p < 0.0001; 36th month 62.7% vs. 86.8%, p = 0.011; 48th month 57.2% vs. 86.1%, p = 0.021 of the HBcAb-positive and HBcAb-negative groups, respectively). Logistic regression analysis showed that the presence of HBcAb positivity (OR 7.46 [95% CI 2.35-14.77], p = 0.004) could favour the emergence of HIV viral rebound by nearly 54% during the entire study follow-up after switching to 2DR-3TC

A Living Cell Repository of the Cranio-/Orofacial Region to Advance Research and Promote Personalized Medicine

The prevalence of congenital anomalies in newborns is estimated to be as high as 6%, many of which involving the cranio-/orofacial region. Such malformations, including several syndromes, are usually identified prenatally, at birth, or rarely later in life. The lack of clinically relevant human cell models of these often very rare conditions, the societal pressure to avoid the use of animal models and the fact that the biological mechanisms between rodents and human are not necessarily identical, makes studying cranio-/orofacial anomalies challenging. To overcome these limitations, we are developing a living cell repository of healthy and diseased cells derived from the cranio-/orofacial region. Ultimately, we aim to make patient-derived cells, which retain the molecular and genetic characteristics of the original anomaly or disease in vitro, available for the scientific community. We report our efforts in establishing a human living cell bank derived from the cranio-/orofacial region of otherwise discarded tissue samples, detail our strategy, processes and quality checks. Such specific cell models have a great potential for discovery and translational research and might lead to a better understanding and management of craniofacial anomalies for the benefit of all affected individuals

COVID-19 Infection in Children and Infants: Current Status on Therapies and Vaccines

Since the beginning in December 2019, the SARS-CoV-2 outbreak appeared to affect mostly the adult population, sparing the vast majority of children who only showed mild symptoms. The purpose of this investigation is to assess the status on the mechanisms that give children and infants this variation in epidemiology compared to the adult population and its impact on therapies and vaccines that are aimed towards them. A literature review, including in vitro studies, reviews, published guidelines and clinical trials was performed. Clinical trials concerned topics that allowed a descriptive synthesis to be produced. Four underlying mechanisms were found that may play a key role in providing COVID-19 protection in babies. No guidelines are available yet for therapy due to insufficient data; support therapy remains the most used. Only two vaccines are approved by the World Health Organization to be used in children from 12 years of age, and there are currently no efficacy or safety data for children below the age of 12 years. The COVID-19 clinical frame infection is milder in children and adolescents. This section of the population can act as vectors and reservoirs and play a key role in the transmission of the infection; therefore, vaccines are paramount. More evidence is required to guide safely the vaccination campaign

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage